Wy14643对缺氧复氧损伤原代大鼠肝细胞的保护作用及机制

Effects of Wy14643 on primary cultured rat hepatocytes injured by hypoxia/reoxygenation and its mechanisms

目的探讨过氧化物酶体增殖物激活受体α(PPARα)选择性激动剂Wy14643对原代大鼠肝细胞缺氧复氧损伤的保护作用及机制。方法采用两步灌流法分离大鼠肝细胞并进行原代培养,将培养细胞随机分成6组:C组(正常组)、H/R组(缺氧复氧组)、D组(对照组)、Ⅰ组、Ⅱ组、Ⅲ组。Ⅰ组、Ⅱ组、Ⅲ组均在缺氧前2 h加入Wy14643(终浓度分别是10、30、100μmol/L)再行复氧处理。将肝细胞缺氧4 h、复氧10 h诱导细胞损伤,然后分别收集细胞培养上清液和肝细胞,测定上清液中谷丙转氨酶(ALT)、天门冬氨酸转氨酶(AST)活性和细胞内谷胱甘肽(GSH)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性,MTT法测定肝细胞的存活率,电子显微镜观察肝细胞损伤情况。结果与C组比较,H/R组ALT、AST、MDA增高(P<0.01),SOD、GSH下降(P<0.01),细胞存活率降低(P<0.01)。Wy14643处理后,肝细胞上清液ALT、AST活性降低,并且呈剂量依赖性(P<0.01);肝细胞内SOD、GSH活性增强(P<0.05),MDA含量降低(P<0.05);细胞存活率升高(P<0.05)。电子显微镜显示H/R组肝细胞比C组损伤明显,Wy14643处理后损伤逐渐减轻。结论 Wy14643对肝细胞缺氧复氧损伤有保护作用,可能与提高肝细胞抗氧化能力有关。

Objective To investigate the effects and mechanisms of peroxisome proliferators activator receptors alpha（PPARα） agonist Wy14643 pretreatment on primary hepatocytes subjected to hypoxia/reoxygenation injury in rats.Methods Primary hepatocytes in rats were isolated by using perfusion method.The primary hepatocytes were randomly divided into 6 groups： normal group（C group）,hypoxia /reoxygenation group（H/R group）,control group（D group）,group Ⅰ,group Ⅱ and group Ⅲ.In group Ⅰ,group Ⅱ and group Ⅲ,Wy14643 was added into culture medium at 2 hours before hypoxia at different Wy14643 concentration of 10,30 and 100 μmol/L,respectively.The content of aminotransferase（ALT）,aspartate aminotransferase（AST）,malondialdehyde（MDA） in culture medium and supeoxide dismutase（SOD）,glutathione（GSH） viability in hepatocytes were measured at the end of the experiments.Cell viability was determined by method of MTT.The appearance and ultramicrstructure changes of hepatocytes were observed by using electron microscope.Results ALT,AST and MDA in H/R group increased significantly as compared to C group（P0.01）,while they were reduced by pretreated with Wy14643 in group Ⅰ,group Ⅱ and group Ⅲ（P0.05）.The activity of SOD and the level of GSH in the hepatocytes decreased in H/R group as compared to C group（P0.01）,while they were enhanced after Wy14643 pretreatment.The ultramicrostructure injury of hepatocytes in H/R group was more serious than that of C group.Conclusion Wy14643 was protective during H/R injury in primary hepatocytes,which was associated with its effect of attenuating oxidative stress.