海兔素对大鼠原代肝细胞酒精性氧化损伤的保护作用

Effects of Aplysin on ethanoHnduced oxidative damage in rat primary hepatocytes

目的探讨海兔素对大鼠原代肝细胞酒精性氧化损伤保护作用。方法门静脉胶原酶Ⅳ原位灌注及密度梯度离心获得大鼠原代肝细胞。MTT实验检测乙醇和海兔素最佳作用剂量及肝细胞活力。酶学实验检测细胞AST、LDH、SOD、MDA、GSH水平;流式细胞术检测细胞凋亡情况;单细胞凝胶电泳观察细胞DNA损伤状况;JC-1荧光探针检测细胞线粒体膜电位水平;比色法及Western blot检测细胞CYP2E1活性及蛋白表达。结果经30 mg·L^(-1)。海兔素预作用2 h,再与300 mmol·L^(-1)。乙醇共作用8 h后,肝细胞活力较酒精模型组明显上升,AST和LDH释放也得到明显抑制;同时,肝细胞SOD和GSH水平明显升高,MDA含量则明显降低,差异均具有显著性(P<0.05)。海兔素干预后,肝细胞凋亡率明显降低,DNA损伤及线粒体膜电位水平明显得到改善。海兔素干预后,肝细胞CYP2E1活性及蛋白表达水平明显受到抑制(P<0.05)。结论海兔素对大鼠原代肝细胞酒精性氧化损伤具有保护作用,其作用机制可能与海兔素抑制酒精对CYP2E1的活化,缓解氧化应激,提高机体抗氧化能力有关。

Aim To investigate the protective effects of Aplysin on ethanol-induced oxidative damage in rat primary hepatocytes.Methods Rat primary hepatocytes were obtained via the portal vein collagenase Ⅳ in situ perfusion technique followed by a Percoll density gradient centrifuge.MTT test was used to determine the optimum dose of Aplysin and ethanol,and detect the cell vitality in primary hepatocytes.Supernatants of primary hepatocytes were harvested to measure AST and LDH level,and the SOD,GSH-PX activities and MDA content in primary hepatocytes were observed.Flow cytometry was used to detect the cell apoptosis rate.DNA damage in primary hepatocytes was detected by singlecell gel electrophoresis assay.The level of mitochondrial membrane potential in primary hepatocytes was tested by fluorogenic probe JC-1.The CYP2E1 activity in primary hepatocytes was detected by colorimetry.The proteins of CYP2E1 were detected by Western blot.Results 300 mmol·L^(-1) dose of ethanol and 30 mg · L^(-1) dose of Aplysin were the optimal dosages and were used in the subsequent experiments.Hepatocyte vitality was significantly increased in Aplysin group compared to that in ethanol group,and Aplysin inhibited the release of AST and LDH(P <0.05).For Aplysin treatment group,the activities of hepatocyte SOD and GSH were significantly increased,and MDA was markedly lowered as compared with those in ethanol group(P < 0.05).Aplysin could alleviate hepatocyte apoptosis significantly,and hepatocyte DNA damage rates of Ⅱ~Ⅲlevel and Ⅳ level were significantly lowered in Aplysin treatment group as compared with those in ethanol group,and Aplysin had evident improvement in alcohol induced mitochondria damage of hepatocyte.Primary hepatocyte activities and protein expression of CYP2E1 were markedly lowered in Aplysin treatment group as compared with those in ethanol group(P <0.05).Conclusion Aplysin has protective effects on liver oxidative damage induced by alcohol of primary cultured rat hepatocytes by blocking CYP2E1 activation,relieving oxidative stress,and sharpening the oxidation resistance ability.