槲皮素对转化生长因子-β_1诱导的肾小球足细胞转分化抑制作用的实验研究

Experimental study of quercetin's inhibiting effect on podocyte EMT induced by TGF-β_1

目的研究槲皮素(Qu)不同给药时间对转化生长因子-β_1(TGF-β_1)诱导的肾小球足细胞向间充质细胞转分化(EMT)的影响。方法取经过鉴定的体外原代培养的足细胞,随机分为对照组、模型组、Qu先干预组、Qu同时干预组和Qu后干预组,共5组。对照组常规培养;模型组采用5 ng/m L TGF-β_1诱导48 h;Qu先干预组采用50μmol/L Qu干预24 h后,再用5 ng/m L TGF-β_1继续诱导24 h;Qu同时干预组采用50μmol/LQu和5 ng/m L TGF-β_1同时干预48 h;Qu后干预组先用5 ng/m L TGF-β_1干预24 h,再用50μmol/L Qu干预24 h。将上述5组细胞分别采用Western blot、反转录聚合酶链反应(RT-PCR)技术检测足细胞裂孔膜蛋白(Nephrin)、Wilms瘤抑癌因子-1(WT-1)和足细胞EMT标志物波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)的蛋白及mRNA表达量,并进行组间比较。结果 1)蛋白表达量:与对照组比较,模型组足细胞Nephrin,WT-1的蛋白表达量明显降低,Vimentin,α-SMA的蛋白表达量明显升高(P<0.01);与模型组比较,3种不同干预方法中,Nephrin的蛋白表达量以Qu后干预组最高,WT-1的蛋白表达量以Qu同时干预组最高,Vimentin的蛋白表达量以Qu先干预组最低,α-SMA的蛋白表达量以Qu先干预组最低(P<0.01)。2)mRNA表达量:与对照组比较,模型组足细胞Nephrin mRNA和WT-1 mRNA的表达量明显降低,Vimentin mRNA和α-SMA mRNA的表达量明显升高(P<0.01);与模型组比较,3种不同干预方法中,Nephrin mRNA的表达量以Qu同时干预组最高(P<0.05),WT-1 mRNA的表达量以Qu先干预组最高(P<0.01),Vimentin mRNA和α-SMA mRNA表达量均以Qu先干预组最低(P<0.01)。结论 Qu能够抑制T GF-β1诱导的足细胞EMT,且Qu先干预组能够更好地抑制TGF-β_1诱导的足细胞EMT,其作用机制可能与抑制TGF-β信号转导通路中的相关信号分子、维持足细胞生理结构的完整性从而保护肾小球滤过屏障有关。

Objective To investigate the quercetin＇s effect on podocytes epithelial mesenchymal transition（ EMT） induced by transforming growth factor-β1（ TGF-β1） at different administration times. Methods The p rimary cultured podocytes in vitro by authenticated were randomly divided into 5 groups： the normal group（ NG）,the model group（ MG）,the pre-administration time group（ PATG）,the same administration time group（ SATG）and the later-administration time group（ LATG）. The podocytes in NG were routinely cultured without any drug i ntervention for 48 h.The MG was only intervened with TGF-β1（ 5 ng/m L） for 48 h.The PATG was incubated with quercetin（ 50 μmol/L） for 24 h first and then intervened with TGF-β1（ 5 ng/m L） for 24 h.The SATG was simultaneously intervened with quercetin（ 50 μmol/L） and TGF-β1（ 5 ng/m L） for 48 h.The LATG was intervened with TGF-β1（ 5 ng/m L） for 24 h and then incubated with quercetin（ 50 μmol/L） for 24 h.Nephrin,Wilms＇ tumor suppressor-1（ WT-1）,Viminten and α-smooth muscle actin（ α-SMA） protein and mRNA levels in podocytes were measured by Western blot and RT-PCR,while comparison was made among groups.Results 1） The protein level：when compared with NG,the expressions of Nephrin and WT-1 in MG decreased significantly（ both P〈0.01）,while the expressions of Viminten and α-SMA increased significantly（ both P 0. 01）; when compared with MG,protein expressions of the groups at different administration time were as follows：（1） The highest expression of Nephrin was in the LATG（ P〈0.01）;（2） The highest expression of WT-1 was in the SATG（ P〈0.01）;（3） The l owest expression of Viminten and α-SMA were in the PATG（ both P〈0.01）.2） The mRNA level： compared with NG,the mRNA expressions of Nephrin and WT-1 in MG were significantly decreased（ both P〈0.01）,while the mRNA expressions of Viminten and α-SMA were significantly increased（ both P〈0.01）; when compared with MG,the mRNA expressions of groups at different administration times were as follows：（1） The highest mRNA expression of Nephrin was in the SATG（ P〈0.05）;（2） The highest mRNA expression of WT-1 was in the PATG（ P〈0.01）;（3）The lowest mRNA expressions of Viminten and α-SMA were in the PATG（ both P〈0.01）.Conclusion Quercetin can inhibit podocytes EMT induced by TGF-β1 and pre-administration of it has much more obvious inhibiting effect.The mechanism of action may be related to the i nhibition of the TGF-β signaling pathway and the maintenance of integrity of the podocytes＇ physiological structure for protecting glomerular filtration barrier.