调肝理脾颗粒剂调节原发性胆汁性胆管炎小鼠TGF-β1/Smads信号通路的研究

Research on Tiaogan Lipi granules regulating TGF-β1/Smads signaling pathway in mice infected with primary biliary cholangitis

[目的]明确调肝理脾颗粒剂对转化生长因子(TGF)-β1/Smads信号通路的调节作用,探讨其防治原发性胆汁性胆管炎(primary biliary cholangitis,PBC)小鼠的分子学机制。[方法]将雌性C57BL/6小鼠随机分为正常组、模型组、调肝理脾组(5.4g·kg^(-1)·d^(-1))和熊去氧胆酸(UDCA)组(0.1g·kg^(-1)·d^(-1))。除正常组外,其余各组采用1%聚肌胞苷酸腹腔注射诱导小鼠PBC模型,同时给药组分别给予相应剂量药物灌胃,每天1次,连续16周。末次给药24h后,检测肝功能及外周血CD4^+CD25^+Foxp3^+含量,取肝脏组织,苏木精-伊红和马松(Masson)染色观察肝脏组织病理学改变情况,免疫组化检测肝组织中Foxp3、TGF-β1、Smad3及Smad7蛋白的表达,实时荧光定量反转录聚合酶链反应(quantitative real-time RT-PCR,qRT-PCR)检测肝组织中TGF-β1、TβRI、TβRII、Smad3及Smad7mRNA的表达。[结果]与正常组比较,模型组小鼠肝功能异常,肝组织病理损伤明显,外周血CD4^+CD25^+Foxp3^+含量下降明显,肝组织中Foxp3、TGF-β1、Smad3蛋白及TGF-β1、TβRI、TβRII、Smad3 mRNA的表达明显增加(P<0.05),Smad7蛋白及mRNA表达减少;与模型组比较,调肝理脾颗粒剂不仅可改善肝功能,增加外周血CD4^+CD25^+Foxp3^+含量,减轻肝组织病理损伤程度,减少胶原沉积,还可显著降低Foxp3、TGF-β1、Smad3蛋白及TGF-β1、TβRI、TβRII、Smad3mRNA的表达(P<0.05),并上调Smad7蛋白及mRNA的表达(P<0.05)。[结论]调肝理脾颗粒剂对PBC小鼠具有一定的保护作用,其机制可能与调控TGF-β1/Smads信号通路,从而抑制Foxp3表达有关。

[Objective]Explicate the regulating effect of Tiaogan Lipi granules in transforming growth factor（TGF）-β1/Smads signaling pathway,and explore its molecular mechanism of preventing and treating primary biliary cholangitis（PBC）mice.[Methods]Female C57 BL/6 mice were randomly divided into the normal group,the model group,the Tiaogan Lipi group（5.4 g·kg^（-1）·d^（-1））and the ursodeoxycholic acid group（0.1 g·kg^（-1）·d^（-1））.The mice of PBC models were induced by intraperitoneal injection of 1% polyI：C.At the same time,the medication groups were given the corresponding dose lavage once a day for 16 weeks.Liver function and peripheral blood content of CD4~＋CD25~＋Foxp3~＋ were detected 24 hours later after the last administration.After getting the liver tissues,we observed its pathology changes with the methods of Hematoxylin-Eosin and Masson staining;and detected the protein expressions of Foxp3,TGF-β1,Smad3 and Smad7 in it with the method of immunohistochemical staining.According to real-time fluorescent quantitative reverse transcription polymerase chain reaction（qRT-PCR）,we detected the mRNA expressions of TGF-β1,TβRI,TβRII,Smad3,Smad7 in the liver tissues.[Results]In the model group,liver function of mice was abnormal compared with the normal group;the liver tissue pathological damages were apparent as well.The content of CD4~＋CD25~＋Foxp3~＋in peripheral blood decreased significantly.The protein expressions of Foxp3,TGF-β1,Smad3 and the mRNA expressions of TGF-β1,TβRI,TβRII,Smad3 increased significantly in the liver tissues（P0.05）.On the contrary,the protein and mRNA expressions of Smad7 reduced.Compared with the model group,Tiaogan Lipi granules could not only improve liver function,increase the content of in peripheral blood,mitigate the pathology damages of liver tissue with reducing collagen deposition,but also could significantly lower the protein expressions of Foxp3,TGF-β1,Smad3 and the mRNA expressions of TGF-β1,TβRI,TβRII,Smad3（P0.05）,and raise the protein and mRNA expressions of Smad7（P0.05）.[Conclusion]Tiaogan Lipi granules has a certain protective effect on PBC mice,its mechanism may be related to regulating TGF-β1/Smads signaling pathway and resulting in suppressing the expression of Foxp3.