痰瘀同治方对缺氧复氧诱导心肌细胞AMPK-mTOR自噬信号通路的影响及机制  

Effect of Tanyu Tongzhi Recipe on Hypoxia/reoxygenation-induced Autophagy Via the AMPK-mTOR Pathway in Cardiomyocytes and Its Mechanism

作　　者：唐丹丽[1,2] 石晓雯 周明眉[3] 朱海燕[4] 张华敏[5] TANG Dan-li;SHI Xiao-wen;ZHOU Ming-mei;ZHU Hai-yan;ZHANG Hua-min(Experimental Research Center of China Acade my of Chinese Medical Sciences, Beijing（100700;Beijing Key Laboratory of Basic Research on TCM Prevention and Treatment of Major Diseases, Beijing （100700;Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, Shanghai （201203;Depart- ment of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, Shanghai （201203;Institute of Basic Theory for Chinese Medicine of China Academy of Chinese Medical Sci- ences, Beijing （100700)

机构地区：[1]中国中医科学院医学实验中心,北京100700 [2]中医药防治重大疾病基础研究北京市重点实验室,北京100700 [3]上海中医药大学中医方证与系统生物学研究中心,上海201203 [4]复旦大学药学院微生物生化教研室,上海200433 [5]中国中医科学院中医药信息研究所,北京100700

出　　处：《中国中西医结合杂志》2018年第4期435-440,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.81373792);中国中医科学院自主选题项目(No.ZZ2017008)

摘　　要：目的研究痰瘀同治方对缺氧/复氧(H/R)心肌细胞自噬的影响及AMPK-mTOR信号通路在其中可能发挥的作用。方法体外培养H9C2心肌细胞,随机分为空白对照组、H/R模型组(缺氧10 h后复氧2 h)、痰瘀同治方组、血府逐瘀汤组及栝蒌薤白半夏汤组,各组给予相应方剂肠吸收液18 mg/m L孵育2 h后H/R处理。电镜下观察细胞自噬体结构,检测细胞上清液中LDH释放量和SOD活性,蛋白免疫印迹测定细胞内LC3、Beclin-1、P-AMPK和P-mTOR蛋白表达。结果心肌细胞缺氧复氧后自噬体形成并增多。与空白对照组比较,H/R模型组心肌细胞LDH含量、LC3及Beclin-1蛋白表达量增加,p-AMPK表达增强,SOD活性下降且p-mTOR表达减弱(P<0.05,P<0.01)。与H/R模型组比较,各中药组干预后均能降低LDH、Beclin-1表达,痰瘀同治方组与血府逐瘀汤组可同时提高SOD活性,抑制LC3高表达(P<0.05,P<0.01)。同时痰瘀同治方组与血府逐瘀汤组可下调p-AMPK水平,并上调p-mTOR蛋白表达(P<0.05,P<0.01)。结论缺氧复氧可激活心肌细胞AMPK-mTOR信号通路,从而诱导自噬过度表达加重心肌损伤,痰瘀同治方可通过调控AMPK-mTOR信号通路、抑制自噬而起到心肌保护作用。Objective To observe the effect of Tanyu Tongzhi（ TYTZ） Recipe on hypoxia/reoxygenation（ H/R） induced autophagy in cardiomyocytes and the possible role of AMPK-mTOR in this development. Methods Myocardial cells were culturedin vitro and randomly divided into control group,H/R group（ 10 hours of hypoxia followed by 2 hours of reoxgenation）,TYTZ Recipe group,Xuefu Zhuyu（ XFZY） Decoction group and Gualou Xiebai Banxia（ GLXB） Decoction group,the cardiomyocytes were incubated with 18 mg/m L corresponding intestinal absorption fluid for 2 hours before H/R treatment. Autophagosome in cellular were observed by electron microscopy. Lactate dehydrogenase（ LDH）,superoxide dismutase（ SOD） activity were assayed for the evaluation of myocardial cell injury. The protein levels of LC3,Beclin-1,PAMPK and P-mTOR were analyzed by Western blot. Results Electron microscopy revealed that the formation of autophagosomes in the H/R group. Compared with control group,the content of LDH was significantly increased and the activity of SOD was decreased in the H/R group. Futhermore,the protein expression of P-AMPK,LC3 and Beclin-1 were all significantly increased（P〈0. 05,P〈0. 01）,while the protein expression of P-mTOR was decreased（P〈0. 05,P〈0. 01） in the H/R group. Compared with the H/R group,the treatments with the intestinal absorption fluid could decrease the content of LDH and the level of Beclin-1 in all treatment groups. Moreover,the levels of P-AMPK,LC3 and Beclin-1 were reduced and the protein expression of P-mTOR was up-regulated（P〈0. 05,P〈0. 01） in both TYTZ Recipe group and XFZY Decoction group. Conclusions The AMPK-mTOR signaling pathway activated by hypoxia reoxygenation induced autophagy and aggravated myocardial damage in cardiomyocytes. TYTZ Recipe could protect myocardium from the injury induced by hypoxia reoxygenation through regulating AMPK-mTOR signaling pathway and inhibiting autophagy.

关 键 词：痰瘀同治方 缺氧复氧 自噬 腺苷单磷酸活化蛋白激酶 哺乳动物雷帕霉素靶蛋白 

分 类 号：R285[医药卫生—中药学]