IL-18通过NF-κB通路调控MRP3对ANIT引起的胆汁淤积模型的影响及其机制

Il-18 regulates the effect of MRP3 on rifampin induced cholestasis model through NF-kappab pathway and its mechanism

目的探讨IL-18通过NF-κB通路调控MRP3对ANIT引起的胆汁淤积模型的影响。方法选取10只大鼠用ANIT诱导建立胆汁淤积模型,另取10只作为对照组,HE染色观察肝组织病理变化,全自动生化仪检测大鼠血清ALP、ALT、ALP、TB、DB、TBA以及肝组织TBA含量。取肝组织培养肝细胞,将正常大鼠肝细胞培养出的细胞作为空白组(A组),胆汁淤积模型大鼠肝细胞培养出的细胞分为低(C组)、中(D组)、高浓度组(E组)和非干预模型组(B组),每组10个样本。C、D、E组肝细胞分别给予IL-18(10、30、100 ng/mL)不同浓度处理,A组和E组给予同体积的生理盐水处理。Western blot和RT-PCR分别检测大鼠肝组织IL-18、p-p65、MRP3、YY1、FXR蛋白及其mRNA。结果与对照组比较,胆汁淤积模型大鼠肝脏出现黄染,边缘变钝,空泡变性,汇管区较多纤维组织增生,可见肝细胞出现脂肪变性、轻度坏死。与对照组比较,胆汁淤积模型组血清ALP、ALT、AST、TB、DB、TBA以及肝组织TBA的含量均升高(均P<0.05)。与对照组比较,胆汁淤积模型组大鼠肝组织IL-18、p-p65、YY1、MRP3蛋白及其mRNA相对表达量均升高(均P<0.05),FXR蛋白及其mRNA相对表达量降低(P<0.05)。五组肝细胞p-p65、YY1、MRP3蛋白及其mRNA相对表达量差异均有统计学意义(均P<0.05),与A组比较,B、C、D、E组均升高(均P<0.05);与B组和C组比较,D和E组均升高(均P<0.05),且E组高于D组。五组肝细胞FXR蛋白及其mRNA相对表达量差异有统计学意义(P<0.05),与A组比较,B、C、D、E组均降低(均P<0.05);与B组和C组比较,D和E组均降低(均P<0.05),且E组低于D组。结论IL-18表达和NF-κB通路作用与胆汁淤积模型发生相关,其可能作用机制为IL-18通过诱导p65磷酸化,激活NF-κB通路调控YY1及FXR促进MRP3表达而影响胆汁淤积模型发病。

Objective To investigate the effect of IL-18 regulating MRP3 through NF-κB pathway on the model of ANIT induced cholestasis.Methods The total of 10 rats were selected to establish the cholestasis model induced by ANIT,and another 10 rats were taken as the control group.HE staining was used to observe the pathological changes of liver tissue,and the serum ALP,alt,ALP,TB,DB,TBA and the content of TBA in liver tissue were detected by automatic biochemical instrument.The hepatocytes of normal rats were taken as the blank group(group A).The hepatocytes of cholestasis model rats were divided into low(group C),medium(group D),high concentration(group E)and non intervention model group(group B).There were 10 samples in each group C.The hepatocytes of group D and group E were treated with IL-18(10,30,100 ng/mL)in different concentrations,and the hepatocytes of group A and group E were treated with normal saline of the same volume.Western blot and RT-PCR were used to detect IL-18,p-p65,MRP3,YY1,fxrprotein and their mRNA in rat liver and liver.Results Compared with the control group,the liver of cholestasis model rats appeared yellow staining,blunt margin,vacuole degeneration,more fibrous tissue proliferation in the portal area,and fatty degeneration and slight necrosis of hepatocytes were observed.Compared with the control group,the contents of ALP,alt,AST,TB,DB,TBA in serum and TBA in liver tissue in cholestasis model group were all increased(P<0.05).Compared with the control group,the relative expression of IL-18,p-p65,YY1,MRP3 protein and their mRNA in the liver tissue of the model group increased(all P<0.05),and the relative expression of fxrprotein and their mRNA decreased(P<0.05).Compared with group A,the expression of p-p65,YY1,MRP3 protein and their mRNA in group B,C,D and e increased(P<0.05);compared with group B and C,the expression of p-p65,YY1,MRP3 protein and their mRNA in group D and e increased(P<0.05),and group E was higher than Group D.Compared with group A,the expression of fxrprotein and its mRNA in liver cells in group B,C,D and e decreased significantly(P<0.05);compared with group B and C,the expression of fxrprotein and its mRNA in group D and e decreased significantly(P<0.05),and group E was lower than Group D.Conclusion The expression of IL-18 and the role of NF-κB pathway are related to the development of cholestasis model.The possible mechanism is that IL-18 can affect the pathogenesis of cholestasis model by inducing p65 phosphorylation,activating NF-κB pathway to regulate the expression of MRP3 by YY1 and FXR.