LI Zi-Rong,HAN Yun-Shn,WU Meng-Yo,LIU Jin,JIN Shi,ZHANG Xi,WANG Yu-Hong*

a.State Key Laboratory of Chinese Medicine Powder and Medicine Innovation in Hunan,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China

b.The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China

c.Qianjin Pharmaceutical Co.,Ltd.,Zhuzhou,Hunan 412000,China

d.Hunan Provincial Brain Hospital,Changsha,Hunan 410007,China

Keywords

Compound Chai Jin Jie Yu Tablets (CJJYT)

Depression

SYN-α/NR

Synaptic plasticity

Cognition

ABSTRACT

Objective To investigate the effectiveness of compound Chai Jin Jie Yu Tablets (CJJYT) in ameliorating cognitive impairment associated with depression and its potential mechanism of action.

Methods In vitro experiments,the hippocampus was isolated from the whole brain of the fetal rat and cultured into hippocampal neuron cells.50 μM corticosterone (CORT) was added to each group 18 h before the experiment for modeling depression,with the exception of the control group.After modeling,the blank serum group was added with 10% blank serum,the CJJYT group and the venlafaxine group were added with the corresponding 10% drug-containing serum,and the control group and the model group were added with equal volumes of culture medium.The intracellular Ca2+ mean fluorescence intensity,miniature excitatory postsynaptic current(mEPSC) current amplitude,and frequency of different hippocampal neurons were evaluated as indicators of synaptic function in the hippocampal neurons.In addition,the expression of synaptic plasticity related proteins,synaptophysin-α (SYN-α),N-methyl-D-aspartate receptor 2A (NR2A),N-methyl-Daspartate receptor 2B (NR2B),post synaptic density 95 protein(PSD-95),calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ) and synaptic associated protein (SynGAP) were detected in the hippocampal neurons by immunofluorescence staining and high content analysis (HCA) system.Then,reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of SYN-α,NR2A,NR2B,PSD-95,CaMKⅡ and SynGAP.For in vivo experiments,except for those in the blank control group,all rats were treated within a single cage for chronic unpredictable stress-induced depression modeling and subjected to corresponding drug interventions.Behavioral tests were used to detect depressive behavior and determine learning,memory and other cognitive abilities,whereas enzyme-linked immunosorbent assay (ELISA) was used to detect the CORT levels.Golgi-Cox staining was used to observe changes in the synaptic morphology of parahippocampal gyrus CA1 area (CA1) and dentategyrus (DG).

Results In vitro, CJJYT treatment reduced the intracellular Ca2+ mean flurorescence intensity in the hippocampal neurons(P < 0.05),causing a reduction in the frequency and current amplitude of mEPSC (P < 0.05),and thus inhibited the excessive activation of post-synaptic receptors.CJJYT treatment reduced the protein and mRNA expression of SYN-α,NR2A,NR2B and PSD-95 in the hippocampal neurons (P < 0.05),increased the mRNA and protein expression of CaMKⅡ and SynGAP (P < 0.05),and thereby improved the synaptic plasticity of the hippocampal neurons.In vivo,CJJYT intervention improved sucrose preference,voluntary activity ,learning and memory ability of Morris water maze test,and suppressed appetite (P < 0.05),and increased the despair feeling of forced swimming test (P < 0.05).The CORT level was reduced (P < 0.05),leading to the repair of synaptic damage in the hippocampal neurons.

Conclusions CJJYT can improve the synaptic function of hippocampal neurons and has obvious protective effects on neurons.It can repair the structural damage in the hippocampal neurons,improving the cognitive ability of the depressed model rats.The mechanism of CJJYT improving cognition in depressed rats may be related to the transmission and function of SYN-α/NR and its downstream neurotransmitters.

1 Introduction

Depression is a chronic disease of the central nervous system,which belongs to the category of emotional disorders.It is characterized by depression,lack of pleasure,lack of interest,slow thinking and impaired cognitive function,and has a high clinical prevalence and recurrence rate.It is the most common central nervous system disease.According to prediction,about 360 million people of all ages have depressive disorders,with an annual incremental growth rate of 113%[1].

Therefore,the diagnosis and treatment of depression becomes especially important.At present,chemically synthesized antidepressants available in the market generally take effect in 2 weeks and cause side effects such as insomnia and severe withdrawal syndrome,which causes the symptoms of depression to recur,causing cognitive impairment[2,3].

Compound Chai Jin Jie Yu Tablets (CJJYT) is a 2017 National Major New Drug Innovation Project,“Pre-clinical Study of a New Type of Antidepressant Traditional Chinese Medicine Compound Chai Jin Jie Yu Tablets” (No.2017ZX09309026).It is based on the classic antidepressant traditional Chinese compound Kai Xin San,and has been selected based on data from scientific experiments (Table 1),which shows effects on soothing liver and strengthening spleen,and relieving depression.CJJYT has clinically significant antidepressant effects and can improve cognitive function.Previous studies[4-6]showed that CJJYT has many antidepressant effect characteristics on monoamine neurotransmitter,hypothalamicpituitary-adrenal (HPA) axis endocrine network and hippocampal plasticity.However,the underlying mechanism by which CJJYT exerts its effects to improve cognitive function is not clear yet.

Hippocampus is the key brain area involved in the process of emotional change,learning and memory,which is manifested in the prominent synaptic plasticity[4].Synapse is the key hub for the transmission of neural information.The miniature excitatory postsynaptic current (mEPSC) of hippocampal neurons is the embodiment of the normal function of neural information transmission in the synapse.Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS),and it plays an important role in the synaptic plasticity[5].After 7 d of culture,neurons can form neural network that shows more active synaptic activity.Spontaneous synaptic activities include excitatory postsynaptic current (EPSC) and inhibitory postsynaptic current (IPSC)[6].

The plasticity of hippocampal synapses and mEPSC are the basis of cognition,emotion,memory and thinking.EPSC frequency is a quantitative index of presynaptic glutamate release,which is directly proportion to glutamate release,whereas EPSC amplitude is a comprehensive result of presynaptic transmitter release and postsynaptic receptor action[7].

Although clinical and animal studies have confirmed that antidepressants can correct the damage in the depressed brain tissue and reduce depression,animal tissue is usually used as a sample to study the mEPSC in the hippocampus or hippocampal neurons unilaterally,without clear specific mechanisms and rules[8].

In recent years,it has been found that the hippocampus is rich in various neurotransmitters and receptors[9].The structure and molecular composition of hippocampal synapse mediate its core role in learning and memory,and its dysfunction is considered to be the crucial pathogenesis of depression[10,11].The synaptic function is recognized to be causally related to the cognitive function of depression,and the recovery of synaptic function is the key to improve the cognitive impairment associated with depression[12,13].

To explore the effect of CJJYT on the cognitive function during depression,in this study,in vivoandin vitroexperiments were conducted to verify whether CJJYT can improve the synaptic function via the downstream neurotransmitter effect of SYN-α/NR,and improve cognition in depressed model animals.

2 Materials and Methods

2.1 In vitro experiments

2.1.1 Experimental animalsIn this study,12 SPF grade Sprague Dawley (SD) female rats (17-19 d of gestation) weighing 350-450 g,and 12 SPF grade SD male rats weighing 180-220 g were used.All rats were provided by Hunan SJA Laboratory Animal Co.,Ltd.(Changsha,Hunan,China),in which female rats were directly used for primary generation of hippocampal neurons.Male rats were raised in SPF experimental animal center at the First Hospital of Hunan University of Chinese Medicine [No.SYXK(Xiang) 2015-0003] at room temperature (24±2)℃,relative humidity (50%±5%) and light/dark cycle(12 h/12 h).

2.1.2 Drugs and main reagentsCJJYT was provided by the First Hospital of Hunan University of Chinese Medicine (Changsha,Hunan,China).Venlafaxine hydrochloride sustained release capsule was obtained from Pfizer Ireland pharmaceuticals (New York,USA).Corticosterone was purchased from sigma Aldrich (St.Louis,Missouri,USA).

Table 1 Components of CJJYT formulation

2.1.3 Preparation of drug containing serumAfter 5 d of adaptive feeding of SPF male SD rats,they were randomly divided into three groups:blank serum,CJJYT and venlafaxine,with four rats in each group.The blank serum group was given the same amount of distilled water by gavage.The other two groups were given CJJYT and venlafaxine (1.35 g/kg and 13.5 mg/kg,ig) 2 times/d for 3 d.The dose of CJJYT is the best dose concentration obtained through previous animal experiment and cell experiment screening.After the last gastric perfusion for 1 h,blood was drawn from abdominal aorta under sterile condition and collected in coagulation promoting tubes.After 2 h,blood was centrifuged at 4 500 rpm for 10 min,supernatant was taken,inactivated at 56 ℃ water bath for 30 min,filtered through 0.22 μM filter membrane,and separately packed in 1.5 mL EP tube,and stored at ? 80℃.

2.1.4 Primary culture of neuronsAccording to previous published method[14],SD rats conceived for 18 d were anesthetized with 10% chloral hydrate,and fetal rats were quickly placed in large petri dishes,fully sterilized with 75% ethanol.Ophthalmic scissors was used to quickly take the whole brain into small cultures containing precooled D-Hanks.Washed thoroughly in the dish,and then used the angled tweezers to carefully lift the surface of the cerebral cortex under the stereo microscope to see the clear“bow” hippocampus.Grasped the hippocampus carefully and separated it from the marginal tissue.Put the newly stripped hippocampus into another small petri dish containing precooled DMEM/F12.Removed the residual meninges and blood vessels carefully,and cut the hippocampal tissue into tissue fragments of about 1 mm3.Added an equal volume of 0.25% trypsin and 0.2% collagenase,mix well,digested at 37 °C for 15-20 min,mixed every 5 min,digested until no obvious tissue precipitation.Added termination culture solution(volume fraction 89% DMEM/F12,10% FBS and 1%Penicillin-Streptomycin) terminate enzyme digestion,gently pipetting 30-50 times,collected supernatant,filtered with 100-mesh screen,collected cell filtrate.Centrifuged at 1 200 r/min for 5 min,discarded supernatant,resuspended,200-mesh screen after sieving the net.Added the inoculation medium (87% DMEM/F12,10% FBS,1% L-glutamine,1% B27 and 1% Penicillin-Streptomycin) to resuspend the cells,and adjusted the cell density to 3.0×105/mL,inoculated in 96-well or 24-well cell culture plate,placed in 37 ℃,5% CO2three gas cell incubator.4 h later,the whole amount was replaced with maintenance medium (volume fraction 96%Neurobas-al,2% B27,1% Glutamax and 1%Penicillin-Streptomycin),and then replaced the maintenance medium every 3 d with half volume.At the same time,after 3-5 d of culture,cytarabine with a final concentration of 5 μmol/L was added as appropriate to inhibit glial cells.In the plate,the plating reagent was replaced with the maintenance solution (Neurobasal:B27:Glutamax:Penicillin-Streptomycin=96:2:1:1) after 4 h.After 5 d of incubation,hippocampal neurons were detected with rabbit anti-rat neuron specific enolase (NSE,Boster Biological Technology Co.,Ltd.,California,USA) and mouse anti-ratβ-tubulin antibodies(Proteintech Group,Inc.,Chicago,USA).Hippocampal neurons were observed to be in good condition with abundant synapses and branches,interconnected into a network with a positive cell rate of more than 95% and with fewer heterogeneous cells.

2.1.5 Model buildingCells were seeded at 2×105cell/mL on 6-,24-and 96-well culture plates,in 4 replicates and the experiments were repeated 3 times.According to a previous study[15],when the neurons were cultured for 5-7 d,the hippocampal neurons showed obvious auras and the protuberations extended to form a more complex network,which could be used for the experiments.CORT (50 μmol/L) was used for 18 h to construct anin vitromodel of hippocampal neurons in a depressive environment.The CJJYT and venlafaxine groups were given CJJYT medicated serum and venlafaxine medicated serum,respectively.The blank serum group was given the same volume of blank serum,as the carrier control for drug-containing serum administration.The control group and model group were both given the same amount of cell culture solution.

2.1.6 Detection of intracellular calcium levelAfter 18 h of model intervention and drug administration in each group,the transwell cuvette was removed and the hippocampal neurons were gently washed with calcium-free D-Hank’s solution 2 times; Furo-3/AM working solution was added to a final concentration of 5 μmol/L,followed by incubation at 37 °C for 45 min in the dark.Fluorescence detection was then performed and the images were acquired on high content analysis system (HCA).The images reflected the changes in Ca2+levels in the hippocampal neurons as a measure of the average fluorescence intensity value of a single cell.

2.1.7 mEPSC recordAll experiments were performed at 22-25 ℃,and the cells of each group were used for patch clamp recording 18 h after dosing.In this experiment,typically healthy hippocampal neurons were selected from 11-14 d,and mEPSC was recorded in the whole cell mode.The microelectrode drawing instrument draws a borosilicate ground glass tube into an experimental recording electrode.The electrode water resistance was 3-8 MΩ,the clamping potential was ? 60 mV,and the membrane was broken after sealing to establish a whole-cell link.The mEPSCs of the hippocampal neurons were recorded in the presence of the calcium channel blocker tetrodotoxin (TTX) (1 μmol/L)andγ-aminobutyric acid (GABA) receptor antagonist picrotoxin (PTX) (10 μmol/L).The patch clamp amplifier Axonpatch 700 B (DL Naturegene Life Sciences,Inc.USA) was used to amplify the current signal,which was recorded by the Clamfit software(DL Naturegene Life Sciences,Inc.USA) after being converted by a Digidata 1 440 A value converter.Records with series resistance of > 20 MΩ,leakage current of > ? 200 pA,and noise of > 20 pA were eliminated.

2.1.8 Detection of the expression levels of synaptic plasticity related proteinAfter 18 h of modeling and drug intervention,the culture medium was aspirated from each well of the culture plate,and the wells were gently rinsed 2 times with phosphate buffer saline (PBS) (0.01 mol/L),followed by fixation with 4% paraformaldehyde at 37 ℃ for 30 min.The liquid was discarded and the wells were rinsed 3 times with PBS before permeabilization with 0.25% triton-100 for 15 min,followed by rinsing 2 times before blocking with 5% bovine serum albumin (BSA) for 30 min.After blocking,primary polyclonal antibodies,anti-synaptophysin-α(SYN-α),anti-Nmethyl-D-aspartate receptor 2A (NR2A),anti-Nmethyl-D-aspartate receptor 2B (NR2B),anti-post synaptic density 95 protein (PSD-95),anti-calcium/calmodulin dependent protein kinase Ⅱ (CaMKⅡ),and anti-synaptic associated protein (SynGAP)polyclonal antibody (50 μL,1:100) were added to each well respectively,followed by incubation with mouse anti ratβ-Tubulin polyclonal antibody (50 μL,1:100).The cell culture plate was placed in a wet box and incubated overnight at 4 °C.After washing 6 times with cold PBS,50 μL fluorescein 5-isothiocyanate (FITC) and retinal pigment epithelium (RPE) secondary anti diluent (1:400) was added to each well,followed by incubation at 37 ℃for 30 min,washing with cold PBS for 5 times,and adding 50 μL of 4',6-diamidino-2-phenylindole(DAPI) (1:800) to each well.The plates were incubated at 22-25 ℃ in dark for 15 min,washed with PBS 2 times,followed by addition of 50 μL PBS to maintain the observation environment,and carry out fluorescence observation and analysis in HCA.

2.1.9 Detection of mRNA expression levels of synaptic plasticity related proteinsTotal RNA from the hippocampus was extracted using the RNA pure Tissue & Cell Kit (Beijing CoWin Biotech,Beijing,China) according to the manufacturer’s instructions.Complementary DNA synthesis of mRNA was performed using the Prime ScriptTMRT Master Mix(Takara Bio,Dalian,Liaoning,China).The reverse transcription-polymerase chain reaction (RT-PCR)was performed using the SYBR? Premix Ex TapTM(Takara Bio,Dalian,Liaoning,China).All real-time assays were carried out under the following conditions:one cycle of pre-denaturation for 3 min at 95 °C,40 cycles of denaturation at 95 °C for 10 s,annealing at 60 °C,and extension at 72 °C for 30 s.Primers were designed using Primer Designer Software (Primer Premier 6.0) (Table 2).All primers were synthesized by Shanghai Sangon Bioengineering Co.,Ltd.(Shanghai,China).Melt curve analysis was performed to confirm the specificity of the amplified products.The expression levels of target genes were calculated using the 2?ΔΔCt method.β-actin was used as internal reference.

Table 2 Primer sequences used for PCR

2.2 In vivo experiments

2.2.1 Experimental animalsSeventy SD male rats weighing 180-220 g were used.All rats were provided by Hunan SJA Laboratory Animal Co.,Ltd.(Changsha,Hunan,China).Rats were raised in SPF Experimental Animal Center at the First Hospital of Hunan University of Chinese Medicine[No.SYXK(Xiang)2015-0003] at room temperature(24±2) ℃,relative humidity (50%±5%) and light/darkcycle (12 h/12 h).

2.2.2 Drugs and main reagentsCORT enzyme linked immunosorbent assay (ELISA) Kit (Enzo Life Sciences,Plymouth Meeting,PA,USA).CJJYT was provided by the First Hospital of Hunan University of Chinese Medicine (Changsha,Hunan,China,Batch No.20180102).Venlafaxine hydrochloride sustained release capsule was obtained from Pfizer Ireland pharmaceuticals (New York,USA,Batch No.T37944D).Corticosterone was purchased from sigma Aldrich (St.Louis,Missouri,USA).

2.2.3 Drug administrationThe rats were adapted for 5 d and fasted for 12 h.The animals were solitary in a single cage.Sucrose water (1%) and distilled water (preliminarily weighed in advance) were placed in the cage.After 30 min,the two bottles of water were exchanged for another 30 min.The 1%sucrose water intake of rats within 1 h was calculated as follows:

Degree of sugar water preference=sucrose water intake/(sucrose water intake+distilled water intake)×100.

The rats were randomly divided into seven groups according to the results of sugar and water preference:(1) control group,4 animals/cage,free access to food and water without any exogenous stimulation;(2) model group,as a model control; (3) venlafaxine group,venlafaxine suspension (13.5 mg/kg) was administered intragastrically for 28 d; (4) CJJYT 4 times,2 times,1 time and 0.5 times dose groups:CJJYT,2.7 g/kg (CJJYT-4×); CJJYT,1.35 g/kg (CJJYT-2×);CJJYT,0.675 g/kg (CJJYT-1×); and CJJYT 0.337 5 g/kg(CJJYT-0.5×); and the above groups were given gastric gavage for 28 d.Except the control group,the other groups of rats were reared in a single cage and given a chronic unpredictable mild stress to establish a depressive rat model.The stimulating factors included day and night inversion for 24 h,4 °C ice water bath for 4 min,fasting for 24 h,water ban for 24 h,dumping at 45 °C for 24 h,tail clamping for 1 min,and noise for 4 h.The 4 °C ice water bath and tail clamping were done once a week,and the rest of the stimulation was carried out alternately for 28 d.Behavioral tests were performed 1 h after the last gastric lavage.

2.2.4 Behavioral tests(1) Sucrose preference:sucrose preference test is a method of measuring a hedonia-like behavior.Rats had access to two 150 mL bottles with different solutions,one containing 1%sucrose solution and the other containing normal drinking water.The location of the two bottles were switched daily.Before the start of the experiment,rat housed individually were acclimated to the cages with sipper tubes for 3 d (days 1-3) with access to food and water,during which baseline measurements were taken.Sucrose preference was calculated as a percentage of the volume of 1%sucrose consumed over the total fluid intake volume.An individual cohort of rats (n=10 per group) was tested.

(2) Morris water maze test:the water maze was divided into four quadrants:1,2,3 and 4.Before the test,the safety platform was placed in one of the quadrants (as shown in Figure 1,the third quadrant)to make the platform invisible to the rats under the light of the experiment.Keeping the room quiet,the objects were placed in the same condition as the lighting,and the water temperature was maintained at around 20 °C[16].

Directional navigation experiment:the rats in each group were trained in learning and memory ability once a day for five consecutive days from day 24 of modeling.① Put the rat into the water (as shown in Figure 1,black solid circle point indicates the location of the animals put into the water),observed each group rats if they could to find security platform within 90 s and recorded the consuming time of climbming up the platform (escape latency),checking in the learning ability of rats.② Space exploration experiment:the safety platform in the water maze was removed,and the rats of each group were still placed in the water maze facing the wall from the first quadrant,and the accumulated stay time (ST) of the rats in the third quadrant was recorded within 30 s to investigate their memory ability.

(3) Open-field test:open field procedure was designed to detect spontaneous locomotor activity with minor modifications[12].A wooden box (80 cm×80 cm×40 cm) with smooth surface was used in the study.For each trial,animals facing the wall of box were placed into one of the corners,and then they were permitted to explore the environment and libitum.The rats were allowed to adapt for 30 s before recording began.The total locomotor activity was recorded for 5 min.

(4) Novelty suppressed feeding (NSF):the NSF assesses stress-induced depressive behavior by measuring the incubation period of an animal to eat a familiar food in an aversive environment.After fasting the rats for 24 h,the bottom of a square open box(specifications:80 cm×80 cm×40 cm) was covered with a layer of 1.5 cm thick wood.The center of the box was placed with 30 pieces of food of equal volume.The animals were placed into the test chamber at the same position facing the wall of the box.After the rats had adapted for 30 s,the incubation period of the rats’ first foraging within 5 min was observed and recorded.The experiment requires an environment with no obvious light source.After the observation of each animal,the inner wall and bottom surface of the box need to be wiped with alcohol to avoid interference from the odor left by the previous animal[17].

(5) Forced swimming (FS) test:the FS test was carried out to observe the behavioral changes in animals in the state of despair.The behavioral cylinder(50 cm high,25 cm in diameter) was filled with water at 24-25 °C to the height of 30 cm.Before the test,rats were allowed to swim for adaption in 1 min.The total immobile time during the 5-min test was recorded.The rats were considered inactive when the limbs of rats were stationary[18].Then,the mouse was removed from the cylinder and dried with towels.

2.2.5 Sample collectionAfter the behavioral tests,three of the rats were anesthetized and perfused with 4% paraformaldehyde.Afterwards,the brain tissues were collected for the Golgi-cox staining.The other rats were anesthetized and sacrificed.The blood sample was harvested and centrifuged at 3 000 rpm for 10 min,then the serum was collected.

2.2.6 Plasma CORT concentrationsThe concentrations of CORT were detected to determine potential differences among treatment groups in this model.Blood was incubated at 4 °C and centrifuged (15 min at 2 000 rpm),and then the plasma was stored at? 80 ℃ until assayed.CORT levels were measured according to the manufacturer’s instructions by ELISA (Enzo Life Sciences,Plymouth Meeting,PA,USA) after 1:40 dilution.

2.2.7 Golgi-Cox stainingThe brain tissue was immersed in mordant solution (concentrated formaldehyde,distilled water,chloral hydrate,potassium dichromate) after double distilled water to remove blood stains,and was incubated in the dark for 3 d.Silver nitrate (1%) was immersed in silver plating,and the silver solution was changed for three consecutive days and placed in a dark place for 3 d.The tissue was sliced and washed firstly,followed by dehydration using concentration gradient ethanol,then the tissue was Transparency with xylene,and finally sealed with neutral glue.Morphological changes in the dendritic spines,dendritic branches,and cell bodies of neurons in the parahippocampal gyrus CA1 area (CA1) and dentategyrus area (DG)were observed under a microscope.

2.3 Statistical analysis

SPSS 21.0 software was used for statistical analysis,and the experimental results are expressed as“mean±SD”.Single factor analysis of variance(ANOVA) was used to compare the differences among groups,least significant difference (LSD) test was used when the variance was uniform,and Dunnett’s T3 test was used to analyze when the variance was uneven.P< 0.05 means the difference is significant,P< 0.01 means the difference is extremely significant,with statistical significance.

3 Results

3.1 In vitro experiment results

3.1.1 Effects of CJJYT on hippocampal neurons intracellular Ca2 + levelsThe intracellular Ca2+levels of hippocampal neurons reflects the strength of synaptic transmission.The hippocampal neurons were fluorescently stained with blue fluorescence as the host-labeled nucleus and green fluorescence as the fluorescence-3/AM-labeled intracellular calcium ion (Figure 2A).The data were analyzed by HCA.Figure 2B shows that the intracellular Ca2+levels in the hippocampal neurons in the CJJYT and venlafaxine groups were significantly reduced compared to that of the blank serum group (P< 0.01).The results show that CJJYT can inhibit the synaptic transmission of neurons.

3.1.2 Effects of CJJYT on hippocampal neurons mEPSC amplitude and frequencyA change in the amplitude of mEPSC suggests postsynaptic changes such as a decrease or increase in the number or density of receptors.In addition,the change in the frequency of mEPSC may be because of the number of active synapses.The mEPSC amplitude and frequency reflect the strength of postsynaptic receptor activity in the hippocampal neurons (Figure 3A).Figure 3B and 3C show that compared with the blank serum group,the mEPSC amplitude and frequency of the CJJYT and venlafaxine groups were significantly reduced(P< 0.05).The results show that CJJYT can control the activity of post-synaptic receptors.

3.1.3 Effects of CJJYT on hippocampal synaptic plasticity-related proteinsThe hippocampal neurons were stained with immunofluorescence,blue fluorescent DAPI labeled neuronal nuclei,orange fluorescent RPE labeled cytoskeletalβ-tubulin,and green fluorescent FITC labeled target proteins SYN-α,NR2A,NR2B,PSD-95,CaMKⅡ and SynGAP (Figure 4A).Figure 4B shows that compared with the blank serum group,the protein expression levels of SYN-α,NR2A and NR2B in the CJJYT and venlafaxine groups were significantly reduced (P< 0.05); the PSD-95 protein expression levels in the CJJYT group were significantly reduced (P< 0.01),and there was no statistical difference in the venlafaxine group.Figure 4C shows that compared with the blank serum group,the expression levels of CaMKⅡ and SynGAP proteins in the CJJYT and venlafaxine groups were significantly increased (P< 0.05).The results indicate that CJJYT can regulate the synaptic plasticity-related proteins in hippocampal neurons and affect synaptic function.Hippocampal neurons,SYN-α,NR2A,NR2B,PSD-95,CaMKⅡ and SynGAP are closely related to synaptic plasticity and have important effects on cognitive activities such as emotions,learning and memory.

3.1.4 Effects of CJJYT on hippocampal synaptic plasticity-related protein’s genes mRNATo study the effects of CJJYT on synaptic plasticity-related proteins,the protein’s genes mRNA expression levels of SYN-α,NR2A,NR2B,PSD-95,CaMKⅡ and SynGAP were determined by HCA and RT-PCR(Figure 5A).Figure 5B shows that compared with the blank serum group,the mRNA expression levels of SYN-α,NR2A,NR2B and PSD-95 in the CJJYT and venlafaxine groups were significantly reduced(P< 0.05).Figure 5C shows that compared with the blank serum group,the mRNA expression levels of CaMKⅡ and SynGAP in the CJJYT and venlafaxine groups were significantly increased (P< 0.05).The results indicate that CJJYT can regulate the mRNA expression of target proteins related to synaptic plasticity in the hippocampal neurons and affect synaptic function.The above results are similar to the results obtained by HCA.

3.2 In vivo experiment results

3.2.1 Effects of CJJYT on depression-like behaviors in depression model ratsFigure 6A shows the effect of CJJYT on sugar preference,which is represented as the core symptom of depression.The sugar water consumption of the model group in the first week was not statistically significant,whereas it significantly decreased starting from the second week to the fourth week (P< 0.05),which suggests that the loss of sugar water consumption decreased as the time goes on to modeling depression.CJJYT-2×group significantly increased the sugar water consumption in the third and fourth weeks (P< 0.05).There was no statistical significance in the second week in the CJJYT-4×and CJJYT-2×groups; and there was no statistical significance in the third week in the venlafaxine,CJJYT-4×,CJJYT-1×and CJJYT-0.5×groups.This suggests that CJJYT formulation could improve the responsiveness to pleasure in the depression model rats.

Figure 6B shows the changes in autonomic activity.There was a statistical decrease in the autonomic activity of model group (P< 0.01).CJJYT-4×and CJJYT-2×groups as well as venlafaxine group showed increase in the times of autonomous activities(P< 0.05).While CJJYT-1×and CJJYT-0.5×groups had a certain influence on the autonomous activities of rat,but there was no statistical significance.It is suggested that CJJYT can significantly improve the depressive autonomic behavior.

Figure 6C shows the motionless time in forcedswimming test,which could reflect the feeling of despair.Compared with the control group,the degree of despair was statistically increased in the model group(P< 0.01).Compared with the model group,there was significant decrease in the degree of despair in the venlafaxine,CJJYT-4×,CJJYT-2×and CJJYT-0.5×groups (P< 0.05),whereas there is no statistically significant difference in the CJJYT-1×group.This data demonstrates that the effects of feeling of despair facing drowning could be effectively relieved by treatment with CJJYT.

3.2.2 Effects of CJJYT on cognitive function of depression model ratsFigure 6D shows the changes in the environmental survey.The escape latency and the stay time may reflect learning and memory cognitive ability.The escape latency and stay time in the model group increased significantly (P< 0.05).The escape latency and stay time of the venlafaxine,CJJYT-2×and CJJYT-1×groups significantly decreased,with statistical significance (P< 0.05).There was no statistical difference between the CJJYT-2×group and CJJYT-0.5×groups.These data demonstrate that effective improvements in learning and memory may be achieved using CJJYT.

Figure 6E shows the changes in environmental inquiry.The feeding latency could reflect the desire to explore and curiosity in the unknown environment.The feeding latency was statistically increased in the model group (P< 0.05).There was significant decrease in the feeding latency in the venlafaxine,CJJYT-2×and CJJYT-1×groups (P< 0.05),whereas there is no statistically significant difference between the CJJYT-4×and CJJYT-0.5×groups.This data shows that treatment with CJJYT can effectively increase curiosity and desire to forage.

3.2.3 Effects of CJJYT on the expression level of CORT in depression model ratsThe levels of CORT in peripheral blood directly reflect the state of the body under stress[19,20].Figure 7A shows that the levels of CORT in the venlafaxine and CJJYT-2×groups were significantly reduced (P< 0.05).This data show that treatment with CJJYT can effectively relieve stress.

3.2.4 Effects of CJJYT on the expression level of CORT in depression model ratsGolgi-Cox staining in the hippocampal CA1 and DG areas can be used to examine the effect of CJJYT on hippocampal neuronal synaptic damage in depression model rats.

Figure 7B shows that in hippocampus CA1,part of the neurons in the model group disappeared,the dendrites were scattered and arranged loosely,and the number of dendritic spines was greatly reduced or absent.The various groups of CJJYT can improve the morphological damage to the synapses of neurons in the hippocampal CA1 region of depression rats,increase the number of dendritic branches and dendritic spines,and restore their density and complexity.Among them,the CJJYT-2×group showed the most effect.

Figure 7B shows that in the hippocampal DG region,the neuronal cell body of the model group almost disappeared,the dendrites were sparse,the morphology changed,and no obvious silver-stained dendritic spines were seen.The various groups of CJJYT damaged the synaptic structure of hippocampal neurons in the model rats.All of them show varying degrees of improvement,especially the compound CJJYT-4×group shows the most significant effect.The results show that CJJYT can repair the synaptic damage of the hippocampal neurons.

4 Discussion

Depression is characterized by cognitive dysfunction,which is caused by genetic and external factors.Depressed people with cognitive impairment often have pessimistic psychology.They often harbor the idea of self-injury or suicide,even give their actions.According to previous studies,the disability rate of depression is about 10.03% of the total disability rate of all diseases.The number of suicides because of depression is about 1 million every year globally,and thus ranking the first in the global burden of disease[21].Therefore,improving the cognitive function of patients with depression is key to the treatment of depression.

Synaptic structure and function of hippocampal neurons directly affect the cognitive behaviors such as learning and memory.Therefore,the interaction of neurotransmitters and receptors related to synaptic plasticity of the hippocampal neurons may be an important pathogenesis of cognitive impairment in depression.

N-methyl-D-aspartate receptor (NR) is an ionic glutamate receptor,which is mainly distributed in the postsynaptic membrane of the nerve cells[22].NR plays an important role in synaptic plasticity,learning,and memory,and regulates the survival of neurons by regulating the structural development of neuronal dendrites and axons.It also plays an important role in the formation of neuronal circuits[23].Previous studies reported by our group showed that under long-term stress,HPA axis was hyperactivated,levels of NR subunits,NR2A and NR2B,and receptor channel binding sites increased,and NR was overactivated[8].On the one hand,Ca2+channel opened causing an increase in the intracellular Ca2+concentration,leading to induction of CaMKⅡ phosphorylation; under the influence of CaMKⅡ,Ca2+very easily induced apoptosis[11]triggering long-term potentiation (LTP) and long-term course inhibition that affects the synaptic plasticity and function.On the other hand,over activation of NR causes the accumulation and release of intracellular glutamate to the extracellular,resulting in neurotoxicity,ultimately leading to hippocampal damage[24].

4.1 Inhibitory effects of CJJYT on the overactivity of hippocampal neurons in depressed environment

Depressive patients show a decrease in cognitive function,with obvious anxiety and motor agitation.Genetic,neurobiochemical,neuroendocrinal and neurogenesis are closely related to the onset of depression[25].

Electrical synapses are particularly important for normal cognitive function.Synapses are dendrites that receive information,integrate in the cell body through action potential,and then propagate along the axons to other neurons in discontinuous space.Electrical synapses are formed by dendritic gap junctions of adjacent neurons.The rapid response of synapses plays a key role by inhibiting glutamate excited neurons[26].It is suggested that the cognitive impairment related to depression may be closely related to the synaptic function of the neurons.

Synaptic function is mediated by neurotransmitters.Neurotransmitters (such as glutamate) come from various sources.Their responses depend on the intracellular calcium concentration,and they are obviously separated because of their obvious time differences.The excitatory neuronal calcium,known as“calcium excitation”,spreads through the gap connection in a wavy way to affect the many surrounding astrocytes,as follows with the calcium wave of astrocytes[27].In this study,CJJYT effectively controlled postsynaptic receptor response to improve the synaptic function of hippocampal neurons.

In our study,CJJYT reduced the Ca2+fluorescence intensity in the hippocampal neurons,reduced the frequency and current amplitude of mEPSC,controlled the response of post-synaptic receptors,and inhibited the over-activation of postsynaptic receptors.The results show that CJJYT has a protective effect on neuronal synapses.

4.2 Regulatory effects of CJJYT on synaptic plasticity-related proteins and synaptic function

The synaptic function is recognized to be directly related to the cognitive function in depression,and the recovery of synaptic function is the key to improve the cognitive impairment in depression[28].In conclusion,the synaptic function of the hippocampal neurons is related to the progressive cognitive impairment of depression.We found that CJJYT acts as an antidepressant and its potential mechanism may be to restore the synaptic plasticity of the hippocampal neurons by reversing the damage to synaptic plasticity touch function.

Synaptic plasticity related proteins are most abundant in the hippocampus,which can regulate the function and activity of the synapses.Interestingly,we found that CJJYT significantly reduced the expression levels of SYN-α,NR2A,NR2B and PSD-95 proteins in the hippocampal neurons,and increased the expression levels of CaMKⅡ and SynGAP proteins in hippocampal neurons.The above results were contrary to the blank serum group,to which CORT (50 μm) was added to prepare the depression model.

SYN-αis a kind of calcium binding glycoprotein located on synaptic vesicles,which is widely expressed in the central nervous system,peripheral nervous system and endocrine cells.It is also the main binding site of Ca2+in the cytoplasm on synaptic vesicles.SYN-αparticipates in the fusion of synaptic vesicles and presynaptic membrane by binding to Ca2+and regulates the release of neurotransmitters through its phosphorylation.In addition,SYN-αregulates the occurrence,development and maturation of neurons,and affects the differentiation of neurons by regulating the assembly of cytoskeleton proteins.SYN-αplays an important role in axonal extension,synaptic connection formation,and synaptic maturation.It has been found that the over release of SYN-αcan activate NR,which leads to excitotoxicity of neurons,resulting in neuronal damage[29].

PSD-95 is composed of a variety of postsynaptic membrane proteins.It contains many receptors,which can control receptor aggregation and regulate receptor function.PSD-95 is the protein marker of postsynaptic membrane,and NR is one of its main components[30].

PSD-95 is the most abundant and important skeleton protein present on the postsynaptic membrane,which mainly exists in the synapse with excitatory glutamatergic energy,but can also be a plasticity marker of synapse.It participates in the formation and reconstruction of synapse and has important functional significance in the aspects of synaptic countability/control synaptic transmission/learning and memory.It has been found that continuous treatment of postnatal mice with ionizing radiation may lead to cognitive dysfunction,accompanied by synaptic plasticity damage.The PSD-95 levels increased in the dentate gyrus of hippocampus was detected 5 protein.ADEOSUN et al.[31]found that the inhibition of postsynaptic protein PSD-95 but not presynaptic protein SYN-αcan lead to dysfunction of postsynaptic transmission in the hippocampus and thus cognitive dysfunction.

SynGAP is a postsynaptic GTPase-activating protein (GTP),which can mediate excitatory synaptic transmission,and is an important protein for synaptic structure maintenance and function regulation[32].The overexpression of glutamate receptor in cultured neurons can significantly reduce mEPSC.

CaMKⅡ is a protein with the highest content in the hippocampus,which is the main binding protein of calcium ions and closely related to the long-term potentiation of synapses and is called “the molecular switch of learning and memory”.Both CaMKⅡ and SynGAP proteins are present in the postsynaptic compact state and are downstream molecules of NR2A and NR2B subtypes.Both CaMKⅡ and PSD-95 dynamically bind to the C-terminal of NR2A protein to regulate the synaptic plasticity of the hippocampus.CaMKⅡ plays a key role in the NR dependent LTP synaptic plasticity,which is essential for the formation of synaptic memory molecule[12].

SynGAP is a synaptic Ras GTPase-activating protein (Ras GTP) that is highly enriched in the excitatory synapses of the brain,and regulates the formation of spine and the transport of glutamate receptor in neurons.During LTP induction,CaMKⅡ phosphorylates SynGAP to rapidly disperse in the dendritic spines of hippocampal neurons,through altering the expression of synaptic plasticity functional proteins,CaMKⅡ and SynGAP,leading to plasticity changes in the synapses of the hippocampal neurons,resulting in neuronal damage,learning and memory dysfunction,and depression[24].

CJJYT improves cognitive impairment by reversing synaptic damage in the hippocampus of depressive rats.CJJYT intervention can improve sucrose preference,voluntary activities,learning and memory ability of Morris water maze test,appetite for novelty suppressed feeding,sense of forced swimming desperation,and reverse CORT levels in the depressed model rats.Interestingly,in the hippocampal CA1 and DG Golgi-Cox staining,CJJYT was found to increase the number of dendritic branches and dendrites,restoring their density and complexity,and repairing the synaptic damage in the hippocampal neurons of the depressed rats.

Considering previousin vitroexperiments,the potential pathogenesis of cognitive impairment in depression may involve over release of SYN-αleading to activation of NR.On the one hand,the over expression of PSD-95 protein,which is the main component of NR,leads to the damage of synaptic plasticity and cognitive impairment; on the other hand,the continuous increase of NR2A and NR2B subtypes leads to a large amount of Ca2+influx and its downstream synaptic plasticity function protein CaMKⅡ.The decrease in the expression of SynGAP in neurons leads to a significant increase in mEPSC and changes in the synaptic plasticity of hippocampal neurons,which leads to the impairment of neurons,learning and memory,and other cognitive dysfunction.

CJJYT controls the post-synaptic receptor response by reversing the expression of the abovementioned hippocampal synaptic plasticity-related proteins,protecting neurons and repairing synaptic damage.Then synaptic function was restored in hippocampal neurons of depressed rats.Briefly,it improves the cognitive behavior such as learning and memory in depressed rats and acts as an antidepressant.

However,it is worth mentioning that the synaptic gene-protein network is related to neuropsychiatric disorder[33].The rare disease variant gene in synapse exists in a series of neurodevelopmental disorders,but its interaction mechanism is not clear.Future studies on synaptic transmission and plasticity should include isogenic controls[34].In our future studies,the synaptic gene protein network will be our follow-up research focus.

Currently,there is no drug available to treat cognitive impairment caused by depression,and the chemical synthesis of antidepressants is one-sided.The Chinese medicine compound is a comprehensive adjustment expert and has remarkable treatment effects.In summary,the potential mechanism of CJJYT antidepressant may be to improve the synaptic plasticity of the hippocampal neurons,repair synaptic damage,and restore synaptic function by regulating the transmission and function of SYN-α/NR and its downstream neurotransmitters.

Acknowledgements

We thank for the funding support from the National Major New Drug Development Project (No.2017ZX09309026) and Provincial Department of Graduate Research Innovation Project of Hunan(No.CX20190565).

Competing Interests

The authors declare no conflict of interest.