T4药物代谢与分子药理学

临床常用普伐他汀钠片大鼠体内药代动力学比较研究

赵 赢1，张 娜2，3，庞晓丛1，生立嵩1，宋俊科1，何 兰3，吕 扬2，杜冠华1

（1.中国医学科学院北京协和医学院药物研究所，北京市药物靶点研究与新药筛选重点实验室，北京 100050；2.中国医学科学院北京协和医学院药物研究所，北京市晶型药物研究重点实验室，北京 100050；3.中国食品药品检定研究院，北京 100050）

T4药物代谢与分子药理学

T4-1

临床常用普伐他汀钠片大鼠体内药代动力学比较研究

赵 赢1，张 娜2，3，庞晓丛1，生立嵩1，宋俊科1，何 兰3，吕 扬2，杜冠华1

（1.中国医学科学院北京协和医学院药物研究所，北京市药物靶点研究与新药筛选重点实验室，北京 100050；2.中国医学科学院北京协和医学院药物研究所，北京市晶型药物研究重点实验室，北京 100050；3.中国食品药品检定研究院，北京 100050）

摘要：目的研究大鼠灌胃不同企业普伐他汀钠片后体内药代动力学的一致性。方法固体灌胃给予SD雄性大鼠普伐他汀钠15 mg·kg-1，采用液质联用法测定不同时间的血药浓度，绘制药时曲线，并用DAS 3.0.7数据处理系统进行主要药代动力学参数的计算和比较。结果三个原料药生产企业的测定结果显示，3号晶F型普伐他汀钠原料药较1、2号晶A型有着更好的药物吸收和相对生物利用度，为优势药物晶型；随机抽取的临床常用7种普伐他汀钠片在大鼠体内药代动力学参数结果呈现显著性差异，Tmax，Cmax，AUC，t1/2的最大差异分别可以达到5.3，3.4，2.4和1.4倍之多。结论不同企业的普伐他汀钠片在大鼠体内的药代动力学过程存在显著性差异，这可能与普伐他汀钠多晶型状态以及药物生产工艺、辅料成分及含量等制剂过程有关。

关键词：普伐他汀；晶型；药代动力学；生物利用度；LC-MS

T4-2

孕三烯酮胶囊多次给药在巴马小型猪体内的药代动力学研究

郭湘洁，李 钊，谢淑武，周洁芸，李国停，钟瑞华，朱 焰（复旦大学生殖与发育研究院上海市计划生育科学研究所生殖药理组，国家人口计生委计划生育药具重点实验室，上海200032）

摘要：目的建立快速灵敏的HPLC-MS/MS法测定巴马小型猪血浆中孕三烯酮的浓度，并研究孕三烯酮胶囊在巴马小型猪体内的药代动力学特征。方法以巴马小型猪为实验动物，连续给药12周，每周给药2次，于给药后不同时间点采集血样。通过测定多次给药后小型猪血浆中孕三烯酮的浓度，计算其药代动力学参数。测定血浆样品以孕二烯酮为内标，经环己烷液液萃取后进行LC-MS/MS分析。质谱检测采用ESI正离子模式，选择检测离子反应对为m/z309.2→241.1（孕三烯酮），m/z 311.2→109.1（孕二烯酮）。结果孕三烯酮在0.1~10 ng·mL－1浓度范围内线性良好（r=0.9999），定量下限为0.1 ng·mL-1。提取回收率均高于83.14%，日间、日内RSD均小于11.27%，专属性良好，在本实验涉及的条件下样品稳定。8只小型猪首次给药后Cmax为（2.85±0.96）μg·L-1，Tmax为（2.06±0.62）h，t1/2为（8.43±4.13）h，AUC（0-85）为（23.81±8.62）μg·L·h-1，AUC（0-∞）为（24.36±8.73）μg·L·h-1，MRT（0-85）为（8.40±2.44）h，MRT（0-∞）为（9.81±3.88）h；末次给药后Cmax为（2.70±0.55）μg·L-1，Tmax为（1.69±0.65）h，t1/2为（8.51±3.72）h，AUC（0-85）为（17.71±4.89）μg·L·h-1，AUC（0-∞）为（18.51±4.48）μg·L·h-1，MRT（0-85）为（8.54±3.50）h，MRT（0-∞）为（9.98±4.41）h。多次给予孕三烯酮胶囊后2 h血药浓度相差不大，且用药后无不良反应，安全性较高。结论该方法准确、快速、灵敏、专属性强，适用于动物实验及临床上测定血浆中孕三烯酮的浓度及其药代动力学研究。

关键词：孕三烯酮；液质联用；巴马小型猪；血药浓度

T4-3

KCNQ钾通道在糖尿病神经病理性疼痛中的作用及机制研究

周 宇，于海波，王晓良

（中国医学科学院药物研究所，北京 100050）

摘要：目的在糖尿病神经病理性疼痛（diabetic neuropathic pain，DNP）发生过程中，KCNQ钾通道所起的作用及其机制。方法首先是在动物整体水平上，建立链脲佐菌素（strep⁃tozocin，STZ）诱导的糖尿病模型以及甲基乙二醛（methylgly⁃oxal，MG）诱导的疼痛模型，通过分离背根神经元，检测两种模型离子通道表达变化是否一致，同时检测糖尿病模型中甲基乙二醛的水平。其次是在细胞水平上，观察MG以及MG+ MG清除剂对离子通道活性和电流的影响，探讨KCNQ3通道开放剂对DNP的缓解作用以及MG是如何调控离子通道改变的。结果在STZ诱导的模型中，血糖明显升高，机械痛和热痛明显降低，其KCNQ3通道的表达也有明显的降低；同时，在STZ诱导的模型中，MG的浓度是明显升高的，而MG模型中KCNQ3的表达量也是明显下降的。结论STZ以及MG诱导的模型，均可诱发KCNQ钾通道表达的降低；MG调控KCNQ活性的变化可能通过与离子通道氨基酸反应，调控通道的活性；KCNQ可作为DNP治疗的药理学靶点。

关键词：神经病理性疼痛；甲基乙二醛；钾离子通道

T4-4

采用双向验证法和斑马鱼模型探索血管生成相关的内源性代谢物及代谢网络的变化

韩利文1，李智平2，何秋霞1，张轩铭1，张 云1，李晓彬1，田青平2，刘可春1，刘昌孝3

（1.山东省科学院生物研究所，山东济南 250014；2.山西医科大学药学院，山西太原 030001；3.天津药物研究院，天津300301）

摘要：目的血管生成与很多疾病有关。目前对于信号传递的下游的代谢了解甚少，探寻与血管相关的新的生物学机制对于疾病的防治意义重大。斑马鱼血管生成评价模型是目前公认的评价血管生成作用的优良模型，能够把血管生成独立出来研究，与移植瘤等模型不同，无须考虑肿瘤等其他因素的干扰。同时借鉴分子生物学科中靶点研究的思路和方法，建立“双向验证”策略，进行血管生成相关的代谢型生物标志物进行研究。方法采用转基因斑马鱼Tg（vegfr2：GFP）幼鱼作为血管生成研究模型，在斑马鱼发育至24h时，采用靶点抑制剂PTK787和中药丹红注射液进行双向处理，观察丹红注射液（DHI）对PTK787造成损伤的逆转作用，用Image J软件对节间血管（ISVs）进行定量分析，作为血管生成评价指标；同步处理后的斑马鱼每20条为一个样本，进行组织破碎、提取、去蛋白后，进行衍生化处理，然后用GC-MS联用仪进行分析，每组6个重复；采用Simca-P软件进行代谢组模式判别分析。结果在本实验条件下，30~60 μL·mL-1的丹红注射液对PTK787造成的斑马鱼节间血管生成的损伤具有明显逆转作用。代谢轮廓分析结果显示，正常组、PTK787处理组、丹红注射液处理在散点图中呈现了明显的损伤和逆转趋势。进一步的分析发现，PTK787处理斑马鱼后，酪氨酸、次黄嘌呤、苯丙氨酸、天冬氨酸、谷氨酰胺、磷酸水平明显升高，肌醇明显降低，而丹红注射液处理后，发现这些代谢物呈现了显著逆转。代谢网络分析显示，这些代谢物可能与缬氨酸-亮氨酸-异亮氨酸生物合成途径、苯丙氨酸-酪氨酸-色氨酸合成途径、苯丙氨酸代谢、甘氨酸-天冬氨酸-谷氨酰胺代谢、磷酸肌醇代谢途径有关。结论本研究利用斑马鱼模型和代谢组学技术对血管生成相关的下游代谢信号进行了探讨，并建立了双向处理法，快速筛选发现了一些相关的代谢物，与常规的药物处理进行模式判别，准确率更高，对利用斑马鱼模型研究疾病相关的代谢途径提供了新的研究方法。

关键词：血管生成；代谢组；GC-MS；生物标志物；斑马鱼

T4-5

液质联用法同时测定新西兰兔血浆中左炔诺孕酮和炔雌醇及其药动学

李 钊，郭湘洁，谢淑武，周洁芸，朱 焰

（复旦大学生殖与发育研究院上海市计划生育科学研究所生殖药理组，国家人口计生委计划生育药具重点实验室，上海200032）

摘要：目的建立液质联用（HPLC-MS/MS）测定新西兰兔血浆中左炔诺孕酮（LNG）和炔雌醇（EE）含量的方法，并研究LNG/EE避孕贴剂多次给药后新西兰兔体内的药动学特征。方法6只雌性新西兰兔给予LNG/EE避孕贴（5 cm× 4 cm，标示量为LNG 5.35 mg，EE 0.11 mg），1次/3 d，连续给药10次，分别于给药后不同时间点进行耳缘静脉采血，血浆样品经丹酰氯衍生化后，采用液质联用方法（HPLC-MS/MS）测定血浆中LNG（内标孕三烯酮）和EE（内标氘代炔雌醇）的血药浓度，并采用DAS3.0软件计算主要药代动力学参数。结果LNG的线性范围为0.1~20 μg·L-1，最低定量限为0.1 μg·L-1，提取回收率均大于92%，日内、日间精密度（RSD）均小于15%。首次给药后LNG的药代动力学参数：Cmax为（8.10±2.38）μg·L-1，Tmax为（2.38±1.45）h，AUC（0-t）为（142.35±36.99）μg·L-1·h-1；末次给药后LNG的药代动力学参数：Cmax为（7.05±1.07）μg·L-1，Tmax为（2.71±1.83）h、AUC（0-t）为（141.95±22.31）μg·L-1·h-1。EE的线性范围为0.02~5 μg·L-1，最低定量限为0.02 μg·L-1，提取回收率均大于85%，日内、日间精密度（RSD）均小于15%。首次给药后EE的药代动力学参数：Cmax为（0.18±0.04）μg·L-1、Tmax为（2.50±1.30）h、AUC（0-t）为（2.65±0.56）μg·L-1·h-1；末次给药后EE的药代动力学参数：Cmax为（0.17±0.07）μg·L-1、Tmax为（2.17±0.26）h、AUC（0-t）为（2.02±0.82）μg·L-1·h-1。结论HPLC-MS/MS法灵敏，准确，可用于LNG和EE同时测定研究，新西兰兔多次给予避孕贴后体内血药浓度无蓄积。

关键词：左炔诺孕酮；炔雌醇；避孕贴；衍生化；药动学

T4-6

基于1H-NMR代谢组学技术研究D-半乳糖致衰老大鼠尿液的动态变化

赵凡凡1，2，周玉枝1，高 丽1，秦雪梅1，杜冠华1，3

（山西大学 1.中医药现代研究中心，2.化学化工学院，山西太原 030006，3.中国医学科学院，北京 100050）

摘要：目的通过代谢组学技术研究D-半乳糖（D-gal）刺激引起大鼠尿液代谢轮廓的动态变化。方法实验分为两组，即正常对照组和模型组；模型组连续皮下注射D-gal溶液（100 mg·kg-1·d-1）10周，对照组皮下注射等量生理盐水，并分别在0,2,4,6,8和10周收集每只实验鼠的尿液。第10周开始采用水迷宫和避暗实验检测空白组与模型组大鼠的学习记忆能力。在行为学实验结束后，将各组实验鼠处死并收集海马。采用代谢组学技术对实验大鼠五次尿液进行NMR数据采集并结合多元统计分析，探讨D-半乳糖致衰老机制。结果结果表明，与空白组相比，D-gal能显著增加大鼠在水迷宫中的潜伏期（P<0.05），减少其在避暗实验中的潜伏期和错误次数（P<0.05）等行为学指标。对2，4，6，8和10周的不同实验点的模型组和空白组大鼠的尿液进行PCA分析，发现造模2~4周时，模型组与空白组的代谢轮廓没有区分，造模6~8周，模型组空白组大鼠逐渐分开，造模10周时，模型组与空白组完全分开。采用OPLS-DA寻找二者之间的差异物，在第10周发现乳酸、丙氨酸、α-酮戊二酸、胆碱等14个峰面积具有显著性差异的潜在生物标志物。结论结果表明，D-gal能够损伤大鼠的学习记忆，衰老模型的建立可能是通过能量代谢、糖代谢、肠菌代谢等代谢途径发挥作用的。

关键词：D-半乳糖；学习记忆；认知障碍；代谢组学

T4-7

抗癫痫新药氯桂丁胺与药物代谢酶CYP450的相互作用

王 昕，程海旭，丁 宇，李 强，王淑梅，李 璞，逯颖媛，楼雅卿，章国良

（北京大学基础医学院药理学系，北京 100191）

摘要：目的研究新型桂皮酰胺类抗癫痫化合物氯桂丁胺体内代谢的CYP450同工酶亚型及其对6种主要CYP450同工酶活性的影响。方法超高速离心法制备大鼠肝微粒体，高效液相色谱法定量分析氯桂丁胺原型药、代谢物、6种CYP450同工酶探针药（CYP1A2/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）代谢物，在大鼠肝微粒体、6种重组人源CYP450同工酶孵育模型中浓度的变化。结果6种重组人源CYP450同工酶分别与氯桂丁胺共孵育，以及6种特异性CYP450同工酶抑制剂（α-奈黄酮/ CYP1A2、磺胺苯吡唑/CYP2C9、奥美拉唑/CYP2C19、奎尼丁/CYP2D6、二乙基二硫氨甲酸/CYP2E1、酮康唑/CYP3A）分别与氯桂丁胺及大鼠肝微粒体共孵育结果显示，参与氯桂丁胺生物转化的主要代谢酶亚型包括代谢物M1（CYP2D6），M2（CYP1A2），M3（CYP2C19和CYP3A4）。氯桂丁胺在0.1~16 μg·mL-1浓度范围内对CYP1A2，CYP2E1和CYP3A酶活性无显著性影响。在2~16 μg·mL-1浓度范围内显著性抑制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%）。结论本研究结果提示氯桂丁胺可经多酶代谢，其自身的代谢清除可能不易受单一CYP450亚型遗传因素所干扰，但由于其抑制CYP2C9，CYP2C19及 CYP3A的活性，如与相应底物类药物合用时应注意药物-药物间相互作用。

关键词：氯桂丁胺；CYP450；肝微粒体；药物-药物相互作用

T4-8

性激素对二乙基亚硝胺（DEN）所致肝癌组织中CYP450酶表达的影响

周滋晶，缪晓洁，尹 航，王 昕，章国良

（北京大学医学部基础医学院药理系，北京 100191）

摘要：目的研究性激素（雌二醇与孕酮）对二乙基亚硝胺（DEN）诱导的大鼠肝癌组织中多种细胞色素P450（CYP450）同工酶表达的影响及可能的作用机制。方法每周1次ip给予DEN（50 mg·kg-1，×12周）制备大鼠肝癌模型，H-E染色及光学显微镜法观察石蜡包埋肝组织切片中病理形态学改变，免疫组织化学法对肝组织中甲胎蛋白（AFP）、CYP1A2，CYP2B1，YP2E1及CYP3A1共5种目的蛋白定性定位观察。结果与生理盐水对照组的H-E染色切片相比，DEN刺激组的肝组织中可见典型的假小叶结构形成，肝癌细胞呈多核巨细胞和核分裂相。DEN+雌二醇（30 mg·kg-1，实验末周1 d×7 d，sc）组、DEN+孕酮（150 mg·kg-1，实验末周1 d×7 d，sc）组均可见更多假小叶及肝细胞内脂肪空泡形成。免疫组化结果显示，DEN组中肝癌发生标志物AFP呈棕色强阳性染色，而CYP1A2，CYP2B1，CYP2E1及CYP3A1阳性棕色染色程度均降低，5种蛋白均定位于细胞质；DEN+雌二醇组中4种CYP450同工酶染色程度较之DEN组略有增强；而DEN+孕酮组可见CYP3A1强阳性棕色染色。结论雌二醇可部分逆转DEN所致肝癌组织中4种CYP450同工酶表达减少，孕酮则显著增加DEN所抑制的CYP3A1表达，其确切的机制有待于进一步探讨。

关键词：雌激素；孕激素；CYP450；DEN肝癌发生模型

T4-9

新型利尿药PU-48在生物样本中LC-MS/MS检测方法的建立与确证

张志远，王 昕，逯颖媛，李 璞，程海旭，杨宝学，章国良

（北京大学医学部基础医学院药理系，北京 100191）

摘要：目的建立和确证以尿素通道蛋白作为药物靶点的噻吩并喹啉类新型利尿药3-甲氧基-6-氨基-噻吩并［2，3-b］喹啉-7-羧酸甲酯（PU-48）在生物样本中的液相色谱串联质谱（LC-MS/MS）检测方法。方法采用Shimadzu LC-20A高效液相色谱串联AB Sciex API 4000 QTRAP质谱仪，Inertsil ODS-4 C18色谱柱（100×2.1 mm，3 μm）。流动相为0.05%的乙腈-水溶液，流速0.3 mL·min-1，梯度洗脱。使用醋酸甲地孕酮作为内标，PU-48和内标分别选择m/z289.1→257.1和m/z385.3→267.1作为定量的离子对。血浆样本使用乙酸乙酯进行液-液萃取，37℃下氮气吹干重溶，取5 μL进样。结果在上述条件下，PU-48的保留时间为6.2 min，内标的保留时间为7.2 min，分离良好且不受内源性物质的干扰。血浆外加药物标准曲线范围0.1~1000 ng·mL-1，相关系数大于0.99，最低检测限（LOD）为0.05 ng·mL-1，定量下限（LLOQ）为0.1 ng·mL-1。大鼠血浆外加低、中、高三个浓度（0.2，10.0,500.0 ng·mL-1）质控点PU-48的日内及日间准确度（RE）分别为<12.95%及<-9.55%，日内及日间精密度（RSD）分别为<4.14%及<8.29%，绝对回收率在85.25~ 98.10%。4℃下放置96 h的准确度精密度分别为<8.12%和<6.07%。结论本研究所建立的LC-MS/MS检测方法的的特异性、灵敏度、精密度、准确度符合《药物非临床药代动力学研究技术指导原则》的要求。

关键词：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）；利尿药；大鼠血浆

T4-10

基于1H-NMR代谢组学的黄芩素干预D-半乳糖致衰老大鼠作用研究

王珂欣1，2，高 丽1，段丹丹1，2，周玉枝1，秦雪梅1

（山西大学1.中医药现代研究中心，山西太原 030006；2.化学化工学院，山西太原 030006）

摘要：目的采用1H-NMR代谢组学方法，探讨黄芩素对D-半乳糖致衰老模型大鼠的干预作用。方法采用皮下注射D-半乳糖（150 mg·kg-1）致大鼠衰老模型，考察黄芩素（50，100和200 mg·kg-1）对衰老大鼠自主活动能力的影响。造模10周后采集大鼠血清进行1H-NMR检测，结合多元统计分析探讨黄芩素的抗衰老作用。结果与空白组相比，D-半乳糖致衰老模型组大鼠穿越格数和直立次数均显著减少（P<0.01），给予黄芩素后大鼠穿越格数和直立次数均逐渐增加，表明黄芩素能显著提高衰老大鼠自主活动能力。与空白组相比，模型组大鼠血清中丙氨酸、赖氨酸、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，苏氨酸，肌醇，α-葡萄糖，组氨酸水平降低，给予黄芩素后能不同程度回调这14种潜在的生物标志物。结论黄芩素可能通过调控氨基酸代谢、能量代谢和糖代谢等途径延缓衰老。

关键词：黄芩素；抗衰老；D-半乳糖；代谢组学

T4-11

杜仲降血压活性成分在正常大鼠和自发性高血压大鼠体内的药代动力学差异研究

巩仔鹏1，3，4，吴林霖1，3，陆 苑1，3，侯靖宇1，3，李月婷1，3，候 佳1，3，黄 勇1，3，李勇军1，2，王永林1，3

（贵州医科大学1.贵州省药物制剂重点实验室，2.民族药与中药开发应用教育部工程研究中，3.药学院，贵州贵阳550004；4.国家苗药工程技术研究中心，贵州贵阳 550004）

摘要：目的鉴于中药主要是用于病理状态下的机体内，因此研究病理状态下的中药的药代动力学较正常机体更有意义，且与临床更相关。本研究旨在比较杜仲降压活性成分（松脂醇二葡萄糖苷和京尼平苷酸）在正常大鼠和自发性高血压大鼠（SHR）大鼠体内的药代动力学行为特征。方法分别给正常大鼠及SHR模型行颈静脉插管手术，术后12 h，灌胃给予杜仲提取物（4.8 g·kg-1，分别相当于松脂醇二葡萄糖苷96 mg·kg-1和巴马汀168.96 mg·kg-1），并于给药前以及给药后5，15，30 min，1，1.5，2，3，4，6，8，10，12，24和36 h通过插管采血0.2mL于肝素化的EP管中，离心，取血浆100μL。然后采用UPLC-MS/MS技术测定血浆中的活性成分的含量。绘制药时曲线，并利用WinNonlin软件计算药代参数（T1/2，Tmax，Cmax，AUC0–t，Vd/F和Cl/F），比较各代表成分在正常大鼠和SHR模型体内的药代动力学差异。结果灌胃杜仲提取物后，与正常大鼠相比，松脂醇二葡萄糖苷和京尼平苷酸在SHR模型体内的T1/2，Cmax和AUC0–t显著增加，Vd/F和Cl/F显著降低。结论自发性高血压的病理状态能够显著改变杜仲降压活性成分（松脂醇二葡萄糖苷和京尼平苷酸）的药代动力学特征，提示临床上在用杜仲治疗高血压时，需要对其剂量进行调整。

关键词：杜仲；松脂醇二葡萄糖苷；京尼平苷酸；自发性高血压；药代动力学

T4-12

伏立康唑血药浓度监测结果评析

颜 苗1，2，王柠柠1，3，李紫薇1，3，蒋梦飞1，王 峰1，2，张毕奎1，2，徐 萍1，2，肖轶雯1，2

（中南大学1.湘雅二医院药学部，2.临床药学研究所，3.药学院，湖南长沙 410208）

摘要：目的通过对不同临床专科伏立康唑进行治疗药物监测（TDM）并比较调整剂量前后的血药谷浓度数据，阐明TDM对伏立康唑合理用药的必要性，并给出相关的临床提示。方法本研究通过回顾性分析伏立康唑TDM的数据，对收治入院的154位患者的435例次伏立康唑血药谷浓度测定结果做一个初期评估。结果肾移植科仅有4.3%的测定结果高于5.5 μg·mL-1，而有26.5%的测定结果低于1.0 μg·mL-1。感染科有52.3%的测定结果高于5.5 μg·mL-1，没有出现低于1.0 μg·mL-1的测定结果。结论TDM对伏立康唑的合理化用药十分必要，肾移植科患者伏立康唑TDM不在建议浓度区间的结果以低于1.0 μg·mL-1居多，而感染科患者则以高于5.5 μg·mL-1居多，临床上应予以重视。临床药师可根据TDM结果更密切的参与伏立康唑临床用药。

关键词：伏立康唑；治疗药物监测；肾移植科；感染科

T4-13

细胞外Ba2+对记录大鼠心肌细胞L型钙通道的影响

孟红旭，姚明江，任钧国，刘建勋

（中国中医科学院西苑医院基础医学研究所，中药药理北京市重点实验室，北京 100091）

摘要：目的观察细胞外Ba2+对记录大鼠心肌细胞L型钙通道的影响。方法采用急性酶解分离法获得大鼠的单个心肌细胞，使用全细胞膜片钳技术记录L型钙通道电流。结果（1）细胞外液中的Ca2+被Ba2+替换后，L型钙通道电流的失活速率明显减慢（时间常数由88 ms增至246 ms，P<0.01）。细胞外液中加入Ba2+（0.2和0.4 mmol·L-1）后，L型钙通道电流的失活速率无明显改变。（2）细胞外液中加入Ba2+（0.2和0.4 mmol·L-1）后，连续记录10和15 min，L-型钙通道电流峰值电流的衰减明显减弱（P<0.01）（3）细胞外液加入Ba2+（0.4 mmol·L-1）使电流电压曲线下移，并改变翻转电位。（4）细胞外液加入Ba2+（0.4 mmol·L-1）后，减弱了丹酚酸A（100 μmol·L-1）对钙电流的抑制强度，并使丹酚酸A量效关系曲线下移。结论细胞外液加入一定浓度的Ba2+能够有效减弱全细胞膜片钳技术记录L型钙通道时出现的“rundown”现象，并减少“rundown”现象对药物量效关系的影响。

关键词：Ba2+；全细胞膜片钳；“rundown”现象；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，FANG 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，FDR=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

基金项目：国家科技部“重大新药创制”科技重大专项（2014ZX09507003-002，2013ZX09102106）；中国食品药品检定研究院中青年发展研究基金课题（2013NA3） 国家自然科学基金（81470163） 国家自然科学基金青年项目（81202584，31400979）；山东省自主创新重大专项（2014ZZCX0215） “十二五”国家科技支撑计划项目（2012BAI31B04） 山西省科技基础条件平台建设项目（2014091022）；山西省科技攻关项目（20140313008-14）；山西省应用基础研究项目（201601D021164）；山西省高校科技创新项目（2016120） 国家自然科学基金（30973585，91029747，81473276）；北京市重点学科基础医学学科建设项目（BMU20110254） 山西大学引进人才事业发展经费（226545008，226545003）；2015年度山西省高等学校科技创新项目（2015118） 国家自然科学基金（81560683）；贵州省中医药管理局中医药、民族医药科学技术研究课题（QZYY-2015-079）；贵州省优秀青年科技人才培养对象专项资金（黔科合人字（2015）11号）；现代药物研究开发协同创新中心（黔教合协同创新字[2013]04） 中南大学临床新技术立项项目 中国中医科学院自助选题项目（zz070821）；中国中医科学院创新团队建设项目（YS1303）；国家重点基础研究发展计划（973计划）（2015CB554405）

通讯作者：杜冠华，E-mail：dugh@imm.ac.cn，Tel：（010） 63165184 朱 焰，E-mail：zhuyan0311@163.com，Tel：（021）64438416 王晓良，Tel：（010）63165173，E-mail：wangxl@ imm.ac.cn；于海波，E-mail：haiboyu@imm.ac.cn 刘可春，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 章国良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802725 章国良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802433 章国良，E-mail：ZhangGL168@bjmu.edu.cn，Tel：（010）82802433 高 丽，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 肖轶雯，E-mail：xiaoyw2005@163.com 刘建勋，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