基于TGR5/NLRP3信号通路探讨活血清解灵对NASH大鼠炎症反应的影响

Research of Influence on Inflammation of Huoxueqingjieling on NASH rats Based on TGR5/NLRP3 Signaling Pathway

关键词:  NASH  活血清解灵  TGR  NLRP3

Key words:  NASH  Huoxueqingjieling  TGR5  NLRP3

摘要:

目的 基于TGR5/NLRP3信号通路，探讨活血清解灵干预非酒精性脂肪性肝炎（NASH）大鼠炎症反应的作用机制。方法 将70只SD雄性大鼠随机分为5组，分别为对照组20只，模型组20只，阿托伐他汀阳性药组、活血清解灵高、低剂量组各10只。对照组给予维持饲料，其余各组均给予高脂饲料，通过模型评价确定于第20周末NASH大鼠模型造模成功。造模成功后，对照组和模型组灌胃生理盐水，阿托伐他汀阳性药组、活血清解灵高、低剂量组灌胃给予相应药物，每日1次，连续4周。第24周末处死大鼠，计算肝指数，全自动生化仪检测血清中甘油三酯（TG）、总胆固醇（TC）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）水平，苏木素-伊红（HE）、油红O染色观察肝脏组织病理变化，蛋白免疫印迹法（Western Blot）、免疫荧光法检测肝脏组织G蛋白偶联胆汁酸受体（TGR5）及核苷酸结合寡聚化结构域样受体3（NLRP3）蛋白表达，酶联免疫吸附测定法（ELISA）试剂盒检测肝脏组织白细胞介素18（IL-18）及白细胞介素1β（IL-1β）含量。结果 活血清解灵能显著改善NASH大鼠一般状态，各剂量组均能显著降低大鼠体质量、肝指数（P<0.01），血清TG、TC含量和ALT、AST活性水平显著下降（P<0.01），病理结果提示活血清解灵能明显改善肝脏脂肪变、炎症及气球样变，TGR5蛋白表达显著上升（P<0.01）、NLRP3蛋白表达及IL-18、IL-1β含量均显著下降（P<0.01，P<0.05）。结论 活血清解灵能够明显改善NASH大鼠状态，抑制其肝脏脂肪变和炎症反应，其作用机制可能与抑制TGR5/NLRP3信号通路传导密切相关。

Abstract:

OBJECTIVE based on the TGR5/NLRP3 signaling pathway, to explore the mechanism of Huoxueqingjieling in improving the inflammatory response of rats with nonalcoholic steatohepatitis(NASH). METHODS a total of 70 male SD rats were randomly divided into 5 groups, 20 rats in the control group, 20 rats in the model group, 10 rats in each of the Atorvastatin positive drug group, the high-dose and low-dose groups of Huoxueqingjieling. The control group was given normal feed, and the rest of the groups were given high-fat diat. Through model evaluation, it was determined that the NASH rat model was successfully established at the end of the 20th week. After successful modeling, the control group and the model group were given with normal saline by intragastric administration, the Atorvastatin positive drug group, and the high and low dose groups of Huoxueqingjieling were given corresponding drugs once a day for 4 weeks. At the end of the 24th week, the rats were killed, and the changes of body weight, wet liver weight and liver index was calculated. The serum was taken to test the triglyceride(TG), total cholesterol(TC), alanine aminotransferase(ALT), and aspartic acid aminotransferase(AST) levels by a automatic biochemical analyzer. Pathological changes of liver tissues were observed by hematoxylin-eosin(HE) staining and Oil Red O staining. The expression levels of G protein-coupled bile acid receptor (TGR5) and nucleotide binding oligomerization domain-like receptor 3 (NLRP3) proteins were detected by Western Blot and immunofluorescence staining. Enzyme-linked immunosorbent assay (Elisa) detects the content of interleukin-18 (IL-18) and interleukin-1β (IL-1β) in liver tissue. RESULTS Huoxueqingjieling can significantly improve the general state of NASH rats. Every dose group can significantly reduce the body weight, liver wet weight and liver index of rats (P<0.01), and TG, TC content and ALT, AST activity levels of serum significantly decreased (P<0.01). The pathological results showed that Huoxueqingjieling can significantly improve liver steatosis, inflammation and balloon-like. The expression of TGR5 protein was significantly increased (P<0.01), the expression of NLRP3 protein and the content of IL-18, IL-1β were significantly decreased (P<0.01, P<0.05). CONCLUSION Huoxueqingjieling can significantly improve the state of NASH rats, inhibit liver steatosis and inflammation, and its mechanism may be closely related to the inhibition of TGR5/NLRP3 signaling pathway.