臨床常用普伐他汀鈉片大鼠體內(nèi)藥代動力學(xué)比較研究

趙 贏1，張 娜2，3，龐曉叢1，生立嵩1，宋俊科1，何 蘭3，呂 揚2，杜冠華1

（1.中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所，北京市藥物靶點研究與新藥篩選重點實驗室，北京 100050；2.中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所，北京市晶型藥物研究重點實驗室，北京 100050；3.中國食品藥品檢定研究院，北京 100050）

T4藥物代謝與分子藥理學(xué)

T4-1

臨床常用普伐他汀鈉片大鼠體內(nèi)藥代動力學(xué)比較研究

趙 贏1，張 娜2，3，龐曉叢1，生立嵩1，宋俊科1，何 蘭3，呂 揚2，杜冠華1

（1.中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所，北京市藥物靶點研究與新藥篩選重點實驗室，北京 100050；2.中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所，北京市晶型藥物研究重點實驗室，北京 100050；3.中國食品藥品檢定研究院，北京 100050）

摘要：目的研究大鼠灌胃不同企業(yè)普伐他汀鈉片后體內(nèi)藥代動力學(xué)的一致性。方法固體灌胃給予SD雄性大鼠普伐他汀鈉15 mg·kg-1，采用液質(zhì)聯(lián)用法測定不同時間的血藥濃度，繪制藥時曲線，并用DAS 3.0.7數(shù)據(jù)處理系統(tǒng)進(jìn)行主要藥代動力學(xué)參數(shù)的計算和比較。結(jié)果三個原料藥生產(chǎn)企業(yè)的測定結(jié)果顯示，3號晶F型普伐他汀鈉原料藥較1、2號晶A型有著更好的藥物吸收和相對生物利用度，為優(yōu)勢藥物晶型；隨機抽取的臨床常用7種普伐他汀鈉片在大鼠體內(nèi)藥代動力學(xué)參數(shù)結(jié)果呈現(xiàn)顯著性差異，Tmax，Cmax，AUC，t1/2的最大差異分別可以達(dá)到5.3，3.4，2.4和1.4倍之多。結(jié)論不同企業(yè)的普伐他汀鈉片在大鼠體內(nèi)的藥代動力學(xué)過程存在顯著性差異，這可能與普伐他汀鈉多晶型狀態(tài)以及藥物生產(chǎn)工藝、輔料成分及含量等制劑過程有關(guān)。

關(guān)鍵詞：普伐他??；晶型；藥代動力學(xué)；生物利用度；LC-MS

T4-2

孕三烯酮膠囊多次給藥在巴馬小型豬體內(nèi)的藥代動力學(xué)研究

郭湘潔，李 釗，謝淑武，周潔蕓，李國停，鐘瑞華，朱 焰（復(fù)旦大學(xué)生殖與發(fā)育研究院上海市計劃生育科學(xué)研究所生殖藥理組，國家人口計生委計劃生育藥具重點實驗室，上海200032）

摘要：目的建立快速靈敏的HPLC-MS/MS法測定巴馬小型豬血漿中孕三烯酮的濃度，并研究孕三烯酮膠囊在巴馬小型豬體內(nèi)的藥代動力學(xué)特征。方法以巴馬小型豬為實驗動物，連續(xù)給藥12周，每周給藥2次，于給藥后不同時間點采集血樣。通過測定多次給藥后小型豬血漿中孕三烯酮的濃度，計算其藥代動力學(xué)參數(shù)。測定血漿樣品以孕二烯酮為內(nèi)標(biāo)，經(jīng)環(huán)己烷液液萃取后進(jìn)行LC-MS/MS分析。質(zhì)譜檢測采用ESI正離子模式，選擇檢測離子反應(yīng)對為m/z309.2→241.1（孕三烯酮），m/z 311.2→109.1（孕二烯酮）。結(jié)果孕三烯酮在0.1~10 ng·mL－1濃度范圍內(nèi)線性良好（r=0.9999），定量下限為0.1 ng·mL-1。提取回收率均高于83.14%，日間、日內(nèi)RSD均小于11.27%，專屬性良好，在本實驗涉及的條件下樣品穩(wěn)定。8只小型豬首次給藥后Cmax為（2.85±0.96）μg·L-1，Tmax為（2.06±0.62）h，t1/2為（8.43±4.13）h，AUC（0-85）為（23.81±8.62）μg·L·h-1，AUC（0-∞）為（24.36±8.73）μg·L·h-1，MRT（0-85）為（8.40±2.44）h，MRT（0-∞）為（9.81±3.88）h；末次給藥后Cmax為（2.70±0.55）μg·L-1，Tmax為（1.69±0.65）h，t1/2為（8.51±3.72）h，AUC（0-85）為（17.71±4.89）μg·L·h-1，AUC（0-∞）為（18.51±4.48）μg·L·h-1，MRT（0-85）為（8.54±3.50）h，MRT（0-∞）為（9.98±4.41）h。多次給予孕三烯酮膠囊后2 h血藥濃度相差不大，且用藥后無不良反應(yīng)，安全性較高。結(jié)論該方法準(zhǔn)確、快速、靈敏、專屬性強，適用于動物實驗及臨床上測定血漿中孕三烯酮的濃度及其藥代動力學(xué)研究。

關(guān)鍵詞：孕三烯酮；液質(zhì)聯(lián)用；巴馬小型豬；血藥濃度

T4-3

KCNQ鉀通道在糖尿病神經(jīng)病理性疼痛中的作用及機制研究

周 宇，于海波，王曉良

（中國醫(yī)學(xué)科學(xué)院藥物研究所，北京 100050）

摘要：目的在糖尿病神經(jīng)病理性疼痛（diabetic neuropathic pain，DNP）發(fā)生過程中，KCNQ鉀通道所起的作用及其機制。方法首先是在動物整體水平上，建立鏈脲佐菌素（strep?tozocin，STZ）誘導(dǎo)的糖尿病模型以及甲基乙二醛（methylgly?oxal，MG）誘導(dǎo)的疼痛模型，通過分離背根神經(jīng)元，檢測兩種模型離子通道表達(dá)變化是否一致，同時檢測糖尿病模型中甲基乙二醛的水平。其次是在細(xì)胞水平上，觀察MG以及MG+ MG清除劑對離子通道活性和電流的影響，探討KCNQ3通道開放劑對DNP的緩解作用以及MG是如何調(diào)控離子通道改變的。結(jié)果在STZ誘導(dǎo)的模型中，血糖明顯升高，機械痛和熱痛明顯降低，其KCNQ3通道的表達(dá)也有明顯的降低；同時，在STZ誘導(dǎo)的模型中，MG的濃度是明顯升高的，而MG模型中KCNQ3的表達(dá)量也是明顯下降的。結(jié)論STZ以及MG誘導(dǎo)的模型，均可誘發(fā)KCNQ鉀通道表達(dá)的降低；MG調(diào)控KCNQ活性的變化可能通過與離子通道氨基酸反應(yīng)，調(diào)控通道的活性；KCNQ可作為DNP治療的藥理學(xué)靶點。

關(guān)鍵詞：神經(jīng)病理性疼痛；甲基乙二醛；鉀離子通道

T4-4

采用雙向驗證法和斑馬魚模型探索血管生成相關(guān)的內(nèi)源性代謝物及代謝網(wǎng)絡(luò)的變化

韓利文1，李智平2，何秋霞1，張軒銘1，張 云1，李曉彬1，田青平2，劉可春1，劉昌孝3

（1.山東省科學(xué)院生物研究所，山東濟(jì)南 250014；2.山西醫(yī)科大學(xué)藥學(xué)院，山西太原 030001；3.天津藥物研究院，天津300301）

摘要：目的血管生成與很多疾病有關(guān)。目前對于信號傳遞的下游的代謝了解甚少，探尋與血管相關(guān)的新的生物學(xué)機制對于疾病的防治意義重大。斑馬魚血管生成評價模型是目前公認(rèn)的評價血管生成作用的優(yōu)良模型，能夠把血管生成獨立出來研究，與移植瘤等模型不同，無須考慮腫瘤等其他因素的干擾。同時借鑒分子生物學(xué)科中靶點研究的思路和方法，建立“雙向驗證”策略，進(jìn)行血管生成相關(guān)的代謝型生物標(biāo)志物進(jìn)行研究。方法采用轉(zhuǎn)基因斑馬魚Tg（vegfr2：GFP）幼魚作為血管生成研究模型，在斑馬魚發(fā)育至24h時，采用靶點抑制劑PTK787和中藥丹紅注射液進(jìn)行雙向處理，觀察丹紅注射液（DHI）對PTK787造成損傷的逆轉(zhuǎn)作用，用Image J軟件對節(jié)間血管（ISVs）進(jìn)行定量分析，作為血管生成評價指標(biāo)；同步處理后的斑馬魚每20條為一個樣本，進(jìn)行組織破碎、提取、去蛋白后，進(jìn)行衍生化處理，然后用GC-MS聯(lián)用儀進(jìn)行分析，每組6個重復(fù)；采用Simca-P軟件進(jìn)行代謝組模式判別分析。結(jié)果在本實驗條件下，30~60 μL·mL-1的丹紅注射液對PTK787造成的斑馬魚節(jié)間血管生成的損傷具有明顯逆轉(zhuǎn)作用。代謝輪廓分析結(jié)果顯示，正常組、PTK787處理組、丹紅注射液處理在散點圖中呈現(xiàn)了明顯的損傷和逆轉(zhuǎn)趨勢。進(jìn)一步的分析發(fā)現(xiàn)，PTK787處理斑馬魚后，酪氨酸、次黃嘌呤、苯丙氨酸、天冬氨酸、谷氨酰胺、磷酸水平明顯升高，肌醇明顯降低，而丹紅注射液處理后，發(fā)現(xiàn)這些代謝物呈現(xiàn)了顯著逆轉(zhuǎn)。代謝網(wǎng)絡(luò)分析顯示，這些代謝物可能與纈氨酸-亮氨酸-異亮氨酸生物合成途徑、苯丙氨酸-酪氨酸-色氨酸合成途徑、苯丙氨酸代謝、甘氨酸-天冬氨酸-谷氨酰胺代謝、磷酸肌醇代謝途徑有關(guān)。結(jié)論本研究利用斑馬魚模型和代謝組學(xué)技術(shù)對血管生成相關(guān)的下游代謝信號進(jìn)行了探討，并建立了雙向處理法，快速篩選發(fā)現(xiàn)了一些相關(guān)的代謝物，與常規(guī)的藥物處理進(jìn)行模式判別，準(zhǔn)確率更高，對利用斑馬魚模型研究疾病相關(guān)的代謝途徑提供了新的研究方法。

關(guān)鍵詞：血管生成；代謝組；GC-MS；生物標(biāo)志物；斑馬魚

T4-5

液質(zhì)聯(lián)用法同時測定新西蘭兔血漿中左炔諾孕酮和炔雌醇及其藥動學(xué)

李 釗，郭湘潔，謝淑武，周潔蕓，朱 焰

（復(fù)旦大學(xué)生殖與發(fā)育研究院上海市計劃生育科學(xué)研究所生殖藥理組，國家人口計生委計劃生育藥具重點實驗室，上海200032）

摘要：目的建立液質(zhì)聯(lián)用（HPLC-MS/MS）測定新西蘭兔血漿中左炔諾孕酮（LNG）和炔雌醇（EE）含量的方法，并研究LNG/EE避孕貼劑多次給藥后新西蘭兔體內(nèi)的藥動學(xué)特征。方法6只雌性新西蘭兔給予LNG/EE避孕貼（5 cm× 4 cm，標(biāo)示量為LNG 5.35 mg，EE 0.11 mg），1次/3 d，連續(xù)給藥10次，分別于給藥后不同時間點進(jìn)行耳緣靜脈采血，血漿樣品經(jīng)丹酰氯衍生化后，采用液質(zhì)聯(lián)用方法（HPLC-MS/MS）測定血漿中LNG（內(nèi)標(biāo)孕三烯酮）和EE（內(nèi)標(biāo)氘代炔雌醇）的血藥濃度，并采用DAS3.0軟件計算主要藥代動力學(xué)參數(shù)。結(jié)果LNG的線性范圍為0.1~20 μg·L-1，最低定量限為0.1 μg·L-1，提取回收率均大于92%，日內(nèi)、日間精密度（RSD）均小于15%。首次給藥后LNG的藥代動力學(xué)參數(shù)：Cmax為（8.10±2.38）μg·L-1，Tmax為（2.38±1.45）h，AUC（0-t）為（142.35±36.99）μg·L-1·h-1；末次給藥后LNG的藥代動力學(xué)參數(shù)：Cmax為（7.05±1.07）μg·L-1，Tmax為（2.71±1.83）h、AUC（0-t）為（141.95±22.31）μg·L-1·h-1。EE的線性范圍為0.02~5 μg·L-1，最低定量限為0.02 μg·L-1，提取回收率均大于85%，日內(nèi)、日間精密度（RSD）均小于15%。首次給藥后EE的藥代動力學(xué)參數(shù)：Cmax為（0.18±0.04）μg·L-1、Tmax為（2.50±1.30）h、AUC（0-t）為（2.65±0.56）μg·L-1·h-1；末次給藥后EE的藥代動力學(xué)參數(shù)：Cmax為（0.17±0.07）μg·L-1、Tmax為（2.17±0.26）h、AUC（0-t）為（2.02±0.82）μg·L-1·h-1。結(jié)論HPLC-MS/MS法靈敏，準(zhǔn)確，可用于LNG和EE同時測定研究，新西蘭兔多次給予避孕貼后體內(nèi)血藥濃度無蓄積。

關(guān)鍵詞：左炔諾孕酮；炔雌醇；避孕貼；衍生化；藥動學(xué)

T4-6

基于1H-NMR代謝組學(xué)技術(shù)研究D-半乳糖致衰老大鼠尿液的動態(tài)變化

趙凡凡1，2，周玉枝1，高 麗1，秦雪梅1，杜冠華1，3

（山西大學(xué) 1.中醫(yī)藥現(xiàn)代研究中心，2.化學(xué)化工學(xué)院，山西太原 030006，3.中國醫(yī)學(xué)科學(xué)院，北京 100050）

摘要：目的通過代謝組學(xué)技術(shù)研究D-半乳糖（D-gal）刺激引起大鼠尿液代謝輪廓的動態(tài)變化。方法實驗分為兩組，即正常對照組和模型組；模型組連續(xù)皮下注射D-gal溶液（100 mg·kg-1·d-1）10周，對照組皮下注射等量生理鹽水，并分別在0,2,4,6,8和10周收集每只實驗鼠的尿液。第10周開始采用水迷宮和避暗實驗檢測空白組與模型組大鼠的學(xué)習(xí)記憶能力。在行為學(xué)實驗結(jié)束后，將各組實驗鼠處死并收集海馬。采用代謝組學(xué)技術(shù)對實驗大鼠五次尿液進(jìn)行NMR數(shù)據(jù)采集并結(jié)合多元統(tǒng)計分析，探討D-半乳糖致衰老機制。結(jié)果結(jié)果表明，與空白組相比，D-gal能顯著增加大鼠在水迷宮中的潛伏期（P<0.05），減少其在避暗實驗中的潛伏期和錯誤次數(shù)（P<0.05）等行為學(xué)指標(biāo)。對2，4，6，8和10周的不同實驗點的模型組和空白組大鼠的尿液進(jìn)行PCA分析，發(fā)現(xiàn)造模2~4周時，模型組與空白組的代謝輪廓沒有區(qū)分，造模6~8周，模型組空白組大鼠逐漸分開，造模10周時，模型組與空白組完全分開。采用OPLS-DA尋找二者之間的差異物，在第10周發(fā)現(xiàn)乳酸、丙氨酸、α-酮戊二酸、膽堿等14個峰面積具有顯著性差異的潛在生物標(biāo)志物。結(jié)論結(jié)果表明，D-gal能夠損傷大鼠的學(xué)習(xí)記憶，衰老模型的建立可能是通過能量代謝、糖代謝、腸菌代謝等代謝途徑發(fā)揮作用的。

關(guān)鍵詞：D-半乳糖；學(xué)習(xí)記憶；認(rèn)知障礙；代謝組學(xué)

T4-7

抗癲癇新藥氯桂丁胺與藥物代謝酶CYP450的相互作用

王 昕，程海旭，丁 宇，李 強，王淑梅，李 璞，逯穎媛，樓雅卿，章國良

（北京大學(xué)基礎(chǔ)醫(yī)學(xué)院藥理學(xué)系，北京 100191）

摘要：目的研究新型桂皮酰胺類抗癲癇化合物氯桂丁胺體內(nèi)代謝的CYP450同工酶亞型及其對6種主要CYP450同工酶活性的影響。方法超高速離心法制備大鼠肝微粒體，高效液相色譜法定量分析氯桂丁胺原型藥、代謝物、6種CYP450同工酶探針?biāo)帲–YP1A2/非那西汀、CYP2E1/氯唑沙宗、CYP3A/硝苯地平、CYP2C9/甲苯磺丁脲、CYP2C19/ S-美芬妥英、CYP2D6/右美沙芬）代謝物，在大鼠肝微粒體、6種重組人源CYP450同工酶孵育模型中濃度的變化。結(jié)果6種重組人源CYP450同工酶分別與氯桂丁胺共孵育，以及6種特異性CYP450同工酶抑制劑（α-奈黃酮/ CYP1A2、磺胺苯吡唑/CYP2C9、奧美拉唑/CYP2C19、奎尼丁/CYP2D6、二乙基二硫氨甲酸/CYP2E1、酮康唑/CYP3A）分別與氯桂丁胺及大鼠肝微粒體共孵育結(jié)果顯示，參與氯桂丁胺生物轉(zhuǎn)化的主要代謝酶亞型包括代謝物M1（CYP2D6），M2（CYP1A2），M3（CYP2C19和CYP3A4）。氯桂丁胺在0.1~16 μg·mL-1濃度范圍內(nèi)對CYP1A2，CYP2E1和CYP3A酶活性無顯著性影響。在2~16 μg·mL-1濃度范圍內(nèi)顯著性抑制CYP2C9酶活性（抑制率60.45%~97.64%）、CYP2C19酶活性（抑制率50.44%~77.44%）、CYP2D6酶活性（抑制率35.92%~71.43%）。結(jié)論本研究結(jié)果提示氯桂丁胺可經(jīng)多酶代謝，其自身的代謝清除可能不易受單一CYP450亞型遺傳因素所干擾，但由于其抑制CYP2C9，CYP2C19及 CYP3A的活性，如與相應(yīng)底物類藥物合用時應(yīng)注意藥物-藥物間相互作用。

關(guān)鍵詞：氯桂丁胺；CYP450；肝微粒體；藥物-藥物相互作用

T4-8

性激素對二乙基亞硝胺（DEN）所致肝癌組織中CYP450酶表達(dá)的影響

周滋晶，繆曉潔，尹 航，王 昕，章國良

（北京大學(xué)醫(yī)學(xué)部基礎(chǔ)醫(yī)學(xué)院藥理系，北京 100191）

摘要：目的研究性激素（雌二醇與孕酮）對二乙基亞硝胺（DEN）誘導(dǎo)的大鼠肝癌組織中多種細(xì)胞色素P450（CYP450）同工酶表達(dá)的影響及可能的作用機制。方法每周1次ip給予DEN（50 mg·kg-1，×12周）制備大鼠肝癌模型，H-E染色及光學(xué)顯微鏡法觀察石蠟包埋肝組織切片中病理形態(tài)學(xué)改變，免疫組織化學(xué)法對肝組織中甲胎蛋白（AFP）、CYP1A2，CYP2B1，YP2E1及CYP3A1共5種目的蛋白定性定位觀察。結(jié)果與生理鹽水對照組的H-E染色切片相比，DEN刺激組的肝組織中可見典型的假小葉結(jié)構(gòu)形成，肝癌細(xì)胞呈多核巨細(xì)胞和核分裂相。DEN+雌二醇（30 mg·kg-1，實驗?zāi)┲? d×7 d，sc）組、DEN+孕酮（150 mg·kg-1，實驗?zāi)┲? d×7 d，sc）組均可見更多假小葉及肝細(xì)胞內(nèi)脂肪空泡形成。免疫組化結(jié)果顯示，DEN組中肝癌發(fā)生標(biāo)志物AFP呈棕色強陽性染色，而CYP1A2，CYP2B1，CYP2E1及CYP3A1陽性棕色染色程度均降低，5種蛋白均定位于細(xì)胞質(zhì)；DEN+雌二醇組中4種CYP450同工酶染色程度較之DEN組略有增強；而DEN+孕酮組可見CYP3A1強陽性棕色染色。結(jié)論雌二醇可部分逆轉(zhuǎn)DEN所致肝癌組織中4種CYP450同工酶表達(dá)減少，孕酮則顯著增加DEN所抑制的CYP3A1表達(dá)，其確切的機制有待于進(jìn)一步探討。

關(guān)鍵詞：雌激素；孕激素；CYP450；DEN肝癌發(fā)生模型

T4-9

新型利尿藥PU-48在生物樣本中LC-MS/MS檢測方法的建立與確證

張志遠(yuǎn)，王 昕，逯穎媛，李 璞，程海旭，楊寶學(xué)，章國良

（北京大學(xué)醫(yī)學(xué)部基礎(chǔ)醫(yī)學(xué)院藥理系，北京 100191）

摘要：目的建立和確證以尿素通道蛋白作為藥物靶點的噻吩并喹啉類新型利尿藥3-甲氧基-6-氨基-噻吩并［2，3-b］喹啉-7-羧酸甲酯（PU-48）在生物樣本中的液相色譜串聯(lián)質(zhì)譜（LC-MS/MS）檢測方法。方法采用Shimadzu LC-20A高效液相色譜串聯(lián)AB Sciex API 4000 QTRAP質(zhì)譜儀，Inertsil ODS-4 C18色譜柱（100×2.1 mm，3 μm）。流動相為0.05%的乙腈-水溶液，流速0.3 mL·min-1，梯度洗脫。使用醋酸甲地孕酮作為內(nèi)標(biāo)，PU-48和內(nèi)標(biāo)分別選擇m/z289.1→257.1和m/z385.3→267.1作為定量的離子對。血漿樣本使用乙酸乙酯進(jìn)行液-液萃取，37℃下氮氣吹干重溶，取5 μL進(jìn)樣。結(jié)果在上述條件下，PU-48的保留時間為6.2 min，內(nèi)標(biāo)的保留時間為7.2 min，分離良好且不受內(nèi)源性物質(zhì)的干擾。血漿外加藥物標(biāo)準(zhǔn)曲線范圍0.1~1000 ng·mL-1，相關(guān)系數(shù)大于0.99，最低檢測限（LOD）為0.05 ng·mL-1，定量下限（LLOQ）為0.1 ng·mL-1。大鼠血漿外加低、中、高三個濃度（0.2，10.0,500.0 ng·mL-1）質(zhì)控點PU-48的日內(nèi)及日間準(zhǔn)確度（RE）分別為<12.95%及<-9.55%，日內(nèi)及日間精密度（RSD）分別為<4.14%及<8.29%，絕對回收率在85.25~ 98.10%。4℃下放置96 h的準(zhǔn)確度精密度分別為<8.12%和<6.07%。結(jié)論本研究所建立的LC-MS/MS檢測方法的的特異性、靈敏度、精密度、準(zhǔn)確度符合《藥物非臨床藥代動力學(xué)研究技術(shù)指導(dǎo)原則》的要求。

關(guān)鍵詞：LC-MS/MS；3-甲氧基-6-氨基-噻吩并［2，3-b］喹啉-7-羧酸甲酯（PU-48）；利尿藥；大鼠血漿

T4-10

基于1H-NMR代謝組學(xué)的黃芩素干預(yù)D-半乳糖致衰老大鼠作用研究

王珂欣1，2，高 麗1，段丹丹1，2，周玉枝1，秦雪梅1

（山西大學(xué)1.中醫(yī)藥現(xiàn)代研究中心，山西太原 030006；2.化學(xué)化工學(xué)院，山西太原 030006）

摘要：目的采用1H-NMR代謝組學(xué)方法，探討黃芩素對D-半乳糖致衰老模型大鼠的干預(yù)作用。方法采用皮下注射D-半乳糖（150 mg·kg-1）致大鼠衰老模型，考察黃芩素（50，100和200 mg·kg-1）對衰老大鼠自主活動能力的影響。造模10周后采集大鼠血清進(jìn)行1H-NMR檢測，結(jié)合多元統(tǒng)計分析探討黃芩素的抗衰老作用。結(jié)果與空白組相比，D-半乳糖致衰老模型組大鼠穿越格數(shù)和直立次數(shù)均顯著減少（P<0.01），給予黃芩素后大鼠穿越格數(shù)和直立次數(shù)均逐漸增加，表明黃芩素能顯著提高衰老大鼠自主活動能力。與空白組相比，模型組大鼠血清中丙氨酸、賴氨酸、醋酸、丙酮酸、酪氨酸水平升高，而O-乙酰糖蛋白，乙酰乙酸，谷氨酰胺，二甲基甘氨酸，甘氨酸，蘇氨酸，肌醇，α-葡萄糖，組氨酸水平降低，給予黃芩素后能不同程度回調(diào)這14種潛在的生物標(biāo)志物。結(jié)論黃芩素可能通過調(diào)控氨基酸代謝、能量代謝和糖代謝等途徑延緩衰老。

關(guān)鍵詞：黃芩素；抗衰老；D-半乳糖；代謝組學(xué)

T4-11

杜仲降血壓活性成分在正常大鼠和自發(fā)性高血壓大鼠體內(nèi)的藥代動力學(xué)差異研究

鞏仔鵬1，3，4，吳林霖1，3，陸 苑1，3，侯靖宇1，3，李月婷1，3，候 佳1，3，黃 勇1，3，李勇軍1，2，王永林1，3

（貴州醫(yī)科大學(xué)1.貴州省藥物制劑重點實驗室，2.民族藥與中藥開發(fā)應(yīng)用教育部工程研究中，3.藥學(xué)院，貴州貴陽550004；4.國家苗藥工程技術(shù)研究中心，貴州貴陽 550004）

摘要：目的鑒于中藥主要是用于病理狀態(tài)下的機體內(nèi)，因此研究病理狀態(tài)下的中藥的藥代動力學(xué)較正常機體更有意義，且與臨床更相關(guān)。本研究旨在比較杜仲降壓活性成分（松脂醇二葡萄糖苷和京尼平苷酸）在正常大鼠和自發(fā)性高血壓大鼠（SHR）大鼠體內(nèi)的藥代動力學(xué)行為特征。方法分別給正常大鼠及SHR模型行頸靜脈插管手術(shù)，術(shù)后12 h，灌胃給予杜仲提取物（4.8 g·kg-1，分別相當(dāng)于松脂醇二葡萄糖苷96 mg·kg-1和巴馬汀168.96 mg·kg-1），并于給藥前以及給藥后5，15，30 min，1，1.5，2，3，4，6，8，10，12，24和36 h通過插管采血0.2mL于肝素化的EP管中，離心，取血漿100μL。然后采用UPLC-MS/MS技術(shù)測定血漿中的活性成分的含量。繪制藥時曲線，并利用WinNonlin軟件計算藥代參數(shù)（T1/2，Tmax，Cmax，AUC0–t，Vd/F和Cl/F），比較各代表成分在正常大鼠和SHR模型體內(nèi)的藥代動力學(xué)差異。結(jié)果灌胃杜仲提取物后，與正常大鼠相比，松脂醇二葡萄糖苷和京尼平苷酸在SHR模型體內(nèi)的T1/2，Cmax和AUC0–t顯著增加，Vd/F和Cl/F顯著降低。結(jié)論自發(fā)性高血壓的病理狀態(tài)能夠顯著改變杜仲降壓活性成分（松脂醇二葡萄糖苷和京尼平苷酸）的藥代動力學(xué)特征，提示臨床上在用杜仲治療高血壓時，需要對其劑量進(jìn)行調(diào)整。

關(guān)鍵詞：杜仲；松脂醇二葡萄糖苷；京尼平苷酸；自發(fā)性高血壓；藥代動力學(xué)

T4-12

伏立康唑血藥濃度監(jiān)測結(jié)果評析

顏 苗1，2，王檸檸1，3，李紫薇1，3，蔣夢飛1，王 峰1，2，張畢奎1，2，徐 萍1，2，肖軼雯1，2

（中南大學(xué)1.湘雅二醫(yī)院藥學(xué)部，2.臨床藥學(xué)研究所，3.藥學(xué)院，湖南長沙 410208）

摘要：目的通過對不同臨床?？品⒖颠蜻M(jìn)行治療藥物監(jiān)測（TDM）并比較調(diào)整劑量前后的血藥谷濃度數(shù)據(jù)，闡明TDM對伏立康唑合理用藥的必要性，并給出相關(guān)的臨床提示。方法本研究通過回顧性分析伏立康唑TDM的數(shù)據(jù)，對收治入院的154位患者的435例次伏立康唑血藥谷濃度測定結(jié)果做一個初期評估。結(jié)果腎移植科僅有4.3%的測定結(jié)果高于5.5 μg·mL-1，而有26.5%的測定結(jié)果低于1.0 μg·mL-1。感染科有52.3%的測定結(jié)果高于5.5 μg·mL-1，沒有出現(xiàn)低于1.0 μg·mL-1的測定結(jié)果。結(jié)論TDM對伏立康唑的合理化用藥十分必要，腎移植科患者伏立康唑TDM不在建議濃度區(qū)間的結(jié)果以低于1.0 μg·mL-1居多，而感染科患者則以高于5.5 μg·mL-1居多，臨床上應(yīng)予以重視。臨床藥師可根據(jù)TDM結(jié)果更密切的參與伏立康唑臨床用藥。

關(guān)鍵詞：伏立康唑；治療藥物監(jiān)測；腎移植科；感染科

T4-13

細(xì)胞外Ba2+對記錄大鼠心肌細(xì)胞L型鈣通道的影響

孟紅旭，姚明江，任鈞國，劉建勛

（中國中醫(yī)科學(xué)院西苑醫(yī)院基礎(chǔ)醫(yī)學(xué)研究所，中藥藥理北京市重點實驗室，北京 100091）

摘要：目的觀察細(xì)胞外Ba2+對記錄大鼠心肌細(xì)胞L型鈣通道的影響。方法采用急性酶解分離法獲得大鼠的單個心肌細(xì)胞，使用全細(xì)胞膜片鉗技術(shù)記錄L型鈣通道電流。結(jié)果（1）細(xì)胞外液中的Ca2+被Ba2+替換后，L型鈣通道電流的失活速率明顯減慢（時間常數(shù)由88 ms增至246 ms，P<0.01）。細(xì)胞外液中加入Ba2+（0.2和0.4 mmol·L-1）后，L型鈣通道電流的失活速率無明顯改變。（2）細(xì)胞外液中加入Ba2+（0.2和0.4 mmol·L-1）后，連續(xù)記錄10和15 min，L-型鈣通道電流峰值電流的衰減明顯減弱（P<0.01）（3）細(xì)胞外液加入Ba2+（0.4 mmol·L-1）使電流電壓曲線下移，并改變翻轉(zhuǎn)電位。（4）細(xì)胞外液加入Ba2+（0.4 mmol·L-1）后，減弱了丹酚酸A（100 μmol·L-1）對鈣電流的抑制強度，并使丹酚酸A量效關(guān)系曲線下移。結(jié)論細(xì)胞外液加入一定濃度的Ba2+能夠有效減弱全細(xì)胞膜片鉗技術(shù)記錄L型鈣通道時出現(xiàn)的“rundown”現(xiàn)象，并減少“rundown”現(xiàn)象對藥物量效關(guān)系的影響。

關(guān)鍵詞：Ba2+；全細(xì)胞膜片鉗；“rundown”現(xiàn)象；丹酚酸A

T4-15

Review on the effects of the traditional Chinese medicine on cytochrome P450

WANG Yan-yan1，2，CHEN Wei-dong1，2

（1.School of Pharmacy，Anhui University of Chinese Medicine，Hefei 230012，China；2.Anhui Academy of Chinese Medicine，Hefei 230012，China）

Abstract：The cytochrome P450 superfamily，one of the most important drug-metabolizing enzyme systems in hu?mans，is responsible for the metabolism of numerous xe?nobiotic substances as well as endogenous substrates.In?duction or inhibition of the cytochrome P450 enzymes，after exposure to different drugs and chemicals，is directly linked to a number of drug-induced toxicity and drug inter?actions leading to treatment failure.As we know，the tra?ditional Chinese medicine（TCM）has complicated com?ponents and is always coadministered with other drugs. Through searching and summing up the literatures about the effects of TCM on cytochrome P450 at home andabroad，the results show that the activity of cytochrome P450 was inhibited or induced by some single herbs，tra?ditional Chinese prescriptions andthe active components of the TCM，such as the flavonoids，flavonoid deriva?tives，alkaloids，saponins，terpenoids，anthraquinones and lignans，and so on，which therefore might slow or speed metabolism of other drugs.The paper tips that much attention should be paid to the research fields of TCM on cytochrome P450，which is meaningful to clini?cal reasonable medicine treatment and new drug devel?opment.

Key words：TCM；cytochrome P450；drug-drug interaction

T4-16

Pharmacokinetic parameters investigation on interaction between Danshen injection and Honghua injection

YU Fan1，3*，LI Guo-zhuan1，3*，GUO Dong-dong1，3，PENG Dai-yin1，2，3，CHEN Wei-dong1，2，3

（1.School of Pharmacy，Anhui University of Chinese Medicine，Hefei 230012，China；2.Syergetic Innovation Center of Anhui Authentic Chinese Medicine Quality，Hefei 230012，China；3.Lab 515，School of Pharmacy，Anhui University of Chinese Medicine，Hefei 230012，China）

Abstract：OBJECTIVEDanshen injection and Honghua injection，traditional Chinese medicine（TCM）injections，made from the extracts of Salvia miltiorrhiza Bge.andCarthamus tinctorius L.，have a potential to be developed into the useful drugs for the.As the common TCM injections，Danshen injection is often combined with Honghua injection to treat cardiovascular disease.The purpose of this study was to investigate the pharmacokinetic parameters of Honghua injection combined with Danshen injection when they were coadministered intravenously in human and rats through the tail vein.METHODSSingle and multiple doses of Danshen injection to study Danshen injection on Honghua injection pharmacokinetics parameters and single and multiple doses of Honghua injection to study Honghua injection on Danshen injection pharmaco?kineticsparameters.Theplasmaconcentrationsof hydroxysafflor A（HSYA）and tanshinol and salvianolic acid B were determined by the reliable high-performance liquid chromatography（HPLC）method.The concentrations of HSYA in urine of rats and human were also deter?mined by HPLC method.DAS 2.1.1software was adopted for calculating the pharmacokinetic parameters.RESULTSThe simultaneous intravenous Honghua injection and salvia miltiorrhiza injiection significantly altered the phar?macokinetic parameters of both injections when com?pared with the individual intravenous administration of each injection.The area under the concentration-time curve（AUC）and maximum plasma concentration（Cmax）of HSYA and tanshinol and salvianolic acid B were signifi?cantly increased.The cumulative urine excretion of HSYA in human and rats during 24 h was decreased after two drugs were administered simultaneously by the intra?venous.CONCLUSIONHonghua injection and Danshen injection interact with each other following simultaneous intravenous and they have a synergistic action.This experiment has identified the pharmacokinetic parameters and provided a rationale for the clinical use of the drug combination.

Keywords：pharmacokineticparameters；Honghua injection；Danshen injection

*Co-first author.

T4-17

Microdosing cocktail study on the determination and pharmacokinetics of six hepatic cytochrome probes and their metabolites in rat

YANG Zhi-hong1，XU Li-yun1，YOU Yu-yang2

（1.Institute of Medicinal Plant Development，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100193，China；2.Beijing Institute of Technology，Beijing 100081，China）

Abstract：OBJECTIVETo describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODSAfter administration of a mixture of six probes（i.e.，a cocktail approach with caffeine 100 μg·kg-1，tolbutamide 100 μg·kg-1，omeprazole 500 μg·kg-1，dextrometho?rphan 500 μg·kg-1，chlorzoxazone 50 μg·kg-1and mid?azolam 100 μg·kg-1）to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column（2.1×50 mm，3.5 μm）.The mobile phase consisted of 0.01%formic acid（1 mmol·L-1ammonium formate）and acetonitrile.The flow rate was 0.3 mL·min-1.The samples were analyzed by LC-20A& 5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0m/zions for caffeine，195.2/138.2m/zfor paraxanthine，269.1/170.0m/zfor tolbutamide，285.1/186.0m/zfor 4-hydroxytolbutamide，346.1/198.1m/zfor omeprazole，362.2/214.2m/zfor 5-hydroxyome?prazole，272.3/147.1m/zfor dextromethorphan，258.2/ 157.0m/zfor dextrorphan，168.1/132.1m/zfor chlorzox?azone，326.1/291.2m/zfor midazolam，and 342.1/324.2m/zfor 1′-hydroxymidazolam.RESULTSThe datashowed that the method was with good linearity in the range of 0.2-200 ng·mL-1for caffeine，0.1-25 ng·mL-1for paraxanthine，0.05-100 ng·mL-1for omeprazole，0.01-25 ng·mL-1for 5-hydroxyomeprazole，0.1-200 ng·mL-1for dextromethorphan，0.05-12.5 ng·mL-1for dextrophan，0.2-200 ng·mL-1for midazolam，and 0.2-25 ng·mL-1for 1′-hydroxymidazolam，respectively.The stability%RSD for all probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSIONThis highly sensitive and quantitative method allowed a phar?macokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms（1A2，2C9，2C19，2D6，2E1 and 3A）research.

Key words：microdosing cocktail；hepatic cytochrome；drug metabolism；probes and metabolites

T4-18

Translational regulation of RPA2 via IRES by UNR and eIF3a

CUI Jia-jia1，2，WANG Lei-yun1，2，YIN Ji-ye1，2

（1.Department of Clinical Pharmacology，Xiangya Hospital，Central South University，Changsha410008，China；2.Institute of Clinical Pharmacology，Central South Uni?versity；Hunan Key Laboratory of Pharmacogenetics，Changsha 410078，China）

Abstract：OBJECTIVETo explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and eIF3a.METHODSBiotin pull down assay was taken to study the binding of RPA2 IRES and UNR.UNR was knocked down and overexpressed in H1299，A549 and SK-MES cell lines.Western blotting and real-time PCR were used to detect protein level and mRNA level respec?tively.CO-IP assay was conducted for the interaction of eIF3a and UNR.GST pull down assay was carried out to ex?plore the exact domains.And the domains of eIF3a and UNR binding to RPA2 IRES were explored with EMSA assay.RESULTSNUR protein can bind to RPA2 IRES as well as eIF3a.UNR regulated the protein expression of RPA2 in H1299，A549 and SK-MES cells，and there was no change in RPA2 mRNA.UNR combined with eIF3a via the first domain of UNR and the first domain of eIF3a.UNR bound to RPA2 IRES with the first domain. And there was no sufficient evidence for the binding do?main of eIF3a with RPA2 IRES yet.CONCLUSIONUNR worked with eIF3a and co-regulate the RPA2 IRES activity and further regulate the expression of protein.This is the possible regulation mechanisms of cellular internal ribo?somal entry site affect translation initiation.

Key words：IRES；RPA2；UNR；eIF3a

T4-19

The anti-nociceptive effect of bufalin，an active ingre?dient from toad venom via modulating voltage-gated sodium channels

SHEN Rui，XU Jian，YIN Pei-hao，TAO Jie

（Department of Neurosurgery and Central Laboratory，PutuoHospital，ShanghaiUniversityofTraditional Chinese Medicine，Shanghai 200062，China）

Abstract：OBJECTIVEToad venom（Venenum Bufonis）isalways used for analgesia in China from ancient to modern times，but the effective component of it remains unclear.METHODSIn the present study，we investigated the anti-nociceptive effect and the underlying mechanism ofbufalin，an active ingredient fromtoad venom by animal behavior，patch clamp and calcium imaging.RESULTSBufalin could significantly relieve formalin-induced spon?taneous flinching and licking response as well as carra?geenan-induced mechanical and thermal hyperalgesia. Using the whole-cell patch-clamp recording，bufalincaused remarkable suppressive effect on the peak currents of Na+channels in dorsal root ganglion neuroblastoma ND7-23 cell line in a U-shaped dependent manner.In addition，bufalinprompted the voltage-dependent activationand caused a negative shift of the fast-state inactivation of Na+channels.However，bufalin produced insignificant ef?fect not onlyon voltage-dependent Kv4.2，Kv4.3 and BK channels，but also on the capsaicin induced Ca2+influx.CONCLUSIONThe present results indicate bufalin is capable of producing remarkable anti-nociceptive effects whichis probably ascribed to its specific modulation of voltage-gated Na+channels.

Key words：bufalin；sodium channels；formalin test；carrageenan test；patch clamp

T4-20

MLKL forms cation channels

XIA Bing-qing，F(xiàn)ANG Sui，CHEN Xue-qin，HU Hong，CHEN Pei-yuan，WANG Hua-yi，GAO Zhao-bing

（State Key Laboratory of Drug Research，Shanghai Institute of Materia Medica，Chinese Academy of Sciences，Shanghai 201203，China）

Abstract：The mixed lineage kinase domain-like（MLKL）protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane dis?ruption.However，our knowledge of how MLKL functions on membrane remains very limited.Here we demon?strate that MLKL forms cation channels that are perme?able preferentially to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover，the N-terminal domain containing six helices（H1-H6）is sufficient to form channels.Using the substituted cysteine accessibility method，we further determine that helix H1，H2，H3，H5 and H6 are trans?membrane segments，while H4 is located in the cyto?plasm.Finally，MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.The Mg2+-preferred permeability and five trans?membrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.

Key words：cation channels； mixed lineage kinase domain-like

T4-21

Characteristics and pharmacokinetics of tripterygium glycosides nano-carries transdermal delivery systems：skin-blood synchronous microdialysis and numerical simulation

LIU Ji-yong1，YANG Meng1，GU Yong-wei2，YANG Di-shun1，LIU Shan-shan1

（1.The Second Military Medical University，Shanghai 200433，China；2.Shandong University of Traditional Chinese Medicine，Jinan 250300，China）

Abstract：The traditional Chinese medicine tripterygium glycosides（TPG）is used clinically to treat some Rheu?matism，Eczema，immunosuppression and tumor，with the activities of hypnosis，antipyretic，analgesic，antiinflammatory，allergy and antitumor.However TPG has low water solubility and low skin permeability，so its clinical use is limited.Transdermal delivery systems can provide a controlled drug release rate that can keep constant con?centrations of drug in the plasma for up to multiple days，improved patient compliance，and the possibility of reducing the rate and severity of side effects.In this study，a fast and sensitive technique skin-blood two sites synchronous microdialysis coupled with LC-MS was used to study the pharmacokinetic parameter of three different formulations（TPG nanoemulsion，TPG nanoemulsion based gels and TPG gel）.Creating a multilayer model，use the model to simulate the three formulations dynamics in transdermal-drug delivery system.The experiment results showed that the TPG nanoemulsion，TPG nano?emulsion based gels can significantly raise the drug con?centrations in skin more than that of TPG gels.The numerical simulation results indicating that TPG gel and TPG nanoemulsion are close to practical measurements，only in the concentration increase phase the numerical simulation result has some difference with the experimental results.TPG nanoemulsion based gels have significant difference with the experimental results，both in concen?trationincreasestageandconcentrationdecreasing stage，but its trend was same.The study shows that the skin-blood synchronous microdialysis technique provided a new method for the pharmacokinetics study of nanocarriers transdermal delivery systems.In addition，the microdialysistechniquecombinedwithmathematical modeling provides a very good platform for the further study of transdermal delivery system.

Key words：tripterygium glycosides；transdermal drug delivery；nano-carriers；microdialysis；numerical simulation

T4-22

A genome-wide association study identifies novel genetic loci that modify pharmacokinetic-pharmaco?dynamic responses to clopidogrel

ZHONG Wan-ping1，2*， WU Hong3*，CHENJi-yan1，2，Li Xin-xin2，LIN Hao-ming3，ZHANG Bin1，2，ZHANG Zhi-wei2，MA Dun-liang1，2，SUN Shuo1，2，LI Han-ping1，2，MAI Li-ping2，HE Gou-dong2，WANG Xi-pei2，LEI He-ping1，2，TANG Lan4，LIU Shu-wen4，ZHONG Shi-long1，2

（1.Guangdong Provincial Key Laboratory of Coronary Heart Disease Prevention，Guangdong Cardiovascular Institute，Guangzhou 510080，China；2.Medical Research Center of Guangdong General Hospital，Guangdong Academy of Medical Sciences，Guangzhou 510080，China；3.Sun Yatsen Memorial Hospital，Sun Yat-sen University，Guangzhou 510020，China；4.Department of Pharmaceutics，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou 510515，China）

Abstract：OBJECTIVEGenetic variants in the pharma?cokinetic（PK）mechanism are the main underlying factors thatmodifytheantiplateletefficacyofclopidogrel. Hence，joint analysis of genetic variants that modify phar?macodynamic（PD）and PK responses to clopidogrel should be effective for identifying the genetic variants affecting the antiplatelet response to the drug.METHODSA genome-wide association study was conducted to identify new genetic loci that modify PD responses to clopidogrel and its active metabolite H4 in 115 Chinese patients with coronary heart disease（CHD）.RESULTSWe identified novel variants in two transporter genes（rs12456693 inSLC14A2and rs2487032 inABCA1）and inN6AMT1（rs2254638）associated with clopidogrel-treated P2Y12 reaction unit（PRU）and plasma H4 concentration.The associations between these single nucleotide polymor?phisms（SNPs）and PK parameters of clopidogrel and H4 were observed in 31 additional CHD patients（P< 0.05）.The new variants，together withCYP2C19*2and clinical factors，dramatically improved the predictability of PRU variability to 37.7%compared with the published value of approximately 20%.The function of these SNPs on the activation of clopidogrel was validated in 32 liver S9 fractions，and theN6AMT1rs2254638 T variant was found to be associated with decreased formation of H4（P=0.0386）.Meanwhile，N6AMT1 rs2254638 was fur?ther identified to exert a marginal risk effect for MACE in an independent CHD patient cohort（OR：1.428，95% CI：0.978-2.086，P=0.0653，F(xiàn)DR=0.4726）.In conclu?sion，we systematically identified new genetic variants as risk factors for the reduced efficacy of clopidogrel.CONCLUSIONOur study findings enhanced the under?standing of the absorption and metabolic mechanisms that influence PD responses to clopidogrel treatment.

Key words：clopidogrel；pharmacokinetics；pharmacody?namics；genome-wide association study；N6AMT1

*Co-first author.

T4-23

Generation and characterization of a novel CYP3A1/2 double knockout rat model using CRISPR-Cas9 system

WANG Xin1，LU Jian1，SHAO Yan-jiao1，QIN Xuan1，LIU Dao-zhi1，CHEN Ang1，LI Da-li1，LIU Ming-yao1，2

（1.Shanghai Key Laboratory of Regulatory Biology，Institute of Biomedical Sciences and School of Life Sciences，East China Normal University，Shanghai，China；2.Center for Cancer and Stem Cell Biology，Institute of Biosciences and Technology，Texas A&M University Health Science Center，Houston，Texas，USA）

Abstract：OBJECTIVECytochrome P450（CYP）3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism，toxicity，and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODSThe CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCRandimmunostaining.Metabolicstudiesofthe CYP3A substrates midazolam and nifedipine bothin vitroandin vivowere conducted to verify that CYP3A1/2 was functionally inactive in KO rats.In addition，compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTSThe Cyp3a1/2 double KO rats were viable and fertile，and had no obvious physiological abnormities.Compared with the wild-type（WT）rat，Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitroandin vivometabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was func?tionally inactive in double KO rats.CONCLUSIONThe Cyp3a1/2 double KO rat model was successfully generat?ed and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study ofthephysiologicalandpharmacologicalrolesof CYP3A， anddeterminingwhethertheabsenceof CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.

Key words：compensatory regulation；CRISPR-Cas9；CYP3A；drug metabolism；gene editing；rat

T4-24

Histone methylation and acethylation contribute to rifampin-mediated induction of CYP3A4 through the interactions between PXR and NCOA6/p300

YAN Liang，ZHANG Li-rong

（Department of Pharmacology，School of Basic Medical Sciences，Zhengzhou University，Zhengzhou 450001，China）

Abstract：OBJECTIVECYP3A4 is one of the majordrug-metabolizing enzymes in humans and is responsible for the metabolism of over 50%of the clinically used drugs.Many drugs have been proved to induce the expression of CYP3A4，which usually causes drug-drug interactions and adverse reactions.The induction by numerous inducers shows significant interindividual differ?ence but genetic factors can not totally explain or esti?mate the difference.Recently，Epigenetic factors have been pointed out to contribute to that difference.Since histone methylation and acethylation are important parts of epigenetics，this study aimed to explore the role of active histone marks（H3K4me3，H3 acethylation）and inactive histone mark（H3K27me3）in rifampin-induced expression of CYP3A4 in LS174T cells.METHODSChromatin immunoprecipitation（ChIP）assay was utilized todeterminethelevelsofhistonemodificationsin CYP3A4 promoter.RNA interference and Co-immunopre?cipitation（Co-IP）assays were performed to determine the role of PXR and interactions between PXR and his?tone methyltransferase/acetylase.RESULTSWe found that the induction of CYP3A4 mRNA by rifampin treat?ment（10 μmol·L-1）in LS174T cells was accompanied by increased levels of H3K4me3 and H3 acethylation and decreased level H3K27me3 in CYP3A4 promoter. Besides，enhanced recruitment of NCOA6，a coactivator of multiple nuclear receptors and transcription factors that is associated with H3K4 methyltransferase，and p300，a histone acetylase，were observed in CYP3A4 promoter.To reveal the relationship between PXR activa?tion and histone methylation/acethylation，gene silence was performed by shRNA transfection.Knockdown of PXR expression not only removed the induction of CYP3A4，but also eliminated the recruitment of NCOA6 and p300 and the decreased levels of H3K4me3 and H3 acethylation in CYP3A4 promoter.Moreover，co-immuno?cipitation（Co-IP）assayshowedthatinteractions between PXR and NCOA6/p300 were occurred upon rifampin stimulation.CONCLUSIONHistone methylation and acethylation contribute to rifampin-mediated induction of CYP3A4 through the interactions between PXR and histone methyltransferase/acetylase.

Key words：CYP3A4；induction；histone modification；PXR

T4-25

TMEM16Acontributestoendothelialdysfunction through accelerating Nox2 NADPH oxidase-derived ROS generation in hypertension

MA Ming-ming1*，GAO Min1，2*，GUO Kai-min3*，LI Xiang-yu1*， WANG Mi4，ZENG Xue-lin1，SUN Lu1，LYU Xiao-fei1，DU Yan-hua1， WANG Guan-lei1，ZHOU Jia-guo1，GUAN Yong-yuan1

（1.Department of Pharmacology，Cardiac and Cerebral Vascular Research Center，Zhongshan School of Medicine，Sun Yat-Sen University，Guangzhou510080，China；2.Department of Pharmacy，the Sixth Affiliated Hospital of Sun Yat-Sen University，Guangzhou 510655，China；3.Department of Obstetrics and Gynecology，Guangzhou Women and Children Medical Center affiliated to Guangzhou MedicalUniversity，Guangzhou510623，China；4.Department of Cardiology，the Second Xiangya Hospital of Central South University，Changsha 410011，China）

Abstract：OBJECTIVEThe Ca2+-activated Cl-channel（CaCC）plays a crucial role in various physiological func?tions.Recent evidences suggest TMEM16A encodes CaCC in various cells，including endothelial cells.However，the role of TMEM16A in the vascular endothelial dysfunc?tion in hypertension is unclear.METHODSIn the study，RT-PCR，Western blotting，co-immunopricipitation，confocal imaging，patch-clamp，and endothelial-specific TMEM16A transgenic and knockout mice were employed.RESULTSWe found that TMEM16A was expressed abundantly and functioned as CaCC in endothelial cells. AngiotensinⅡ（AngⅡ）induced endothelial dysfunction with an increase in TMEM16A expression，which was al?leviated by TMEM16A inhibitor.Further studies revealed that TMEM16A endothelial-specific knockout significantly lowered the blood pressure and ameliorated endothelial dysfunction in AngⅡ-induced hypertension，whereas，TMEM16A endothelial-specific overexpression showed the opposite effects.These results were related to the increased reactive oxygen species（ROS）generation，NADPH oxidase activation，and Nox2，p22phox expres?sion facilitated by TMEM16A upon AngⅡ-induced hyper?tensive challenges.Moreover，TMEM16A directly inter?acted with Nox2 monomer and reduced the degradation of Nox2 through the proteasome-dependent endoplasmic recticulum-associated degradation pathway.TMEM16A alsopotentiatedthetranslocationofp47phoxand p67phox from cytosol to cell membrane and the subse?quent interaction with Nox2.CONCLUSIONOur results demonstrated that TMEM16A，as CaCC，is a positive regulator of ROS generation via upregulating the activa?tion of Nox2 NADPH oxidase in the vascular endotheli?um，and therefore facilitates endothelial dysfunction and hypertension.Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated cardiovascular diseases.

Key words：TMEM16A；endothelial dysfunction；ROS；NADPH oxidase；Nox2；angiotensinⅡ

*Co-first author.

T4-26

Effects of FOXO1 mediated regulation of mitochondri?al function in diabetic wound healing

SHI Yun-di，WANG Di，LI Xue-jun，TIE Lu

（State Key Laboratory of Natural&Biomimetic Drugs，Department of Pharmacology，School of Basic Medical Sciences，and Institute of System Biomedicine，Peking University，Beijing 100191，China）

Abstract：OBJECTIVERefractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens.Recent studies have shown that the imbalance of mitochondrial dynamics was associated with the increased reactive oxy?gen species（ROS） production in endothelial cells，which is a significant contributor to the microvascular complications of diabetes.The present study was designed to determine the involvement of transcription factor FOXO1 in diabetic wound healing and investigate underlying mechanisms.METHODS&RESULTSImpaired mito?chondrial networks and increased phosphorylation of dynaminrelated protein-1（Drp1）at ser616，a protein required for mitochondrial fission，were observed in human umbilical vein endothelial cells（HUVECs）24 h after exposure to high concentrations of glucose.Inhibition of FOXO1bysiRNAorbyFOXO1selectiveinhibitor AS1842856 abrogated high glucos-induced alterations in mitochondrial networks and phosphorylation of Drp1. Treatment with AS1842856 or siRNA of FOXO1 could significantly increase the mitochondrial membrane poten?tial and suppress the overproduction of ROS induced by high glucose.Addition of AS1842856 inhibited glucoseinduced apoptosis，ameliorated capillary tube formation in HUVECs.In vivo，AS1842856 dose-dependently rescued the delay of wound closure in diabetic mice，and 5 mg·kg-1of AS1842856 treatment significantly increased the mean perfusion rate.CONCLUSIONThese findings suggested that FOXO1 is critical to preserve mitochondrial quantity and function in endothelial cells，inhibition of FOXO1 rescued the delayed wound healing and improved wound angiogenesis in diabetic mice.

Key words：diabetes；FOXO1；endothelial；mitochondri?al；fission

T4-27

Translational regulation of DNA repair systems by eIF3a in cancer chemotherapeutic response

CHEN Juan1，2，CUI Jia-jia1，2，ZHANG Jian-ting3，4，ZHOU Hong-hao1，2，LIU Zhao-qian1，2，YIN Ji-ye1，2

（1.Department of Clinical Pharmacology，Xiangya Hospital，Central South University，Changsha410008，China；2.Institute of Clinical Pharmacology，Central South University；Hunan Key Laboratory of Pharmacogenetics，Changsha 410078，China；3.Department of Pharmacology and Toxicology,4.IU Simon Cancer Center，Indiana Uni?versity School of Medicine，980 W.Walnut Street，Indianapolis，Indiana 46202，USA）

Abstract：OBJECTIVETo investigate the role of eIF3a in the regulation of DNA repair pathways in cancer che?motherapeutic response.METHODSImmunohistochem?istry was used to determine the expression of eIF3a in lung and breast cancer tissues followed by association analysis of eIF3a expression with patient′s response to chemotherapy.Ectopic overexpression and RNA interfer?ence knockdown of eIF3a were carried out in NIH3T3 and H1299 cell lines，respectively，to determine the ef?fect of altered eIF3a expression on cellular response to chemotherapeutic drugs by using MTT assay.The DNA repair capacity of these cells was evaluated by using host-cell reactivation，NHEJ and HR assay.Real-time reverse transcriptase PCR and Western Blot analyses were carried out to determine the effect of eIF3a on the DNA repair genes by using cells with altered eIF3a ex?pression.RESULTSeIF3a expression associates with response of lung and breast cancer patients to platinum and anthracycline.eIF3a knockdown or overexpression，respectively，increased and decreased the cellular resis?tance to cisplatin and anthracycline anticancer drugs，DNA repair activity，and expression of NER and NHEJ DNA repair proteins.CONCLUSIONeIF3a plays an important role in regulating the expression of NER and NHEJ DNA repair proteins which，in turn，contributes to cellular response to DNA-damaging anticancer drugs and patients′response to platinum and anthracycline chemotherapy.

Key words：eIF3a；DNA repair；translation regulation；platinum；anthracycline；cancer chemotherapy

基金項目：國家科技部“重大新藥創(chuàng)制”科技重大專項（2014ZX09507003-002，2013ZX09102106）；中國食品藥品檢定研究院中青年發(fā)展研究基金課題（2013NA3） 國家自然科學(xué)基金（81470163） 國家自然科學(xué)基金青年項目（81202584，31400979）；山東省自主創(chuàng)新重大專項（2014ZZCX0215） “十二五”國家科技支撐計劃項目（2012BAI31B04） 山西省科技基礎(chǔ)條件平臺建設(shè)項目（2014091022）；山西省科技攻關(guān)項目（20140313008-14）；山西省應(yīng)用基礎(chǔ)研究項目（201601D021164）；山西省高校科技創(chuàng)新項目（2016120） 國家自然科學(xué)基金（30973585，91029747，81473276）；北京市重點學(xué)科基礎(chǔ)醫(yī)學(xué)學(xué)科建設(shè)項目（BMU20110254） 山西大學(xué)引進(jìn)人才事業(yè)發(fā)展經(jīng)費（226545008，226545003）；2015年度山西省高等學(xué)?？萍紕?chuàng)新項目（2015118） 國家自然科學(xué)基金（81560683）；貴州省中醫(yī)藥管理局中醫(yī)藥、民族醫(yī)藥科學(xué)技術(shù)研究課題（QZYY-2015-079）；貴州省優(yōu)秀青年科技人才培養(yǎng)對象專項資金（黔科合人字（2015）11號）；現(xiàn)代藥物研究開發(fā)協(xié)同創(chuàng)新中心（黔教合協(xié)同創(chuàng)新字[2013]04） 中南大學(xué)臨床新技術(shù)立項項目 中國中醫(yī)科學(xué)院自助選題項目（zz070821）；中國中醫(yī)科學(xué)院創(chuàng)新團(tuán)隊建設(shè)項目（YS1303）；國家重點基礎(chǔ)研究發(fā)展計劃（973計劃）（2015CB554405）

通訊作者：杜冠華，E-mail：dugh@imm.ac.cn，Tel：（010） 63165184 朱 焰，E-mail：zhuyan0311@163.com，Tel：（021）64438416 王曉良，Tel：（010）63165173，E-mail：wangxl@ imm.ac.cn；于海波，E-mail：haiboyu@imm.ac.cn 劉可春，E-mail：hliukch@sdas.org，Tel：（0531）82605352 朱 焰，E-mail：zhuyan0311@163.com，Tel：（021）64438416 周玉枝，E-mail：zhouyuzhi@sxu.edu.cn，Tel：（0351）7019178 章國良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802725 章國良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802433 章國良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802433 高 麗，E-mail：gaoli87@sxu.edu.cn，Tel：（0351）7018379；秦雪梅，Tel：（0351）7011501，E-mail：qinxm@sxu.edu.cn 王永林，E-mail：gywyl@gmc.edu.cn，Tel：（0851）86908899 肖軼雯，E-mail：xiaoyw2005@163.com 劉建勛，E-mail：liujx0324@sina.com，Tel：（010）62835601

Foundation item：The project supported by National Natural Science Foundation of China（81301908）；and the Science andTechnologyCommissionofShanghaiMunicipality（15140904700，13ZR1412600 and 14DZ2270100） WANG Xin，E-mail：xwang@bio. ecnu.edu.cn CHEN Wei-dong， E-mail：anzhongdong@126.com，Tel：15156091053 The project supported by 2016-2018 Anhui University Research Platform Innovation Team s：PENG Dai-yin，CHEN Wei-dong，E-mail：Pengdy@ahtcm.edu.cn，anzhongdong@126.com The project supported by National NaturalScienceFoundationofChina（81473579，81273654，81102879）；and the National Science and Technology Major Projects for“Major New Drugs Innova?tion and Development”（2013ZX09103002-022） YOU Yu-yang，E-mail：youyuyang@ bit.edu.cn Theproject supported by National Natural Science Foundation of China（81573463） YIN Ji-ye，E-mail：yinjiye2005@ sina.com The project supported by Innovation Program of Shanghai Municipal Education Commission（15ZZ063）；and by Research Projectof Putuo Hospital，Shanghai University of Traditional Chinese Medicine（2014YJ002） TAO Jie，E-mail：jietao_putuo@ foxmail.com；YIN Pei-hao，E-mail：yinpeihao1975@hotmail.com GAO zhao-bing，Tel：（021）20239067，E-mail：zbgao@simm.ac.cn The project supported by National Natural Science Foundation of China（81573613，81373896）；the Major Program for the Fundamental Research of Shanghai Committee of Science and Technology（14JC1491300）；and Open Fund of State Key Laboratory of Natural Medicines（SKLNMKF201612） LIU Ji-yong，Tel：（021）31162316，E-mail：liujiyong999@126.com Theproject supported by National Natural Science Foundation of China（81373486）；Science and Technology Development Projects of Guangdong Prov?ince，China（2016B090918114，2013B021800157）；and Science and Technology Development Projects of Guangzhou，Guangdong，China（201510010236，201604020096） ZHONG Shi-long，E-mail：zhongsl@ hotmail.com The project supported by National Natural Science Foundation of China（81301908）；and the Science and Technology CommissionofShanghaiMunicipality（15140904700，13ZR1412600，14DZ2270100） WANG Xin，E-mail：xwang@bio. ecnu.edu.cn The project supported by National Nat?ural Science Foundation of China（81173127，81273581） ZHANG Li-rong，Tel：（0371）67781855；E-mail：zhanglirongzzu@126.com The project supported by National Natural Science Foundation of China（81230082，81302771，81525025，81573422，81500226）；Natural Science Foundation of Guangdong Province（2014A030313087）；and by Science and Technology program of Guangzhou City（201607010255） s：MA Ming-ming，Tel：（020）87331155，E-mail：mamm3@mail.sysu.edu.cn；GUAN Yong-yuan，Tel：（020）87331857，E-mail：guanyy@mail.sysu.edu.cn The project supported by National Natural Science Foundation of China（81373405，30901803）；and Beijing Higher Education Young Elite Teacher Project（YETP0053） TIE Lu，E-mail：tielu@bjmu.edu.cn The project supported by National High-tech R&D Program of China 863 Program Grant（2009AA022704）；National Natural Science Foundation of China（81573463，81173129，81202595 and NIHGrant CA 94961） YIN Ji-ye，E-mail：yinjiye@csu.edu.cn

T4-14

Inhibitory effect of plumbagin，a potential anticancer natural compound，on cytochrome P450 2J2 in humansLU Jian1，LIU Dao-zhi1，ZHOU Xiao-jing1，CHEN Ang1，LIU Ming-yao1，2，WANG Xin1

（1.Shanghai Key Laboratory of Regulatory Biology，Institute of Biomedical Sciences and School of Life Sciences，East China Normal University，Shanghai，China；2.Center for Cancer and Stem Cell Biology，Institute of Biosciences and Technology，Texas A&M University Health Science Center，Houston，Texas，USA）

Abstract：OBJECTIVECytochrome P450（CYP）2J2 is highly expressed in many kinds of human tumors and promotes tumor cell growth via regulating the metabolism of arachidonic acids.The purposes of this study were to identify the new inhibitor of CYP2J2 from natural com?pounds and evaluate its potential to inhibit hepatoma car?cinoma cells.METHODSTotal fifty natural products were screened for the inhibitory potency against the activity of CYP2J2-mediated astemizole O-demethylation via LCMS/MS analysis.Enzyme kinetic and molecular docking studies were also carried out.RESULTSOur data found that plumbagin potently inhibited CYP2J2 with IC50value at 3.42，3.37 and 1.17 μmol·L-1in rat liver microsomes，humanlivermicrosomes（HLMs） andrecombinant CYP2J2（rCYP2J2），respectively.Further enzyme kinetic studies showed that plumbagin was a mixed-type inhibitor of CYP2J2 in HLMs and rCYP2J2 with Kivalues of 1.88 and 0.92 μmol·L-1，respectively.Docking data presented that plumbagin interacted with CYP2J2 mainly through GLU222 and ALA223，which were crucial residues for substrates binding.At the same time，plumbagin showed cytotoxicity effects on hepatic carcinoma cell lines，such as HepG2 and SMMC-7721，with IC50values at 11.55±1.06 and（13.15±1.11）μmol·L-1，respectively.CONCLUSIONThese results indicated that plumbagin was a potent CYP2J2 inhibitor and potential anticancer agent.Further studies are needed to cover the mechanism of its antitumor activity.

Key words：plumbagin；astemizole；CYP2J2；antitumor；LC-MS/MS；cytotoxicity