益气养阴活血通络法干预肺成纤维细胞TGF-β_1/Smads信号传导通路的机制

Intervention Mechanism of Method of Supplementing Qi,Nourishing Yin,Activating Blood Circulation and Dredging Collaterals on TGF-β_1/Smads Signaling Pathway in Lung Fibroblasts

目的:探讨益气养阴活血通络法调控肺成纤维细胞转化生长因子-β_1(TGF-β_1)/Smads信号传导通路机制。方法:SPF级雄性Wistar大鼠30只,采用随机数字法,将30只大鼠随机分为3组,中药含药血清组,西药含药血清组,正常血清组,第4天末次给药后提取含药血清。气管内注入博来霉素造肺纤维化大鼠动物模型,采用胰蛋白酶消化液消化法培养肺成纤维细胞。细胞分为模型组,中药组,西药组,抑制剂组。免疫荧光法鉴定肺成纤维细胞,并分别于第24,48,72 h取材,采用逆转录聚合酶链式反应(RT-PCR)检测TGF-β_1,Smad2,Smad4,Smad7 mRNA的表达及流式细胞仪检测细胞凋亡率。结果:第24,48,72 h检测结果显示,与模型组比较,中药组、西药组、抑制剂组细胞内TGF-β_1,Smad2,Smad4 mRNA表达水平明显下调(P<0.01);第24 h西药组TGF-β_1mRNA表达水平明显低于中药组(P<0.05);第48,72 h检测结果均显示与中药组比较,西药组、抑制剂组TGF-β_1mRNA表达水平均明显下调(P<0.05,P<0.01);第24,48 h检测结果显示与模型组比较,中药、西药、抑制剂各组细胞内Smad2 mRNA表达水平下调明显(P<0.01);中药组、西药组、抑制剂组3者表达水平相近,无统计学意义。第72 h检测结果显示与中药组比较,西药组、抑制剂组Smad2 mRNA表达水平均明显下调(P<0.05);第24,48,72 h检测结果均显示与中药组比较,西药组、抑制剂组Smad7 mRNA表达水平均明显下调(P<0.01)。第24,48,72 h检测结果均显示与模型组比较,中药组、西药组与抑制剂组凋亡率明显上调(P<0.01);中药组细胞凋亡率明显高于抑制剂组(P<0.01)。结论:益气养阴活血通络法可能通过下调肺成纤维细胞TGF-β_1,Smad2,Smad4含量和上调Smad7含量调控TGF-β_1/Smads信号通路,从而延缓特发性肺纤维化(IPF)进展。

Objective： To discuss the mechanism of the method of supplementing Qi,nourishing Yin,activating blood circulation and dredging collaterals in regulating transforming growth factor-β1（ TGF-β1）/Smads signaling pathway in lung fibroblasts. Method： A total of 30 SPF male rats were divided into three groups by the method of random number table,namely traditional Chinese medicine（ TCM） serum group,western medicine serum group and normal serum group. Serums were extracted at the end of the fourth day after administration.Bleomycin was intratracheally injected to make animal model of pulmonary fibrosis in rats,and the lung fibroblasts were cultured by trypsin digestion method. The cells were divided into model group,TCM group,western medicine group and inhibitor group. Lung fibroblasts were identified by immunofluorescence,and materials were collected at24,48,72 h. Reverse transcription-polymerase chain reaction was used to detect the mRNA expressions of TGF-β1,Smad2,Smad4,and Smad7,and flow cytometry was used to detect apoptosis rate. Result： The results of24,48,72 h showed that compared with model group,the mRNA expressions of TGF-β1,Smad2,and Smad4 in the TCM group,western medicine group and inhibitor group were obviously lower（ P〈0. 01）. The mRNA expressions of TGF-β1in 24 h western medicine group was lower than that of TCM group（ P〈0. 05）. The results of48,72 h showed that compared with the TCM group,the mRNA expressions of TGF-β1in the western medicine group and the inhibitor group were all obviously lower（ P〈0. 05,P〈0. 01）. The results of 24,48 h showed that compared with the model group,the Smad2 mRNA expression in TCM group,western medicine group and inhibitor group were obviously lower（ P〈0. 01）. The expressions of TCM group,western medicine group and inhibitor group were similar,with no statistical significance. The results of 72 h showed that compared with the TCM group,the Smad2 mRNA expressions in the western medicine group and the inhibitor group were all obviously lower（ P〈0. 05）. The results of 24,48,72 h showed that compared with the TCM group,the Smad7 mRNA expressions in the western medicine group and the inhibitor group were all obviously lower（ P〈0. 01）. The results of 24,48,72 h showed that compared with the model group,the apoptosis rates of TCM group,western medicine group and inhibitor group were obviously increased（ P〈0. 01）. The apoptosis rate of TCM group was higher than that of inhibitor group（ P〈0. 01）. Conclusion： The method of supplementing Qi,nourishing Yin,activating blood circulation and dredging collaterals may delay the progress of idiopathic pulmonary fibrosis by decreasing the content of TGF-β1,Smad2 and Smad4,increasing the content of Smad7 and regulating the TGF-β1/Smads signaling pathway.