基于HMGB1-TLR4信号通路探讨黄芩甲苷对DDC诱导胆汁淤积性肝病小鼠的保护作用研究

Protective effect of astragaloside IV on DDC-induced cholestatic liver disease based on HMGB1-TLR4 signaling pathway

目的探讨黄芩甲苷对胆汁淤积性肝病小鼠的影响。方法40只雄性SPF级C57BL/6小鼠随机、平均分为正常对照组、模型组、熊去氧胆酸组和黄芩甲苷组。动物通过自由喂食0.1%利用喂食3,5-二乙氧基羰基-1,4-二氢-2,4,6-三甲基吡啶(DDC)饲料建立小鼠胆汁淤积性肝病模型,于造模5 d后以灌胃方式给药,连续给药3 d,1次/d。观察各组小鼠肝组织病理形态学变化,检测肝组织炎症因子[肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、白介素6(IL-6)、和氧化应激指标[丙二醛(MDA)]超氧化歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)]水平,检测各组小鼠血清肝功能酶[丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)]、胆红素[总胆红素(T-BIL)、直接胆红素(D-BIL)、间接胆红素(I-BIL)]和胆汁酸(TBA)的水平,Western blot法检测HMGB1-TLR4信号通路高迁移率族蛋白B1(HMGB1)和Toll样受体4(TLR4)蛋白的表达水平。结果与正常对照组比较,模型组肝组织损伤严重,可见较多炎性细胞浸润以及肝细胞凋亡、坏死,肝组织SOD和GSH-PX活力显著降低(P<0.01),TNF-α、IL-1β、IL-6和MDA水平显著升高(P<0.01),血清T-BIL、D-BIL、I-BIL、TBA、ALT、AST、ALP水平显著升高(P<0.01),肝组织HMGB1和TLR4蛋白表达水平显著上调(P<0.01)。与模型组比较,黄芩甲苷组和熊去氧胆酸组均能改善肝组织损伤程度,减少炎症细胞浸润和肝细胞凋亡、坏死,显著升高肝组织SOD和GSH-PX活力(P<0.01)以及降低TNF-α、IL-1β、IL-6和MDA水平(P<0.01),显著降低血清T-BIL、D-BIL、I-BIL、TBA、ALT、AST、ALP水平(P<0.01),下调肝组织HMGB1和TLR4蛋白表达水平(P<0.01)。结论黄芪甲苷对DDC诱发的小鼠胆汁淤积性肝病具有明显的保护作用,其保护作用可能与调节HMGB1-TLR4信号通路相关。

Objective To investigate the effect of baicalin IV on mice with cholestatic liver disease.Methods Forty male SPF C57BL/6 mice were randomly divided into normal control group,model group,ursodeoxycholic acid group and astragaloside IV group.The mice model of cholestatic liver disease was established by feeding 0.1%DDC feed freely.After 5 days of modeling,the mice were given the drug by gavage for 3 days,once a day.The levels of TNF-α,IL-1β,IL-6 and MDA and the activities of SOD and GSH-PX in liver tissues were detected.The levels of liver function enzyme(ALT,AST,ALP),bilirubin(T-BIL,D-BIL,I-BIL)and bile acid(TBA)in serum were detected.The expression levels of HMGB1 and TLR4 proteins in HMGB1-TLR4 signaling pathway were analyzed by Western blot.Results Compared with the normal control group,the liver tissue in model group was severely damaged,with more inflammatory cell infiltration,apoptosis and necrosis of liver cells,the activities of SOD and GSH-PX in liver tissue were significantly decreased(P<0.01),and the levels of TNF-α,IL-1β,IL-6 and MDA were significantly increased(P<0.01).The levels of T-BIL,D-BIL,I-BIL,TBA,ALT,AST and ALP in serum were significantly increased(P<0.01),and the protein expressions of HMGB1 and TLR4 in liver tissue were significantly up-regulated(P<0.01).Compared with model group,astragaloside IV group and ursodeoxycholic acid group could improve the degree of liver tissue injury,reduce inflammatory cell infiltration,apoptosis and necrosis of liver cells,significantly increase the activities of SOD and GSH-PX in liver tissue(P<0.01),and reduce the levels of TNF-α,IL-1β,IL-6 and MDA in liver tissue(P<0.01).The serum T-BIL,D-BIL,I-BIL,TBA,ALT,AST and ALP levels were significantly decreased(P<0.01),and the protein expressions of HMGB1 and TLR4 in liver tissue were down-regulated(P<0.01).Conclusion Astragaloside IV has a significant protective effect on DDC-induced cholestatic liver disease in mice,and its protective effect may be related to the regulation of HMGB1-TLR4 signaling pathway.