丙型肝炎病毒核心蛋白活化内源性大麻素系统诱导不完全线粒体自噬

Hepatitis C virus core protein induces incomplete mitophagy via activation of endocannabinoids system

目的研究丙型肝炎病毒(HCV)核心蛋白活化内源性大麻素系统诱导不完全线粒体自噬的机制。方法HepG2细胞或表达HCV核心蛋白的HepG2细胞与肝星状细胞(LX-2细胞)共培养。共培养后高效液相色谱-质谱联用技术检测2-AG水平,分光光度法测定检测线粒体呼吸链酶复合体活性,流式细胞仪检测细胞活性氧(ROS)水平,激光共聚焦显微镜检测Ca^(2+)浓度和线粒体膜电位,试剂盒检测线粒体丙二醛(MDA)和超氧化物歧化酶(SOD)含量,蛋白质免疫检测大麻素受体1(CB1R)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和p62蛋白表达。计量资料采用t检验。结果与LX-2细胞和HepG2细胞共培养组相比,LX-2细胞和表达HCV核心蛋白HepG2细胞共培养组,2-AG水平增加,线粒体呼吸链酶复合体活性下降,ROS水平和Ca^(2+)浓度升高,线粒体膜电位下降,MDA升高而SOD下降,CB1R水平升高,p-Akt和p-mTOR表达下降,LC3-Ⅱ表达增加,而p62蛋白表达无明显差异。结论 HCV核心蛋白增加2-AG水平,上调CB1R表达;增加线粒体ROS水平和Ca^(2+)浓度,降低线粒体膜电位;下调Akt和mTOR活性引起不完全线粒体自噬。

Objective To investigate the mechanism of hepatitis C virus（HCV）core protein inducing incomplete mitophagy via activation of endocannabinoids system.Methods Hepatic stellate X-2 cells were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein.After co-culture,2-arachidonoylglycerol（2-AG）level,reactive oxygen species（ROS）and activity of mitochondrial respiratory chain enzyme complexes were measured with liquid chromatography/mass spectrometry,flow cytometer and spectrophotometric method,respectively.Mitochondria membrane potential and calcium（Ca^（2＋））concentration were detected with laser scanning confocal microscope.The levels of cannabinoid receptor 1（CB1 R）,phosphorylated serine-threonine kinase（p-Akt）,mammalian target of rapamycin（mTOR）,microtubuleassociated protein 1 light chain 3（LC3）-Ⅱ and p62 protein were measured using western blot.The levels of malondialdehyde（MDA）and superoxide dismutase（SOD）were assayed with kit.All quantitative data was analyzed withttest.Results In LX-2 cells co-cultured with HepG2 cells expressing HCV core protein,levels of 2-AG,ROS,Ca^（2＋）concentration,MDA,CB1 Rprotein and expression of LC3-Ⅱ protein were increased,while the activity of mitochondrial respiratory chain enzyme complexes,mitochondria membrane potential,SOD level,expression of p-Akt and p-mTOR protein were decreased compared with those in LX-2 cells co-cultured with HepG2 cells.However,the expression level of p62 protein did not change.Conclusion HCV core protein may not only increase 2-AG content,ROS level and Ca^（2＋）concentration,but also up-regulate the expression of CB1R.Meanwhile,it may decrease mitochondria membrane potential,and down-regulate p-Akt and p-mTOR protein to induce incomplete mitophagy.