黄芪甲苷联合丹参酚酸B对肝细胞过氧化损伤的保护作用及其机制研究

Protective Mechanism of Astragalus Ⅳ Combined with Salvianolic Acid B on Hepatocyte (HHL-5) Peroxidation Injury

目的:探讨黄芪甲苷联合丹参酚酸B对HHL-5肝细胞过氧化损伤的保护作用机理。方法:采用1640完全培养基培养HHL-5细胞株,3次传代后分为正常组、过氧化损伤模型组、黄芪甲苷组、丹参酚酸B组、黄芪甲苷联合丹参酚酸B组。采用过氧化氢诱导细胞过氧化损伤,丹参酚酸B和黄芪甲苷给药浓度均是10-5mol/L。qRT-PCR法检测细胞TLR4、NF-κB mRNA的表达水平,细胞爬片免疫组化法检测TLR4、NF-κB蛋白的表达,ELISA法检测细胞上清液中TNFα和SOD的含量。结果:与正常组比较,模型组TLR4、NF-κB mRNA和蛋白的表达水平显著增加,细胞上清液中TNFα的含量明显升高,SOD的含量明显降低(P<0.001)。与模型组比较,丹参酚酸B和黄芪甲苷干预组TLR4、NF-κB mRNA和蛋白的表达水平显著下降,细胞上清液中TNFα的含量明显降低,SOD的含量明显升高(P<0.01)。与黄芪甲苷或丹参酚酸B组比较,黄芪甲苷联合丹参酚酸B干预组TLR4、NF-κB mRNA和蛋白的表达水平显著下降,细胞上清液中TNFα的含量明显降低,SOD的含量明显升高(P<0.05)。结论:丹参酚酸B和黄芪甲苷能保护肝细胞的过氧化损伤且二者具有协同作用,该保护作用与抑制TLR4-NF-κB-TNFα炎症损伤通路相关。

Objective:To investigate the protective effects and mechanism of AstragalusⅣcombined with salvianolic acid B on peroxidation injury of HHL-5 liver cells.Methods:HHL-5 cells were cultured in 1640 complete medium.After 3 passages,they were divided into normal group,peroxidative injury model group,AstragalusⅣgroup,salvianolic acid B group,AstragalusⅣcombined with salvianolic acid B group.Hydrogen peroxide was used to the induce cell peroxidation injury.The concentration of AstragalusⅣand salvianolic acid B were 10-5 mol/L.The expression levels of TLR4 and NF-κB mRNA were detected by qRT-PCR method.The expression levels of TLR4 and NF-κB protein were detected by immunohistochemistry method.The contents of TNF-αand SOD in cell supernatant were detected by ELISA method.Results:Compared with the normal group,the expression levels of TLR4,NF-κB mRNA and protein increased significantly.The content of TNF-αincreased while SOD decreased significantly in supernatant in the model group(P<0.001).Compared with the model group,the expression levels of TLR4,NF-κB mRNA and protein decreased significantly and the content of TNF-αdecreased while SOD increased significantly in Astragalus IV group and salvianolic acid B group(P<0.01).Compared with Astragalus IV group or salvianolic acid B group,the expression levels of TLR4,NF-κB,mRNA and protein decreased significantly and the content of TNF-αdecreased while SOD increased significantly in Astragaloside IV combined with salvianolic acid B group(P<0.05).Conclusion:Astragaloside IV and salvianolic acid B can protect hepatocytes from peroxidative injury and two drugs have synergistic effects.The protective effects of Astragaloside IV and salvianolic acid B were partly by inhibiting TLR4-NF-κB-TNFαpathway.