目的:探讨茵陈蒿汤方剂挥发油(简称茵陈挥发油)对肝纤维化(HF)模型小鼠的治疗效果及可能机制.方法:70只昆明小鼠随机分7组(n=10):正常组、模型组、秋水仙碱组(0.2 mg/kg)、茵陈挥发油低、中、高剂量组(2、4、8 g/kg)和自噬抑制剂组(3MA,30 mg/kg).除正常组外,每组均以腹腔注射10%CCl4玉米油的方法构建HF小鼠模型,每周2次,持续8周.此外,各给药组在造模的同时均给予相对应的药物治疗,每天1次,持续6周.末次给药结束后,用HE染色法观察肝脏和肾脏组织的病理变化.用试剂盒法测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)活性,ELISA法测肝纤维化标志物Ⅳ型胶原(Col-Ⅳ)、Ⅲ型前胶原(PC-Ⅲ)、透明质酸(HA)、丙二醛(MDA)和层粘连蛋白(LN)水平,肝脏组织炎症因子TNF-α、IL-6的水平.使用免疫组化法或Western blot法来检测肝组织中蛋白质的表达水平,包括Smad3、组织转化生长因子-β1(TGF-β1)、自噬关键分子酵母Atg6同系物(Beclin1)及微管相关蛋白1轻链3(LC3B).结果:与正常组比较,模型组肝组织出现大量胶原纤维堆积,炎症细胞浸润,肝小叶结构不清,有明显纤维间隔形成,肝细胞变性坏死,血清活性ALT、AST及Col-Ⅳ、PC-Ⅲ、HA、LN、MDA水平显著增加(P<0.01),肝组织TNF-α、IL-6蛋白显著增加(P<0.01),肝组织Smad3、TGF-β1水平显著增加(P<0.01)及LC3B、Beclin1水平明显增加(P<0.05,P<0.01).与模型组比,茵陈挥发油各剂量组小鼠肝组织胶原纤维沉积程度明显减轻,纤维化程度有不同程度改善,血清活性ALT、AST及Col-Ⅳ、PC-Ⅲ、HA、LN、MDA水平明显减少(P<0.05,P<0.01),肝组织TNF-α、IL-6蛋白明显减少(P<0.05,P<0.01),肝组织Smad3、TGF-β1水平明显减少(P<0.05,P<0.01),茵陈挥发油高剂量组LC3B、Beclin1水平显著减少(P<0.01).结论:茵陈蒿汤方剂挥发油对CCl4致小鼠纤维化具有明显的治疗作用,能明显改善肝功能,减轻HF病变,机制通过TGF-β1/Smad3介导的信号转导途径,减少HF自噬,实现抗纤维化.
Objective:The aim of this study was to investigate the therapeutic effect and possible mechanism of essential oil extracted from Yinchenhao Tang Formula(essential oil extracted from Yinchenhao)on hepatic fibrosis model mice.Methods:Seventy Kunming mice were randomly divided into 7 groups(n=10):normal group,model group,colchicine group(0.2 mg/kg),essential oils extracted from Yinchenhao in different doses-low,medium and high dose group(2,4,8 g/kg)and autophagy inhibited group(3MA,30 mg/kg).Except for the normal group,each group was treated with intraperitoneal injection of 10%CCl4 corn oil to construct hepatic fibrosis mouse model twice a week for 8 weeks.In addition,each administration group was given the corresponding drug treatment once a day for 6 weeks.After the last dose,Hematoxylin-eosin(HE)staining was used to observe the pathological changes of the liver and kidney tissue in each group.The active levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by the kit method,while the levels of liver fibrosis markers type Ⅳ collagen(Col-Ⅳ),type Ⅲ Procollagen(PC-Ⅲ),hyaluronic acid(HA),laminin(LN),malondialdehyde(MDA),and inflammatory cytokines including TNF-α,and IL-6 in the liver tissue were measured by ELISA.Besides,the immunohistochemistry method or western blot method was adopted to measure the expression levels of proteins in the liver tissue,including Smad3,TGF-β1,LC3B and Beclin1.Results:Compared with the normal group,the liver tissue in the model group appeared significant symptoms:large amount of collagen fiber deposition,infiltration of inflammatory cell,unclear liver lobule structure,with obvious fibrous septum forming and liver cells degeneration and necrosis.Besides,it was found that while the active levels of ALT and AST in serum,the levels of Col-Ⅳ,HA,LN,PC-Ⅲ,MDA,LN(P<0.01),and the protein level of liver tissue TNF-α and IL-6 increased significantly(P<0.01),Smad3 and TGF-β1 levels in liver tissue(P<0.01)and LC3B and Beclin1 levels(P<0.05,P<0.01)increased dramatically.Moreover,comparing with the model group,it was found that the collagen deposition in liver of each dose essential oils groups were found significantly reduced,with the degree of fibrosis were alleviated in different levels and remarkably reduce in the levels of ALT and AST in serum,levels of Col-Ⅳ,HA,LN,PC-Ⅲ,MDA,LN(P<0.05,P<0.01),liver tissue TNF-α and IL-6 levels were observed(P<0.05,P<0.01).In addition,liver tissue Smad3 and TGF-β1 levels(P<0.05,P<0.01)were also found reduced significantly while the evident reduction in LC3B and Beclin1 levels(P<0.01)was found in oils extracted from Yinchenhao high dose group.Conclusion:Essential oil extracted from Yinchenhao Tang Formula have significant therapeutic effects on carbon tetrachloride(CCl4)-induced mice with liver fibrosis model,which is able to obviously improve liver function,mitigate the liver fibrosis lesions.Through TGF-β1/Smad3-mediated signal transduction pathway,the mechanism is able to reduce autophagy of liver cells fibrosis and realize the anti-fibrosis.