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目的观察大麻二酚(CBD)对棕榈酸(PA)诱导的肝细胞损伤的保护作用,并考察其与自噬流相关的潜在机制。方法原代培养大鼠肝细胞,分别给予1μmol/L和5μmol/L的CBD处理24 h,采用蛋白质印迹法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)和p62的表达,考察CBD对细胞自噬的影响。将细胞分为4组并分别给予不同处理:PA组(800μmol/L PA处理细胞)、PA+CBD组(800μmol/L PA和5μmol/L CBD联合作用)、PA+CBD+CQ组[800μmol/L PA、5μmol/L CBD和50 nmol/L自噬抑制剂氯喹(CQ)共同作用]和阴性对照组(加入等体积0.03%DMSO处理细胞),每组作用时间均为24 h;采用蛋白质印迹法检测LC3和p62的蛋白表达,流式细胞术考察细胞凋亡情况,q PCR法检测内质网应激相关因子CCAAT/增强子结合蛋白同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)和X盒结合蛋白1(XBP-1)mRNA的表达,Rh123和lucigenin荧光探针分别检测线粒体的膜电位和活性氧簇(ROS)含量。结果 1μmol/L和5μmol/L CBD均不影响肝细胞LC3-Ⅱ/LC3-Ⅰ的比值以及p62蛋白的表达。与阴性对照组相比,PA组肝细胞LC3-Ⅱ/LC3-Ⅰ的比值和p62蛋白表达增加(P<0.05),细胞凋亡增加(P<0.05),CHOP、GRP78、XBP-1mRNA表达增加(P<0.05),线粒体膜电位降低(P<0.05),线粒体ROS的生成增加(P<0.05);与PA组相比,PA+CBD组肝细胞内的自噬流恢复,细胞凋亡减少,内质网应激和线粒体失常减轻(P<0.05);同时给予CQ处理可以逆转CBD的保护作用(P<0.05)。结论 CBD能够通过促进自噬流减轻PA诱导的肝细胞损伤,改善内质网应激和线粒体功能。
Objective To observe the protective effect of cannabidiol (CPP) on hepatocellular injury induced by palmitic acid (PA) and investigate its potential mechanism of autophagic flow. Methods Primary cultured rat hepatocytes were treated with 1μmol / L and 5μmol / L CBD for 24 h, respectively. Western blotting was used to detect the expressions of LC3 and p62, CBD on cell autophagy. The cells were divided into 4 groups and given different treatments: PA group (800μmol / L PA-treated cells), PA + CBD group (800μmol / L PA and 5μmol / L PA, 5μmol / L CBD and 50 nmol / L autophagy inhibitor chloroquine (CQ)] and negative control group (treated with an equal volume of 0.03% DMSO) for 24 h. Western blot LC3 and p62 were detected by flow cytometry. Flow cytometry was used to detect apoptosis. QPCR method was used to detect endoplasmic reticulum stress-related factors CCAAT / CHOP, GRP78, And X box-binding protein 1 (XBP-1) mRNA. Rh123 and lucigenin fluorescent probes were used to detect mitochondrial membrane potential and reactive oxygen species (ROS) content. Results Both 1 μmol / L and 5 μmol / L CBD did not affect the ratio of LC3-Ⅱ / LC3-Ⅰ and the expression of p62 protein in hepatocytes. Compared with the negative control group, the ratio of LC3-Ⅱ / LC3-Ⅰ and the expression of p62 protein in PA group increased (P <0.05) and the expression of CHOP, GRP78 and XBP-1 mRNA increased (P <0.05), mitochondrial membrane potential (P <0.05) and mitochondrial ROS production increased (P <0.05). Compared with PA group, the autophagic flux in hepatocytes of PA + CBD group recovered and the apoptosis decreased , Endoplasmic reticulum stress and mitochondrial dysfunction (P <0.05). At the same time, CQ treatment reversed the protective effects of CBD (P <0.05). Conclusion CBD can alleviate PA-induced hepatocellular injury and promote endoplasmic reticulum stress and mitochondrial function by promoting autophagic flow.