miR-20a通过ATG16L1影响细胞自噬并参与调节雷公藤甲素肝毒性  

miR-20a regulates autophagy by targeting ATG16L1 and involved in triptolide-induced hepatotoxicity

作　　者：张彩霞 王伟艳 李会芳 杜晨晖 刘珊 魏砚明 ZHANG Cai-xia;WANG Wei-yan;LI Hui-fang;DU Chen-hui;LIU Shan;WEI Yan-ming(College of Chinese Medicine and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学中药与食品工程学院,山西晋中030619

出　　处：《中草药》2023年第13期4214-4223,共10页Chinese Traditional and Herbal Drugs

基　　金：山西省科技厅基础研究课题资助项目(202103021224295);山西中医药大学毒效关系研究创新团队(2022TD1016);山西中医药大学科技创新能力培养计划“基础研究专项”课题资助项目(2020PY-JC-17)。

摘　　要：目的观察雷公藤甲素对miR-20a及自噬相关16样蛋白L1(autophagy related 16 like protein1,ATG16L1)表达的影响,探讨mi R-20a对雷公藤甲素所致肝细胞毒性的调控作用。方法利用雷公藤甲素处理人正常肝细胞株HL7702和C57BL/6J小鼠,通过qRT-PCR和Western blotting检测miR-20a、ATG16L1及自噬标记物微管相关蛋白1轻链3II(microtubuleassociated protein 1 light 3II,LC3II)的表达。利用雷公藤甲素和miR-20a模拟物或抑制物共同处理HL7702细胞,通过qRTPCR和Western blotting检测ATG16L1及LC3II的表达;CCK-8法检测细胞存活率;试剂盒检测乳酸脱氢酶(lactate dehydrogenase,LDH)释放量;ELISA检测半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)和Caspase-9的活性。结果雷公藤甲素显著下调人正常肝细胞或小鼠肝组织中miR-20a表达(P<0.05),上调ATG16L1和LC3II的表达(P<0.05)。mi R-20a模拟物可抑制雷公藤甲素引起的ATG16L1、LC3II表达升高(P<0.05),进一步降低细胞存活率(P<0.05),提高LDH释放量(P<0.05),上调Caspase-3和Caspase-9活性(P<0.05),而miR-20a抑制物具有相反作用。结论miR-20a通过ATG16L1影响自噬过程,参与调节雷公藤甲素肝细胞毒性。Objective To validate the effect of triptolide on miR-20a and autophagy related 16 like protein1(ATG16L1)expressions and evaluate the regulatory role of miR-20a in triptolide-induced hepatotoxicity.Methods HL7702 cells and C57BL/6J mice were treated with triptolide,and then miR-20a and ATG16L1 levels were detected by qRT-PCR and Western blotting.After HL7702 cells exposure to triptolide and simultaneous transfection with miR-20a mimics or inhibitor,ATG16L1 and microtubule-associated protein 1 light chain 3(LC3Ⅱ)expressions were detected by qRT-PCR and Western blotting;Cell survival rate was detected by CCK-8 method;The release of lactate dehydrogenase(LDH)was detected by kit;The activities of cystein-asparate protease-3(Caspase-3)and Caspase-9 were detected by ELISA.Results Triptolide significantly down-regulated miR-20a expression in normal liver cells or mouse liver tissues(P<0.05),as well as upregulated ATG16L1 and LC3Ⅱexpressions(P<0.05).miR-20a mimics significantly reversed the elevated ATG16L1 and LC3Ⅱexpressions induced by triptolide(P<0.05),further exacerbated triptolide-elicited decrease in cell viability(P<0.05),increase in lactate dehydrogenase leakage and activation of apoptosis proteases Caspase-3 and Caspase-9(P<0.05),whereas miR-20a inhibitor exhibited opposite effects.Conclusion miR-20a plays a regulatory role on triptolide-mediated hepatotoxicity by targeting ATG16L1 to affect autophagy.

关 键 词：雷公藤甲素 肝细胞毒性 细胞自噬 miR-20a ATG16L1 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]