基于肝窦毛细血管化探讨清毒调肝方治疗肝纤维化的作用机制

To explore mechanism of Qingdu Tiaogan decoction in treating hepatic fibrosis based on sinusoid capillarization

目的探讨清毒调肝方通过改善肝窦毛细血管化在治疗肝纤维化中的作用机制。方法选取60只SD雄性大鼠,10只作为空白组,其余50只通过尾静脉注射刀豆蛋白A(ConA)建立肝纤维化模型。造模成功后,将模型大鼠随机分为模型对照组、索拉非尼组和清毒调肝方低、中、高剂量组,每组10只。空白组和模型对照组不予干预,其余各组大鼠分别灌胃索拉非尼或不同剂量的清毒调肝方,每天灌胃1次,连续5周。通过光镜观察大鼠肝组织形态学变化,扫描电镜观察肝窦内皮细胞窗孔变化,并通过酶联免疫吸附法(ELISA)检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和免疫球蛋白G(IgG)水平,免疫组织化学(IHC)检测肝组织中层粘连蛋白(LN)、Ⅳ型胶原(Col-Ⅳ)表达,免疫印迹(WB)技术检测肝组织中血小板-内皮细胞黏附分子(CD31)、血管性血友病因子(vWF)表达,实时荧光定量聚合酶链式反应(qPCR)检测血管内皮生长因子(VEGF)mRNA表达。结果与空白组比较,模型对照组大鼠肝组织损伤严重,表现为肝细胞坏死、空泡形成、纤维组织增生、肝窦内皮细胞窗孔减少及基底膜增加,血清ALT、AST、IgG水平和LN、Col-Ⅳ、CD31、vWF、VEGFmRNA表达显著升高(P<0.05)。与模型对照组比较,清毒调肝方低、中、高剂量组均能减轻肝组织损伤,干预肝窦内皮细胞去窗孔化,显著降低ALT、AST、IgG水平和LN、Col-Ⅳ、CD31、vWF、VEGFmRNA表达(P<0.05)。结论清毒调肝方可以减轻肝损伤,改善肝功能,提升免疫水平,干预肝窦内皮细胞去窗孔化和内皮下基底膜沉积,下调LN、Col-Ⅳ、CD31、vWF和VEGFmRNA表达,从而抑制血管生成,改善肝窦毛细血管化,减少肝纤维化发生。

ObjectiveTo explore the mechanism of Qingdu Tiaogan decoction in treating liver fibrosis by improving hepatic sinusoid capillarization.Methods60 male SD rats were selected,with 10 as the blank group and the remaining 50 rats established a liver fibrosis model by tail vein injection of ConA.After successful modeling,the model rats were randomly divided into a model control group,a sorafenib group,and low,medium,and high dose groups of Qingdu Tiaogan decoction,with 10 rats in each group.The blank group and model control group were not intervened,while the other groups of rats were orally administered sorafenib or different doses of Qingdu Tiaogan decoction once a day for 5 consecutive weeks.By observing the morphological changes of rat liver tissue under light microscopy,scanning electron microscopy was used to observe the changes in the hepatic sinusoidal endothelial cell window,and enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and immunoglobulin G(IgG).Immunohistochemistry(IHC)was used to detect the expression of laminin(LN)and type IV collagen(Col IV)in liver tissue.Immunoblotting(WB)technology was used to detect the expression of platelet endothelial cell adhesion molecule(CD31)and von Willebrand factor(vWF)in liver tissue,and real-time fluorescence quantitative polymerase chain reaction(qPCR)was used to detect the expression of vascular endothelial growth factor(VEGF)mRNA.ResultsCompared with the blank group,the model control group showed severe liver tissue damage in rats,manifested as hepatocyte necrosis,vacuolar formation,fibrous tissue proliferation,reduced hepatic sinusoidal endothelial cell fenestration,and increased basement membrane.Serum ALT,AST,IgG levels,and LN,Col IV,CD31,vWF,VEGF mRNA expression were significantly increased(P<0.05).Compared with the model control group,the low,medium,and high dose groups of Qingdu Tiaogan decoction can alleviate liver tissue damage,intervene in the de fenestration of liver sinusoidal endothelial cells,significantly reduce ALT,AST,IgG levels,and LN,Col IV,CD31,vWF,VEGF mRNA expression(P<0.05).ConclusionQingdu Tiaogan decoction can alleviate liver injury,improve liver function,enhance immune levels,intervene in the de fenestration of hepatic sinusoidal endothelial cells and deposition of subendothelial basement membrane,downregulate the expression of LN,Col IV,CD31,vWF,and VEGF mRNA,thereby inhibiting angiogenesis,improving hepatic sinusoidal capillarization,and reducing the occurrence of liver fibrosis.