三七皂苷对TGF-β_1诱导的大鼠肾小管上皮细胞EMT的影响

Effect of Panax Notoginseng Saponins on EMT of Rat Renal Proximal Tubular Epithelial Cells Induced by TGF-β_1

目的:研究三七皂苷(PNS)对转化生长因子-β_1(TGF-β_1)诱导的大鼠肾小管细胞上皮-间充质转化(EMT)的干预作用,并从沉默信息调节因子2相关酶1(SIRT1)/TGF-β_1/Smad通路分析其可能机制。方法:在DMEM培养基中,用10%胎牛血清培养大鼠肾小管上皮细胞(NRK-52E),分为正常组,TGF-β_1组(5μg·L^(-1)),白藜芦醇组(50 mg·L^(-1)),SIRT1阻断剂EX527组(10μmol·L^(-1)),PNS组(100 mg·L^(-1)),EX527+PNS组(EX527 10μmol·L^(-1),PNS 100 mg·L^(-1)),48 h后收集细胞;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测SIRT1,α-平滑肌肌动蛋白(α-SMA),E-钙黏附蛋白(E-cadherin),TGF-β_1,Smad3,Smad4 mRNA表达;蛋白免疫印迹法(Western blot)检测SIRT1,α-SMA,E-cadherin,TGF-β_1蛋白表达。结果:与正常组比较,TGF-β_1组α-SMA mRNA及蛋白表达明显增多(P <0. 05),E-cadherin表达显著减少(P <0. 01);与TGF-β_1组比较,PNS,白藜芦醇组,E-cadherin mRNA及蛋白表达均显著增加(P <0. 01),α-SMA表达显著减少(P <0. 01),EMT发生被抑制,同时,SIRT1表达显著增加(P <0. 01),TGF-β_1,Smad3,Smad4表达显著降低(P <0. 01)。在SIRT1阻断剂EX527的干预下,PNS则不能发挥明显抑制作用。结论:PNS可以阻止TGF-β_1诱导的肾小管上皮细胞EMT的发生,其机制可能是通过激活SIRT1进而抑制TGF-β_1/Smad通路实现的。

Objective: To investigate the intervention effect of panax notoginseng saponins( PNS) on epithelial-mesenchymal transition( EMT) of rat renal tubular epithelial cells( NRK-52 E) induced by transforming growth factor-β1( TGF-β1),and analyze the mechanism based on the silent information regulation 2 homolog 1( SIRT1)/TGF-β1/Smad signaling pathway. Method: NRK-52 E were cultured in DMEM medium with 10% fetal bovine serum,and divided into normal control group,TGF-β1 group( 5 μg·L-1),resveratrol( RSV) group( 50 mg·L-1),EX527 group( 10 μmol·L-1),Panax notoginseng saponins( PNS) group( 100 mg·L-1),and EX527 + PNS group( 10 μmol·L-1+ 100 mg·L-1). Then cells were collected after drug intervention for 48 h. The expressions of α-SMA,E-cadherin,SIRT1,TGF-β1,Smad3,Smad4 mRNA in each group were detected by Real-time PCR. The protein expressions of α-SMA,E-cadherin,SIRT1 and TGF-β1 were detected by Western blot. Result: Compared with normal group,mRNA and protein expressions of α-SMA increased obviously( P <0. 05),but E-cadherin decreased significantly( P < 0. 01) in TGF-β1 group. Compared with TGF-β1 group,mRNA and protein expressions of α-SMA decreased significantly( P < 0. 01),while E-cadherin increased( P <0. 01) in resveratrol and PNS groups,and EMT was inhibited. Meanwhile,mRNA and protein expressions of SIRT1 increased significantly( P < 0. 01),while mRNA expressions of TGF-β1,Smad3,and Smad4 decreased( P < 0. 01). Under the intervention of SIRT1 blocker EX527,PNS could not play a significant inhibitory effect on the cells. Conclusion: PNS can prevent the occurrence of EMT of renal tubular epithelial cells induced by TGF-β1,and the mechanism may be related to active SIRT1 to inhibit TGF-β1/Smad pathway.