目的 观察葛黄颗粒含药血清对乙醇损伤的人肝细胞L-02修复作用,并探讨其可能机制.方法 ①将L-02细胞分为A、B、C、D、E、F组.除F组外,其余各组均加入乙醇培养基培养24 h制备肝细胞损伤模型,造模后吸走培养液.D、F组加入空白血清(10％正常大鼠血清),A组加入1.25％葛黄颗粒含药血清+8.75％正常大鼠血清,B组加入2.5％葛黄颗粒含药血清+7.5％正常大鼠血清,C组加入5％葛黄颗粒含药血清+5％正常大鼠血清,E组加入5％美他多辛含药血清+5％正常大鼠血清,继续培养6、12、24h.比较各组细胞上清液谷草转氨酶(AST)、乳酸脱氢酶(LDH)活力.②将L-02细胞按2×104个/孔接种于6孔板中,将其分为a、b、c、d组.除d组外,其余各组均加入乙醇制备肝细胞损伤模型,造模后吸走培养液.b、d组加入空白血清,a组加入5％葛黄颗粒含药血清,c组加入5％美他多辛含药血清,继续培养24h.比较各组丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)、细胞活性氧(ROS)、线粒体膜电位(MMP)、三磷酸腺苷(ATP).结果 与F组比较,D组各时点AST、LDH升高;与D组比较,B组24 hAST、LDH降低,C、E组12 h和24h的AST、LDH降低;与A组比较,B组24 h AST降低,C组24 h AST及12 h LDH降低,E组12、24 hAST和LDH降低;与B组比较,C、E组12 h AST、LDH降低;与同组6h比较,C组24 h AST降低,12、24 h LDH降低;与同组12 h比较,B、C、E组24 hAST降低,C组24 h LDH降低;P均＜0.05.与d组比较,b组SOD、GSH-px、MMP、ATP降低,MDA、ROS升高(P均＜0.05);与b组比较,a组和c组SOD、GSH-px、MMP、ATP升高,MDA、ROS降低(P均＜0.05).结论 葛黄颗粒含药血清对乙醇致L-02细胞损伤有一定修复作用,其机制可能是调整细胞内氧化应激水平和线粒体功能.%Objective To observe the effects of different dosages of Gehuang granule medicated serum (GHG) on the ethanol-induced injury of human hepatocytes L-02 and to explore its possible mechanism.Methods ① L-02 cells were divided into groups A,B,C,D,E,and F.Except the group F,the other groups were cultured in ethanol for 24 h,and the medium was taken away after the model was established.The groups D and F were added with the blank serum (10％ normal rat serum),the rest were added with the corresponding drug-containing serum:group A was added with 1.25％ GHG + 8.75％ normal rat serum,group B was added with 2.5％ GHG + 7.5％ normal rat serum,groups C was added with 5％ GHG +5％ normal rat serum,group E was added with 5％ metatoxin medicated serum +5％ normal rat serum and then we continued to culture the cells for 6,12,and 24 h.The activities of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the supernatant of each group were compared.② The L-02 cells were inoculated into 2 × 104 cells/6-well plate,and then were divided into groups a,b,c,and d.In addition to the group d,the remaining groups were added with the ethanol modeling,after that,the medium was taken away.The groups b and d were added with the blank serum,group a was added with 5％ GHG,group c was added with 5％ metatoxin medicated serum and then we continued to culture the cells for 24 h.The levels of malondialdehyde (MDA),superoxide dismutase (SOD),glutathione peroxidase (GSH-px),reactive oxygen species (ROS),mitochondrial membrane potential (MMP),and adenosine triphosphate (ATP) were compared.Results Compared with group F,AST and LDH increased at each time point in the group D;compared with group D,AST and LDH in the group B decreased at 24 h,AST and LDH decreased at 12 and 24 h in the groups C and E,respectively;compared with group A,24-hour AST (24 h AST) decreased in the group B,24 h AST and 12 h LDH decreased in the group C,while AST and LDH decreased at 12 and 24 h in the group E;compared with the group B,AST and LDH decreased at 12 h in the group C and E;compared with the same group at 6 h,the 24 h AST in the group C decreased and the LDH decreased at 12 and 24 h;compared with the same group at 12 h,AST decreased at 24 h in the groups B,C,and E,and the LDH decreased at 24 h,all P ＜ 0.05.Compared with the group d,the levels of SOD,GSHpx,MMP,and ATP decreased,and MDA and ROS increased in the serum of group b (all P ＜ 0.05).Compared with the group b,the levels of SOD,GSH-px,MMP,and ATP increased,and MDA and ROS decreased in the groups a and c (all P ＜ 0.05).Conclusion GHG has a protective effect on ethanol-induced L-02 cell injury by regulating the level of intracellular oxidative stress and protecting mitochondrial function.
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