June 14～17, 2019, Tai’an, China

· Other Abstracts·

OA-4 Thioredoxin-1 and Geranylgeranylacetone Resist Neurotoxicity of Amyloid-β

BAI Li-ping1，2，LI Ye2，ZHOU Xiao-shuang2，ZHANG Xian-wen2，SUN Bo2，YAN Chen2，DENG Ru-hua2，BAI Jie2，*
1. Faculty of Environmental Science and Engineering，Kunming University of Science and Technology，Kunming，650500，China
2. Medical School，Kunming University of Science and Technology，Kunming，650500，China

【ABSTRACT】Objective：Alzheimer’s disease （AD） is the most common neurodegenerative disorder which is characterized by amyloid-β （Aβ） aggradation in the brain and impairment of cognitive function.Thioredoxin-1 （Trx-1） is a redox regulating protein，and plays roles in resisting the oxidative stress and protecting neurons.Our previous study found that Trx-1 improved the cognitive function of Parkinson’s Disease （PD） mice.Geranylgeranylacetone （GGA） is an antiulcer drug and induces the expression of Trx-1 in vivo and in vitro.However，whether Trx-1 improves cognitive functions in mice of APP/PS1 or GGA protects SH-SY5Y cells from cytotoxicity induced by Aβ is still unknown.The objective of present is to investigate the roles of Trx-1 and GGA in inhibiting neurotoxicity of Aβ.Methods：We used MTT assay to test the cell viability induced by Aβ（25-35）and western blot to detect the expression of Trx-1 in SH-SY5Y cells.Trx-1 overexpression transgenic mice were hybridized with APP/PS1 transgenic mice to get control，Trx-1，Tx-1/APP/PS1 and APP/PS1 mice.Then we used Morris water maze，high plus maze and object recognition test to detect the cognitive function of different kinds of mice.We also used RT-PCR and western blot to test the mRNA level and expression of Trx-1，APP，PS1 and Aβ.Results：In our present study，we demonstrated that Aβ（25-35）decreased the cell viability and the expression of Trx-1 in SH-SY5Y cells.The cell viability and the expression of Trx-1 were reversed by GGA.Our results showed that the escape latency in APP/PS1 mice was longer when compared with the Trx-1/APP/PS1 mice in Morris water maze and high plus maze.Whereas navigational experiments in Morris water maze result showed that the total number of crossings and the percentage of time spent in the target quadrant were significantly decreased in APP/PS1 mice when compared to Trx-1/APP/PS1 mice.Object recognition test the discrimination index was significantly decreased in APP/PS1 mice when compared with Trx-1/APP/PS1 mice.The mRNA levels and the expression of APP，PS1 and Aβ were decreased in Trx-1/APP/PS1 mice when compared to APP/PS1 mice.Conclusion：These results suggest that GGA protects SH-SY5Y cells from cytotoxicity induced by Aβ（25-35）and restored the expression of Trx-1.Trx-1 overexpression improves cognitive function of APP/PS1 mice.Trx-1 may be a potential therapeutic target for the clinical management of AD.

【KEY WORDS】thioredoxin-1；Alzheimer’s disease；geranylgeranylacetone；amyloid-β；APP/PS1；cognitive function

Corresponding author：Medical Faculty，Kunming University of Science and Technology，No.727 Jingming South Road，Kunming 650500，China，E-mail：jiebai662001@126.com

OA-5 Isolation and Purification of Pruritic Compounds from Yam (Dioscoreae Rhizoma)

SUN Yu-ling，YANG Yan，QIAN Lin-nan，GUAN Dong-lang，JIAN Tun-yu，TANG Zong-xiang*
School of Medicine and Life Sciences，Nanjing University of Chinese Medicine，Nanjing，210023，China

【ABSTRACT】Backgrounds：Chinese yam is a traditional Chinese medicine.It has many benefits for people，such as antidiarrhea，anti-inflammation，antidiabetic，hypocholesterolemia，antioxidation，antitumor，and immunomodulation.The mucus of the fresh yam could induce itch in the skin，however，the pruritus compound in yam is very unclear.Objective：1） To isolate and purify the pruritus compounds from fresh Chinese yam；2） To test the activity of the pruritus compound-allantoin；3） To study the cellular mechanism of allantoin-induced itching.Methods：We extracted allantoin crystal from fresh Chinese yam using suspension drop method.Multidimensional chromatographic separation methods including membrane filtration，ion exchange chromatography，C18 reverse phase chromatography，silica column and preparation liquid phase，mass spectrometry analysis were used to purify the active compounds.Scratch numbers of mice were used to evaluate the degree of pruritus induced by allantoin in behavior tests.We cultured DRG neurons from Pirt-GCaMP3 heterozygotes examined their responses to allantoin using calcium imaging techniques.We also examined the electrophysiological characteristics of the neurons induced by allantoin using whole cell recording.Results：We got the allantoin crystal by simple ethanol extraction.The content of allantoin extracted from yam （origin from Jiaozuo，Henan） is about 3.567 mg·g-1.Our results show that allantoin could induce more scratch numbers in mice than control group.Allantoin also directly activated DRG neurons to induce calcium influx and inward current.Conclusion：This is the first time to obtain allantoin crystal from fresh yam.Allantoin is widely found in many natural plants，but the effect of allantoin on DRG neurons and animal itching behavior was the first time.Our research shows that allantoin not only activated small DRG neurons，but also caused itch in mice.Allantoin could induce the itching behavior of mice，which not only help us understand the cause of yam to induce itch，more importantly，we found a natural itch causing compound.This discovery will have implications for our future research on the mechanism of itch.

【KEY WORDS】allantoin；calcium influx；inward current；dorsal horn ganglion；itch

Corresponding author：Tang Zong-Xiang，E-mail：zongxiangtang1@163.com

OA-6 β-arrestin 2 Negatively Regulates NOD2 Signaling Pathway through Association with TRAF6 in Microglia after Cerebral Ischemia/Reperfusion Injury

CHEN Lin1#，KONG Ling-jun1#，WEI Xin-bing1，WANG Yi-meng1，WANG Bing2，ZHANG Xiu-mei1，SUN Jinpeng3，LIU Hui-qing1*
1. Department of Pharmacology，School of Basic Medical Sciences，Shandong University，Jinan，250012，China
2. Department of Emergency，The people’s Hospital of Huaiyin，Jinan，250021，China
3. Key Laboratory Experimental Teratology of the Ministry of Education，Department of Biochemistry and Molecular Biology，School of Medicine，Shandong University，Jinan，250012，China

【ABSTRACT】 We previously reported that nucleotide binding oligomerization domaincontaining protein （NOD） 2 was involved in the inflammatory responses to cerebral ischemia/reperfusion （I/R） insult.However，the mechanism by which NOD2 participates in brain ischemic injury and the regulation of NOD2 in the process are still obscure.Increased β-arrestin 2（ARRB2） expression was observed in microglia following cerebral I/R in wild type mice besides the upregulation of NOD2 and TRAF6.Stimulation of NOD2 by muramyl dipeptide （MDP）in BV2 cells induced the activation of NF-κB by the phosphorylation of p65 subunit and the degradation of IκBα.Meanwhile，the protein level of COX-2，the protein expression and activity of MMP-9 were significantly increased in BV2 cells after administration of MDP.Furthermore，overexpression of ARRB2 significantly suppressed the inflammation induced by MDP，silence of ARRB2 significantly enhanced the inflammation induced by MDP in BV2 cells.In addition，we observed endogenous interaction of TRAF6 and ARRB2 after stimulation of MDP or cerebral I/R insult，indicating ARRB2 negatively regulates NOD2-triggered inflammatory signaling pathway by associating with TRAF6 in microglia after cerebral I/R injury.Finally，the in vivo study clearly confirmed that ARRB2 negatively regulated NOD2-induced inflammatory response，as ARRB2 deficiency exacerbated stroke outcomes and aggravated the NF-κB signaling pathway induced by NOD2 stimulation after cerebral I/R injury.These findings revealed ARRB2 negatively regulated NOD2 signaling pathway through the association with TRAF6 in cerebral I/R injury.

【KEY WORDS】cerebral ischemia/reperfusion；β-arrestin 2；NOD2；inflammation

#Lin Chen and Lingjun Kong contributed equally to this work

Corresponding author：Huiqing Liu，Ph.D，E-mail：liuhuiqing@sdu.edu.cn

OA-7 Effect of Paeoniflorin on Chronic Depression Model Mice based on Endoplasmic Reticulum Stress

DOU Wen-xiao，XU Rong-rong，MU Cheng-sen，ZHANG Xin-yue，GUO Ting-ting，LI Na，WANG Yan，XU Xiao-yan1*
Shandong First Medical University & Shandong Academy of Medical Sciences，Taian，271016，China

【ABSTRACT】Objective：To observe the behavioral effects of paeoniflorin on chronic unpredictable depression model mice，and to explore its antidepressant-like effects and possible mechanisms.Methods：KM mice were randomly divided into control group，model group，paeoniflorin group and fluoxetine group.Model group，paeoniflorin group and fluoxetine group were given chronic unpredictable stimulation for 28 days to establish chronic depression model.The fluoxetine group was intragastrically administered with fluoxetine 10 mg·kg-1，and the paeoniflorin group was intragastrically administered with paeoniflorin 50 mg·kg-1and 100 mg·kg-1，respectively.The control group and the model group were intragastrically administered with the same amount of normal saline for 21 days.The effects of paeoniflorin on the behavior of mice with chronic depression were observed by detecting the sucrose preference，forced swimming test and tail suspension test.Western Blot was used to detect the express of brain-derived neurotrophic factor （BDNF） and glucose-regulated protein 78 （GRP78） in hippocampus and cortex.Results：Compared with the model group，paeoniflorin significantly improved the sucrose intake of mice，prolonged the immobility time in forced swimming and tail suspension experiments （P＜0.05，P＜0.01），and significantly increased the expression of BDNF protein （P＜0.05），reducing the expression of GRP78 protein.Conclusion：Paeoniflorin has antidepressant effect on chronic depression mice，and its mechanism may be related to the inhibition of excessive endoplasmic reticulum stress.

【KEY WORDS】paeoniflorin；chronic unpredictable depression model mice；endoplasmic reticulum stress

OA-8 Seselin Targeted Jak2 to Ameliorate Inflammation

FENG Li-li1；2，WU Xu-dong1，XU Qiang1，*，HAN Feng2，*
1. State Key Laboratory of Pharmaceutical Biotechnology，School of Life Sciences，Nanjing University，Nanjing，210023，China
2. Key Laboratory of Cardiovascular & Cerebrovascular Medicine，School of Pharmacy，Nanjing Medical University，Nanjing，211166，China

【ABSTRACT】Objective：Sepsis is a serious clinical condition with high mortality rate.Emerging evidence proved that inhibition of inflammation was beneficial for the treatment of sepsis.Here，we evaluated the anti-inflammation activity of seselin in septic mice and further detected underlying molecular mechanism.Methods：The in vivo therapeutic effect of seselin was evaluated in septic C57BL/6 mice.Sepsis was induced by cecal ligation and puncture or injection of LPS.The in virto anti-inflammation activity of seselin was determined in LPS and IFN-γ stimulated macrophages.The mechanism was investigated by co-immunoprecipitation，cellular thermal shift assay and molecular docking.Results：In vivo study showed that seselin obviously ameliorated cecal ligation and puncture induced sepsis in mice.In septic mice lung tissue and cultured macrophages，seselin downregulated level of pro-inflammatory factors and activity of STAT1 and p65，the master signal pathway molecules for proinflammatory macrophages polarization.Importantly，adoptive transferred with seselin pretreated BMDM significantly descended system proinflammatory factors in LPS challenged mice.The further mechanism was that seselin targeted Jak2 to block interaction with IFN-R and downstream STAT1.Conclusion：Our study suggested the anti-inflammatory activity of seselin and identified its molecular target，Jak2.These results indicated the potential possibility of seselin in treatment of inflammatory disease via blocking the proinflammatory phenotype of macrophages.

【KEY WORDS】anti-inflammation；Jak2 inhibition；sepsis；seselin；macrophage

OA-9 新型化合物 YY-21 快速抗抑郁機(jī)制研究

郭飛1*，張兵2*，李揚(yáng)1
1.中國(guó)科學(xué)院上海藥物研究所，上海，201203，中國(guó)
2.同濟(jì)大學(xué)醫(yī)學(xué)院，上海，201204，中國(guó)

【摘要】 目的：抑郁癥以及抑郁-躁狂雙向情感障礙等精神類疾病，嚴(yán)重影響了人們健康和生活質(zhì)量。經(jīng)典的臨床一線藥物主要以單胺類藥物為主，但存在起效慢，個(gè)體差異大，總體治愈率低以及副作用等多種不利因素，因此開(kāi)發(fā)出快速起效，療效顯著、低毒副作用的新機(jī)制藥物是精神疾病的重中之重。方法：我們從古籍中發(fā)現(xiàn)中藥知母百合湯有顯著地抗抑郁作用，進(jìn)一步經(jīng)有效成分分析和純化，發(fā)現(xiàn)了知母皂苷及經(jīng)過(guò)結(jié)構(gòu)改造的新化合物YY-21。并應(yīng)用多種抑郁癥模型動(dòng)物進(jìn)行藥效評(píng)價(jià)，并研究藥理作用集中。結(jié)果：研究發(fā)現(xiàn)YY-21 抗抑郁癥作用顯著，最快一周起效，比臨床一線藥物氟西汀（百憂解）快3-4 周。對(duì)其藥理機(jī)制研究發(fā)現(xiàn)，YY-21 可能是非單胺類靶點(diǎn)藥物，對(duì)興奮性谷氨酸神經(jīng)遞質(zhì)的NMDA 受體抑制作用顯著。進(jìn)一步研究顯示YY-21 增強(qiáng)突觸前谷氨酸神經(jīng)遞質(zhì)傳遞，誘發(fā)興奮性突觸后電位的長(zhǎng)時(shí)程增強(qiáng)效應(yīng)（LTP）。胞內(nèi)信號(hào)通路的研究發(fā)現(xiàn)YY-21 促進(jìn)神經(jīng)生長(zhǎng)因子 BDNF 釋放，增加mTOR 和GSK3 的磷酸化水平，促進(jìn)突觸生長(zhǎng)相關(guān)蛋白的表達(dá)（如PSD-95，Synapcin）等。結(jié)論：研究結(jié)果提示YY-21 的快速抗抑郁作用可能與增強(qiáng)皮層谷氨酸神經(jīng)遞質(zhì)傳遞，改善大腦前額葉皮層的高級(jí)認(rèn)知功能有關(guān)。

【關(guān)鍵詞】抑郁癥；抗抑郁藥；突觸傳遞；YY-21

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目（No.31200771，No.81030065，No.81274055，No.3137106601，No.3117101101）* 共同第一作者

通訊作者：李揚(yáng)，E-mail：liyang@simm.ac.cn

OA-10 Resting-State Functional Connectivity of the Hippocampus in Suicide Ideal Patients with Major Depressive Disorder

HE Can-can1，GONG Liang1，XIE Chun-ming1，2，*，ZHANG Zhi-jun1，2
1. Department of Neurology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，210009，China
2. Department of Psychology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，210009，China

【ABSTRACT】 Objective：Suicide is highly-prevalent and the most serious outcome in major depressive disorder （MDD），yet the disturbances in neural functional system that confer suicide risk remain obscure.In this study，we seek to investigate the neural functional differences between healthy participants with suicidal ideal （SI） MDD patients.Methods：Seventy-seven healthy controls （HC） and 124 well-matched MDD patients with different degrees of SI were recruited.Based on suicide factors of 17-items Hamilton Depression Scale （HAMD-17），We divided MDD patients into four groups，MDD_0 （n=55），MDD patients without SI；MDD_1 （n=41），MDD patients with mild SI，MDD_2 （n=20），MDD patients with moderate SI，MDD_3 （n=8），MDD patients with severe SI.Bilateral hippocampus were selected to construct hippocampus memory functional connectivity networks （HFCN）.One-way ANCOVA was applied to investigate the difference in HFCN between different level of SI patients with MDD and HC.Furthermore，the correlation analyses were conducted to identify the correlation between HFCN and HAMD-17 scores in the SI groups.Results：Compared with CN subjects，MDD patients showed severer SI.We found significant group differences on the bilateral HFCN in bilateral supramarginal gyrus（SMG），right middle prefrontal cortex （mPFC），orbital frontal cortex （OFC），angular cortex，as well as left middle temporal gyrus （MTG），superior parietal gyrus （SPG），and precuneus.Interestingly，an inverted U-shaped modulation was showed in the right mPFC （control system），right IPG，left SPG and SMG，while U-shaped modulations were found in the right hippocampus.The correlation analysis showed a significant correlation between the HAMD-17 scores and functional connectivity of the right hippocampus with the right mPFC area in the SI groups（r= -2.12，P=0.018）.Conclusion：Our study manifested a nonlinear modulation of interacting between suicide ideal and depression on brain function.These findings expand our understanding of the functional neural basis of different level of SI in MDD patients and will help to reveal the pathophysiology underlying suicidality in patients with MDD.

【KEY WORDS】major depressive disorder；suicide ideal；hippocampus；neural circuit；functional connectivity

Fig.1 Group differences of right hippocampus functional connectivity network （HFCN）

Corresponding authors：Xie Chun-ming M.D.&Ph.D，E-mail：chmxie@163.com

OA-11 Identification of MiR-9 Mediating the Effects of Maltreat in Childhood on Depression Using Brain Connectivity

HE Can-can1，ZAN Wang1，2，QING Wang1，XIE Chun-ming1，2，*，ZHANG Zhi-jun1，2
1 Department of Neurology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，210009，China
2 Department of Psychology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，210009，China

【ABSTRACT】Objective：MicroRNA 9 （miR-9） has been suggested to be involved in the pathophysiology of major depressive disorder （MDD） via regulate the major triggers of depression.However，it is unclear how miR-9 dysregulation mediates the effects of neural circuits associated with emotional regulation in MDD patients.Methods and materials：Forty unmedicated MDD patients and 34 demographically-matched healthy controls （HC） were recruited and conducted a comprehensive neuropsychological assessment，resting-state functional magnetic resonance imaging （R-fMRI） scan，and the miR-9 level from the peripheral blood was obtained.Bilateral amygdala were selected to construct the amygdala functional connectivity （AFC）network.Multivariate linear regression analysis was performed to investigate the neural substrates of miR-9，depression，and environment factors，which reflected by the Childhood Trauma events Questionnaires （CTQ），as well as their interactive effects on the AFC network in MDD patients.In addition，mediation analysis was employed to explore whether intrinsic AFC network mediates the association among CTQ，miR-9 and depression severity in MDD patients.Support vector classifier （SVC） model of machine learning was used to distinguish the MDD patients from HC.Results：MiR-9 level was significantly increased in MDD subjects，compared to HC.Higher miR-9 levels in MDD patients are negatively correlated to Hamilton Depression Scale （HAMD-17）scores and CTQ scores.Interestingly，interactive effects among CTQ，miR-9，and HAMD-17，on bilateral AFC networks in MDD patients，were primarily located in fronto-limbic network.These identified brain regions linked with increased miR-9 levels were also associated with severer depression symptoms and maltreat in childhood.Furthermore，mediation analysis suggested that amygdala connectivity strength involved in the fronto-limbic system mediated the relationship among CTQ，miR-9 and depression severity in MDD patients.Particularly，SVC model of miR-9 for MDD diagnosis represented a good power of differentiating mild and moderate MDD patients from HC subjects （an area under the curve of 0.82）.Conclusion：We demonstrated that miR-9 played a crucial role in the occurrence of depression associated with adverse environmental factors.Also，miR-9 dysregulation in MDD patients was associated with brain dysfunction in fronto-limbic network （the key network for emotional regulation and memory），which could mediate the relationship among adverse environmental factors，miR-9，and depression.Notably，miR-9 may consider as a potential biomarker for distinguishing mild and moderate MDD from HC.These results deepen our understanding of the pathophysiology of MDD，and provide new insight into the objective index of early diagnosis of depression.

Fig.1 Neural basis of miR-9 and HAMD-17 with regard to the amygdala functional connectivity （AFC） networks in MDD patients

Corresponding authors：Xie Chun-ming M.D.&Ph.D.，E-mail：chmxie@163.com

OA-12 Learning and Memory Impairment and Abnormal Blood-Brain Barrier Function in Rats Induced by Increased Blood Pressure Variability

JIA Xiao-li，WANG Wei，ZHOU Yan-meng，TAN Rui，ZHANG Fang-fang，ZHOU Li-jun，MIAO Shen，JI Wei，ZHU De-rong，ZHAO Xiao-min*，ZHANG Han-ting
Institute of Pharmacology，Shandong First Medical University & Shandong Academy of Medical Sciences，Taian，271016，China

【ABSTRACT】Objective：Clinical reports have shown that increased blood pressure variability can lead to decreased learning and memory，but the mechanism is still unclear.Based on the dysfunction of blood-brain barrier is one of the mechanisms of learning and memory disorder，we hypothesize that the mechanism of learning and memory impairment caused by elevated blood pressure variability is related to abnormal blood-brain barrier function.Method：The rat model of increased blood pressure variability was established by sinoaortic denervation （SAD）in rats.Morris Water Maze was used to evaluate learning and memory ability of rats.Then the blood-brain barrier function was evaluated by the permeability of Evans blue and FITC-dextran.Results：Compared with sham-operated （Sham） rats，the escape latency of Morris Water Maze was significantly extended in rats two，four，eight and sixteen weeks after SAD（P＜0.05，P＜0.01）.Similarly，the blood-brain barrier permeability in SAD group was markedly increased than that in Sham group （P＜0.05，P＜0.01） from two weeks to sixteen weeks after SAD.Additionally，Western blot result showed that，compared with Sham group，occludin expression was lower in SAD group （P＜0.05，P＜0.01）.Conclusion：Increased blood pressure variability leads to learning and memory dysfunction in rats and this dysfunction may be associated with the impaired blood-brain barrier function.

【KEY WORDS】blood pressure variability；rat；learning and memory；blood-brain barrier

Acknowledgements：This work was supported by the Natural Science Foundation of Shandong Province of China（ZR2017MH048），the Foundation of Overseas Distinguished Taishan Scholars of Shandong Province of China （to H-TZ）and Collaborative Innovation Center for Research and Development of Traditional Chinese Medicine in Mount Tai.

Corresponding author：zhaoxiaominty@hotmail.com

OA-13 Reinforcing Effects and Discriminative Stimulus Effects of Three Pyrrolidine-Containing Synthetic Cathinones Derivatives in Rats

LAI Miao-jun1，F(xiàn)U Dan1，XU Wen-jin1，LIU Hui-fen1，XU Peng2，ZHOU Wen-hua1*
1. Laboratory of Behavioral Neuroscience，Ningbo Addiction Research and Treatment Center，School of Medicine，Ningbo University，Ningbo，China
2. Drug Intelligence and Forensic Center，Ministry of Public Security，Beijing，China

【ABSTRACT】 A synthetic cathinones，α-pyrrolidinopentiophenone （α-PVP），is commonly identified in abused “bath salt” preparations.It has been reported that people who consumed α-PVP sometimes lose consciousness，develop difficulty breathing，and，in the worst case，die.The molecular and behavior aspects of α-PVP have been characterized.However，the other analogs of α-PVP have not received the same attention and little is known about how structural modification of α-PVP influences the abuse liability.The current study aims to characterize the behavioral pharmacology of three pyrrolidinylated second-generation cathinones：α-PVP，4-chloro-αpyrrolidinopentiophenone （4cl-α-PVP） and 4-chloro-α-Pyrrolidinopropiophenone （4cl-α-PPP）.The rewarding effects of these drugs were assessed in rats using self-administration model.The discriminative stimulus effects of these compounds were tested in rats trained to discriminate either heoin or methamphetamine.Both 4cl-α-PVP and a-PVP produced self-administration in an inverted U-shaped dose effect，whereas 4cl-α-PPP did not produce self-administration.α-PVP fully substituted for the discriminative stimulus effects of both heroin and methamphetamine，whereas 4cl-α-PVP substituted partly for the discriminative stimulus effects of methamphetamine only，and 4cl-α-PPP couldn’t substitute for the discriminative stimulus effects of both heroin and methamphetamine.These findings suggest that 4cl-α-PVP and α-PVP are likely to be recreationally used and have potential for addiction and abuse，but 4cl-α-PPP may not.

【KEY WORDS】synthetic cathinones；α-pyrrolidinopentiophenone；abuse

OA-14 SB-334867 Improves Anxiety-Like Behavior in Social Defeat Mouse by Regulating HPA Axis

ZHANG Yun-peng1，2，CHEN Long2，3，CHEN Dan-mei1，2，WANG Cui-ting1，2，WANG Xiao-kun1，2，LI Bing1，2，*
1. Research Center for Clinical Medicine，Jinshan Hospital Affiliated to Fudan University，Shanghai，201500，China
2. The Institute of Neurology，The Academy of Integrative Medicine of Fudan University，Shanghai，201500，China
3. Department of Neurology，Jinshan Hospital Affiliated to Fudan University，Shanghai，201500，China

【ABSTRACT】Objective：Depression is one of the major disease and becomes a public medical health problem worldwide.Current antidepressant drugs typically require weeks to months before a response to the treatment can be observed in the patients with major depressive disorders （MDD）.Therefore，development of novel agents that produce a rapid antidepressant response represents a major unmet medical need for the treatment of MDD.Recently，several studies indicate that Orexin antagonists，and in particular selective OxR1 （Orexin-1 receptor） antagonists，can reduce drug-seeking at doses that have minimal effects on natural reward-seeking behavior.With respect to the potential use of orexin receptor antagonists for anxiety and depression，we proposed the use of OxR1 antagonist （SB-334867） in the treatment of depressive disorder.We observed the effects of SB-334867 on behavioral stress reactivity and the plasma levels of ACTH and cortisol in social defeat mouse depression model，confirming that SB-334867 can improve the anxiety-like behavior of socially failing mice by modulating the HPA axis.Methods：The social defeat mouse（C57 BL/6J） model was prepared according to the method of Tatsuhiko Goto et al.，and SB-334867 （2 mg·kg-1） was injected through the tail vein.Elevated O maze test and Social Avoidance Test were performed on the social defeat mouse，and the plasma levels of ACTH and cortisol were detected by Elisa.Besides，Western blot was used to detect the changes of Orexin-A and Orexin-1 receptor in the hypothalamus.Results：The social interaction ratio （SI ratio） illustrated that the depression animal model induced by modified chronic social defeat stress is reliable.The SI ratio of the model group，the vehicle group and the drug-administered group were significantly lower than those of the control group；however，after 14 days of intravenous injection of SB-334867，the SI ratio of the mice in the drug-administered group was significantly higher than that in the model group and the vehicle group.The percentage of time spent on the open arms of the elevated O-maze test in the model group，the vehicle group，and the drug-administered group after model establishment was significantly lower than that in the control group，but the percentage of time spent on the open arms of the elevated O-maze test in mice treated with SB-334867 was significantly higher than that in the model group and the vehicle group.Western blot results showed that Orexin-A and Orexin-1 receptor in the hypothalamus of the model group，the vehicle group and the drug-administered group were significantly higher than that of the control group.There were significantly increase of plasma ACTH and corticosterone levels after mice were submitted to chronic social defeat stress as compared to the control mice.Compared with the model group and the vehicle group，subchronic administration of SB-334867 decreased of plasma ACTH and corticosterone levels.Conclusion：By using a depression animal model induced by modified chronic social defeat stress，we found an abnormal increase in the expression of Orexin-A and Orexin-1 receptor in the hypothalamus of social defeat mice.Subchronic administration of SB-334867 can improve the social disorder behavior and anxiety-like behavior caused by mild chronic social defeat stress；meanwhile，subchronic administration of SB-334867 restored stress-induced disturbances in the HPA axis.Therefore，we hypothesized that SB-334867 improved the abnormality of the HPA axis by inhibiting Orexin-1 receptor，and ultimately alleviated the anxiety-like behavior of social defeat mice.

【KEY WORDS】social defeat；anxiety；Orexin；receptor；HPA axis

Corresponding author：LI Bing，libing22@126.com

OA-15 Dendrobium nobile Lindl alkaloids Produces Neuroprotection against 6-OHDA-Induced Dopamine Neurotoxicity

LI Dai-di，WANG Guo-qing，SHI Jing-shan，ZHANG Feng*
Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education，Zunyi Medical University，Zunyi，China

【ABSTRACT】Background：Parkinson’s disease （PD） is one of the most common neurodegenerative diseases characterized with a gradual loss of midbrain substantia nigra（SN） dopamine （DA） neurons.An excessive evidence demonstrated that microglia-mediated inflammation might be involved in the pathogenesis of PD.Thus，inhibition of neuroinflammation might possess a promising potential for PD treatment.Dendrobium nobile Lindl alkaloids（DNLA），an active component extracted from the Dendrobium nobile Lindl presents amounts of pharmacological properties，such as anti-inflammation，anti-oxidant，and anti-aging.Recent studies show DNLA produced neuroprotection against brain dysfunction.However，the mechanisms underlying DNLA-exerted DA neuroprotection are fully illuminated.Objective：To research the protective effects of Dendrobium nobile Lindl （DNLA） on dopaminergic（DA） neuronal injury induced by 6-hydroxydopamine （6-OHDA）.Methods：In the present study，6-OHDA-induced rat midbrain DA neuronal damage were applied to investigate the neuroprotective effects of DNLA.The rats were randomly divided into control，DNLA （10 mg·kg-1），6-OHDA （8 ug），6-OHDA+DNLA （10 mg·kg-1） groups.Rats were received a single 6-OHDA unilateral injection into the SN pars compacts，after 14 daily intragastric administration of DNLA，rats’ behavior was analyzed by Rotarod test.Immunohistochemical staining was used to detect the anti-tyrosine hydroxylase （TH） and anti-OX42，then observed the quantitative changes of DA neurons.In addition，primary rat midbrain neuron-glia co-cultures were performed to explore the mechanisms underlying DNLA-mediated DA neuroprotection，primary neuronglia cultures were performed on the ventral midbrain tissues of 14～15 days gestational age SD rats.The cultures were randomly divided into control，DNLA （25 ng·L-1），6-OHDA （40 μmoL），6-OHDA+DNLA （25 ng·L-1） groups.DA neuronal lesion and the activation of microglia in the substantia nigra were tested by immunocytochemical staining with anti-TH antibody and anti-OX-42 antibody.The levels of TNF-α，IL-1β and nitric oxide （NO） in the culture supernatant were measured with Griess reagent and enzyme link immunosorbent assay （ELISA）.Results：In vivo data showed that DNLA significantly attenuated 6-OHDA-induced DA neuronal loss and subsequent animal behavior changes，then significantly reduced microglia activation.In vitro data showed that DNLA protected DA neurons from 6-OHDA-induced DA neuronal damage and inhibited microglia activation and pro-inflammatory factors （TNF-α，IL-1β，NO） production.Conclusion：DNLA could protect DA neurons against 6-OHDA-induced neurotoxicity both in vivo and in vitro.These findings suggest that DNLA might hold potential therapeutic effects on PD.

【KEY WORDS】Parkinson’s disease；DNLA；6-hydroxydopamine；dopaminergic neuronal；neuroinflammation

Corresponding author：Feng Zhang，E-mail：zhangfengzmc@163.com

OA-16 蜂膠對(duì)酒精暴露小鼠認(rèn)知功能損傷的改善效果研究

李學(xué)龍，梁惠*1，姜雨杉，劉穎
青島大學(xué)，青島，中國(guó)

【摘要】 目的：探討蜂膠對(duì)酒精暴露小鼠認(rèn)知功能損傷的改善效果。方法：30 只10 周齡雄性C57 BL/6J 小鼠，體質(zhì)量24～26 g。以酒精偏愛(ài)度為基礎(chǔ)將小鼠隨機(jī)分為三組（10 只/組），即：健康對(duì)照組、酒精模型組和蜂膠干預(yù)組。于實(shí)驗(yàn)第0 周對(duì)各組小鼠進(jìn)行新物體識(shí)別實(shí)驗(yàn)、Y 迷宮自由交替實(shí)驗(yàn)、Y 迷宮新奇臂探索實(shí)驗(yàn)、礦場(chǎng)實(shí)驗(yàn)、高架十字迷宮實(shí)驗(yàn)、糖水實(shí)驗(yàn)以及強(qiáng)迫游泳實(shí)驗(yàn)等行為學(xué)基線測(cè)試。適應(yīng)性喂養(yǎng)一周后，對(duì)小鼠實(shí)施酒精暴露及蜂膠干預(yù)。具體方案如下：酒精模型組和蜂膠干預(yù)組小鼠周一至周四每日8：00～16：00 給予自由飲用自來(lái)水，16：00～次日8：00 給予每日新鮮配置的15%酒精水溶液自由飲用，周五至周六切斷一切水源，周日全天給予自來(lái)水自由飲用；健康對(duì)照組小鼠每周日至周四給予自來(lái)水自由飲用，周五至周六斷飲。各組小鼠全程自由進(jìn)食，并于每日中午12：00 進(jìn)行灌胃，其中健康對(duì)照組與酒精模型組小鼠給予0.1 mL/只大豆油灌胃，蜂膠干預(yù)組給予100 mg·kg-1蜂膠灌胃。實(shí)驗(yàn)持續(xù)10 周后，再次對(duì)各組小鼠進(jìn)行行為學(xué)測(cè)試。采用Western blot 檢測(cè)各組小鼠前額葉皮質(zhì)腦源性神經(jīng)營(yíng)養(yǎng)因子（BDNF）及其受體TrkB 蛋白表達(dá)水平。結(jié)果：酒精暴露小鼠對(duì)新物體的探索時(shí)間及Y 迷宮有效進(jìn)臂次數(shù)與健康對(duì)照組比較無(wú)顯著性差異（P＞0.05），提示酒精暴露對(duì)小鼠學(xué)習(xí)記憶和空間記憶能力無(wú)顯著影響。礦場(chǎng)實(shí)驗(yàn)發(fā)現(xiàn)，模型組小鼠在中心區(qū)域的運(yùn)動(dòng)量和運(yùn)動(dòng)速度有所減少；Y 迷宮新奇臂探索實(shí)驗(yàn)則顯示，模型組小鼠在新奇臂的進(jìn)臂次數(shù)和停留時(shí)間均顯著減少，與健康對(duì)照組比較，均具有顯著性差異（P＜0.05），提示酒精暴露小鼠探索能力及其對(duì)新環(huán)境的好奇程度受到抑制。高架十字迷宮實(shí)驗(yàn)結(jié)果表明，小鼠在開(kāi)放臂的進(jìn)臂次數(shù)和停留時(shí)間明顯縮短；糖水偏愛(ài)實(shí)驗(yàn)顯示，酒精暴露小鼠糖水偏愛(ài)度顯著降低，強(qiáng)迫游泳實(shí)驗(yàn)也發(fā)現(xiàn)，模型組小鼠在水中的不動(dòng)時(shí)間明顯增多（P＜0.05），提示酒精暴露導(dǎo)致小鼠焦慮及抑郁行為的發(fā)生。蜂膠干預(yù)后，小鼠的探索能力及其對(duì)新環(huán)境的好奇程度較模型組明顯提高，其焦慮及抑郁樣行為得到顯著改善（P＜0.05）。Western blot 結(jié)果顯示，蜂膠干預(yù)使酒精暴露小鼠BDNF 及TrkB 蛋白表達(dá)水平降低現(xiàn)象得到有效抑制（P＜0.05）。結(jié)論：蜂膠對(duì)酒精暴露小鼠認(rèn)知功能損傷具有一定改善效果，其作用機(jī)制可能與蜂膠上調(diào)BDNF 及其受體TrkB 蛋白表達(dá)水平有關(guān)。

【關(guān)鍵詞】蜂膠；酒精暴露；小鼠；認(rèn)知功能；BDNF

通訊作者：qdlianghui@126.com

OA-17 NOD2 Promotes Dopaminergic Degeneration Regulated by NADPH Oxidase 2 in 6-Hydroxydopamine Model of Parkinson’s Disease

CHENG Li1#，CHEN Lin1#，WEI Xin-bing1，WANG Yi-meng1，REN Zhi-ping1，ZENG Sheng-lan1，ZHANG Xiumei1，WEN Hai-tao2，GAO Cheng-jiang3，LIU Hui-qing1*
1. Department of Pharmacology，School of Basic Medical Sciences，Shandong University，Jinan，250012，China
2. Department of Microbial Infection and Immunity，Infectious Disease Institute，The Ohio State University，Columbus，Ohio，USA.
3. State Key Laboratory of Microbial technology，Key Laboratory of Infection and Immunity of Shandong Province & Department of Immunology，the School of Basic Medical Sciences，Shandong University，Jinan，250012，China

【ABSTRACT】 Background：In Parkinson’s disease （PD），loss of striatal dopaminergic （DA）terminals and degeneration of DA neurons in the substantia nigra （SN） are associated with inflammation.Nucleotide-binding oligomerization domaincontaining protein （NOD）2，one of the first discovered NOD-like receptors，plays an important role in inflammation.However，the role of NOD2 has not been elucidated in PD.Methods：NOD2 mRNA and protein expression in the SN and the striatum of C57BL/6 mice treated with 6-hydroxydopamine （6-OHDA） was measured.We next investigated the potential contribution of the NOD2-dependent pathway to 6-OHDA-induced DA degeneration using NOD2-deficient （NOD2-/-） mice.Assays examining DA degeneration and inflammation include HPLC，Western blot，immunohistochemistry，TUNEL staining，and cytometric bead array.To further explore a possible link between NADPH oxidase 2 （NOX2） and NOD2 signaling in PD，microglia were transfected with shRNA specific to NOX2 in vitro and apocynin were given to mice subjected to 6-OHDA and muramyl dipeptide （MDP）striatal injection.Results：The expression of NOD2 was upregulated in an experimental PD model induced by the neurotoxin 6-OHDA.NOD2 deficiency resulted in a protective effect against 6-OHDA-induced DA degeneration and neuronal death，which was associated with the attenuated inflammatory response.Moreover，silencing of NOX2 in microglia suppressed the expression of NOD2 and the inflammatory response induced by 6-OHDA and attenuated the toxicity of conditioned medium from 6-OHDA or MDP-stimulated microglia to neuronal cells.Furthermore，apocynin treatment inhibited NOD2 upregulation and DA degeneration in the SN of WT mice induced by 6-OHDA and MDP.Conclusion：This study provides the direct evidence that NOD2 is related to 6-OHDA-induced DA degeneration through NOX2-mediated oxidative stress，indicating NOD2 is a novel innate immune signaling molecule participating in PD inflammatory response.

【KEY WORDS】nucleotide-binding oligomerization domain-containing protein 2 （NOD2）；neuroinflammation；microglia；cytokine；apoptosis；reactive oxygen species （ROS）；NADPH oxidase 2 （NOX2）；Parkinson’s disease

#Lin Chen and Lingjun Kong contributed equally to this work

Corresponding author：Huiqing Liu，Ph.D.E-mail：liuhuiqing@sdu.edu.cn

OA-18 Dynamic Functional Connectivity Strength within Different Frequency-Band in Schizophrenia

LUO Yu-ling，HE Hui，DUAN Ming-jun，YAO De-zhong，LUO Cheng*
The Clinical Hospital of Chengdu Brain Science Institute，MOE Key Lab for Neuroinformation，Center for Information in Medicine，School of life Science and technology，University of Electronic Science and Technology of China，Chengdu，610054，China

【ABSTRACT】 Objective：As a complex psychiatric disorder，schizophrenia is interpreted as a“dysconnection” syndrome，which is linked to abnormal integrations in between distal brain regions［1］.Recently，neuroimaging has been widely adopted to investigate how schizophrenia affects brain networks.Furthermore，some studies reported frequency dependence of the abnormalities of functional network in schizophrenia［2］.In previous study based on graph theory，functional connectivity strength （FCS），a voxel-wise data-driven method，is used to investigate functional integration among brain regions in various frequency bands［3］.The current study aimed to assess the difference of frequency-specific whole brain dynamic functional connectivity in patients with schizophrenia.Methods：In the current study，96 patients with schizophrenia and 121 gender and aged-matched healthy controls were recruited.Resting state functional images were performed on 3-T MRI scanner.Within four different frequency bands，we assessed whole brain static and dynamic functional connectivity strength （dFCS） respectively.Four frequency bands are included：slow-5：0.01-0.027 Hz，slow-4：0.027-0.073 Hz，slow-3：0.073-0.198 Hz，slow-2：0.198-0.25 Hz［4］.At each frequency band，firstly，we calculated the static FCS map of each subject（f），and then using sliding windows analysis，we divided the whole time series into n non-overlapping temporal windows （100 s，interval 10 s），then we defined the dynamic index of a voxel k as［5］：

Where ftis the FCS of the k at the time window t，t = 1，2，…n.Furthermore，individual voxelwise static and dynamic FCS maps were standardized to z-scores，and spatially smoothed（FWHM=6 mm）.Subsequently，the voxel-wise dynamic FCS maps was compared between patients and controls through two-sample t-test.FDR correction （P＜0.05） was used for multiple comparisons.Results：Overall altered dynamic FCS of schizophrenic subjects was revealed across the four frequency bands （Fig.1）.In slow-5，schizophrenic subjects showed increased dFCS within cerebellum and basal ganglia network （thalamus and caudate），while decreased dFCS in sensorimotor network and auditory network，such as posterior insula and superior temporal gyrus.Meanwhile，in slow-4，schizophrenic subjects showed increased dFCS in cerebellum，orbitofrontal gyrus and middle occipital gyrus，and reduced dFCS mainly in the visual network.Furthermore，increased dFCS in cerebellum and limbic system，such as orbital frontal cortex，were observed in slow-3，as well as decreased dFCS in sensorimotor and visual area of schizophrenia.There was no difference of schizophrenic subjects in slow-2 compared to healthy controls.Conclusion：These results revealed altered dFCS in cerebellum，basal ganglia，sensorimotor，visual networks and limbic system in schizophrenia.Many brain areas showed significant difference in specific frequency bands.Especially，in the BG network，some brain regions of schizophrenic patients exhibited enhanced dynamic FCS at low frequency （slow-5），while reduced dynamic FCS at high frequency （slow-3）.Our findings suggest that the abnormalities of dFCS in schizophrenia is dependent on the frequency band and may provide potential implications for exploring the neuropathological mechanism of schizophrenia.

Fig.1

【KEY WORDS】schizophrenia；fMRI；frequency band；functional connectivity strength

OA-19 Increased Blood Pressure Variability Induces Depression-Like Behavior and Gap Junction Protein Changes in Rats

MIAO Shen#，ZHOU Li-jun#，WANG Wei，JIA Xiao-li，TAN Rui，ZHANG Fang-fang，GAO Shan，JI Wei，ZHU Derong，ZHAO Xiao-min*，ZHANG Han-ting
Institute of Pharmacology，Shandong First Medical University & Shandong Academy of Medical Sciences，Taian，271016，China

【ABSTRACT】 Objective：To study depression-like behavior caused by the increase of blood pressure varibalityby and mechanism underlying gap junction protein.Methods：Male SD rats were divided into Sham group and sinoaortic denervation （SAD） group，and behavioral （Sucrose preference test，F(xiàn)orced swimming test，light and dark shuttle） was detected two，four，eight and sixteen weeks after SAD.The expression levels of CX40，CX43，CX45，CREB and BDNF were detected by Western blotting in the left front prefrontal cortex and hippocampus of each group.Result：Compared with the Sham group，the sucrose preference rate of the SAD group was significantly low，the forced swimming time was markedly long，and the retention time of the clear box was obviously short.The expression of CX40 and CX45 in the SAD group was increased and the expression of CX43，CREB and BDN was decreased.All the above changes existed from two to sixteen weeks after SAD.Conclusion：Increased blood pressure variability can cause depression-like behavior in rats，and the underlying mechanism may be related to gap junction protein changes.

【KEY WORDS】blood pressure variability；depression；hippocampus；prefrontal cortex；gap junction protein；rat

Acknowledgements：This work was supported by the Natural Science Foundation of Shandong Province of China（ZR2017MH048），the Foundation of Overseas Distinguished Taishan Scholars of Shandong Province of China （to H-TZ） and Collaborative Innovation Center for Research and Development of Traditional Chinese Medicine in Mount Tai.

#These two authors contributed equally to this work.

Corresponding author：zhaoxiaominty@hotmail.com.

OA-20 The Study of the Effect and Mechanism of Danggui Buxue Tang on Angiogenesis in Mice with Acute Cerebral Ischemia

MO You-sheng1，WEN Hui-hong1，2，XU Er-jin3，HUANG Shui-qing3，WANG Qi1
1.廣州中醫(yī)藥大學(xué)臨床藥理研究所，廣州，510405，中國(guó)
2.廣東省人民醫(yī)院/廣東省醫(yī)學(xué)科學(xué)院，廣州，510000，中國(guó)
3.廣州中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院，廣州，510405，中國(guó)

【ABSTRACT】Objective：Emerging evidences have shown that pro-angiogenic therapy targeting Endothelial Progenitor Cells （EPCs） has potential advantages in the treatment of ischemic stroke.Danggui Buxue Decoction （DBT） is a classic prescription for the treatment of blood circulation，although previous studies have demonstrated that DBT is effective to treat acute ischemic stroke.However，it is still unclear whether DBT can alleviate ischemic stroke via regulating the biological function of EPCs to promote angiogenesis.In this study，we aimed to investigate the effects of DBT on EPCs function and angiogenesis in mice with cerebral ischemia-reperfusion injury，in order to explore the protective mechanism of DBT on acute cerebral ischemia injury.Method：Forty-five male C57BL/6J mice weighing 26～30 g were divided into Shame group and Model group，mouse in model group received a Middle cerebral artery occlusion （MCAO） operation according to the Koizumi method，while mice in Shame group given an shame operation.After that，mice in DBT treatment group were given DBT （6 g·kg-1） by intragastric administration twice a day，mice in the Sham group and the MCAO group were given an equal volume of normal saline.The mice were scored with mMSN Scale on the 1st，3rd and 5th day after surgery.At 24 hours after operation，3 animals were randomly selected from each group for TTC staining.On the 5th day after surgery，6 animals in each group were subjected for immunohistochemical staining and Western blotting assays.Results：After DBT treatment，the Bederson neurological function scores of mice in the model group were significantly reduced on the 1st，3rd and 5th day after operation and the infarct volume of the ischemic side was significantly reduced （P＜0.01）.In addition，DBT reduced the number of injured neurons of model mice and increased the expression of Bcl-2 protein in ischemic brain tissue significantly（P＜0.01）.Moreover，DBT significantly increase the homing of EPCs and increase the expression of vWF and CD31 on the ischemic injury side of model mice.Conclusion：DBT could alleviate the ischemia-reperfusion injury of MCAO mice model and promote the angiogenesis of ischemic injury，therefore，DBT might be a potential therapy for acute ischemic stroke.

【KEY WORDS】Danggui Buxue Tang；ischemic stroke；EPCs；angiogenesis

Corresponding to：WANG Qi，E-mail：wangqi@gzucm.edu.cn

OA-21 Transmembrane AMPA Receptor Regulatory Protein γ-8 is Associated with Antisocial Personality Disorder

PENG Shi-xiao1，2，WANG Yue-ying1，2，LIU Na3*，Yun Stone Shi1，2，4*
1. State Key Laboratory of Pharmaceutical Biotechnology，Department of Neurology，Drum Tower Hospital，Medical School
2. MOE Key Laboratory of Model Animal for Disease Study，Model Animal Research Center，Nanjing University，Nanjing，210032，China
3. Department of Medical Psychology，Nanjing Medical University affiliated Nanjing Brain Hospital，Nanjing，210029，China
4. Institute for Brain Sciences，Nanjing University，Nanjing，210032，China

【ABSTRACT】Antisocial personality disorder （ASPD），a common subtype of personality disorder characterized by irresponsible behavior often leading to criminal behavior，has heritability.In current study，we found SNP rs10420324 in the 1st intron of CACNG8，the gene encoding transmembrane AMPA receptor regulatory protein γ-8 （TARP γ-8），is associated with ASPD in a male prisoner sample in Chinese Han people.The distribution of rs10420324G is significantly higher in ASPD subjects relative to the normal healthy males.Using a reporter molecule in which the sequence covering rs10420324 were fused to the N-terminal of GFP protein，we found the expression level of the reporting GFP protein，mRNA and pre-mRNA relied on the rs10420324 variants.Furthermore，in 120 fore-cortical brain samples from a Chinese Han brain library，the expression level of TARP γ-8 was associated with the SNP rs10420324.The SNP is located in a guanine-rich region.We found it modulated the conformation and stability of a local G-quadruplex DNA structure，which appeared to regulate the transcription of TARP γ-8.Using TARP γ-8 knockout mice，we found that the decrease of TARP γ-8 lead to abnormal social behaviors and aggression.Therefore，the AMPA receptor auxiliary subunit TARP γ-8 contributes to a risk factor for ASPD.

【KEY WORDS】TARP γ-8；SNP；G-quadruplex；ASPD；aggression

Corresponding author：happyliun@126.com，yunshi@nju.edu.cn

OA-22 莫諾苷調(diào)節(jié)體外神經(jīng)干細(xì)胞增殖分化的作用機(jī)制研究

孫芳玲1*，王文，劉婷婷，吳錚，郭敏
首都醫(yī)科大學(xué)宣武醫(yī)院，北京，100053，中國(guó)
北京市老年病醫(yī)療研究中心，北京，100053，中國(guó)

【摘要】 目的：干細(xì)胞治療是國(guó)際神經(jīng)科學(xué)研究的熱點(diǎn)和重點(diǎn)，腦卒中能刺激內(nèi)源性神經(jīng)干細(xì)胞增殖短時(shí)間內(nèi)躍遷，其潛在的機(jī)制受多條信號(hào)通路調(diào)控。課題組前期研究表明，EphB4 過(guò)表達(dá)能促進(jìn)體外培養(yǎng)人胚胎神經(jīng)干細(xì)胞的增殖以及向神經(jīng)元的分化，抑制干細(xì)胞向膠質(zhì)細(xì)胞分化，推測(cè)EphB4 很可能是疾病后神經(jīng)元再生修復(fù)的關(guān)鍵靶點(diǎn)之一。體內(nèi)研究顯示我們自行提取的中藥單體化合物莫諾苷對(duì)腦卒中后神經(jīng)血管再生修復(fù)具有明顯的促進(jìn)作用。本研究擬在體外水平進(jìn)一步明確莫諾苷對(duì)胚胎神經(jīng)干細(xì)胞增殖、分化的影響，尤其是EphB4 是否是其調(diào)控干細(xì)胞增殖分化活動(dòng)的關(guān)鍵機(jī)制。方法：體外培養(yǎng)并經(jīng)免疫熒光染色鑒定后的神經(jīng)干細(xì)胞（NSCs），加入莫諾苷1、10、100 μmol·L-1孵育24 h。進(jìn)行氧糖剝奪處理 12 h，之后采用Ki67 免疫熒光染色檢測(cè)NSCs 的增殖，14 d 后Map2、GFAP 免疫熒光染色檢測(cè)NSCs 的分化。進(jìn)一步地，使用攜帶綠色熒光蛋白的短發(fā)卡RNA（shRNA）慢病毒載體轉(zhuǎn)染NSC，采用實(shí)時(shí)熒光定量qRT-PCR和免疫蛋白印跡檢測(cè)干擾效果。轉(zhuǎn)染后細(xì)胞加入莫諾苷100 μmol·L-1孵育2 d 后，采用Ki67免疫熒光染色檢測(cè)NSCs 的增殖，莫諾苷孵育9 d 后Map2、GFAP 免疫熒光染色檢測(cè)NSCs 的分化。結(jié)果：莫諾苷能顯著增加氧糖剝奪刺激后Ki67+細(xì)胞數(shù)量，并能顯著增加Map2+神經(jīng)元數(shù)量，減少GFAP+神經(jīng)膠質(zhì)細(xì)胞數(shù)量。轉(zhuǎn)染LV-shRNA-EphB4 后給予莫諾苷（100 μmol·L-1）作用2 d，Ki67+/GFP+細(xì)胞仍有所增加；莫諾苷（100 μmol·L-1）作用9 d，Map2+神經(jīng)元數(shù)量有所增加而GFAP+細(xì)胞數(shù)量減少。結(jié)論：莫諾苷能顯著促進(jìn)NSCs 的增殖以及向神經(jīng)元方向的分化，其機(jī)制可能部分通過(guò)并不局限于EphB4 信號(hào)通路。

【關(guān)鍵詞】莫諾苷；神經(jīng)干細(xì)胞；氧糖剝奪；EphB4；增殖；分化

通訊作者：孫芳玲，sun_fangling@163.com

OA-23 Inhibitory Effect of Xiaofeng Powder on Pruritus in AEW Mice and Its Mechanism

SUN Meng-han1，YUAN Xiao-lin2，TANG Zong-xiang1
1.School of Medicine and Life Sciences，Nanjing University of Chinese Medicine，Nanjng，210023，China
2.Basic Medical College，Nanjing University of Chinese Medicine，Nanjng，210023，China

【ABSTRACT】 Objective：To investigate the antipruritic effect and antipruritic mechanism of Xiaofeng Powder in AEW animal itching model.Methods：Male C57/BL6 mice were randomly divided into AEW models （modeling method：after shaving on the back，acetone∶ether=1∶1 wet compress for 15 seconds，wet water for 30 seconds，morning and evening 2 times，for 7 days）According to the saline group，the AEW model Xiaofeng Powder low-dose group，and the AEW model Xiaofeng Powder high-dose group，the behavioral video recording method was used to evaluate the degree of itchiness of the mouse hind paws scratching the back modeling area；HE staining observation Morphological changes of skin lesions；correlation analysis of skin and DRG tissues in mouse model area by fluorescence real-time quantitative PCR，and observation of changes in mRNA expression of TRPA1，TRPV1 and TRPV4，and evaluation of itching Efficacy；Western blotting was used to observe the phosphorylation levels of the above three itch-related proteins，and the antipruritic effect of Xiaofeng Powder was evaluated.Results：The preventive administration of Xiaofeng Powder low-dose and high-dose groups reduced the scratching behavior of AEW model mice.The low-dose group had significant difference from the 4th day to the 7th day of the model （P＜0.05）.The high-dose group showed significant difference from the third day to the seventh day of the model （P＜0.01），and it was dose-dependent.The anti-itch effect of the high-dose group was better than that of the low-dose group；the low-dose，high-dose group The thickness of the epidermis and the tight junction of the skin tissue were all lighter than that of the control saline group.The results of Q-RT-PCR showed that the mRNA expression level of TRPA1/V1/V4 was up-regulated in the saline group after modeling，and the wind was low.The high-dose group was down-regulated，and there was a significant difference （P＜0.05）.The WB results showed that the phosphorylation level of TRPA1/V1/V4 was up-regulated in the saline group after model establishment，while the low-dose，high-dose group was down-regulated.There was a significant difference （P＜0.05）.Conclusion：Xiaofeng Powder has an inhibitory effect on pruritus caused by AEW model in a dose-dependent manner.The mechanism of Xiaofeng Powder inhibiting pruritus in AEW model may be related to the mediation of TRPA1，TRPV1 and TRPV4.

【KEY WORDS】Xiaofeng Powder；AEW itching model；antipruritic mechanism；TRPA1/V1/V4

Corresponding author：zongxiangtang1@163.com

OA-24 Role of Acute Phase Protein ORM in A Mice Model of Ischemic Stroke

WAN Jing-jing1，WANG Peng-yuan1，ZHANG Yu，HU Bo-han，SUN Yang，XU Dong-ping*，LIU Xia*
Department of Pharmacology，School of Pharmacy，Second Military Medical University，Shanghai，China

【ABSTRACT】 Objective：The only FDA-approved treatment for acute ischemic stroke is tissue plasminogen activator，and the discovery of novel therapeutic targets is critical.Here，we found ORM，an acute phase protein mainly produced by the liver，might act as a treatment candidate for ischemic stroke.Our aim is to explore the role and underlying mechanism of ORM in ischemic stroke.Methods：Ischemic stroke was set up by middle cerebral artery occlusion （MCAO） in mice.Brain microvascular endothelial cells （bEnd.3） were exposed to oxygen-glucose-deprivation（OGD） for 4 h and then treated with 10 μg·mL-1of ORM for another 4 h.Purified ORM was injected via tail vein 0.5 h before，immediately，or 1 h，3 h，4.5 h and 6 h after MCAO in C57BL/6 mice，and infarct size was detected 24 h after surgery by TCC staining.ORM subtypes were determined by real-time PCR，and also pro-inflammatory factors，including IL-1β，IL-6 and TNF-α，24 h after surgery.Cerebrovascular permeability was evaluated by Evans blue staining and immunofluorescence.Briefly，22 h after MCAO，2% Evans Blue dye （0.1 mL·g-1） was injected via the tail vein.2 h later，the brain was rapidly harvested，detecting Evans blue staining and immunofluorescence results.Blood-brain barrier-associated proteins expression of ZO-1，claudin-5，occludin VE-cadherin and caveolin-1 were detected by immunoblotting.Oxidative stress was clarified by ELISA.Cell apoptosis and caspase-3 activity were detected by ELISA，TUNEL and flowcytometry.Results：The results showed that ORM2 is the dominant subtype in mice normal brain tissue.After middle cerebral artery occlusion （MCAO），the level of ORM2 is significantly increased in the ischemic penumbra compared to the contralateral normal brain tissue，while ORM1 knockout did not affect infarct size.Exogenous ORM could significantly decrease infarct size and neurological deficit score.Inspiringly，the best administration timepoint was at 4.5 h and 6 h after MCAO.ORM treatment could significantly reduce the area of Evans blue staining in MCAO mice，and the content of Evans blue in the supernatant of ischemic penumbra homogenyates.ORM could improve blood-brain barrier-associated proteins expression，MCAO significantly decreased the junction protein expression of ZO-1，claudin-5，occludin and VE-cadherin，while significantly increased the endocytic transport protein level of caveolin-1 in the ischemic penumbra.Exogenous ORM administration could significantly reverse these changes，Similar results were obtained in OGD-treated bEnd3 cell.Meanwhile，ORM could significantly alleviate inflammation by inhibiting the mRNA levels of IL-1β，IL-6 and TNF-α，and reduce oxidative stress by improving the balance of MDA and SOD.ORM treatment also significantly reduced cell apoptosis and the activity of caspase-3 in the ischemic area of MCAO mice and OGD-treated bEnd.3 cells.Conclusion：In general，our studies identified ORM as a novel protein involved in the development of ischemic stroke.It can effectively protect against ischemic stroke，especially with the relative longer time window.Considering ORM is classified as a protective protein in ischemic stroke，its elevation may represent a negative feedback mechanism.Due to its protective role at multiple levels，ORM might be a promising therapeutic target for ischemic stroke.

【KEY WORDS】ORM；ischemic stroke；blood-brain barrier；inflammation；oxidative stress；apoptosis

1The two authors contributed equally to this work.

Corresponding author：LIU Xia or XU Dong-Ping，Department of Pharmacology，School of Pharmacy，Second Military Medical University，325 Guohe Road，Shanghai，China.Email：lxflying@aliyun.com

OA-25 PLTP 過(guò)表達(dá)對(duì)3×Tg-AD 小鼠學(xué)習(xí)記憶的影響及機(jī)制研究

李明偉，付華容，夏增輝，王文芝，陳瑩，王浩*，張繼國(guó)*
山東第一醫(yī)科大學(xué)，泰安，271016，中國(guó)

【摘要】 目的：阿爾茨海默?。ˋlzheimer’s disease，AD）是中樞神經(jīng)系統(tǒng)退行性疾病，其發(fā)病機(jī)制至今仍未被完全闡明，進(jìn)而缺少有效的治療措施。磷脂轉(zhuǎn)運(yùn)蛋白（phospholipid transfer protein，PLTP）是一種廣泛表達(dá)于中樞神經(jīng)系統(tǒng)的糖蛋白。近幾年，體內(nèi)、外研究均發(fā)現(xiàn)，PLTP與AD 密切相關(guān)。本課題組前期研究發(fā)現(xiàn)PLTP 缺乏可促進(jìn)老年小鼠認(rèn)知功能的下降，而對(duì)于PLTP 過(guò)表達(dá)是否具有AD 改善作用，目前還鮮有報(bào)道。本課題擬以APP/PS1/Tau 轉(zhuǎn)基因（3×Tg-AD） 小鼠作為AD 模型，側(cè)腦室注射攜帶PLTP 基因與增強(qiáng)型綠色熒光蛋白報(bào)告基因的腺相關(guān)病毒（AAV-PLTP-EGFP）構(gòu)建腦組織特異性PLTP 過(guò)表達(dá)模型，觀察PLTP 過(guò)表達(dá)對(duì)AD小鼠學(xué)習(xí)記憶的影響及可能的機(jī)制。方法：選取10 月齡3×Tg-AD 雄鼠及與之具有相同遺傳背景的野生型（wide type，WT）小鼠進(jìn)行實(shí)驗(yàn)，側(cè)腦室分別注射AAV-PLTP-EGFP 和AAV-EGFP。水迷宮實(shí)驗(yàn)和穿梭被動(dòng)回避實(shí)驗(yàn)檢測(cè)小鼠的學(xué)習(xí)記憶能力；行為學(xué)實(shí)驗(yàn)后，處死小鼠，分離皮質(zhì)和海馬，ELISA 法檢測(cè)海馬及皮質(zhì)Aβ1-40及Aβ1-42水平；western blot 法檢測(cè)海馬及皮質(zhì)與Aβ生成相關(guān)蛋白APP、PS1、BACE1 的表達(dá)。結(jié)果：（1）行為學(xué)結(jié)果顯示：水迷宮定位航行實(shí)驗(yàn)中，與WT 小鼠相比，3×Tg-AD 小鼠逃避潛伏期明顯延長(zhǎng)，而給予PLTP 能翻轉(zhuǎn)此作用；在水迷宮空間探索實(shí)驗(yàn)中，與WT 小鼠相比，3×Tg-AD 進(jìn)入平臺(tái)所在區(qū)域的潛伏期明顯延長(zhǎng)、60 s 內(nèi)穿越平臺(tái)的次數(shù)明顯減少，同樣給予PLTP 后也能翻轉(zhuǎn)此作用；穿梭被動(dòng)回避實(shí)驗(yàn)中，與WT 小鼠相比，3×Tg-AD 暗箱停留時(shí)間長(zhǎng)，進(jìn)入暗箱的潛伏期縮短，給予PLTP 后明顯改善。（2） ELISA法實(shí)驗(yàn)結(jié)果：與WT 小鼠相比，3×Tg-AD 小鼠大腦皮質(zhì)及海馬 Aβ1-40及Aβ1-42蛋白表達(dá)均明顯增高，而給予PLTP 可以降低兩種Aβ的高表達(dá)。（3）Western blot 結(jié)果顯示：與WT 小鼠相比，3×Tg-AD 小鼠大腦皮質(zhì)及海馬APP、PS1 蛋白表達(dá)均明顯升高，而給予PLTP 可降低其表達(dá)，但BACE1 蛋白表達(dá)各組相比無(wú)明顯變化。結(jié)論：PLTP 過(guò)表達(dá)可改善3×Tg- AD 小鼠的學(xué)習(xí)記憶能力，其機(jī)制可能與降低Aβ產(chǎn)生相關(guān)。

【關(guān)鍵詞】阿爾茨海默??；磷脂轉(zhuǎn)運(yùn)蛋白；β-淀粉樣蛋白；學(xué)習(xí)記憶

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目（No.81441111，No.81601229）

通訊作者：tywanghao_2005@163.com，jgzhang@tsmc.edu.cn

OA-26 Discovery of A Subtype-Selective Inhibitor Targeting TRPV2 Ion Channel

LI Tian-yu1，2，CHAI Hao1，2，LI Yang1，2*
1. Key Laboratory of Receptor Research，Shanghai Institute of Materia Medical，Chinese Academy of Sciences，Shanghai，201203，China
2. University of Chinese Academy of Sciences，Beijing，100049，China

【ABSTRACT】 Objective：Calcium-permeable cation channel TRPV2 is expressed in various regions of the brain.Lack of selective inhibitors makes it difficult to study the function of TRPV2.We discovered TRP subtype-selective inhibitors （TRPV2 inhibitors） and investigated the role of TRPV2 in the brain.Methods：We discovered TRPV2 inhibitors using a strategy combining structural and evolutionary information，and tested the inhibitory effects of compounds by electrophysiology.In whole-cell voltage clamp experiments on HEK293T cells transiently transfected with TRPV2，we recorded and analyzed the compounds’ inhibitory effects.Results：We unveiled three ligand-binding sites of TRPV2 and compounds that bind to these sites.Structural optimization of the best-hit compound provided a potent selective TRPV2 inhibitor，SET2.Residues Phe467，Gln525，and Leu470 stabilized the orientation of the SET2 pyrimidine group in the transmembrane pocket.None of TRPV1，TRPV3，or TRPV4 was sensitive to SET2.Conclusion：We optimized the best hit and developed a highly potent selective antaonist SET2.The molecular basis and subtype-selective inhibition mechanism were quantitatively characterized and experimentally verified.

【KEY WORDS】TRPV2；subtype-selective inhibitor；ligand-binding sites

Corresponding author：Yang Li，liyang@simm.ac.cn

OA-27 CXCR2 Antagonist Efficiently Promotes Oligodendrocyte Precursor Cell Differentiation and Enhances Remyelination in A Mouse Model of Multiple Sclerosis

WANG Lu，YANG Han-yu，ZANG Cai-xia，DONG Yi，SHANG Jun-mei，CHEN Jia-jing，WANG Yue，LIU Hui，ZHANG Zi-hong，XU Heng*，BAO Xiu-qi*，ZHANG Dan*
Department of Pharmacology，Institute of Materia Medica，Chinese Academy of Medical Sciences and Peking Union Medical College.State Key Laboratory of Bioactive Substance and Function of Natural Medicine，Institute of Materia Medica，Chinese Academy of Medical Sciences and Peking Union Medical College

【ABSTRACT】Multiple sclerosis （MS） is a chronic autoimmune demyelinating disease characterized by the autoimmune attack of oligodendrocytes （OLs），leading to demyelination and progressive functional deficits.The pathological hallmarks of MS are inflammation，immunization，demyelination and axonal injury，which appear throughout the CNS and are most easily recognized in white matter.CXC chemokine receptor 2 （CXCR2），which is expressed by both inflammatory myeloid cells and OLs in the CNS，is a member of the chemokine receptor family and primarily modulates immune coordination and inflammation.CXCR2 is a developmental molecule known to orchestrate the migration，proliferation and differentiation of oligodendrocyte precursor cells （OPCs） in the developing brain.CXCR2 increases in chronic demyelinating disease，marking it as a potential pharmacological target of MS.Objective：To study a CXCR2 antagonist，compound 2，for its therapeutic efficacy in MS and investigate the mechanisms involved in its remyelinating effect.Methods：Co-incubated compound 2 with primary cultured OPCs to investigate whether it can directly act on OPCs and to study whether it promotes differentiation and maturation of OPCs.Cuprizone （CPZ） is a copper ion chelating agent that can cause oxidative stress and destroy the myelin sheath.After intoxication for 6 weeks with 0.2% CPZ feed，C57/BL mice were orally administered compound 2 at doses of 25，50，and 100 mg/kg or the positive control drug cyclosporin A （CyA） 70 mg/kg twice per day for 9 consecutive days without CPZ.Then，we evaluated the exercise ability and neurological function of mice by roller test and neurological function score，and detected the key markers in the process of myelination both in vitro and in vivo，included Ki67，Olig2 and Caspr.Based on this，we use transcriptome analysis to further study the mechanism of compound 2.Results：The results showed that compound 2 significantly promoted OPCs proliferation and differentiation，as indicated by the decreased expression of platelet-derived growth factor receptor α （PDGFRα）and A2B5，and the increased expression of myelin basic protein （MBP）.Further examination of CPZ-intoxicated mice suggested that compound 2 ameliorated neurological impairment and improved motor function.Additionally，in the demyelinated lesions of CPZ-intoxicated mice treated with compound 2，OPCs proliferation was more vigorous and myelin repair was more efficient compared with tissues from untreated mice.Subsequent investigation of the underlying mechanisms identified that upon inhibition of CXCR2，compound 2 activated PI3K/AKT/mTOR signaling，thereby upregulating Ki67，transcription factor 2 （Olig2） and Caspr expression，ultimately promoting OPCs differentiation and enhancing remyelination in MS.Conclusion：In this manuscript，we reported that compound 2 could act directly on OPCs and result in a promotion in the morphological changes associated with proliferation and differentiation of OPCs.And the treatment of compound 2 could improve motor behavior and alleviate neurological impairment，and thereafter enhanced myelin repair and remyelination in a MS mouse model caused by CPZ challenge.Our results demonstrated CXCR2 as a promising therapeutic target for the treatment of chronic demyelinating diseases such as MS.

【KEY WORDS】multiple sclerosis；CXC chemokine receptor 2；oligodendrocytes；differentiation；PI3K/AKT/mTOR pathway

Corresponding author：Zhang Dan，E-mail：danzhang@imm.ac.cn

OA-28 Cortical Atrophy Mediating the Accumulating Effects of Vascular Risk Factors on Cognition in Alzheimer’s Disease Spectrum

WANG Qing1，HE Can-can1，ZHANG Zhi-jun1，2，XIE Chun-ming1，2*
1. Department of Neurology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，210009，China
2. Neuropsychiatric Institute，Affiliated ZhongDa Hospital，Southeast University，Nanjing，210009，China，

【ABSTRACT】 Objective：Vascular risk factors （VRFs） might increase the risk of Alzheimer’s disease （AD） and contribute to neurodegenerative processes.The purpose of this study was to investigate whether the accumulating effects of VRFs contributes to gray matter volume and cognitive function and also examine the mediation effect of cortical atrophy linking the VRFs and cognition in AD spectrum population.Methods：We selected 30 AD patients，41 patients with LMCI，52 patients with EMCI，and 69 cognitively normal （CN） subjects from the Alzheimer’s Disease Neuroimaging Initiative 1 database.All participants completed vascular risk factor acquisition，including fMRI scans，T1 MRI scans，and cognitive function assessment （MMSE and ADAS-Cog scores）.ANOVA was used to examine group differences of VRF scores across all subjects.Partial correlation analysis was employed to detect the association between VRFs and cognition.Multivariate linear regression analysis was used to examine main effects of VRFs and cognitive function on the gray matter volume.In addition，mediation analysis was employed to explore whether cortical atrophy mediates the association between VFR and cognition in AD spectrum.Results：We found that subjects with higher VRFs suffered cognitive decline and severe cortical atrophyespecially in right ventromedial prefrontal cortex and medial temporal gyrus.Furthermore，VRFs and cognitive function presented some overlapped brain regions，such as the right temporal gyrus，prefrontal cortex，left superior parietal gyrus and angular gyrus.More importantly，mediation analysis identified that these cortical atrophy mediated the relationship between VRF and cognition in AD spectrum.Conclusions：These findings extend our understanding ofVRFs accounted for cortical atrophy and cognitive decline in AD spectrum.And the mediation analysis demonstrated that cortical atrophy mediated the association between VFR and cognition in AD spectrum.

Corresponding author：Chun-ming Xie，M.D.&Ph.D，E-mail：chmxie@163.com.

OA-29 The Role of NLRP3 Inflammasome Activation in Learning and Memory Impairment Induced by Increased Blood Pressure Variability in Rats

WANG Wei，JIA Xiao-li，ZHANG Fang-fang，TAN Rui，ZHOU Yan-meng，ZHOU Li-jun，MIAO Shen，JI Wei，ZHU De-rong，HOU Xue-qin，WANG Hao，ZHAO Xiao-min*，ZHANG Han-ting
Institute of Pharmacology，Shandong First Medical University & Shandong Academy of Medical Sciences，Taian，271016，China

【ABSTRACT】 Objective：Clinical studies have shown that increased blood pressure variability can lead to learning and memory impairment，but the mechanism is not clear.Based on previous studies，target organ damage caused by increased blood pressure variability is closely related to inflammation，and learning and memory impairment is also closely related to inflammation and NLRP3 inflammasome activation.Therefore，we hypothesize that the learning and memory impairment caused by increased blood pressure variability is related to the activation of NLRP3 inflammasome.Methods：Male SD rats underwent sinoaortic denervation （SAD） to establish an animal model of increased blood pressure variability.Morris water maze，and passive avoidance trial were used to evaluate learning and memory ability.Western blotting was used to analyze protein changes in the hippocampus.Results：Sixteen weeks after SAD operation，compared with

the Sham group rats，in the Morris water maze experiment，SAD rats showed significant long escape latency （P＜0.05），obviously decreased number of times of crossing platform （P＜0.05）；In the passive avoidance trial experimental，the escape latency was decreased significantly （P＜0.05），the dark time and number of errors was increased markedly （P＜0.05） in SAD rats.Moreover，Western blot result indicated that，the levels of Pro-IL-18，mature-IL-18，Pro-IL-1beta，mature-IL-1beta，NLRP3，ASC，Pro-caspase-1 and caspase-1P20 of SAD rats was increased significantly（P＜0.05，P＜0.01） .Conclusions：The mechanism of learning and memory impairment induced by increased blood pressure variability in rats may be related to the activation of NLRP3 Inflammasome.

【KEY WORDS】blood pressure variability；learning and memory；inflammation；NLRP3 inflammasome

Acknowledgements：This work was supported by the Natural Science Foundation of Shandong Province of China（ZR2017MH048），the Foundation of Overseas Distinguished Taishan Scholars of Shandong Province of China （to H-TZ） and Collaborative Innovation Center for Research and Development of Traditional Chinese Medicine in Mount Tai

Corresponding author：zhaoxiaominty@hotmail.com

OA-30 Functional Connection between Anterior Cingulate Cortex and Orbital Frontal Cortex in Risk-Based Decision Making in Rats

WANG Yu-chen1，YANG Sha-sha1，XU Rui1，HUANG Wen-qiang1，HONG Dan-dan1，GAO Wen-jun2，ZHOU Ning3*，YU Ping1*

【ABSTRACT】Risk-based decision making is the selection of a behavior option when its potential value and the probability in redeeming its value are already known.Both anterior cingulate cortex （ACC） and orbital frontal cortex （OFC） participate in risk-based decision making.However，whether ACC and OFC play different roles and how they cooperate in this process remains unclear.In this study，by using probability-discounting task paradigm and multiple channel recordings，we simultaneously recorded the neuronal discharges and local filed potential in ACC and OFC when the rat was performing the risk-based decision-making task.We found that both ACC and OFC contained three types of neurons exhibiting specific discharging patterns，including prediction neurons，action + award neurons，and award neurons.Particularly，ACC and OFC prediction neurons exhibit different discharging patterns between risky and conservative choices and between the different probabilities of large/risky award.It means that they were both sensitive to the information about the probability of award.We also find rhythmic oscillation in the ACC and OFC exhibit specific coherent activities among the different probabilities of large/risky award at decision-making point.The tendency of low-risky blocks （50% block and 100%block） increased sharply before pressing，while rose slowly in 50% block and keep the baseline level in 100% block.Moreover，the average coherence showed that the average coherence was no significant difference in low-probability blocks but showed significant difference in highprobability blocks.These findings provide the first evidence of different roles of ACC and OFC in risky decision making and reveals the functional connection between the two brain areas during risk-based decision making.

【KEY WORDS】anterior cingulate cortex；orbital frontal cortex；decision making；risk-based decision-making task；in vivo recording；rat

Corresponding author：YU Ping，E-mail：pingyu@cnu.edu.cn

OA-31 羅氟司特對(duì)CUMS 小鼠抑郁行為的影響

夏增輝，張毅偉，陳瑩，王文芝，王浩，王德才*，張漢霆*
山東第一醫(yī)科大學(xué)（山東省醫(yī)學(xué)科學(xué)院），泰安，271016，中國(guó)

【摘要】 目的：抑郁癥（depression）是一類復(fù)發(fā)率高、嚴(yán)重危害人類身心健康的慢性綜合征。其確切發(fā)病機(jī)制還未完全闡明，也未有有效的治療措施。抑制磷酸二酯酶（phosphodiesteras，PDE）4 可改善抑郁，因此PDE4 成為抗抑郁作用的有效靶點(diǎn)。羅氟司特為PDE4 抑制劑，近年發(fā)現(xiàn)，其可以透過(guò)血腦屏障進(jìn)入中樞，并對(duì)認(rèn)知具有一定的改善作用，但目前對(duì)其抗抑郁作用還鮮有報(bào)道。本研究以慢性不可預(yù)見(jiàn)性應(yīng)激（chronic unpredictable mild stress，CUMS）誘導(dǎo)抑郁，觀察了羅氟司特的抗抑郁作用，并對(duì)其作用機(jī)制進(jìn)行了初步探索。方法：選取22～22 g 雄性C57BL/6J 小鼠，隨機(jī)分為空白組、CUMS 模型組、羅氟司特低劑量（0.1 mg·kg-1）組、羅氟司特高劑量（1.0 mg·kg-1）組、氟西?。?0 mg·kg-1）組。除空白組外，其余各組單籠飼養(yǎng)，給予各種不可預(yù)知刺激，復(fù)制CUMS 模型，三周后開(kāi)始灌胃給藥，除空白組和模型組給予等容量溶劑外，其余各組給予相應(yīng)劑量的藥物。給藥三周后，依次進(jìn)行糖水偏好測(cè)試、曠場(chǎng)實(shí)驗(yàn)、懸尾實(shí)驗(yàn)、強(qiáng)迫游泳實(shí)驗(yàn)，觀察藥物對(duì)小鼠抑郁行為的影響。行為學(xué)實(shí)驗(yàn)后，將小鼠處死，取前額皮質(zhì)、海馬，Western Blot 檢測(cè)相關(guān)蛋白的表達(dá)。結(jié)果：（1）糖水偏好結(jié)果顯示：與空白組相比，CUMS 模型組小鼠糖水偏好率明顯下降，而給予羅氟司特和氟西汀均后能明顯提高其糖水偏好率；（2）曠場(chǎng)實(shí)驗(yàn)、懸尾實(shí)驗(yàn)結(jié)果顯示：與空白組相比，CUMS 模型組小鼠，不動(dòng)時(shí)間明顯延長(zhǎng)，而給予羅氟司特及氟西汀后，其不動(dòng)時(shí)間明顯縮短；（3）Western Blot 結(jié)果：與空白組相比，CUMS 模型組小鼠皮質(zhì)和海馬PDE4D、PDE4B 表達(dá)明顯升高，而給予羅氟司特及氟西汀后，兩種蛋白表達(dá)明顯下降，而PDE4A 變化不明顯。結(jié)論：羅氟司特對(duì)CUMS 誘導(dǎo)小鼠抑郁模型的抑郁樣具有改善作用，可能與抑制PDE4D、PDE4B 蛋白表達(dá)有關(guān)。

【關(guān)鍵詞】抑郁癥；羅氟司特；CUMS

通訊作者：dcwang@tsmc.edu.cn；hzhang@hsc.wvu.edu

OA-32 The Neuroprotective Effects of Osthol Nanoemulsions against Glutamate-Induced Damage in SY5Y Cells

XIAN Xin1，WANG Mu-fang1，LI Jie1，HOU Zhe，GUO Le-zong，HOU Xue-qin*1
1. Institute of Pharmacology，Shandong First Medical University& Shang dong Academy of Medical Sciences，Tai’an，271016，China

【ABSTRACT】Osthole （OST），a natural coumarin derived from C.monnieri，has been found to possess potential benefits for the prevention and treatment of Alzheimer’s disease （AD）.However，the poor water solubility of OST becomes the the main bottleneck to restrain its application.This study investigated the feasibility of using nanoemulsions for delivery of OST to prevent the damage induced by glutamate in SY5Y cells.At first，the OST nanoemulsions were tailored with ethyl oleate，Cremophor RH35/ HS-15，and PEG-400 after screening the formulation by pseudoternary phase diagrams and determination the solubility of OST in the lipids and emulsifiers.The physicochemical properties，such as the particle size，morphology of the developed nanoemulsions，were then evaluated.The neuroprotective effects of OST nanoemulsions were assessed by measuring cell viability，Immunofluorescence，biochemical reaction and Western blot.The optimum composition was as follows：3.6% of ethyl oleate as oil phase，11.4% of the surfactant （Polyoxyethylene 35 castor oil and Polyethylene glycol-15-hydroxystearate ratio of 1∶1），and 3% of Polyethylene glycol 400 as cosurfactant and 82% of the aqueous phase.The average particle size was about 22 nm.The transmission electron microscope（TEM） studies revealed almost homogeneous spherical globules without aggregation.In order to further study of SY5Y against L - Glu induced nerve injury mechanism，this study further examined the active enzyme levels in cells，and cells themselves REDOX state detection of SOD，MDA，GSH-px，GSH change and Western blot method examined SY5Y neuron apoptosis proteins Bax and change trend of caspase-3，revealing the rationality of cnidium monnieri element nano emulsion，provide theoretical basis for the development of Chinese medicine effective component animal experiments.Results：Osthole microemulsion prepared by droplet method had good physical stability，particle size distribution was about 22 nm，and PDI was 0.05.Osthol showed a good linear relationship in the range of 5.0～75 μg·mL-1，R2=0.999 9.In vitro efficacy evaluation，according to the results of common cnidium lactone，nano emulsion in 10 to 30 umol·L-1，but dose dependence to fight L-Glu sample cell damage caused by neurons，maintain normal morphology of cells and synapses and improve cell survival rate，and significantly improve the L-Glu reduced damage of intracellular GSH content and the expression of apoptosis proteins Bax，caspase-3，Tunel staining showed significantly nerve protective effect.Osthol nanoemulsion can play an important role in the protection of L-Glu injured neurons by affecting the oxidative stress apoptosis pathway.Conclusion：In this study，the classical in vitro Alzheimer’s disease cell model was used，and ostholin nanoemulsion was first found to have a good protection effect on Gluinduced neurotoxicity.At the same time，according to the further exploration of the mechanism，osthole nanoemulsion plays an important role in protecting the nerve cells damaged by AD mainly by affecting the apoptosis pathway of oxidative stress.It is suggested that osthol nanoemulsion may have potential curative effect in the prevention and/or treatment of AD.

【KEY WORDS】osthole nanoemulsion；oxidative stress；Alzheimer’s disease；SH-SY5Y cells；L-Glu

Acknowledgments：The study was supported by research grants from the National Natural Science Foundation of China （NO.81703901），the Shandong Province Natural Science Foundation of China （ZR2016HB56）

Corresponding author：Ji-fu Hao Professors and HOU Xue-qin，PhD，Shandong First Medical University，619 Chang Cheng Road，Taian，Shandong 271016，P.R.China.

OA-33 硫酸軟骨素納米硒對(duì)D-gal 和AlCl3誘導(dǎo)的AD 小鼠學(xué)習(xí)記憶障礙的改善作用

姬東生，劉平，肖玉良*1
山東第一醫(yī)科大學(xué)（山東省醫(yī)學(xué)科學(xué)院）藥學(xué)院藥理研究所，泰安，271000，中國(guó)

【摘要】 目的：考察硫酸軟骨素納米硒（CS@Se）對(duì)D-gal 和AlCl3誘導(dǎo)的癡呆（AD）小鼠學(xué)習(xí)記憶障礙的改善作用。方法：雄性的C57BL/6 小鼠隨機(jī)分為8 組（n=14），分別為空白組、模型組、Vc 組、CS@Se 高劑量組、CS@Se 中劑量組、CS@Se 低劑量組、CS 組和納米Se 組。采用皮下注射D-gal 和AlCl3灌胃法構(gòu)建癡呆小鼠模型，皮下注射D-gal 和AlCl3連續(xù)給藥30 天后，給藥干預(yù)30 天后，曠場(chǎng)、十字高架、新物體識(shí)別和 Morris 水迷宮等行為學(xué)方法考察CS@Se 對(duì)AD小鼠的空間學(xué)習(xí)記憶能力和探索能力以精神狀況改善作用。行為學(xué)測(cè)試束后處死小鼠，取大腦組織石蠟切片和海馬組織超薄切片，HE 染色觀察小鼠海馬神經(jīng)細(xì)胞和神經(jīng)髓鞘的形態(tài)學(xué)變化情況，透射電鏡觀察小鼠海馬體神經(jīng)元形態(tài)結(jié)構(gòu)、神經(jīng)元之間突觸的數(shù)目和線粒體的形態(tài)結(jié)構(gòu)。結(jié)果：新物體識(shí)別和 Morris 水迷宮實(shí)驗(yàn)結(jié)果表明高劑量的CS@Se 可以明顯改善小鼠空間學(xué)習(xí)記憶和探索能力，且效果優(yōu)于其他治療組。曠場(chǎng)、十字高架實(shí)驗(yàn)結(jié)果表明高中低三個(gè)劑量的CS@Se 均可不同程度的緩解AD 小鼠的焦慮情緒。 通過(guò)HE 染色可以觀察到對(duì)照組海馬CA1區(qū)細(xì)胞排列整齊且細(xì)胞形態(tài)完整，結(jié)構(gòu)正常，核仁清晰可見(jiàn)；而模型組海馬組織呈現(xiàn)典型的AD病變，CA1 區(qū)細(xì)胞出現(xiàn)明顯的核固縮現(xiàn)象且部分出現(xiàn)細(xì)胞水腫，CA1 區(qū)細(xì)胞帶寬度較對(duì)照組明顯變窄，缺失現(xiàn)象明顯，層次少而不清晰，排列較為紊亂，細(xì)胞核固縮形成三角形或者多角形，呈現(xiàn)濃染。CS@Se 給藥后，CS@Se 高劑量組小鼠的CA1 區(qū)神經(jīng)元損傷程度明顯減輕，基本無(wú)水腫現(xiàn)象，神經(jīng)細(xì)胞的數(shù)目明顯增加，排列較為整齊，細(xì)胞形態(tài)較為完整，細(xì)胞核的固縮情況明顯改善。透射電鏡結(jié)果顯示模型組海馬神經(jīng)元細(xì)胞有出現(xiàn)核固縮現(xiàn)象，突觸結(jié)構(gòu)受損，突觸后致密帶變薄，相對(duì)面變短，突觸曲界面曲度減小，偏平無(wú)活力；小鼠海馬神經(jīng)元內(nèi)線粒體出現(xiàn)腫脹，膜破裂，嵴退化現(xiàn)象。CS@Se 高劑量組小鼠海馬神經(jīng)元突觸的突觸后致密帶、突觸相對(duì)面、突觸曲界面曲率均有所改善；小鼠海馬神經(jīng)元線粒體嵴較為完整，較少出現(xiàn)線粒體腫脹、膜破裂現(xiàn)象。結(jié)論：CS@Se 可以顯著改善D-gal 和AlCl3誘導(dǎo)的AD 小鼠的學(xué)習(xí)記憶障礙，緩解AD 小鼠的焦慮情緒，改善小鼠海馬神經(jīng)元存活及突觸超微結(jié)構(gòu)，本論文的研究為進(jìn)一步闡明CS@Se改善學(xué)習(xí)記憶障礙的作用特點(diǎn)及機(jī)制奠定了理論基礎(chǔ)。

【關(guān)鍵詞】硫酸軟骨素；納米硒；阿爾茨海默?。籇-gal；AlCl3

通訊作者：肖玉良，男，教授，博士，主要從事多糖類藥物研究，E-mail：111925wj@163.com

OA-34 FCPR03，A Novel Phosphodiesterase 4 Inhibitor，Alleviates Cerebral Ischemia/Reperfusion Injury through Activation of the AKT/GSK3β/β-Catenin Signaling Pathway

XU Bing-tian1，WANG Tian-tian1，QIN Yun-yun1，CAI Ning-bo1，ZHOU Zhong-zhen1，WANG Hai-tao1，3，4*，XU Jiang-ping1，2，3，4*
1. Department of Neuropharmacology and Drug Discovery，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou，510515，China

2. Central Laboratory，Southern Medical University，Guangzhou，510515，China
3. Center for Brain Science and Brain-Inspired Intelligence，Guangdong-Hong Kong-Macao Greater Bay Area，Southern Medical University，Guangzhou，510515，China
4. Key Laboratory of Mental Health of the Ministry of Education，Southern Medical University，Guangzhou，510515，China

【ABSTRACT】 Objective：Inhibition of phosphodiesterase 4 （PDE4） is a promising strategy for the treatment of ischemic stroke.However，the side effects of nausea and vomiting from the current PDE4 inhibitors have limited their clinical applications.FCPR03 is a novel PDE4 inhibitor with little emetic potential.This study aimed to investigate the effects of FCPR03 on neuronal injury after cerebral ischemia/reperfusion and the underlying signaling pathway.Methods：The effects of FCPR03 on cellular apoptosis，intracellular accumulation of reactive oxygen species （ROS），and mitochondrial membrane potential （MMP） were evaluated in HT-22 neuronal cells and cortical neurons exposed to oxygen-glucose deprivation （OGD）.The impact of FCPR03 on brain injury，neurological scores and behavioral performance was investigated in rats subjected to middle cerebral artery occlusion （MCAO）.The protein kinase B （AKT） inhibitor MK-2206 and β-catenin siRNA were used to investigate the underlying pathways.Results：FCPR03 dose-dependently protected against OGD-induced cellular apoptosis in both HT-22 cells and cortical neurons.The levels of MMP and ROS were also restored by FCPR03.FCPR03 increased the levels of phosphorylated AKT，glycogen synthase kinase-3β （GSK3β），and β-catenin.Interestingly，the role of FCPR03 was reversed by MK-2206 and β-catenin siRNA.Consistently，F(xiàn)CPR03 reduced the infarct volume and improved neurobehavioral outcomes in rats following MCAO.Moreover，F(xiàn)CPR03 increased the levels of phosphorylated AKT，GSK3β and β-catenin within the ischemic penumbra of rats following cerebral ischemia-reperfusion.Conclusion：Taken together，F(xiàn)CPR03 has therapeutic potential in cerebral ischemia-reperfusion.The neuroprotective effects of FCPR03 are mediated through activation of the AKT/GSK3β/β-catenin pathway.

【KEY WORDS】ischemic stroke；phosphodiesterase 4；FCPR03；akt；neuroprotection

Corresponding author：Dr.WANG Hai-Tao，Ph.D.，E-mail：wht821@smu.edu.cn.Prof.XU Jiang-Ping，M.D.，Ph.D.，E-mail：jpx@smu.edu.cn

OA-35 Inhibition of Src Kinase Attenuates Neuroinflammation and Protects Dopaminergic Neurons in Parkinson’s Disease Models

YANG Han-yu，WANG Lu，ZANG Cai-xia，WANG Yue，SHANG Jun-mei，BAO Xiu-qi，WANG Xiao-liang，ZHANG Dan*
State Key Laboratory of Natural Products and Functions，Department of Pharmacology，Institute of Materia Medica，Chinese Academy of Medical Sciences & Peking Union Medical College，Beijing，100050，China

【ABSTRACT】 Background：Chronic neuroinflammation plays an important role in the pathogenesis of Parkinson’s disease （PD）.During the process of neuroinflammation，over-activated microglia release many pro-inflammatory factors，which eventually induce neurodegeneration.Inhibition of excessive microglial activation is regarded as a promising strategy for PD treatment.Src is a non-receptor tyrosine kinase and its role is mostly reported to be related to tumors.Recently，some reports indicated that Src is a central mediator in multiple signaling pathways including neuroinflammation.Objective：The aim of the present study was to demonstrate the role of Src in microglial regulation and neuroinflammation.Methods：The lipopolysaccharide （LPS） -stimulated BV-2 microglia in vitro model and the classic 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （MPTP） -induced in vivo PD model were applied to evaluate the effect of Src inhibition on neuroinflammation，neuron survival and PD motor symptom.Western blot analysis was used to measure the protein expression.Immunocytochemistry assay was used to measure the protein expression and cell morphology.Immunohistochemistry assay was used to observe pathological change.qRT-PCR analysis was used to measure the mRNA expression.Griess method was used to measure NO concentration in cell cultures.The rotarod test is used to measure coordinated motor skills.Results：The results showed that inhibition of Src could significantly relieve microgliosis and decrease levels of inflammatory factors both in vitro and in vivo.Src inhibition significantly reduced protein expressions of iNOS and COX2 and decrease mRNA expressions of TNF-α，and IL-6 in both LPS-stimulated BV-2 microglia and MPTP-treated mice.We also found that Src is critical for microglia to exert neurotoxic effect.SH-SY5Y cell viability was dramatically reduced after incubating with conditioned medium from LPS-treated BV2 cells，and the morphological change of SH-SY5Y cells could be observed.Src inhibition could partially block the effect of neurotoxicity mediated by LPS-stimulated microglia.Besides，inhibition of Src function could reduce the loss of dopaminergic neurons and improve the motor behavior of the MPTP-treated mice.TH-positive neurons in SNpc showed a significant reduction in MPTP-treated mice，which was prevented by PP2 treatment.The mRNA and protein level of TH significantly decreased in SNpc of MPTP-induced PD model mice，and Src inhibitor administration markedly increased TH expression.As for behavior test，our findings revealed that behavioral dysfunction had occurred 7 days post MPTP injection.Compared with control group，MPTP treated mice stayed less time on the rod during all the 3 tests.At day 7，the performance of Src inhibitor treated mice had no improvement，but it enhanced significantly in the tests at day 10 and 12.Conclusion：This study not only verified the critical role of Src tyrosine kinase in neuroinflammation，but also further proved that interfering neuroinflammation is beneficial for PD treatment.More importantly，this study shed a light on the hypothesis that Src tyrosine kinase might be a potential therapeutic target for PD and other neuroinflammation-related diseases.

【KEY WORDS】Src；microglia；neuroinflammation；Parkinson’s disease；neuroprotection；BV2 cells

Corresponding author：Zhang Dan，E-mail：danzhang@imm.ac.cn

OA-36 Attenuation of Cerebral Ischemia-Reperfusion Injury by Specific Activation of Kv7/KCNQ/M-Channels

YANG Pan，LIU Ya-ni，WANG Ke-wei*
Department of Pharmacology，Qingdao University School of Pharmacy，Qingdao 266021，China

【ABSTRACT】 Objective：Stroke is the second common cause of death and a leading cause of adult disability.During ischemic stroke，energy depletion induces anoxic depolarization and excitotoxicity in neurons characterized by progressive cell death and development of permanent local brain damage.Ischemic stroke is unmet medical need，representing a major challenge of how to develop an effective stroke drug therapy.Over the past twenty years，all the NMDA receptor antagonists aimed at stroke treatment have completely failed in clinical trials.This outcome strongly suggests that the neuronal excitotoxicity is likely mediated by the non-NMDA mechanism.Voltage-gated KCNQ/Kv7/M- potassium channel is important in regulating neuronal excitability by stabilizing the resting membrane potential.The aim of this study was to test any neuroprotective effect of KCNQ channel openers on ischemic injury in mice.Methods：Mouse model of ischemic-reperfusion （I/R） was established by transient middle cerebral artery occlusion（tMCAO）.Neurologic deficit scores and infarct volume measurement were assessed.Open field，accelerating rotarod and grip strength were performed to assess neurological deficit behavior.The model of oxygen-glucose deprivation （OGD）-induced injury in mouse primary culture of cortical neurons was used.Cell viability was determined by measuring the release of lactate dehydrogenase （LDH） and the number of viable cells was evaluated using Cell Counting Kit-8 （CCK-8） after OGD injury.Immunofluorescence staining was used for evaluating expression of KCNQ2 subunits in cultured cortical neurons before and after OGD injury.The whole-cell clamp recordings of cortical neurons after OGD injury were carried out.Results：The tMCAO led to rapid，predictable and consistently sized necrotic brain lesions and behavioral deficits.Administration of KCNQ channel openers 30 min before surgery significantly decreased neurological scores and brain infarct size in male Kunming mice （8～10 weeks） at 24 h after tMCAO.The following behavioral test results support the histological finding.Administration of Kv7 opener retagabine （10 mg·kg-1） or SCR2862 （1 mg·kg-1） improved the track length and speed of mice in open filed task，and increased the latency that mice stayed on an accelerating rotarod.The grip strength of affected side forelimb was also enhanced by KCNQ channel openers.Furthermore，neurons incubated with KCNQ channel openers 1 h before OGD exposure showed an increased cell vitality through decreased release of LDH when subjected to OGD injury for 20 min.Electrophysiological recordings showed that OGD exposure for 10 min caused significant depolarization of cortical neurons with decreased in AP amplitude and firing rates.The depolarization of membrane potential was blocked by Kv7 channel openers，retagabine at 10 μmol·L-1or SCR2862 compound at 1 μmol·L-1.Conclusions：An obvious improvement in the reduction of brain injury and functional impairment was observed in mouse model of ischemic injury after administration of KCNQ channel openers.Activation of Kv7 channel function also exhibited protective effects on OGD induced injury in primary cultured cortical neurons.The mechanism maybe that KCNQ channel openers blocked the depolarization after OGD in primary cortical neurons and declined the excitability of damaged neurons.

【KEY WORDS】ischemic stroke；KCNQ channel openers；tMCAO model；oxygen-glucose deprivation；electrophysiology

Corresponding author：WANG Ke-wei，E-mail：wangkw@qdu.edu.cn

OA-37 Study on the Activity of Melatonin Flexible Liposomes against Skin Photoaging in Animal Model

YU Wan-qing1，LIU Hong-bo1，ZHOU Ming-hong1，HU Hai-ping1，ZHANG Yang，HOU Xue-qin*1
1. Institute of Pharmacology，Shandong First Medical University& Shang dong Academy of Medical Sciences，Tai’an，271016，China

【ABSTRACT】 Objective：To establish a skin photoaging （SP） mouse model using female Kunming （KM） mice and compare the level of oxygen free radicals in the skin of normal mice with that in mice treating with melatonin liposome，so as to discuss the pharmacological activity of melatonin liposome.Methods：Skin aging includes endogenous aging and exogenous aging.Endogenous aging，also known as intrinsic aging or time aging，is an irreversible process，which is only controlled by time and genetic information.Exogenous aging refers to the degeneration of the skin caused by environmental factors.Among many environmental factors，ultraviolet （UV） is the leading one，which is also known as photoaging.It is a kind of skin aging disease that can be prevented or treated.Therefore，the research on anti-aging in the research field generally refers to the research on anti-skin exogenous aging （photoaging）.In this study，long wave ultraviolet （UVA）combined with medium wave ultraviolet （UVB） were used as the light source，and KM mice were used as experimental animals to prepare the skin exogenous photoaging model.Based on this model，the skin was coated with melatonin flexible liposomes for treatment.The macroscopic evaluation of dorsal skin，tensile test and weight measurement were employed to evaluate skin hydration and the degree of skin elasticity，and to explore the protective effect of melatonin flexible liposomes on light skin aging model mice induced by UV.In order to further study the mechanism of melatonin flexible liposome against skin photoaging，we further analyzed the activities of a series of antioxidant enzymes，including superoxide dismutase （SOD），glutathione peroxidase （GSH-Px） and catalase （CAT），and malondialdehyde （MDA） in the mouse skin tissues by biochemical quantitative analysis.In addition，this study also observed and analyzed the degree of irregular epidermal hyperplasia，the morphology，content and distribution of collagen fibers and elastic fibers in animals through the experimental method of HE staining，and made an overall evaluation on the changes of skin histology.Results：The results showed that melatonin flexible liposome could significantly improve UV-induced skin dryness，flabbiness，coarse and deep wrinkles，nodules，pigmentation spots，and leather-like appearance.In addition，it could significantly shorten the recovery time after skin stretching，suggesting that melatonin flexible liposome can significantly increase skin hydration，maintain skin elasticity，and maintain the skin macroscopic normal characterization.The results showed that melatonin flexible liposome could significantly increase the activity of SOD，GSH-Px and CAT in skin tissue of photoaging mice，significantly reduce the content of MDA.At the same time，histopathological results showed that melatonin flexible liposome could significantly improve the deformability of skin structure caused by UV （especially the distortion and fracture of collagen fiber and elastic fiber），and maintain the normal distribution of collagen fiber and elastic fiber.It is suggested that the external application of melatonin flexible liposome can reverse the skin photoaging damage caused by repeated UV radiation via the antioxidant effect.Conclusion：In this study，it was found that melatonin flexible liposome could significantly increase the elasticity of animal skin and maintain the macroscopic normal characterization of skin through in vivo anti-aging experiments.According to the further exploration of the mechanism，it was found that the skin coated with melatonin flexible liposome could play an anti-aging role through anti-oxidation.It is suggested that melatonin flexible liposome has potential efficacy in preventing skin photoaging.This study is beneficial to the development and application of melatonin flexible liposome in anti-aging diseases in the future，and provides a reference and basis for the development of melatonin cosmetics.

【KEY WORDS】melatonin；flexible liposome；photoaging of skin；oxidation radical；ultraviolet（UV）

Acknowledgments：The study was supported by research grants from the National Natural Science Foundation of China （NO.81703901），the Shandong Province Natural Science Foundation of China （ZR2016HB56）

Corresponding author：HOU Xue-qin，MD，PhD，Institute of Pharmacology，Shandong First Medical University，619 Chang Cheng Road，Taian，Shandong 271016，P.R.China；E-mail：houxueqin09@126.com

OA-38 The α4β2 nAChRs Contribute to Regulating the Emotion in CUMS Mice by Balancing Neurotransmitters

YU Zi-ru，LIANG Yu，SONG Jun-ke，SHEN Yan-jia，ZHAO Xiao-yue，DU Guan-hua*
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines，Beijing Key Laboratory of Drug Target and Screening Research，Institute of Materia Medica，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100050，China

【ABSTRACT】Depression is a public health threat with high suicide rate，and the World Health Organization predicted that it will become the second largest burden of disease in the world by 2020.Although a great progress have been made in illustrating the pathophysiology of depression，many limitations still exist.The clinical antidepressants can only provide 2/3 of these patients with some relief.Cholinergic-adrenergic theory has received more and more attention in recent years，and the central role of different subtypes has also received attention.Objective：To study the role of α4β2 nAChRs in the antidepressant process in CUMS mice and to explore the mechanism preliminarily.Methods：We established a mouse model of chronic mild unpredictable stress （CUMS），and we antagonized Nicotine using the α4β2 subtype acetylcholine receptor specific antagonist DHβE，also，DHβE administration alone group was set up to rule out the effect of DHβE itself on mice at this dose.Subsequently，the depression-related behaviors，neuronal morphological function，neurotransmitter level，receptor expression level and related signaling pathway protein expression in mice of different administration groups were investigated.Results：Nicotine showed significant antidepressant effects in CUMS mice，which could effectively improve the depression and anxiety behaviors of CUMS mice in tail suspension，forced swimming，sucrose preference test，open field experiment，elevated plus maze test.But the behavior changes of Nicotine were antagonized when α4β2 nAChRs were blocked.Meanwhile，Nicotine improved the neurological function，regulated the ACh and NE contents，decreased β2 nAChRs expression，and activated the PKA-CREB-BDNF signaling pathway.And all these effects were weakened when Nicotine was combined with DHβE，in another word，when α4β2 nAChRs were antagonized.Conclusion：It showed that nicotinic acetylcholine receptor agonists and antagonists were effective

in regulating animal mood both during normal state and after environmental stimulation，and α4β2 nAChRs played an essential role in antidepressant process in CUMS mice.This article will provide theoretical basis and experimental data support of cholinergic receptor-related compounds，and provide a reference for researchers to develop new antidepressants.

【KEY WORDS】depression；chronic unpredictable minor stress （CUMS）；α4β2 nAChRs；nicotine；DHβE；PKA-CREB-BDNF signaling pathway

Corresponding author：dugh@imm.ac.cn（G.Du）

OA-39 Effects of the rs1799986 Polymorphism of the LRP1 Gene on the Default Mode Network among Individuals in Preclinical and Prodromal Stage of Alzheimer’s Disease

ZANG Fei-fei1，ZHU Yao1，ZHANG Qian-qian1，TAN Chang1，XIE Chun-ming1*，Alzheimer’s Disease Neuroimaging Initiative2
1. Department of Neurology，Affiliated ZhongDa Hospital，School of Medicine，Southeast University，Nanjing，Jiangsu，China
2. Data in present study were obtained from the Alzheimer’s Disease Neuroimaging Initiative （ADNI） database（adni.loni.usc.edu）

【ABSTRACT】 Objective：The low-density lipoprotein receptor-related protein 1 （LRP1）as the receptor of apolipoprotein E，plays a critical role in the clearance of Amyloidβ-peptide.The present study aims to explore the effects of the LRP1 gene rs1799986 variant on the default mode network （DMN） among subjects from subjective cognitive decline （SCD） to mild cognitive impairment （MCI） and eventually to Alzheimer’s disease （AD）.Methods：Twenty-six AD subjects，42 MCI subjects，45 SCD subjects，and 55 cognitively normal controls （CN） from Alzheimer’s Disease Neuroimaging Initiative （ADNI） database were included in this study.Restingstate functional MRI scans and neuropsychological assessments were collected at baseline.Grouplevel independent component analysis （ICA） was used to obtain the DMN patterns.Multivariate linear regression analysis was employed to measure the effects of gene，disease and gene×disease on the default mode network.Results：The LRP1 gene×disease interaction for four-group subjects was mainly detected in the bilateral middle frontal gyrus （bMFG），posterior cingulate cortex （PCC）as well as retrosplenial cortex （RSC）.While，the brain areas such as left temporoparietal joint （TPJ），inferior parietal cortex （IPC），PCC and RSC represent the main effect of disease，and left middle frontal gyrus （lMFG） and PCC were responsible for the main effect of LRP1 gene.Furthermore，the Z-transformed mean functional connectivity （FC） of the interactive PCC region was correlated with the Z-transformation score of mini-mental state examination （MMSE） among non-normal subjects.Conclusion：The LRP1 gene could consistently affect the DMN patterns among individuals in preclinical and prodromal stage of Alzheimer’s disease.

【KEY WORDS】Alzheimer’s disease；low-density lipoprotein receptor related protein 1；LRP1；default mode network；subjective cognitive decline；mild cognitive impairment；DMN；ADNI

Corresponding authors：chmxie@163.com

OA-40 Sirt6 Regulates Circadian Rhythm through Directly Interacting with the Clock Components

ZHANG Feng1，GUO Bao-qiang2，ZHANG Ji-guo1*，MENG Qing-jun2*
1. Institute of Pharmacology，Shandong First Medical University & Shandong Academy of Medical Science，Taian，271000，China
2. Faculty of Life Sciences，University of Manchester，Oxford Road，Manchester，M13 9PT，UK

【ABSTRACT】 Background：In mammals，almost all physiological and behavioral activities are oscillated ～24 h rhythms by an internal conserved circadian clock.At molecular level，circadian clock depending on a transcriptional/translational feed-back loop.CLOCK and BMAL1，two key transcription factors，heterodimerize and activate transcription of two repressors PERs （PER1，PER2） and CRYs.NAD+-dependent deacylase Sirt1 deacetylates BMAL1 and PER2 to regulate circadian rhythms.Sirt6，another NAD+-dependent deacylase，is also involved in maintenance of circadian rhythms.However，the precise function and targets of Sirt6 on the regulation of circadian clock are elusive.Objective：To investigate the precise mechanism of Sirt6 on the regulation of circadian clock.Methods：The gene expressions of Sirt1 or Sirt6 in mouse different tissues were analyzed by Real-time PCR.For Sirt6 knockdown，small interfering RNA （siRNA）targeting Sirt6 was designed and transient transfection was performed with RNAiMax transfection reagent.Circadian rhythms in bioluminescence from per2：：luc in SW1353 cells were recorded in real time using photomultiplier tube （PMT） devices.The capability of Sirt6 to interact with several core clock components in HEK293T cells was assessed by Co-Immunoprecipitation （CoIP） assay.The degradation of PER2 after Sirt6 knockdown was measured by western blotting.Results：（1） Sirt6 is widely distributed in the body，especially highly expression in the hypothalamus and pituitary in brain.（2） SiRNA-mediated depletion of SIRT6 led to significant changes in clock oscillations，with a period lengthening （～113%；P＜0.01） and amplitude reduction （～50%；P＜0.01）.（3） Sirt6 directly interacted with core clock component including BMAL1，CLOCK，and PER2.（4） Sirt6 knockdown repressed the degradation of PER2.Conclusion：In summary，we found that Sirt6 directly interacted with the clock components，i.e.CLOCK，BMAL1 and PER2，to participate in the regulation of circadian clock.Furthermore，Sirt6 regulates the degradation of PER2，which may be the key to its role.

【KEY WORDS】sirt6；CLOCK；BMAL1；PER2；circadian clock

Corresponding authors：jgzhang@tsmc.edu.cn；Qing-Jun.Meng@manchester.ac.uk

OA-41 鹽酸羥哌吡酮（YL-0919）抗精神-認(rèn)知損傷的效應(yīng)及可能機(jī)制研究

張黎明，李云峰*
軍事科學(xué)院軍事醫(yī)學(xué)研究院毒物藥物研究所，北京，100850，中國(guó)

【摘要】 目的：研究鹽酸羥哌吡酮（YL-0919）抗精神-認(rèn)知損傷的行為學(xué)效應(yīng)及靶標(biāo)調(diào)控機(jī)制。方法：采用行為絕望模型、慢性應(yīng)激模型、小鼠孔板、爬梯實(shí)驗(yàn)、高架十字迷宮等模型評(píng)價(jià)YL-0919 的抗抑郁、抗焦慮活性，采用新物體識(shí)別、Morris 水迷宮和小鼠跳臺(tái)等模型評(píng)價(jià)YL-0919的促認(rèn)知活性。采用放射配體競(jìng)爭(zhēng)結(jié)合抑制實(shí)驗(yàn)和［35S］GTPγS 結(jié)合實(shí)驗(yàn)，研究YL-0919 的靶標(biāo)活性。采用清醒大鼠腦微透析技術(shù)結(jié)合高效液相-電化學(xué)檢測(cè)技術(shù)、電生理技術(shù)、蛋白免疫印跡法等技術(shù)考察YL0919 抗精神-認(rèn)知障礙的可能機(jī)制。結(jié)果：（1） YL-0919 抗抑郁效應(yīng)研究：在小鼠懸尾、小鼠強(qiáng)迫游泳和大鼠強(qiáng)迫游泳行為絕望模型上，YL-0919 急性給藥具有顯著抗抑郁活性且在其有效劑量范圍內(nèi)無(wú)中樞興奮和抑制作用。YL-0919 慢性和亞慢性灌胃給藥，在小鼠和大/小鼠多種經(jīng)典行為學(xué)模型上具有顯著的抗抑郁作用：在小鼠新奇抑制攝食模型上，YL-0919（2.5 mg·kg-1）起效時(shí)間較氟西汀快速（1 周vs.2 周），提示YL-0919 可能具有快速起效的優(yōu)勢(shì)。在小鼠獲得性無(wú)助模型上，YL-0919（1.25～5 mg·kg-1）亞慢性灌胃給藥，減少逃避失敗次數(shù)，縮短逃避潛伏期，與對(duì)照組比較有顯著性差異。在大鼠慢性不可預(yù)知應(yīng)激模型上，YL-0919（0.312 5～2.5 mg·kg-1）慢性灌胃給藥，提高慢性應(yīng)激模型大鼠蔗糖偏嗜度，增加慢性應(yīng)激模型大鼠的水平跨格次數(shù)及垂直站立次數(shù)，縮短慢性應(yīng)激模型大鼠攝食潛伏期。在大鼠嗅球切除模型上YL-0919（0.312 5～2.5 mg·kg-1）慢性灌胃給藥，顯著減少嗅球切除大鼠的開(kāi)場(chǎng)實(shí)驗(yàn)活動(dòng)性與電擊逃避失敗次數(shù)。（2） YL-0919 抗焦慮效應(yīng)研究：YL-0919 急性或慢性灌胃給藥在多個(gè)焦慮模型上具有顯著的抗焦慮作用，表現(xiàn)在：大鼠高架十字迷宮模型上，YL-0919（2.5 mg·kg-1）慢性給藥，在不影響入臂總次數(shù)和總時(shí)間的情況下，增加大鼠進(jìn)入開(kāi)臂次數(shù)百分比和在開(kāi)臂停留時(shí)間百分比。在大鼠新奇抑制攝食實(shí)驗(yàn)上，YL-0919（1.25，2.5 mg·kg-1）慢性給藥，顯著縮短攝食潛伏期，效應(yīng)與氟西汀相當(dāng)，但其有效劑量較之氟西汀更低。大鼠Vogel 飲水沖突實(shí)驗(yàn)上，YL-0919（0.625，1.25 mg·kg-1）慢性給藥能夠增加大鼠電擊飲水次數(shù)，與對(duì)照組比較有顯著性差異。（3） YL-0919 促認(rèn)知效應(yīng)研究：亞慢性灌胃給予YL-0919（1.25～2.5 mg·kg-1）可顯著增加小鼠在新物體識(shí)別實(shí)驗(yàn)中的識(shí)別指數(shù)，增加小鼠在Morris 水迷宮模型中的目標(biāo)象限穿越次數(shù)、目標(biāo)象限累計(jì)停留時(shí)間、穿越平臺(tái)次數(shù)，并縮短首次穿越平臺(tái)時(shí)間，顯著縮短小鼠在跳臺(tái)實(shí)驗(yàn)中的跳臺(tái)潛伏期，提示YL-0919 具有顯著的增強(qiáng)認(rèn)知行為活性，而5-HT1A部分激動(dòng)和5-HT 重?cái)z取抑制劑維拉佐酮（2～4 mg·kg-1）并沒(méi)有表現(xiàn)出促認(rèn)知活性；進(jìn)一步研究表明，5-HT6受體拮抗劑SB271046（10 mg·kg-1）在不影響認(rèn)知行為的情況下，可完全阻斷YL-0919 的促認(rèn)知活性。上述結(jié)果提示，5-HT6激動(dòng)活性是YL-0919 促認(rèn)知的重要機(jī)制。（4） YL-0919 的性功能副反應(yīng)評(píng)價(jià)：YL-0919 和維拉唑酮亞慢性和慢性給藥在其抗抑郁有效劑量范圍內(nèi)對(duì)雄性大鼠性功能無(wú)影響；氟西汀亞慢性和慢性給藥可導(dǎo)致雄性大鼠性功能障礙，這與其臨床副作用表現(xiàn)是一致的。（5）作用機(jī)制研究：YL-0919 靶標(biāo)新穎明確，靶標(biāo)活性更強(qiáng)。放射性配體競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)顯示，YL-0919與5-HT 轉(zhuǎn)運(yùn)蛋白（SERT）和5-HT1A/6 有高親和力和高選擇性結(jié)合，35S-GTPγ-S 結(jié)合實(shí)驗(yàn)顯示YL-0919 對(duì)5-HT1A 受體具有選擇性部分激動(dòng)活性，并對(duì)5-HT6 受體表現(xiàn)出全激動(dòng)劑特征。清醒大鼠腦微透析技術(shù)結(jié)合高效液相-電化學(xué)檢測(cè)（HPLC-ECD）方法顯示，單次灌胃給予YL-0919（1.25，2.5 mg·kg-1），劑量依賴且持續(xù)增加清醒大鼠腹側(cè)海馬5-HT 水平，并且該作用顯著強(qiáng)于維拉唑酮和氟西汀。重復(fù)灌胃YL-0919（5 mg·kg-1，7 d）或（1.25～5 mg·kg-1，21 d）顯著增強(qiáng)活體大鼠海馬長(zhǎng)時(shí)程增強(qiáng)效應(yīng)（LTP），表現(xiàn)為群峰電位（PS）幅值和抑制性突觸后電位（EPSP）斜率顯著增加，而陽(yáng)性藥氟西?。?0 mg·kg-1）僅在21 d 增強(qiáng)海馬LTP，提示YL-0919 可增強(qiáng)海馬神經(jīng)可塑性，并且較之氟西汀起效更迅速。結(jié)論：YL-0919 是是兼有5-HT1A/6激動(dòng)劑和5-HT重?cái)z取抑制劑活性的抗抑郁新藥，在多種動(dòng)物模型上，表現(xiàn)出顯著的抗抑郁、抗焦慮及增強(qiáng)認(rèn)知行為作用，并且較之于臨床一線抗抑郁藥，具有快速起效、增強(qiáng)認(rèn)知、無(wú)性功能障礙等優(yōu)點(diǎn)，很可能成為兼有促認(rèn)知活性的新一代抗抑郁新藥。

【關(guān)鍵詞】YL-0919；抗抑郁；抗焦慮；促認(rèn)知；5-HT1A 受體；5-HT6 受體；5-HT 轉(zhuǎn)運(yùn)蛋白

OA-42 Phytochemical and Pharmacological Characterization of Lily-Rehmannia Decoction，A Classic Chinese Herbal Formula，for Menopausal Syndrome in Ovariectomized Mice：A Novel Brain-Uterus Mechanism Distinct from Estrogen Therapy

ZHANG Zhang-jin
School of Chinese Medicine，F(xiàn)LS Faculty of Medicine，the University of Hong Kong，Hong Kong，China

【ABSTRACT】 Background：Although estrogen therapy is the mainstay for management of menopause syndrome，long-term estrogen therapy increases the risk of breast and ovarian cancer，stroke，and cardiovascular disease.A search of alternative therapy particularly from herbal medicine is therefore highly desired.Lily-Rehmannia Decoction （LRD，百合地黃湯） is a classic Chinese medicine formula which consists of Bulbus Lilli （百合） and Radix Rehmanniae （地黃）and is often used for the treatment of depression，dysphoria，and insomnia.Here，we examined whether LRD and its components AEBL and AERR （aqueous extract of them），could ameliorate the menopause-associated psychiatric disorders.Methods：Ovariectomized （OVX） mice were treated with 1.8 g·kg-1AELB，2.6 g·kg-1AERR，2.3 g·kg-1LRD or 0.3 mg·kg-1estradiol for 5 weeks.Multiple behavioral paradigms were conducted.Uterus and brain were collected for the measurement of neurotransmitters and their related biomarkers，neurotrophins，and estrogen receptors （ERα and ERβ）.Results：The three herbal agents showed similar anxiolytic and antidepressant effects with estrogen therapy，which seemed to be achieved via the predominant protection of neurotransmitters，neurotrophins and ERβ receptors in the related brain regions.While estradiol limited OVX-induced weight gains and prevented uterine shrinkage and the drop of serum estrogen level，the herbal agents had no or minor effects on these indices.Moreover，the herbal agents might exert a better safety profile than estrogen therapy because they had minor effects on uterine nerve growth factor （NGF） and ERα expression levels.However，AEBL，but not AERR or LRD，exerted similar effects on the OVX-induced cognitive deficit with estrogen therapy，which was possibly associated with the upregulation of cortical ERα.Conclusions：AEBL is a promising agent that may serve as a novel therapy for reducing menopause-associated neuropsychological disorders.

Acknowledgements：This study was supported by General Research Fund （GRF） of Research Grant Council of Hong Kong （17115017）

OA-43 藁本內(nèi)酯通過(guò)調(diào)節(jié)腦微血管內(nèi)皮細(xì)胞GLUT1 改善APP/PS1 小鼠Aβ轉(zhuǎn)運(yùn)

章時(shí)杰1，許婷婷1，王奇，1*
1.廣州中醫(yī)藥大學(xué)臨床藥理研究所，廣州，510405，中國(guó)

【摘要】 目的：阿爾茨海默病是一種最常見(jiàn)類型的癡呆，臨床上病理表現(xiàn)為大腦內(nèi)神經(jīng)元周圍淀粉樣斑塊沉積和神經(jīng)元內(nèi)神經(jīng)纖維纏結(jié)。阿爾茨海默病的真正病因至今仍然不明確。對(duì)于AD 的治療，目前尚沒(méi)有有效的治療藥物，迫切需要尋找有效的治療藥物。流行病學(xué)研究發(fā)現(xiàn)，在西方發(fā)達(dá)國(guó)家，由于增強(qiáng)了對(duì)血管以及大腦健康狀況的關(guān)注，AD 的發(fā)病率呈一定程度的下降趨勢(shì)。對(duì)于腦血管的保護(hù)，從腦微血管的角度入手，轉(zhuǎn)運(yùn)清除腦內(nèi)的淀粉樣斑塊沉積作為另外一種的研究思路，可能可以為治療AD 提供新的手段。本課題旨在從淀粉樣斑塊沉積轉(zhuǎn)運(yùn)清除的角度探討藁本內(nèi)酯改善阿爾茨海默病模型動(dòng)物學(xué)習(xí)記憶能力的藥理作用。方法：在本研究中，我們使用經(jīng)典的APP/PS1 雙轉(zhuǎn)基因小鼠模型來(lái)探索藁本內(nèi)酯的作用。3 月齡雄性APP/PS1 雙轉(zhuǎn)基因小鼠 48 只，同時(shí)選擇相同月齡轉(zhuǎn)基因陰性小鼠 16 只。分組：轉(zhuǎn)基因陰性鼠作為正常對(duì)照組，APP/PS 雙轉(zhuǎn)基因鼠隨機(jī)分成 3 個(gè)組，分別是模型組、藁本內(nèi)酯（ligustilide，LIG）低（10 mg·kg-1）、高劑量（20 mg·kg-1）。從10 月齡開(kāi)始，給予小鼠每天灌胃給藥，每24 h 給藥一次。對(duì)照組及模型組均給予DMSO 溶劑，給藥 8 周后開(kāi)始行為學(xué)測(cè)試。機(jī)制研究：用Western blotting 等方法，測(cè)定Aβ代謝相關(guān)通路及其下游級(jí)聯(lián)反應(yīng)相關(guān)蛋白。結(jié)果：Morris 水迷宮和Y 迷宮測(cè)試結(jié)果表明，在APP/PS1 雙轉(zhuǎn)基因小鼠的灌胃給藥（10 mg·kg-1·d-1和20 mg·kg-1·d-1）改善了學(xué)習(xí)和記憶能力。藁本內(nèi)酯主要通過(guò)直接作用于腦微血管內(nèi)皮細(xì)胞GLUT1/LRP1/RAGE，提高了Aβ的轉(zhuǎn)運(yùn)清除，減少了Aβ在腦內(nèi)的沉積。藁本內(nèi)酯同時(shí)也改善了Aβ代謝相關(guān)通路（ADAM10，BACE1，NEP，IDE，sAPPα，RAGE and LRP1），減少了神經(jīng)退行性病變（NGF、SYN、PSD93、PSD95、BDNF）、氧化應(yīng)激（ROS、MDA）、神經(jīng)凋亡（BAX、BCL-2、CASPASE-3）、神經(jīng)炎癥（IL-1β、IL-6、NFκB、NLRP3）和內(nèi)質(zhì)網(wǎng)應(yīng)激的水平。結(jié)論：這些結(jié)果表明，藁本內(nèi)酯有可能通過(guò)直接作用于腦微血管內(nèi)皮細(xì)胞GLUT1 促進(jìn) Aβ 的轉(zhuǎn)運(yùn)與清除，從而達(dá)到改善 AD 模型動(dòng)物的學(xué)習(xí)記憶能力，為AD 患者提供一個(gè)新的治療策略。

【關(guān)鍵詞】阿爾茨海默??；藁本內(nèi)酯；血腦屏障；GLUT1；Aβ轉(zhuǎn)運(yùn)

通訊作者：王奇，E-mail：wangqi@gzucm.edu.cn

OA-44 Inhibition of PDE4 by FCPR16 Induces AMPKDependent Autophagy and Confers Neuroprotection in SH-SY5Y Cells and Neurons Exposed to MPP+-Induced Oxidative Insult

ZHONG Jia-hong1，XIE Jin-feng1，XIAO-Jiao1，ZHOU Zhong-zhen1，WANG Hai-tao*1，XU Jiang-ping*1，2
1. Department of Neuropharmacology and Drug Discovery，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou，510515，China
2. Guangdong Provincial Key Laboratory of New Drug Screening，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou，510515，China

【ABSTRACT】 Objective：The cause of Parkinson disease （PD） is generally not clear，but it is considered to be related to excessive oxidative damage.Therefore，the identification of therapeutic targets and compounds with antioxidant damage is a reasonable strategy to slow down the progress of PD.FCPR16 is a novel phosphodiesterase4 inhibitor with little emetic potential.Our previous studies showed that FCPR16 was an effective compound for blocking 1-Methyl-4-phenylpyridine （MPP+）-induced oxidative damage in SH-SY5Y cells and neurons.However，the detailed mechanisms underlying its protective effect have not been investigated.The level of oxidative stress in neurons is closely related to the balance of mitochondria mass，while autophagy strongly regulates mitochondrial activity in neurons.Our previous study indicated that inhibition of PDE4 or PDE4 knockdown enhanced the activation of autophagy in microglial cells.While whether PDE4 inhibition mediates autophagy in neurons is largely unknown.As described above，autophagy plays a pivotal role in maintaining redox and mitochondrial homeostasis.We were interested in exploring the impact of PDE4 inhibition on autophagy in neurons.Methods：SH-SY5Y cells and neurons was induced with 1-methyl-4-phenylpyridinium （MPP+） to mimic Parkinson’s disease cell injury in vitro，and MTT assay was used to investigate the viability effects of FCPR16 （50 μmol·L-1） with or without different autophagy inhibitors on MPP+-injured SH-SY5Y cells.Detection of apoptosis was performed by PI staining fluorescence.Lysosomes are essential in autophagy，so LYT Red Stain was used to detect lysosomes in SY5Y cells and neurons.Cells were exposed to CellROX Deep Red Reagent to detect intracellular reactive oxygen species （ROS）.Mitochondrial membrane potential （Δψm） measurement was executed by 5，5′，6，6′-tetrachloro-1，1′，3，3′-tetraethylbenzimidazolyl- carbocyanineiodide （JC-1）.To better detect intracellular autophagy，we used the CYTO-ID Autophagy detection kit to detect the autophagic vacuoles and monitor autophagic flux.The expression of autophagy related proteins and other related signal molecules were demonstrated by Western blot.As relevant indicators of oxidative stress，3-nitrotyrosine （3-NT） and highly toxic peroxide 4-hydroxynonenal （4-HNE）were detected with 3-NT and 4-HNE ELISA kits.Results：FCPR16 could significantly decrease the expression of p62，an autophagy substrate，at 6 h and 12 h，while FCPR16 enhanced the level of LC3-Ⅱ.Similarly，F(xiàn)CPR16 increased the lysosomes fluorescence and CYTO-ID signal in cells and neurons，while it could be block by 3-methyladenine （3-MA） and hydroxychloroquine sulfate（HCQ）.Simultaneously，Treatment of SH-SY5Y cells with FCPR16 prevented MPP+-induced production of reactive oxygen species （ROS） and the decline of mitochondrial membrane potential（Δψm）.Importantly，we also found that FCPR16 phosphorylated and thus activated AMPK in SHSY5Y cells treated with MPP+.In contrast，blockade of the AMPK pathway with compound C blocked the role of FCPR16 in autophagy enhancement.MPP+-induced a significant increase in PI-positive cells，while FCPR16 decreased the ratio of PI positive cells and 3-MA and compound C could block the protective effect.Additionally，F(xiàn)CPR16 reduced MPP+-induced decline of cell viability，and 3-MA and compound C could block the protective effect.Conclusion：Deficits in autophagy have been proven to participate the pathology of PD and targeting autophagic function has been viewed as a potential therapeutic strategy for the clearance of toxic proteins （such as α-synuclein） and of impaired mitochondria.this is the first time that PDE4 inhibition has been shown to induce autophagic enhancement both in SH-SY5Y cells and in primary cultured neurons.In addition，our findings indicate that inhibition of PDE4 by FCPR16 protects against MPP+-induced oxidative stress and cellular injury in SH-SY5Y cells and neurons through the activation of AMPK-dependent autophagy.Taken together，these results show that PDE4 is a promising target for developing novel drugs against neuronal apoptosis and FCPR16 may be a potential compound for the prevention and treatment of PD.

【KEY WORDS】phosphodiesterase 4；FCPR16；oxidative stress；mitochondrial membrane potential；Parkinson’s disease

Foundation item：This work was supported by National Natural Science Fund of China （No.81773698） and the Funding from Guangzhou Science and Technology Department （No.2015B020211007，201604020112）.

OA-45 Comparison of PDE4 Expression in Different Brain Regions of C57BL/6J and DBA/2J Mice and the Association with Ethanol Drinking Behavior

DU Xian1，WANG Dong1，ZHANG Fang-fang1，ZHOU Yan-meng1，WANG Hao1，F(xiàn)U Hua-rong1，ZHANG Hanting*1，2
1. Institute of Pharmacology，Shandong First Medical University & Shandong Academy of Sciences，Tai’an，271016，China
2. Departments of Behavioral Medicine & Psychiatry，Physiology & Pharmacology，and Neuroscience，the Rockefeller Neurosciences Institute，West Virginia University Health Sciences Center，Morgantown，WV 26506，USA

【ABSTRACT】 Background：Alcohol dependence is not only the most common public health problem worldwide，but also the cause of many alcohol-related diseases，yet the mechanisms still remain unclear.Increasing evidence suggests that genetic factors play an important role in alcohol drinking behavior.C57BL/6J （B6） mice innately consume larger amounts of alcohol compared to DBA/2J （DBA） mice，which drink little alcohol.Our recent studies have demonstrated that phosphodiesterase-4 （PDE4），an enzyme that catalyzes the hydrolysis of cyclic AMP （cAMP），is an important target for regulating alcohol consumption.However，it is not known which of the four PDE4 subtypes （A-D） is involved.Previous studies demonstrate that lower levels of cAMP signaling are observed in specific brain regions of alcohol-preferring rodents；inhibition of cAMP signaling increases alcohol consumption，while activation of cAMP signaling in the brain decreases alcohol intake，in particularly brain regions such as the striatum and amygdala.Thus，we hypothesized that PDE4 expression in the brain of B6 and DBA mice is different and relevant to alcohol drinking.Objective：To compare the expression of PDE4 subtypes in the ethanol drinking-related brain regions of B6 and DBA mice and their responses to ethanol drinking in the absence or presence of the PDE4 inhibitor.Methods：Adult male and female B6 and DBA mice were tested for ethanol （7% and 10%，v/v） intake and preference using the twobottle choice.In addition，a separate set of B6 and DBA mice was examined for PDE4 subtype expression levels in the striatum，hippocampus，cerebral cortex，and amygdala of the brain using Western blotting.Further，PDE4 subtype expression in brain regions of B6 mice in response to ethanol drinking was examined.Finally，the PDE4 inhibitor rolipram was administered to B6 mice to detect alcohol drinking behavior changes.Results：1.Differences in ethanol drinking behavior between B6 and DBA mice.Compared to DBA mice，B6 mice did not show significant difference in body weights，sucrose intake and preference，and quinine intake and preference，but their alcohol intake and preference were significantly higher.2.Differences in the expression of PDE4 subtypes in drinking-related brain regions of B6 and DBA mice.Compared to the DBA strain，naive B6 mice showed significant higher expression of PDE4B in the striatum （P＜0.05，female），cerebral cortex （P＜0.05，female），and amygdala （P＜0.05，male；P＜0.01，female）.3.PDE4B in the drinking-related brain regions of naive and alcohol-drinking B6 mice.Expression of PDE4B in the striatum （P＜0.05，female），hippocampus （P＜0.05，male and female），cerebral cortex （P＜0.05，female），and amygdala （P＜0.05，female；P＜0.001，male） was significantly increased relative to naive controls.4.Effect of rolipram on alcohol drinking behavior in B6 mice.Rolipram reduced alcohol intake （P＜0.05 at 0.5 mg·kg-1；P＜0.01 at 1 mg·kg-1） and preference （P＜0.05，0.5 and 1 mg·kg-1）without altering the total fluid intake in B6 mice.Conclusion：These results demonstrate that mice with excessive alcohol drinking （such as B6） exhibit higher levels of PDE4B expression in the brain relative to mice innately disliking alcohol （such as DBA）；PDE4B expression can be further increased following ethanol consumption.Thus，PDE4B is an important player in the regulation of alcohol consumption.Drugs inhibiting PDE4B can be novel treatments for alcoholism.

【KEY WORDS】phosphodiesterase-4 （PDE4）；alcohol drinking；C57BL/6J mice；DBA/2J mice；cAMP

OA-46 Research for Early Recognition of Alzheimer’s Disease Based on Hippocampal Subfield Volume and Regional Cerebral Blood Flow

TAN Chang，XIE Chun-ming*1，ZHANG Zhi-jun
Department of Neurology，Zhongda Hospital，Southeast University，Nanjing，210009，China

【ABSTRACT】 Objectives：We intended to describe the reduction of hippocampal subfields volume as well as the changes of regional CBF in individuals at the risk of AD.We hypothesized that combination of hippocampal subfields volumes and regional CBF can acquire higher accuracy than either method alone in discriminating mild cognitive impairment （MCI） patients from cognitively normal elderly adults.Methods：44 early mild cognitive impairment （EMCI） patients，50 late mild cognitive impairment （LMCI） patients and 40 healthy controls （HC） underwent sMRI and ASL-MRI scan along with a battery of neuropsychological tests.Hippocampal subfields volume was acquired by institutional specially-design software，with total intracranial volume normalization.Normalized regional and whole-brain voxel-based CBF differences were calculated using voxel-based analysis software.We compared the differences in hippocampal subfields volumes and regional CBF among the three groups.Combined with the results of multidimensional cognitive psychology evaluation，we analyzed the significant correlation between hippocampal subfield volumes and regional CBF with behavioral performance.Finally，Various combinations of these parameters were used to create the Receiver Operating Characteristic curves （ROC） in distinguishing MCI patients from the controls.Results：1.There were statistically significant differences in episodic memory score and executive function score among the three groups.2.The normalized volume of multiple subfields in bilateral hippocampus was significantly lower in LMCI patients than in the controls （P＜0.05）.3.The normalized volumes of each hippocampus，left DG，right CA2，right CA3 and right DG were significantly positive correlated with performance in episodic memory （P＜0.001） and Logical Memory Test-Delay recall（LMT-DR）（P＜0.001） in all subjects.4.Left medial frontal gyrus CBF was significantly higher in the EMCI patients than the controls （P＜0.01）.Right anterior/meddle cingulate gyrus CBF was significantly lower in LMCI patients than the EMCI patients （P＜0.01）.5.Right anterior cingulate gyrus CBF was significantly positive correlated with performance in Visuospatial function（P=0.003 1） and Clock Drawing Test （CDT） （P=0.002 76） in HC.6.For predicting the LMCI patients，the highest area under curve value was 0.857，by combining normalized right CA3 subfield volume，normalized CBF in the right anterior/middle cingulate gyrus and Left medial frontal gyrus.Conclusion：Regional CBF changes precede the hippocampal subfields atrophy.Both them reflect the cognitive function status of patients at an early stage of AD.Also，they can be used as important biomarkers to evaluate the cognitive impairment.Combination of hippocampal subfield volume and regional CBF could improve the efficacy in early recognition of high-risk individuals of AD.

【KEY WORDS】Alzheimer’s disease；magnetic resonance imaging；hippocampal subfields；gray matter volume；cerebral blood flow

Corresponding author：Xie Chun-ming，E-mail：chmxie@163.com

OA-47 The Effect of PDE4B1 Overexpression on Ethanol Drinking Behavior in C57BL/6J Mice

XU Bing-bing1，ZHOU Yan-meng1，ZHANG Han-ting2
1. Shandong First Medical University & Shandong Academy of Sciences，Tai’an，Shandong，271016，China
2. Departments of Behavioral Medicine & Psychiatry，Physiology & Pharmacology，and Neuroscience，The Rockefeller Neuroscience Institute，West Virginia University Health Sciences Center，Morgantown，WV 26506，USA

【ABSTRACT】 Objective：To observe the effects of high expression of PDE4B1 in the striatum on ethanol drinking behavior and the cAMP-CREB signaling pathway in mice.Methods：Thirtytwo adult male C57BL/6J mice were used and divided into 4 groups：three of the groups were microinfused with lentiviral vectors containing full-length PDE4B1 or negative control （NC）sequence，or normal saline into bilateral striata （0.7 μL/side，8×108TU·mL-1） according to the following coordinates：AP，-2.5 mm from bregma；ML，±0.5 mm from the midline；DV，-2.7 mm from the dura；the forth group of mice was infused with the same volume of lenti-PDE4B1 into bilateral prefrontal cortices （PFC；AP，-1.5 mm from bregma；ML，±0.5 mm from the midline；DV -1.2 mm from the dura）.Two weeks later，alcohol consumption and preference were determined using the two-bottle choice test，followed 30 min later by the open-field test for spontaneous activity.The striatum and cerebral cortex were dissected for biochemical assays，including Western blotting for expressions of PDE4B1 and its downstream signaling molecules such as CREB/pCREB/BDNF/NPY，and ELISA for cAMP levels.Results：Compared to the NCor saline-treated animals，lenti-PDE4B1-treated mice displayed overexpression of PDE4B1 in the striatum and PFC，but only the former showed significant increases in alcohol consumption and preference，in addition to an early increase in locomotor activity.Western blotting also revealed that the expression of the signaling molecules such as pCREB，CREB，BDNF，and NPY was reduced in the striatum，but not significantly changed in the PFC.Consistently，the cAMP levels were reduced in the striatum of lenti-PDE4B1-treated mice.Conclusion：The results provide additional demonstration that PDE4B，in particular its PDE4B1 isoform，plays a unique role in the regulation of ethanol consumption.

【KEY WORDS】phosphodiesterase-4B1 （PDE4B1）；alcohol consumption；cAMP signaling；mice

OA-48 The Pharmacological Effect and Mechanism Study of Cynomorium Songaricum on Alzheimer’s Disease Based on Mitochondrial Dynamic Balance

CHENG Dan，LI Ling-Ling，LI Xin-Jie，SU Lei，LU Yi*
School of Chinese Medicine，Beijing University of Chinese Medicine，Beijing，100029，China

【ABSTRACT】 Objective：Cynomorium songaricum is a traditional Chinese herb，which could tonify kidney and benefit essence.Our previous studies found the ethyl acetate extract from cynomorium songaricum （ECS） could improve the behavior and morphology of Alzheimer’s disease （AD） model.In this research，the pharmacological effect and mechanism of ECS on the AD model in vitro based on mitochondrial dynamic balance was studied.Method：1.Testing ECS on survival rate and morphology of these two kinds of model cells：HT22 cells were used to establish AD models with H2O2and Aβ25-35respectively.The cell survival rate was measured by CCK-8 method at different concentration and time.The cells were allocated to control，ECS，Aβ，Aβ+ECS，H2O2，H2O2+ECS group.The morphological changes of cells in each group were observed by light microscope.2.ECS effects on mitochondrial dysfunction：After the intervention of ECS on cell models of Aβ and H2O2，the ultrastructural changes of mitochondria were observed by transmission electron microscopy （TEM）；Western Blot was used to detect the expression of mitochondrial kinetics-related proteins of Drp1，p-Drp1（ser 637），F(xiàn)is1，Mfn1，Mfn2 and Opa1；The cytosolic reactive oxygen species （ROS） content was measured by flow cytometer；ATP in cells was measured using luminescent fluorescent microplate reader of multi-wavelength measurement system；fluorescence microscope was used to detect the mitochondrial membrane potential （MMP）.3.Explore the mechanism of ECS improves mitochondrial dysfunction：Calmodulin phosphatase inhibitor （FK506） was added to different groups，and intracellular calcium expression was detected by fluorescence microscopy；calmodulin phosphatase （CaN），p-Drp1 and Drp1 were detected by Western Blot.Result：1.20 μmol·L-1Aβ was used to establish the model for 24 hours，and 200 μmol·L-1H2O2was used to establish the model for 2 hours.ECS with the concentration of 200 μg·mL-1was used to intervene two models for 24 hours respectively，and the cell survival rate of these two model groups were significantly improved after being intervened by ECS.The morphological changes of cells were observed via optical microscopes，and it was discovered that cell was shrinking in the two models，and neurite retraction as well.After the intervention of ECS，the cells in the model group became normal in morphology，and neurite became longer，and the cells were tightly connected.As shown in TEM，mitochondria were swell，and mitochondrial crista was blurred and irregular in the two model groups，which compared with control group.After the intervention of ECS，the mitochondrial morphology basically returned to normal.ECS could significantly decreased ROS，increased ATP and enhanced MMP in the two models of Aβ and H2O2.Western Blot results showed that the expressions of Drp1 and Fis1 were significantly increased，while p-Drp1/Drp1 were decreased in the Aβ and H2O2model groups compared with the control group.There was no significant difference in the expression of Mfn1，Mfn2 and Opa1 between the model and the control groups.ECS could significantly reduce the expression of Drp1 and Fis1，and increased the p-Drp1/Drp1.3.The results showed that ECS and Calcineurin inhibitors （KF-506） could significantly reduce intracellular calcium expression，decrease the content of Calcineurin （CaN）in the models of Aβ and H2O2，and increase the ratio of p-Drp1/Drp1，thus ensuring the normal function of mitochondria.Conclusion：ECS could improve the cell survival rate in AD models of Aβ and H2O2in vitro and inhibit the expression of CaN，thus decreasing the intracellular calcium concentration and increasing the ratio of p-Drp1/Drp1，and regulates the expression of mitochondrial dynamic related proteins，and maintains the dynamic balance of mitochondria；improves morphology and mitochondrial ultrastructure of model cell；reduces the concentration of ROS and intracellular calcium ions in AD model cells；increases intracellular ATP and MMP，thereby improving mitochondrial dysfunction in Alzheimer’s disease.

【KEY WORDS】Alzheimer’s disease；mitochondrial dysfunction；mitochondrial fission/fusion；the extract of cynomorium songaricum

Corresponding author：luyi@bucm.edu.cn

OA-49 Antidepressant-Like Effects of Ginsenoside Rg1 in Chronic Restraint Stressed Rats

JIANG Ninga，LV Jing-weia，HUANG Hongb，LU Conga，WANG Qiongc，YANG Yu-jiec，WANG Hai-xiaa，LIU Xinmina*
a. Research Center for Pharmacology and Toxicology，Institute of Medicinal Plant Development （IMPLAD），Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing，China
b. Key Laboratory of Standardization of Chinese Herbal Medicine in Ministry of Education，School of Pharmacy，Chengdu University of Traditional Chinese Medicine，Chengdu，611137，China
c. Affiliated TCM Hospital/School of Pharmacy/Sino-Portugal TCM International Cooperation Center，Southwest Medical University，Luzhou，646000，China

【ABSTRACT】Depression is a common but serious mental illness，with a huge socioeconomic burden，which adversely affects human mood and cognition.Ginsenosides Rg1，a major bioactive component enriched in ginseng，has potent neuroprotective effect.Chronic restraint stress （CRS）has been proposed as a classical animal model of depression and could mimic the behavioral and physiological symptoms of clinical depression well.In this study，the CRS protocol （6 h·day-1，for 28 days） was utilized to induce depression-like behavior in rats；Ginsenoside Rg1 on chronic restraint stress-induced depression was investigated for the first time.Ginsenoside Rg1 （12.5 μmol·kg-1/i.p） administrated for 2 weeks significantly attenuated depression-like behaviors （anhedonia and behavioral despair） as shown in sucrose preference test and forced swimming tests.Moreover，CRS exposure noticeably altered oxidative parameters，neurotransmitter（5-HT，NE，GABA and Glu）levels in prefrontal cortex as well as corticosterone levels in the serum，while Ginsenoside Rg1 treatment normalized these levels.Meanwhile，Ginsenoside Rg1 treatment significantly upregulated the decreased BDNF and Trkb levels induced by CRS.In conclusion，Ginsenoside Rg1 exerts an antidepressant-like effect in CRS rat possibly by regulating neurotransmitters levels and HPA function，antagonizing oxidative stress and restoring BDNF-TrkB signaling in the prefrontal cortex.This study suggested that Ginsenoside Rg1 may be a new candidate natural therapy for the prevention against stress-induced depression.

【KEY WORDS】ginsenosides Rg1；depression；chronic restraint stress；neurotransmitter；oxidative stress；BDNF

Corresponding author：Prof.Xin-min Liu，E-mail：liuxinmin@hotmail.com

OA-50 Mdivi-1，A Mitochondrial Fission Inhibitor，Modulates T Helper Cells and Suppresses the Development of Experimental Autoimmune Encephalomyelitis

LI Yan-huaa，b，1，XU Fangc，1，Rodolfo Thomea，GUO Min-fangb，SUN Man-luanb，SONG Guo-binb，LI Rui-lanb，CHAI Zhic，Bogoljub Cirica，A.M.Rostamia，MA Cun-genb，c*，ZHANG Guang-xiana*
a. Department of Neurology，Thomas Jefferson University，Philadelphia，PA 19107，USA
b. Institute of Brain Science，Shanxi Key Laboratory of Inflammatory Neurodegenerative Diseases，Shanxi Datong University，Datong，037009，China
c. “2011” Collaborative Innovation Center/Research Center of Neurobiology，Shanxi University of Traditional Chinese Medicine，Taiyuan，030024，China

【ABSTRACT】 Background：Unrestrained activation of Th1 and Th17 cells is associated with experimental autoimmune encephalomyelitis （EAE），an animal model of multiple sclerosis.While inactivation of dynamin-related protein 1 （Drp1），a GTPase that regulates mitochondrial fission，is known to protect myelin from demyelination and to reduce severity of EAE，its effect on T cells is not yet known.Methods：We investigated the effect of Mdivi-1，a small molecule inhibitor of Drp1，on EAE.Clinical scores，inflammation，demyelination and p-Drp1 expression in the central nervous system （CNS），and T cell responses in both CNS and periphery were determined.Results：Mdivi-1 effectively suppressed EAE severity by reducing demyelination and cellular infiltration in the CNS.Mdivi-1 treatment decreased the phosphorylation of Drp1 （ser616） on CD4+T cells，reduced the numbers of Th1 and Th17 cells and increased Foxp3+regulatory T cells in the CNS.Moreover，Mdivi-1 treatment effectively inhibited IFN-γ+，IL-17+and GM-CSF+CD4+T cells，while it induced CD4+Foxp3+，CD4+IL-10+regulatory T cells in the spleen.Conclusions：Together，our results demonstrate that Mdivi-1 has therapeutic potential in EAE by modulating the balance between Th1/Th17 and regulatory T cells.

【KEY WORDS】experimental autoimmune encephalomyelitis；dynamin-related protein 1；Mdivi-1；T cells

These authors contributed equally to this work.

Corresponding author：macungen2001@163.com；guang-xian.zhang@jefferson.edu

OA-51 卵巢摘除對(duì)小鼠學(xué)習(xí)記憶及海馬相關(guān)蛋白的動(dòng)態(tài)影響

陶雪，張夢(mèng)荻，王麗莎，周云豐，王智，劉新民，常琪*
中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥用植物研究所，北京，100193，中國(guó)

【摘要】 目的：婦女進(jìn)入更年期后由于雌激素水平急劇降低，導(dǎo)致學(xué)習(xí)記憶水平有所下降，嚴(yán)重影響其晚年的生活質(zhì)量。去卵巢（OVX）小鼠可以較好模擬更年期認(rèn)知行為及相關(guān)指標(biāo)的改變，盡管文獻(xiàn)對(duì)摘除卵巢引起的認(rèn)知行為改變有所報(bào)道，但由于摘除時(shí)間長(zhǎng)短不一，認(rèn)知行為學(xué)檢測(cè)結(jié)果良莠不齊。近年來(lái)在認(rèn)知障礙模型中關(guān)于BDNF/TrkB 信號(hào)通路在突觸可塑性以及自噬相關(guān)蛋白在細(xì)胞環(huán)境穩(wěn)態(tài)方面的作用研究越來(lái)越引起科學(xué)工作者的重視。本研究將從行為學(xué)和海馬相關(guān)蛋白的變化對(duì)摘除卵巢時(shí)長(zhǎng)對(duì)學(xué)習(xí)記憶的影響進(jìn)行研究，以建立穩(wěn)定的模擬婦女更年期認(rèn)知功能下降的動(dòng)物模型，更好的指導(dǎo)后續(xù)藥效評(píng)價(jià)及機(jī)理研究。方法：對(duì)三組7～8 周齡的ICR 小鼠摘除卵巢后分別于術(shù)后2W、4W 及8W 進(jìn)行空?qǐng)?、新物體識(shí)別、水迷宮以及避暗實(shí)驗(yàn)。行為學(xué)檢測(cè)結(jié)束后進(jìn)行腦組織內(nèi)海馬部分的解剖分離，通過(guò)Western blot 的方法對(duì)海馬組織中的TrkB、ULK 和P62 進(jìn)行含量測(cè)定。結(jié)果：研究結(jié)果顯示，與假手術(shù)組（Sham 組）相比，OVX小鼠4W 時(shí)在避暗實(shí)驗(yàn)中其入暗潛伏期顯著降低，錯(cuò)誤次數(shù)、暗室時(shí)間和明室近口區(qū)時(shí)間顯著增加；OVX 小鼠8W 時(shí)在新物體識(shí)別實(shí)驗(yàn)中其相對(duì)辨別指數(shù)顯著降低，水迷宮工作記憶實(shí)驗(yàn)的潛伏期顯著增加。值得注意的是，OVX 小鼠2W 時(shí)各項(xiàng)檢測(cè)并沒(méi)有與sham 組表現(xiàn)出顯著差異，并且水迷宮定位航行實(shí)驗(yàn)的相關(guān)檢測(cè)指標(biāo)在2W、4W 及8W 均沒(méi)有表現(xiàn)出顯著變化。海馬組織的蛋白含量測(cè)定結(jié)果顯示，與Sham 組相比，OVX 小鼠海馬中的TrkB 蛋白從術(shù)后4W 即發(fā)生顯著性降低，這一改變?cè)谛g(shù)后8W 仍然存在；而OVX 小鼠海馬中的自噬相關(guān)蛋白ULK 和P62 均是在術(shù)后8W 才表現(xiàn)出顯著降低，術(shù)后兩周的行為學(xué)結(jié)果和海馬相關(guān)蛋白并未表現(xiàn)出顯著性變化。結(jié)論：小鼠卵巢摘除后學(xué)習(xí)記憶損傷隨時(shí)間延長(zhǎng)逐漸加重，4W 時(shí)產(chǎn)生一定程度的記憶受損，8W 時(shí)損傷進(jìn)一步加深，工作記憶受損。該記憶障礙發(fā)生的進(jìn)程可能和神經(jīng)營(yíng)養(yǎng)以及自噬通路的先后改變有關(guān)。

【關(guān)鍵詞】卵巢摘除；造模時(shí)長(zhǎng)；學(xué)習(xí)記憶

基金項(xiàng)目：中國(guó)醫(yī)學(xué)科學(xué)院創(chuàng)新工程項(xiàng)目：人類疾病動(dòng)物模型平臺(tái)（2016-I2M-2-006）

通訊作者：qchang@implad.ac.cn

OA-52 質(zhì)譜成像技術(shù)在神經(jīng)系統(tǒng)疾病藥物研發(fā)中的應(yīng)用

劉佳琦1，楊琪1，王沛1，應(yīng)明耀1，張建民1，2
1.國(guó)典（北京）醫(yī)藥科技有限公司，北京，100176，中國(guó)
2.中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所，北京協(xié)和醫(yī)學(xué)院基礎(chǔ)學(xué)院，北京，100730，中國(guó)

【摘要】近幾年質(zhì)譜成像技術(shù)（MSI）發(fā)展迅速，在藥物研發(fā)和新型生物標(biāo)志物鑒定方面表現(xiàn)出獨(dú)特的技術(shù)優(yōu)勢(shì)，MSI 可以在組織、細(xì)胞或亞細(xì)胞水平上對(duì)小分子化合物、多肽、蛋白、核酸、脂類等多種分子及其代謝物進(jìn)行精確分析，無(wú)需復(fù)雜前處理即可實(shí)現(xiàn)組織樣本中多種分子的原位定性、定量分析，繪制出它們的空間分布圖。我們利用德國(guó)Bruker 公司最新一代的rapifleX基質(zhì)輔助激光解吸電離（MALDI）質(zhì)譜成像技術(shù)，研究了多種藥物分子在小鼠腦、肝臟、腎臟和脾臟等組織的空間分布情況，在海馬、皮層、小腦等不同腦區(qū)，我們繪制了藥物分子在多種類型神經(jīng)元中的分布圖，分辨率達(dá)到亞細(xì)胞水平（10 μm）。同時(shí)，我們分析了這些藥物分子與各組織內(nèi)代謝成分的相關(guān)性，建立了藥物分布與代謝組學(xué)進(jìn)行協(xié)同研究的高效方法。 我們的研究證明，MALDI 質(zhì)譜成像技術(shù)在神經(jīng)系統(tǒng)疾病靶向藥物的超高通量篩選（uHTS）、藥物代謝研究（ADME）、藥效（pharmacodynamic）及毒理評(píng)價(jià)（toxicology）、生物標(biāo)志物（biomarker）的發(fā)現(xiàn)及檢測(cè)、藥物蛋白質(zhì)組學(xué)（pharmacoproteinomics）探討及固體制劑評(píng)價(jià)等方面具有獨(dú)特優(yōu)勢(shì)，顯著提高神經(jīng)系統(tǒng)疾病藥物的研發(fā)效率及成功率。

【關(guān)鍵詞】質(zhì)譜成像；神經(jīng)系統(tǒng)疾?。凰幬镅邪l(fā)；藥物代謝

OA-53 嗅球切除大鼠尿液代謝物的改變和氟西汀的調(diào)節(jié)作用

周云豐1，馮利2，陶雪1，王麗莎1，張夢(mèng)荻1，王智1，劉新民1，常琪1，*
1.中國(guó)醫(yī)學(xué)科學(xué)院&北京協(xié)和醫(yī)學(xué)院 藥用植物研究所，北京，100193，中國(guó)
2.國(guó)家開(kāi)放大學(xué)，北京，100039，中國(guó)

【摘要】 目的：采用代謝組學(xué)技術(shù)嗅球切除（olfactory bulbectomy，OBX）抑郁模型大鼠的體內(nèi)代謝物改變，探究其抑郁發(fā)生的生物學(xué)物質(zhì)基礎(chǔ)，并觀察氟西汀對(duì)OBX 大鼠代謝物的調(diào)節(jié)作用。方法：SD 雄性大鼠購(gòu)入后適應(yīng)環(huán)境7 天，按照體重將大鼠分為假手術(shù)（Sham）組、OBX 組和OBX+Flu 組。OBX 組和OBX+Flu 組大鼠的嗅球通過(guò)手術(shù)被吸除，Sham 組大鼠除不吸除嗅球外其他操作與OBX 組相同?；謴?fù)2 周后，OBX+Flu 組大鼠給予氟西汀（10 mg·kg-1），OBX 組大鼠給予同等體積的蒸餾水。連續(xù)灌胃給藥14 天后收集尿液并進(jìn)行行為學(xué)檢測(cè)。利用UFLCMS 技術(shù)對(duì)大鼠前額葉皮層中色氨酸代謝物和相關(guān)的神經(jīng)遞質(zhì)進(jìn)行測(cè)定。采用UPLC-Q-TOFMS 技術(shù)對(duì)尿液代謝物進(jìn)行非靶向代謝組學(xué)分析，利用XCMS 和CAMERA 軟件包對(duì)原始數(shù)據(jù)進(jìn)行峰提取、非線性保留時(shí)間校正和峰注釋，然后將數(shù)據(jù)導(dǎo)入SIMCA-13 軟件，采用主成分分析（PCA）和偏最小二乘法判別分析（PLS-DA）進(jìn)行多元數(shù)據(jù)分析。選擇VIP＞1 和P＜0.05 的代謝物進(jìn)行鑒定，利用KEGG 軟件進(jìn)行代謝通路的分析。結(jié)果：與Sham 組相比，OBX 大鼠表現(xiàn)出高活性、抗焦慮樣行為和絕望狀態(tài)，體現(xiàn)在OBX 大鼠在空?qǐng)鲋械倪\(yùn)動(dòng)路程和排便數(shù)顯著增加；在高架十字迷宮檢測(cè)中開(kāi)臂停留時(shí)間和開(kāi)臂進(jìn)入率顯著增加；在強(qiáng)迫游泳實(shí)驗(yàn)中的不動(dòng)時(shí)間顯著延長(zhǎng)。氟西汀能夠很好地逆轉(zhuǎn)這些行為學(xué)異常。以上結(jié)果表明OBX 抑郁大鼠模型建立成功。與Sham 組相比，OBX 大鼠前額葉皮層中TRP、5-HIAA、5-HIAA/5-HT 和DA 含量顯著升高，而KYNA 和5-HT 水平顯著降低。在OBX 大鼠尿液中，鑒定到26 個(gè)與抑郁相關(guān)的生物標(biāo)志物，氟西汀能夠不同程度的逆轉(zhuǎn)其中15 個(gè)代謝物的異常改變。與OBX 大鼠尿液代謝物紊亂相關(guān)的通路包括色氨酸代謝、腸道微生物代謝、能量代謝、嘌呤代謝、抗壞血酸和醛糖二酸代謝以及酪氨酸代謝。結(jié)論：本實(shí)驗(yàn)首次通過(guò)代謝組學(xué)技術(shù)揭示OBX 大鼠尿液代謝物的改變，鑒定的26個(gè)潛在生物標(biāo)志物可能有助于臨床抑郁癥的診斷，尤其是以行為運(yùn)動(dòng)激越為特征的抑郁病人。

【關(guān)鍵詞】抑郁癥；嗅球切除；氟西??；尿液代謝組學(xué)

基金項(xiàng)目：中國(guó)醫(yī)學(xué)科學(xué)院創(chuàng)新工程項(xiàng)目（No.2016-I2M-1-012），國(guó)家重大新藥創(chuàng)制專項(xiàng)（No.2017ZX09301029）

通訊作者：qchang@implad.ac.cn

OA-54 Cerebellar Neuron Derived Exosomes Improve Pathological Phenotype of Alzheimer’s Disease

YIN Yue-miao1，2，HU Kai-qiang1，GAO Yu-qi1，HUA rui1，WANG zhao1，*
1. MOE Key Laboratory of Protein Sciences，School of Pharmaceutical Sciences，Tsinghua University，Beijing，100084，China
2. Department of Pharmaceutics，Medical College of Qinghai University，Xi’ning，810001，China

【ABSTRACT】 Objective：Exosomes involved in various pathological processes of AD，such as regulating the metabolism of Aβ，including extracellular clearance or aggregation and intercellular delivery.The aim of this study was to investigate the mechanism of cerebellar neuron derived exosomes on rescuing SH-SY5Y cell from injure induced by Aβ and improving the learning and memory ability of APP/PS1 mice.Methods：1） Primary cerebellar neurons were obtained from 18 days rat fetus.Cerebellar neuron derived exosomes were isolated from the cell culture medium.2） Transmission electron microscope and NTA test were performed to identify the physical features of cerebellar neurons derived exosomes.3） AD cell model was established by inducing SH-SY5Y cells injury with Aβ42.Cerebellar neuron derived exosomes was added into the AD cell model medium.Then we used CCK-8 Kit to detect cell viability.4） Cerebellar neuron derived exosomes was injected into lateral ventricle of APP/PS1 mice as the AD animal model.7 days after cerebellar neuron derived exosomes injection，MWM test was performed to test learning and memory function of APP/PS1 mice，then we sacrificed the mice and dissected the brain.Cerebral Aβ level was detected by Immunofluorescence and imaged.Results：Image of transmission electron microscope showed typical cup-shaped morphology.NTA test confirmed that the diameter of the exosomes we isolated were between 30～200 nm，both results confirmed the physical features of exosomes.In AD cell model，CCK-8 test showed Aβ42（25 mmol·L-1）significantly induced cell viability decline and cerebellar neuron derived exosomes significantly increase the cell viability，which implied that cerebellar neuron derived exosomes could protect SH-SY5Y cell from the harm of Aβ42.In AD animal model，immunofluorescence results showed that lateral ventricle injection of cerebellar neuron derived exosomes can significantly reduce total Aβ level of cerebral in APP/PS1 mice.MWM test also confirmed that learning and memory function of APP/PS1 mice were significantly improved.Conclusion：Cerebellar neuron derived exosomes rescued SH-SY5Y cell from injure induced by Aβ and improving the learning and memory ability of APP/PS1 mice，may providing a new perspective of clinical AD treating strategy.

【KEY WORDS】cerebellar neurons；exosomes；Alzheimer’s disease

Corresponding author：Dr.WANG Zhao，E-mail：zwang@tsinghua.edu.cn

OA-55 人參皂苷Rg1 改善皮質(zhì)酮致星形膠質(zhì)細(xì)胞縫隙連接損傷的機(jī)制研究

夏聰媛，王真真，陳乃宏*
中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所，北京 100050，中國(guó)

【摘要】 目的：人參皂苷Rg1 是傳統(tǒng)補(bǔ)益中藥人參（Panax Ginseng C.A.Mey）的主要成分之一，對(duì)多種抑郁動(dòng)物模型具有抗抑郁作用，但其分子機(jī)制仍未完全闡明。最新報(bào)道，抑郁模型動(dòng)物出現(xiàn)星形膠質(zhì)細(xì)胞縫隙連接損傷現(xiàn)象，但機(jī)制尚未闡明。本文旨在建立皮質(zhì)酮損傷星形膠質(zhì)細(xì)胞縫隙連接功能損傷模型，闡明皮質(zhì)酮損傷星形膠質(zhì)細(xì)胞縫隙連接的作用機(jī)制，并評(píng)價(jià)Rg1是否通過(guò)星形膠質(zhì)細(xì)胞縫隙連接發(fā)揮抗抑郁作用。方法：首先分離培養(yǎng)新生大鼠前額皮層和海馬組織中的原代星形膠質(zhì)細(xì)胞，利用皮質(zhì)酮（50 μmol·L-1，24 h）模擬應(yīng)激條件。通過(guò)劃痕標(biāo)記免疫示蹤實(shí)驗(yàn)檢測(cè)縫隙連接功能，蛋白免疫印跡實(shí)驗(yàn)檢測(cè)Cx43 蛋白及磷酸化水平，免疫熒光實(shí)驗(yàn)檢測(cè)Cx43 蛋白胞漿胞膜分布，免疫共沉淀實(shí)驗(yàn)檢測(cè)Cx43 與膜蛋白間相互作用，PCR 實(shí)驗(yàn)檢測(cè)Cx43 生成階段的變化。結(jié)果：皮質(zhì)酮顯著減少原代星形膠質(zhì)細(xì)胞中Cx43 的全蛋白含量，減少Cx43 在細(xì)胞膜上的分布，上調(diào)Cx43 Serine368 位點(diǎn)磷酸化水平。皮質(zhì)酮顯著降低Cx43 的mRNA 水平，并且促進(jìn)Cx43 的降解。前額皮質(zhì)腦區(qū)星形膠質(zhì)細(xì)胞：皮質(zhì)酮促進(jìn)Cx43 與ZO-1/Drebrin 的相互作用，增加N-cadherin 的含量；海馬腦區(qū)星形膠質(zhì)細(xì)胞：皮質(zhì)酮促進(jìn)Cx43 與ZO-1/Drebrin 的相互作用，降低N-cadherin 的表達(dá)。皮質(zhì)酮刺激前，Rg1（0.1，1，10 μmol·L-1）預(yù)孵育1 小時(shí)。1 μmol·L-1Rg1 和10 μmol·L-1Rg1 顯著增加星形膠質(zhì)細(xì)胞間熒光黃染料的擴(kuò)散面積，顯著增加 Cx43 全蛋白含量，并下調(diào)Cx43 Serine368 位點(diǎn)磷酸化水平。結(jié)論：本實(shí)驗(yàn)首次利用皮質(zhì)酮建立縫隙連接功能損傷模型，提供了抑郁癥相關(guān)的體外模型。皮質(zhì)酮應(yīng)激條件下，星形膠質(zhì)細(xì)胞縫隙連接功能被損傷，該過(guò)程與Cx43 生命周期紊亂有關(guān)。Rg1 改善皮質(zhì)酮介導(dǎo)的星形膠質(zhì)細(xì)胞縫隙連接功能損傷，為進(jìn)一步闡明了Rg1 的抗抑郁作用機(jī)制提供了基礎(chǔ)。

【關(guān)鍵詞】抑郁癥；星形膠質(zhì)細(xì)胞；縫隙連接；人參皂苷Rg1；Cx43

通訊作者：chennh@imm.ac.cn

OA-56 Radix Rehmanniae Preventing Aging in Caenorhabditis Elegans through Insulin Signalling Pathway via DAF-16 for Neurodegenerative Diseases

ZHAO Jia-hui1，ZHANG Xun-le1，HUANG Jia-qing1，WEN Wu1，WANG Shan-shan1，Christopher Fang-Yen2，GONG Yue-song1*
1. Jiangsu Key Laboratory for Functional Substance of Chinese Medicine，Department of Biopharmaceutics and Food Science，Nanjing University Chinese Medicine，Nanjing，210023，China
2. Department of Bioengineering and Neuroscience，University of Pennsylvania，Philadelphia，PA 190104，USA

【ABSTRACT】 Objective：Aging is one of the main risk factors for neurodegenerative conditions.Intriguingly，both by tinkering with particular signaling pathways and by balancing nutrition can extend the lifespan of many organisms，and prevent aging and aging-related diseases.A number of herbal medicines have been used traditionally to increase longevity and health.The traditional Chinese medicine Radix Rehmanniae （RR） a known hypoglycemic activities that prevents redox imbalance in organisms，and the Radix Rehmanniae Prepared （RRP） is that RR is processed through nine cycles of steaming and drying.This processing is considered as an important role in the therapeutic application.In this study，we will explore whether RR have anti-aging effect and its related mechanism，as well as its processed product RRP with the similarities and differences of efficacy，which could be used for preventing neurodegenerative diseases in future.Methods：We used water or 70% ethanol to extract RR and RRP for this antiaging examination.Wild or mutant C.elegens were selected as model animals，to evaluate their bioactivities of anti-aging through life expectancy，stress resistance，dietary restriction and DAF-16 nuclear localization.Results：The results showed that these extracts of RR and RRP can increase the lifespans of N2 wild-type C.elegans significantly，RRP showed stronger bioactivities than RR did，which could be related to the resistance enhancement in heat stress and oxidative stress，caloric restriction and lipofuscin accumulation.However，these extracts can’t effect on the lifespan of C.elegans with daf-2 or daf-16 mutation.Meanwhile，we found the extracts of RR and RRP could increase the nuclear localization of the core transcription factor DAF-16.The longevity effect of RR and RRP could promote the prolongation of life mainly through insulin/insulin-like growth factor pathway in which daf-2/daf-16 is involved.Conclusion：Overall，these results indicate that RR and RRP could be used for anti-aging to prolong the lifespan of C.elegans by enhancing translocation of DAF-16 nuclear through insulin signaling pathway.RRP showed stronger bioactivities for anti-aging than RRP.Based on daf-2/daf-16 age-related pathway involved are evolutionarily conserved，these tweaks could eventually benefit human health for preventing aging-related disease such as neurodegenerative diseases.

Corresponding author：Yuesong Gong，E-mail：ygong@njucm.edu.cn

OA-57 Correlation Analysis between Taurine Content of Brain Tissue Determined by Amino Acid Analyzer and the Age of Growth in Mice

WU Chun-yang1，ZHANG Jian-mei2，LI Zhao-zhen1，LI Wei1，ZHANG Dan-shen2*，ZHANG Li1*
1. Department of Pharmacy，Hebei North University，Hebei Provincial Key Laboratory of Neuropharmacology，Zhangjiakou，075000，China
2. Hebei University of Science and Technology，Shijiazhuang，050018，China

【ABSTRACT】Objective：To observe the correlation between taurine（Tau）content in mouse brain tissue and the age of growth，and to provide normal reference data for the change of Tau content of brain under different disease state.Methods：We selected SPF level and Kunming species mice，the age range of which was from 1 week to 34 weeks（1 w，2 w，3 w，4 w，5 w，6 w，7 w，8 w，9 w，10 w，15 w，18 w，22 w，26 w，31 w，and 34 w），using the amino acid analyzer A300，continuously measured the content of Tau in the mice brain，and observed the correlation between the level of Tau in the brain tissue of male and female mice and the age of growth.Then we compared the differences of Tau content of male and female mice，and also compared the difference of Tau content in cerebral cortex and hippocampus.Results：The change trend of Tau content in the brain of mice was as follows：Tau content was declining continuously in 1～4 weeks（0.266→0.127 mg），fluctuating in a certain range（0.119→0.158 mg） in 4～26 weeks，and stable in 26～34 weeks（0.110→0.125 mg）.Among the 16 time points in this study，compared with the female mouse cortex，the Tau content in the cortex of male mice was lower at 31 weeks.While compared with the hippocampus of female mice，the Tau content in the hippocampus of male mice was lower at 2 weeks and higher at 15 weeks（P＜0.05）.And compared with male mice hippocampus，the content of Tau in the cortex of male mice was higher at 2 and 22 weeks，and lower at 3，4，6，and 15 weeks.Also compared with the hippocampus of female mice，the content of Tau in the cortex of female mice was higher at 26 weeks，and lower at 2 to 4 and 18 weeks（P＜0.05）.Conclusions：The Tau content first decreased，then fluctuated，and reached a trough at 4～6 weeks，and remained a low value from 26 weeks.There was no significant difference between Tau content of male and female mice.But compared with hippocampus，the cortical tissue showed significant differences of Tau content before 6 weeks.The reasons and mechanisms for the change of Tau content need to be further studied.

【KEY WORDS】 taurine；amino acid；cortex；hippocampus；content；age

Corresponding authors：ZHANG Dan-shen，E-mail：zhangds2011@126.com；ZHANG Li，E-mail：hbnulzhang@126.com.