摘要
目的研究黄连碱(coptisine)对正常人肝细胞L02的细胞毒性及作用机制,为临床安全用药提供参考。方法分别以不同浓度黄连碱作用于L02细胞24 h,通过CCK-8法检测黄连碱对L02细胞存活率的影响,流式细胞仪检测细胞凋亡、线粒体膜电位和细胞内活性氧(ROS)含量的改变,激光扫描共聚焦显微镜观察线粒体膜电位和细胞内钙离子水平的变化,Western印迹检测L02细胞内Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)、细胞色素C(cytochrome C)、免疫球蛋白结合蛋白(BIP)、蛋白激酶R样内质网激酶(PERK)、CCAAT增强子结合蛋白(CHOP)、激活转录因子4(ATF4)、真核生物启动因子2α(eIF2α)、caspase-12蛋白表达水平,探讨黄连碱细胞毒性的可能作用机制。结果黄连碱能以浓度依赖方式抑制细胞存活,诱导L02细胞凋亡,可促使细胞内ROS大量蓄积,线粒体膜电位下降,细胞色素C、Bax蛋白表达量上升;同时,还可激活内质网应激PERK-ATF4-CHOP通路,显著影响相关蛋白的表达,其中BIP、CHOP、ATF4蛋白表达量升高,PERK、eIF2α蛋白表达量下降,明显诱导细胞内钙离子含量升高,激活下游因子caspase-12、caspase-3蛋白表达。结论黄连碱可通过线粒体途径和内质网应激(ERS)途径诱导L02肝细胞凋亡,其潜在的肝毒性风险及对中药黄连安全性的影响有待进一步研究。
Objective To investigate the cytotoxicity and the underlying mechanism of coptisine on human normal hepatocytes cells(L02)in order to provide reference for clinical drug safety.Methods The viabilities of L02 cells were detected by CCK-8 assay after being treated with coptisine at different concentrations for 24 h.The apoptosis rate,reactive oxygen species(ROS)level and mitochondrial membrane potential(MMP)were measured by flow cytometry.A laser scanning confocal microscope was used to observe the changes in MMP and Ca2+levels.The protein expressions of Bax,caspase-3,cytochrome C,BIP,PERK,CHOP,ATF4,eIF2α,and caspase-12 were analyzed by Western blotting.Results The proliferation activity of L02 cells was decreased by coptisine in a dose-dependent manner,with the apoptosis promoted.Coptisine could promote the accumulation of ROS in cells and the decline of MMP while increasing the protein expression of cytochrome C and Bax.Coptisine could also activate the endoplasmic reticulum stress(ERS)PERK-ATF4-CHOP pathway and affect the expressions of related proteins significantly.Among them,BIP,CHOP,and ATF4 protein levels were improved,while PERK and eIF2αprotein levels were decreased.The treatment of coptisine caused the increase in intracellular Ca2+content and activated the expressions of caspase-3 and caspase-12.Conclusion Coptisine can cause apoptosis of L02 cells via the mitochondrial pathway and the endoplasmic reticulum stress pathway.Its potential hepatotoxicity risk and the effect on the safe use of the traditional Chinese medicine Coptidis need to be further studied.
作者
连闻雨
杨春启
林毅
谭洪玲
肖成荣
马增春
王宇光
高月
LIAN Wen-yu;YANG Chun-qi;LIN-Yi;TAN Hong-ling;XIAO Cheng-rong;MA Zeng-chun;WANG Yu-guang;GAO Yue(Guangdong Pharmaceutical University,Guangzhou 510006,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Anhui Medical University,Hefei 230032,China)
出处
《军事医学》
CAS
2021年第11期834-842,共9页
Military Medical Sciences
基金
国家重点研发计划(2018YFC1704504)。
