藿朴夏苓汤对CCl4诱导肝纤维化模型小鼠的作用及机制研究

Study on the Effect and Mechanism of Huo Pu Xia Ling Tang on CCl4-Induced Liver Fibrotic Model Mice

目的 基于TGF-β/Smad通路探讨藿朴夏苓汤(HPXLT)对四氯化碳(CCl4)诱导肝纤维化模型小鼠的保护作用及机制。方法 采用连续12周腹腔注射CCl4制备肝纤维化小鼠模型。将C57BL/6J小鼠随机分为对照组、模型组、HPXLT高剂量组(1.0 g·kg^(-1))、HPXLT低剂量组(0.5 g·kg^(-1)),每组8只。每日1次,连续灌胃给药30 d。采用HE染色法观察小鼠肝脏组织病理形态变化;Masson染色法观察小鼠肝脏组织纤维化情况;全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平;ELISA法测定血清透明质酸(HA)和层粘连蛋白(LN)含量;qRT-PCR法检测肝脏组织TGF-β、Smad2、Smad3、α-SMA、COL1A1、FN1、TIMP-1、MMP-2、MMP-9、Smad7 mRNA表达水平。结果 与对照组比较,模型组小鼠的血清肝功指标ALT、AST水平和纤维化指标HA、LN水平均显著升高(P<0.01);肝组织病理评分及纤维化水平显著升高(P<0.01);肝组织的纤维化基因TGF-β、Smad2、Smad3、α-SMA、COL1A1、FN1、TIMP-1 mRNA表达显著上调(P<0.01),而纤维化抑制基因MMP-9、Smad7 mRNA表达则显著下调(P<0.01)。与模型组比较,HPXLT高、低剂量组小鼠的血清ALT、AST及HA、LN水平均明显降低(P<0.05,P<0.01),肝组织病理评分及纤维化水平明显降低(P<0.05,P<0.01);HPXLT高剂量组小鼠肝组织TGF-β、Smad2、Smad3、α-SMA、COL1A1、FN1、TIMP-1 mRNA表达明显下调(P<0.05,P<0.01),MMP-2、MMP-9、Smad7 mRNA表达则明显上调(P<0.05)。结论 藿朴夏苓汤能够改善CCl4诱导的肝纤维化小鼠的血清肝功能及纤维化水平,减少肝脏胶原沉积,改善肝纤维化病理损伤,其机制可能与抑制TGF-β/Smad通路,下调肝脏胶原合成相关基因的表达有关。

Objective To explore the protective effect and mechanism of Huo Pu Xia Ling Tang(HPXLT)on CCl4-induced liver fibrotic model mice based on TGF-β/Smad pathway.Methods A mouse model of liver fibrosis was prepared by continuous intraperitoneal injection of CCl4for 12 weeks.The mice were randomly divided into the control group,the model group,the HPXLT high-dose group(1.0 g·kg^(-1))and the low-dose group(0.5 g·kg^(-1)).All the groups were administered by gavage once daily for 30 days.The histopathological changes in the liver were observed by HE staining;the liver fibrosis was observed by Masson staining;serum alanine aminotransferase(ALT)and aspartate transaminase(AST)levels were detected by automatic biochemical analyzer;serum hyaluronic acid(HA)and laminin(LN)levels were detected by ELISA;mRNA expression levels of TGF-β,Smad2,Smad3,α-SMA,COL1A1,FN1,TIMP-1,MMP-2,MMP-9 and Smad7 were detected by qRT-PCR.Results Compared with the control group,the serum liver function indexes ALT and AST levels and fibrosis indexes HA and LN levels were significantly increased in the model mice(P<0.01);the liver histopathology score and fibrosis level were significantly increased(P<0.01);the mRNA expressions of the fibrosis genes TGF-β,Smad2,Smad3,α-SMA,COL1A1,FN1,TIMP-1 were significantly up-regulated(P<0.01),while mRNA expressions of fibrosis inhibitory genes MMP-9,Smad7 were significantly down-regulated(P<0.01).Compared with the model group,the serum ALT,AST,HA and LN levels of mice in the high-and low-dose groups of HPXLT were significantly reduced(P<0.05,P<0.01),and the liver histopathological score and fibrosis level were significantly reduced(P<0.05,P<0.01);the mRNA expression levels of TGF-β,Smad2,Smad3,α-SMA,COL1A1,FN1,TIMP-1 in liver tissue of mice in the HPXLT high-dose group were significantly down-regulated(P<0.05,P<0.01),while the m RNA expressions of MMP-2,MMP-9 and Smad7 were significantly up-regulated(P<0.05).Conclusion HPXLT can improve the serum liver function and fibrosis level of CCl4-induced liver fibrosis mice,reduce liver collagen deposition and improve the pathological injury of liver fibrosis,and the mechanism may be related to the inhibition of TGF-β/Smad pathway and the decrease on the expression of genes related to liver collagen synthesis.