罗格列酮对肾小管上皮细胞间质转化的影响

Effect of Rosiglitazone on the Epithelial-mesenchymal Transition Process of Renal Tubular Epithelial Cell

目的探讨罗格列酮对转化生长因子-β(TGF-β)诱导的肾小管上皮细胞间质转化(EMT)过程的影响。方法体外培养人肾小管上皮细胞HK-2,并给予不同浓度TGF-β及罗格列酮处理,观察HK-2形态学变化,利用免疫印迹检测过氧化物酶体增殖激活物受体γ(PPARγ)、SMAD家族成员2/3、钙粘附蛋白-E(E-cadherin)及波形蛋白(Vimentin)水平变化,通过实时荧光定量聚合酶链反应(PCR)术检测PPARγ、E-cadherin、Vimentin、锌指转录因子Snail及Slug的mRNA水平变化,并利用双荧光素酶报告基因检测TGF-β及罗格列酮对E-cadherin启动子活性的影响。结果 TGF-β可诱导HK-2细胞发生伪足增多变长、细胞间隙变大等EMT样形态学变化,进而激活TGF-β下游的SMAD2/3信号通路,导致上皮细胞标志E-cadherin表达明显减少,伴有间质细胞标志的Vimentin表达增高,转录因子Snail及Slug的mRNA水平分别升高6倍以上,伴随E-cadherin启动子活性下降70%。罗格列酮可以显著抑制上述TGF-β诱导的EMT过程,表现为HK-2细胞足减少变短,细胞间隙变小等,同时伴有E-cadherin表达增加,Vimentin表达降低,Snail及Slug的mRNA水平明显降低,E-cadherin启动子活性恢复到对照组水平。结论罗格列酮可通过激活PPARγ促进E-cadherin转录活性及蛋白表达,拮抗TGF-β诱导的EMT过程。

Objective To investigate the effect of rosiglitazone on the epithelial-mesenchymal transition process of renal tubular epithelial cell induced by transforming growth factor-β（TGF-β）. Methods The human renal tubular epithelial cell line HK-2 was cultured in vitro and treated with TGF-β at the presence or absence of rosiglitazone, and the morphological changes of HK-2 cells were observed. Then PPARγ,SMAD family number 2/ 3,E-cadherin and Vimentin were detected by western blot, and the mRNA expression of PPARγ, E-cadherin, Vimentin, zinc finger transcription factor Snail and Slug were measured by realtime quantitative PCR. Finally,dual luciferase reporter assay was performed to detect the effect of TGF-β and rosiglitazone on the tran-scription activity of E-cadherin promoter. Results TGF-β could induce the EMT process of HK-2, including morphological changes such as longer pseudopod and broader cell space. TGF-β also activated the SMAD2/ 3 signaling pathway, with expression changes of E-cadherin and Vimentin. Rosiglitazone reversed the morphological changes of HK-2 cells induced by TGF-β, with shor-ter pseudopod and narrower cell space. Rosiglitazone also inhibited the mRNA and protein expressions of Vimentin, and restored the mRNA and protein expression of E-cadherin by up-regulating PPARγ. The activity of E-cadherin promoter was enhanced under the treatment of rosiglitazone. Conclusion Rosiglitazone can antagonize the EMT process of renal tubular epithelial cell induced by TGF-β, through the activation of PPARγ and stimulating transcription and protein expression of E-cadherin.