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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 investigate the effect of Shuganjianpi Decoction on TLR4 / p38MAPK signaling pathway in hepatocytes of nonalcoholic steatohepatitis (NASH) rats and to reveal the mechanism of action of Shugan Jianpi Decoction against rat NASH from the perspective of inflammation. Methods 120 SPF SD rats were randomly divided into 8 groups. The rat model of NASH was established by feeding with high fat diet. The liver tissue was stained with HE staining, oil red O staining and automatic biochemical analysis of hepatic dyslipidemia after 26 weeks. Serum levels of IL-1, IL-6, The content of TNF-α was determined. The isolated hepatocytes were isolated by ex vivo perfusion with type Ⅳ collagenase and the purity and purity of hepatocytes were identified by flow cytometry (FCM). The expression of TLR4 and p38MAPK mRNA in hepatocytes was detected by real-time PCR. The expression of TLR4, p38MAPK and phospho-p38MAPK were detected by Western blotting. Results NASH rat model was established successfully on a 26 w high fat diet. The HE staining and oil red O staining confirmed the typical NASH pathological changes in the rat model. The levels of IL-1, IL-6 and TNF-α in the model group were significantly higher than those in the normal group (P <0.01). Compared with the model group, the levels of IL-1 and IL- (P <0.05, P <0.01). The levels of IL-1 and IL-6 in high-dose spleen group were significantly decreased (P <0.05). Compared with the normal group, the expression of TLR4 and p38MAPK mRNA and phosphorylated protein p38MAPK in the model group were significantly increased (P <0.01). Compared with the model group, the high-dose and low-dose group The expression of TLR4, p38MAPK mRNA and phosphorylated protein p38MAPK in the dose group were significantly decreased (P <0.05, P <0.01). Conclusion Shuganjianpi Decoction can reduce the levels of serum inflammatory cytokines in NASH rats and improve the pathological changes in the liver of NASH rats. TLR4 / p38MAPK may be the drug target of Shugan Jianpi Prescription for anti-NASH action.