益肺汤调控TGF-β1/Smad2通路抗肺纤维化机制研究

Mechanism of Yifei decoction regulating TGF-β1/Smad2 signaling pathway against pulmonary fibrosis

目的:研究益肺汤含药血清对血管紧张素Ⅱ(AngⅡ)诱导的肺成纤维细胞(NIH-3T3)的影响,探讨其抑制肺纤维化的作用机制。方法:采用SPF级C57小鼠灌胃益肺汤,1次/d,连续给药3 d制备含药血清。实验分为正常对照组、模型组、模型+正常血清组、模型+含药血清组,给药48 h后0.1%FBS的培养基饥饿处理24 h,100 ng/μl的AngⅡ诱导造模24 h。免疫组化检测α-平滑肌肌动蛋白(α-SMA);免疫荧光检测Smad2;Western blot检测Ⅰ型胶原蛋白(ColⅠ)、α-SMA、纤维连接蛋白(FN)、Smad2;ELISA检测细胞上清转化生长因子-β1(TGF-β1)的表达情况。结果:Western blot和免疫荧光显示,造模后,α-SMA、FN、ColⅠ、Smad2的蛋白相对表达显著升高,差异具有统计学意义(均P<0.05)。益肺汤含药血清干预48 h后造模,α-SMA、FN、Smad2、ColⅠ的相对量较模型组下降(均P<0.05)。ELISA结果显示,造模后,TGF-β1的相对表达显著升高(P<0.05),益肺汤含药血清干预48 h后,TGF-β1相对量较模型组下降(P<0.05)。结论:益肺汤含药血清对AngⅡ诱导的NIH-3T3有一定影响,可有效减轻肺纤维化,其作用机制可能与调控TGF-β1/Smad2信号通路,降低α-SMA、ColⅠ、FN的表达有关。

Objective:To study the effect of serum containing Yifei decoction on NIH-3T3 induced by angiotensin Ⅱ(Ang Ⅱ),and to explore its mechanism of inhibiting pulmonary fibrosis.Methods:SPF C57 mice were given Yifei decoction once a day for 3 days to prepare medicated serum.The experiment was divided into normal control group,model group,model+normal serum group and model+medicated serum group.After 48 hours of administration,0.1%FBS was starved for 24 hours,and 100 ng/μl Ang Ⅱ was used to induce the model for 24 hours.α-smooth muscle actin(α-SMA) was detected by immunohistochemistry.Smad2 was detected by immunofluorescence.Collagen Ⅰ(Col Ⅰ),α-SMA,fibronectin(FN) and Smad2 were detected by Western blot.The expression of transforming growth factor-β1(TGF-β1) in cell supernatant was detected by ELISA.Results:Western blot and immunofluorescence showed that the relative expression of α-SMA,FN,Col Ⅰ and Smad2 increased significantly after modeling,with statistical significance(all P<0.05).After 48 hours of intervention with medicated serum,the relative amounts of α-SMA,FN,Smad2 and Col Ⅰ decreased locally compared with the model group(all P<0.05).The results of ELISA showed that the relative expression of TGF-β1 increased significantly after modeling(P<0.05),the relative amount of TGF-β1 decreased locally compared with the model group after 48 hours of intervention with drug-containing serum(P<0.05).Conclusion:The serum containing Yifei decoction has certain effect on NIH-3T3 induced by Ang Ⅱ,which can effectively alleviate pulmonary fibrosis.This mechanism may be related to the regulation of TGF-β1/Smad2 signaling pathway and the reduction of α-SMA,Col Ⅰ and FN expression.