青藤碱对缺氧诱导的脑神经元损伤和内质网应激性自噬的作用  

Effects of Sinomenine on Hypoxia-induced Neuronal Injury and Endoplasmic Reticulum Stress Autophagy

作　　者：白毅婕 王强 周嘉鑫 何剑波 BAIYijie;WANG Qiang;ZHOU Jiaxin;HE Jianbo(Department of Neurology,the Second Clinical Medical College of Shaanxi University of Chinese Medicine,Xianyang,Shaanxi,712046,China;Innovation Research Center of Acupuncture Combined With Medicine,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi,712046,China;Baoji Chen Cang hospital,Baoji,Shaanxi,721000,China;Department of Neurology Western Electric Group Hospital,Xi＇an,710077,China)

机构地区：[1]陕西中医药大学第二临床医学院,陕西省咸阳市712046 [2]陕西中医药大学针药结合创新研究中心,陕西省咸阳市712046 [3]宝鸡市陈仓医院,陕西省宝鸡市721000 [4]西电集团医院神经内科,西安市710077

出　　处：《医学分子生物学杂志》2018年第5期302-308,共7页Journal of Medical Molecular Biology

基　　金：陕西省卫生和计划生育委员会项目（No.2016E006）

摘　　要：目的探究青藤碱（sinomenine，SN）对缺氧诱导的神经元损伤和内质网应激（endoplasmicre-ticulumstress，ERS）性自噬的作用及作用机制。方法分离培养原代神经元，用不同浓度sN处理细胞，MTF检测细胞存活率；复制神经元缺氧模型，观察细胞形态变化，流式检测细胞凋亡，Western印迹检测ERS、自噬标记蛋白和磷酸肌醇3激酶（phosphofvlinosnal3kinase，P13K）／Akt／雷帕霉素靶蛋白（mamma-liantargetofrapamycin，mTOR）通路蛋白的表达。结果sN浓度高于20μmol／L时，细胞存活率低于80％，因此，选择5、10、20μmol／L3个浓度进行后续实验；SN可显著改善缺氧诱导神经元形态的改变，促进神经元存活，还可显著抑制神经元凋亡；同时，SN能明显抑制ERS相关促凋亡分子C／EBP同源蛋白（C／EBP-homologousprotein，CHOP）和Caspase-12表达，上调促存活分子生长抑制DNA损伤蛋白34（growth arrestand DNA damage protein34．GADD34）和免疫球蛋白重链结合蛋白（immunoglobulin heavychain binding protein，BiP）表达；此外，SN能显著减弱缺氧下调LC3U／LC3I比值和自噬标记蛋白Beclinl表达水平的作用，并抑制P62表达；SN还可明显反转缺氧对p-P13K和p-Akt表达的抑制作用，并降低mTOR表达水平。3．甲基腺嘌呤（3-methyladenine，3-MA）抑制自噬后，sN对自噬、P13K／Ak/mTOR通路标记蛋白的作用明显被反转。结论SN可诱导ERS性自噬抑制，抑制神经元凋亡，其作用机制与激活P13K／Akt／mTOR通路有关。Objective To investigate the anti-apoptotic effect and the underlying mechanism of sinomenine （SN） working on hypoxia-induced neuronal injury and endoplasmie reticulum stress au- tophagy. Methods Primary neurons were cultured and exposed to various levels of SN- MTT assay was performed to determine cell viability. Then, neurons were exposed to hypoxia for 24 h, and the cell phenotype and viable cells were calculated. Apoptosis rate was flow cytometrically detected and expression levels of ERS-related, autopahgy-related and phosphotylinosital 3 kinase （PI3 K ） / Akt/mammalian target of rapamycin （mTOR） pathway-related proteins were measured by Western blotting. Results The cell viability was decreased to less than 80 % by SN at a concentration rangefrom 50 μmol/L to 400 μmol/L. Thus, the concentrations of 5, 10 and 20 μmol/L were selected for further experiment. SN dose-dependently attenuated morphological and survival changes of neurons and inhibited hypoxia-induced apoptosis of neurons. Meanwhile, SN treatment inhibited ERS in a dose-dependent manner, as demonstrated by the decreased C/EBP-homologous protein （CHOP）, Caspase-12 and increased growth arrest and DNA damage protein 34 （GADD34）, immunoglobulin heavy chain binding protein （BiP） . Moreover, post-conditioning with SN dramatically increased Beclinl and LC3 Ⅱ/LC3 I expression with P62 expression decreased in hypoxia-induced neurons. In addition, SN notably ameliorated hypoxia-induced reduction of p-PI3 K, p-Akt expression and in- crease of mTOR. However, 3-MA partially reversed the effects of SN on autophagy and PI3 K/Akt/ mTOR pathway. Conclusion SN inhibits apoptosis and promoting ERS-induced autophagy, and the mechanism may involve PI3 K/Akt/mTOR signaling pathway.

关 键 词：青藤碱 内质网应激 自噬 凋亡 脑缺血 

分 类 号：R349.7[医药卫生—基础医学]