疏肝健脾方药对非酒精性脂肪性肝病大鼠肝细胞TLR4/p38MAPK信号通路的影响

Effects of Shugan-Jianpi prescriptions on TLR4/p38MAPK signaling pathway in hepatocytes of rats with NASH

目的探讨疏肝健脾方药对非酒精性脂肪性肝炎(NASH)大鼠肝细胞TLR4/p38MAPK信号通路的干预作用,从炎症角度揭示疏肝健脾方药抗大鼠NASH的作用机制。方法选用SPF级SD大鼠120只,随机分为8组。采用高脂饲料喂养建立大鼠NASH模型,26 w后对肝组织进行HE染色,油红O染色,全自动生化分析肝功血脂改变,ELISA检测血清白介素(IL)-1、IL-6、肿瘤坏死因子(TNF)-α含量;采用离体循环灌注Ⅳ型胶原酶法分离肝细胞,流式细胞术(FCM)对肝细胞纯度进行活性和纯度鉴定。荧光定量PCR检测肝细胞TLR4、p38MAPK mRNA表达量;Western印迹法检测肝细胞蛋白TLR4、p38MAPK及磷酸化蛋白p38MAPK表达水平。结果 26 w高脂饮食成功建立了NASH大鼠模型,HE染色和油红O染色证实大鼠模型存在典型的NASH病理改变。与正常组比较,模型组血清IL-1、IL-6、TNF-α含量显著升高(P<0.01);各用药组与模型组比较,合方高、低剂量组IL-1、IL-6、TNF-α含量显著降低(P<0.05,P<0.01),健脾高剂量组IL-1、IL-6含量降低有统计学差异(P<0.05)。与正常组比较,模型组大鼠肝细胞TLR4、p38MAPK mRNA表达及磷酸化蛋白p38MAPK表达量显著增加(P<0.01);各用药组与模型组比较,合方高、低剂量组,健脾高剂量组TLR4、p38MAPK mRNA蛋白及磷酸化蛋白p38MAPK表达量显著降低(P<0.05,P<0.01)。结论疏肝健脾方药能够降低NASH大鼠血清炎症因子水平,改善NASH大鼠肝脏的病理变化,TLR4/p38MAPK可能是疏肝健脾方药发挥抗NASH作用的药物靶点。

Objective To discuss the influence of Shugan and Jianpi methods and prescriptions on TLR4 /p38 MAPK signaling pathways in hepatocytes of rats with NASH and reveal the mechanisms. Methods 120 SPF SD male rats were divided into 8 groups randomly.After 26 weeks,liver pathology of the rats was analyzed by HE and Oil red O staining. The levels of TC,TG,LDL-C,HDL-C in blood serum were detected. Then the levels of IL-1,IL-6,TNF-α of blood serum were analyzed by ELISA. Cell activity and purity were appraised by FCM technical. TLR4 and p38 MAPK mRNA and protein expressions of hepatocytes were analyzed by fluorescence quantitative PCR and Western blot. Results The levels of serum IL-1,IL-6,TNF-α were increased in normal group than those in model group( P<0. 01). Comparing with those of model group,the levels of IL-1,IL-6,TNF-α were reduced significantly in high and low integrated prescriptions groups( P<0. 05,P<0. 01),the levels of IL-1,IL-6 were decreased in high Jianpi prescriptions group( P<0. 05). The levels of TLR4,p38 MAPK mRNA,protein phosphorylation p38 MAPK in rats hepatocyte were increased in model group than those in normal group( P<0. 01),and they were decreased in high and low integrated prescriptions,high Jianpi recipe groups than those in model group( P < 0. 05,P < 0. 01). Conclusions ShuganJianpi prescriptions could reduce the serum inflammatory factors and prevent liver's steatosis of the NASH rats,and TLR4 / p38 MAPK might be the targets of Shugan-Jianpi prescriptions in the treatment of NASH.