补肾柔肝方对肝纤维化大鼠肝组织TGF-β_1-Smads信号通路的干预作用

Intervention of Bushen Rougan Fang on TGF-β_1-Smads signaling pathway in liver tissue of rats with hepatic fibrosis

目的探讨补肾柔肝方对肝纤维化大鼠肝组织TGF-β_1-Smads信号通路的影响。方法将53只Wistar大鼠随机分成空白组8只、模型组15只、补肾柔肝方组15只和秋水仙碱组15只。除空白组外,其余组均首次在背部皮下注射纯四氯化碳5 m L/kg,3 d后注射40%CCl4花生油3 m L/kg,每周注射2次,连续8周。第9周开始,补肾柔肝方组和秋水仙碱组分别给予补肾柔肝方流浸膏5 m L/(kg·d)、秋水仙碱溶液5 m L/(kg·d)灌胃,空白组和模型组给予等体积生理盐水灌胃,共8周。然后取各组大鼠肝组织,分别采用免疫组化法和逆转录多聚酶链式反应检测肝组织中TβRⅠ、TβRⅡ蛋白,Smad2和Smad3磷酸化水平及TGF-β_1、TβRⅠ、TβRⅡmRNA的表达情况。结果与空白组比较,模型组肝组织中TβRⅠ、TβRⅡ蛋白及TGF-β_1、TβRI、TβRⅡmRNA表达水平均显著升高(P均<0.05),Smad2和Smad3磷酸化水平无明显变化(P均>0.05);与模型组比较,补肾柔肝方组和秋水仙碱组TβRⅠ、TβRⅡ蛋白以及TGF-β_1、TβRⅠ、TβRⅡmRNA表达水平均显著降低(P均<0.05),Smad2和Smad3磷酸化水平无明显变化(P均>0.05)。结论在TGF-β_1-Smads信号通路中,补肾柔肝方主要是通过抑制TβRⅠ、TβRⅡ表达,无法磷酸化的Smad2和Smad3失去活性,从而阻断信号通路传导发挥抗纤维化的作用。

Objective It is to investigate the effect of Bushen Rougan Fang（ BSRG） on TGF-1-Smads signaling pathway in liver tissue of rats with hepatic fibrosis. Methods Fifty-three Wistar rats were randomly divided into blank group（ 8 rats）,model group（ 15 rats）,BSRG group（ 15 rats） and colchicine group（ 15 rats）. Except for the blank group,the other groups were injected with pure carbon tetrachloride 5 m L/kg subcutaneously for the first time,and injected with 40%CCl4 peanut oil 3 m L/kg after 3 days,2 times a week for 8 weeks. At the beginning of the ninth weeks,the animals in the BSRG group and colchicine group were given intragastric administration of BSRG extract 5 m L/（ kg·d） and colchicine solution 5 m L/（ kg·d） respectively,but the ones in the blank group and model group were given the same volume of normal saline,the treatment lasted for 8 weeks. Then the rat liver tissues were taken from each group. The TβRI,TβRII protein,the phosphorylation level of Smad2 and Smad3,and the expression of TGF-β1,TβRI,TβRII mRNA in liver tissues were detected by immunohistochemistry and reverse transcription polymerase chain reaction（ RT-PCR）,respectively. Results Compared with the blank group,the expression levels of TβRⅠ and TβRⅡ protein,TGF-β1,TβRⅠ and TβRⅡ mRNA in the liver tissues of the model group were significantly increased（ all P〈0. 05）,but there was no significant change in the phosphorylation level of Smad2 and Smad3（ all P〈0. 05）. Compared with the model group,the expression levels of TβRⅠ and TβRⅡ protein,TGF-β1,TβRⅠ and TβRⅡ mRNA were significantly decreased in the BSRG group and colchicine group（ all P〈0. 05）,but there was no significant change in the phosphorylation level of Smad2 and Smad3（ all P〈0. 05）. Conclusion In the TGF-β1-Smads signaling pathway,BSRG can makes Smad2 and Smad3 which can not be phosphorylated inactive by inhibit the expression of TβRⅠ and TβRⅡ,thus blocking the transmission of the signal pathway,and play the role of anti fibrosis.