复原活血汤影响TGF-β1/Smad3及TGF-β1/p38通路抗小鼠肝纤维化的作用及机制

Effect and its mechanism of Fuyuan Huoxue Decoction on TGF-β1/Smad3 and TGF-β1/p38 pathway against liver fibrosis in mice

目的:探讨复原活血汤通过转化生长因子-β1(TGF-β1)/Smad3及TGF-β1/p38通路抗小鼠肝纤维化的作用及机制。方法:24只C57BL/6雄性小鼠随机分为空白组、复原活血汤组、模型组、CCl4+复原活血汤组。模型组和CCl4+复原活血汤组小鼠腹腔注射含25%CCl4的玉米油溶液,每周2次,连续6周。空白组、复原活血汤组腹腔注射等量玉米油。从造模第3周开始,复原活血汤组、CCl4+复原活血汤组每天以复原活血汤灌胃,共4周。后进行HE染色、天狼猩红染色,羟脯氨酸(Hyp)含量检测,血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平检测,以评价肝组织炎症损伤和胶原沉积变化;Western Blot及免疫组化法检测α-平滑肌激动蛋白(α-SMA)、Ⅰ型胶原(Collagen-Ⅰ)蛋白表达;Western Blot检测TGF-β1、p38、p-p38、JNK、p-JNK、Smad3、p-Smad3蛋白表达。结果:与空白组比较,模型组纤维沉积加重,血清中ALT、AST含量显著升高(P<0.01)。与模型组比较,CCl4+复原活血汤组小鼠肝细胞变性、坏死及纤维化组织增生程度均明显减轻,血清ALT、AST、Hyp含量显著降低(P<0.05),肝组织中α-SMA、Collagen-Ⅰ、TGF-β1、p-p38、p-Smad3、p-JNK蛋白水平均显著降低(P<0.01)。结论:复原活血汤具有较好的抗CCl4诱导小鼠肝纤维化的作用,其作用机制可能与下调TGF-β1/Smad3及TGF-β1/p38通路抑制肝星状细胞活化相关。

Objective:To explore the effect and mechanism of Fuyuan Huoxue Decoction(FYHX)on the effect of TGF-β1/Smad3 and TGF-β1/p38 pathway against liver fibrosis in mice.Methods:Twenty-four C57BL/6 male mice were divided into four groups:blank group,FYHX group,model group and CCl4+FYHX group.Mice in the model group and CCl4+FYHX group were intraperitoneally injected with 25%CCl4 corn oil solution,twice a week for 6 consecutive weeks.The blank group and the FYHX group were intraperitoneally injected with the same amount of corn oil.Starting from the third week of modeling,the FYHX group and the CCl4+FYHX group were given FYHX by gavage every day for 4 weeks.HE staining,Sirius red staining,hydroxyproline(Hyp)content detection,serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)levels were evaluated.Western Blot and immunohistochemical methods were used to detectα-smooth muscle agonist(α-SMA)and type I collagen(Collagen-I)protein expression;Western Blot was used to detect TGF-β1,p38,p-p38,JNK,p-JNK,Smad3 and p-Smad 3 protein expression.Results:Compared with the blank group,fiber deposition in model group increased,and ALT and AST increased significantly(P<0.01).Compared with the model group,the degree of hepatocyte degeneration,necrosis,and fibrotic tissue proliferation in the CCl4+FYHX group was reduced,and the serum ALT,AST and Hyp content were significantly reduced(P<0.05);α-SMA,Collagen-I,TGF-β1,p-p38,p-Smad3 and p-JNK protein levels in liver tissue were significantly reduced(P<0.01).Conclusion:FYHX has good anti-CCl4-induced liver fibrosis in mice,and its mechanism of action may be related to the inhibition of hepatic stellate cell activation by TGF-β1/Smad3 and TGF-β1/p38 pathways.