缬沙坦对人肾近端小管上皮细胞TGF-β/Smad信号途径的影响

目的观察缬沙坦对TGF-β刺激人肾近端小管上皮细胞表达Smad的影响。方法以人肾小管上皮细胞(HKC)为对象,分为正常对照组;TGF-β1刺激组;缬沙坦组。Western blot检测p-Smad2/3、smad2/3、Smad7以及细胞上清ColⅠ蛋白含量;RT-PCR检测Smad2 mRNA和Smad7 mRNA水平。MTT法检测刺激组及缬沙坦组在不同时间、不同药物浓度对细胞增殖状态的影响。结果①Western blot显示,与TGF-β1刺激组相比,缬沙坦使Smad2/3、p-Smad2/3蛋白的表达下降,ColⅠ分泌显示相同趋势,而Smad7蛋白的表达没有变化。②半定量RT-PCR显示,与TGF-β1刺激组相比,缬沙坦组Smad2 mRNA降低,Smad7 mRNA无变化。③MTT法检测显示不同浓度缬沙坦对TGF-β1刺激所引起的细胞增殖受抑制现象均有改善,并且随药物浓度的增加该作用增强。结论提示缬沙坦的肾脏保护作用可能部分是通过抑制TGF-β/Smads信号途径实现的。

Effect of valsartan on expression of TGF-β/Smads signaling pathway in human kidney proximal tubular epithelial cell

Aim To observe the effect of valsartan on expression of Smads of HKCs stimulated by TGF-β1.Methods HKCs were divided into three groups:control group,TGF-β1 group and valsartan group.Total protein was firstly abstracted at 30 min after stimulation to detect p-Smad2/3 protein by Western blot.Total RNA and protein were abstracted at 48 hours after stimulation.The expressions of Smad2/3,Smad7 were measured by Western blot.Smad2mRNA and Smad7mRNA were measured by RT-PCR.Meanwhile, The protein synthesis of Col Ⅰ in the supernatants of the HKCs was detected by Western blot.MTT assay was used to investigate the effect of valsartan on proliferation of HKCs stimulated by 5 μg·L-1 TGF-β1 at different time and different drug concentration.Results Western blot analysis indicated that TGF-β1 stimulation could increase the expression of Smad2/3 and activate phosphorylation of Smad2/3 at 48 hour.At the same time,Smad7 decreased.Valsartan could reduce the level of Smad2/3 and p-Smad2/3 but it did not have any effect on the expression of Smad7 protein.RT-PCR analysis also showed that the expression of Smad2mRNA or Smad7mRNA was higher after TGF-β1 stimulation than control group,while valsartan could down-regulate the expression of Smad2mRNA.There was no difference of the expression of Smad7mRNA between the TGF-β1 stimulation group and valsartan treatment group.Valsartan could also greatly ameliorate the secretion of Col Ⅰ in the supernatants of the HKCs stimulated by TGF-β1 detected by Western blot.Compared with control group,TGF-β1 supressed the proliferation of HKCs,While 1,10 and 100 μmol·L-1 valsartan ameliorated it and the effect was dependent of dose.Conclusion Valsartan has some renal protective effects on renal interstitial fibrosis,partly through inhibiting TGF-β/Smads signaling pathway.