p38MAPK信号通路与醛糖还原酶对转化生长因子β1诱导人系膜细胞纤连蛋白表达的影响

Effects of p38 MAPK signaling pathway and aldose reductase on transforming growth factor-β1 induced expression of fibronectin in cultured human mesangial cells

目的探讨p38MAPK信号通路与醛糖还原酶（AR）对转化生长因子131（TGF·131）诱导人系膜细胞（HMC）细胞外基质成分纤连蛋白（FN）表达的影响。方法应用醛糖还原酶抑制剂和p38MAPK信号通路抑制剂、转染及RNA干扰技术分别作用于HMC后，再用TGF-1刺激，观察刺激前后HMC表达FN的情况。Westernblot和免疫荧光检测HMC的FN、AR、p38的变化及real—time聚合酶链反应（PCR）鉴定转染和RNA干扰效果。结果HMC在TGF—β1作用后，AR和FN蛋白表达升高；单独使用醛糖还原酶抑制剂Sorbinil、Zopolrestat并不能下调FN蛋白的表达；但预先使用Sorbinil或Zopolrestat孵育后，再用TGF－β1刺激，FN蛋白表达下降（P〈0．05）；单独使用p38MAPK信号通路抑制剂后，FN蛋白表达下降；各组细胞预先使用Sorbinil、Zopolrestat、SB203580孵育后，再用TGF-β1刺激，FN蛋白表达下降（P〈0．05）；转染pCDNA3-AR后，HMC中ARmRNA及FN蛋白表达增多，再用TGF—β1刺激，FN蛋白表达明显上调（P〈0．05）；用AR—siRNA干扰AR基因后，HMC中ARmRNA及FN蛋白表达减少，即使用TGF-β1刺激，FN蛋白表达仍然下降（P〈0．05）。结论AR基因作为TGF-β1反应性基因，参与了HMC中TGF-Bl诱导的FN表达的调控，p38MAPK信号通路在这一过程中起着重要作用。

Objective To study the effects of p38 MAPK signaling pathway and aldose reductase （AR） on the transforming growth factor （TGF）-β1-induced expression of fibronectin （FN）. Methods Human mesangial cells （HMCs） were cultured, and transfected with pCDNA3-AR. AR gene silencing was induced by small interfering RNA （siRNA）. AR expression in HMCs was examined by immunofluorescence analysis. RT-PCR and real-time PCR were performed to detect the mRNA expression of AR in the HMCs and Western blotting was used to detect the protein expression of AR, FN and p38. AR inhibitors （ ARIs）, Sorbinil and Zopolrestat were added and co-incubated, followed by addition of TGF-β1. Western blotting was used to document protein expression of FN and p38 mitogen-activated protein kinases （p38 MAPKs） in the HMCs. Results Immunofluorescence analysis showed a stronger expression of AR in HMCs transfected with AR than that of normal HMCs and HMCs transfected with blank vector. In comparison with normal HMCs and those transfected with blank vector, HMCs transfected with AR showed stronger protein expression of FN （P 〈 0. 05）. After incubation of ARIs, protein expression of FN decreased in HMCs transfected with AR （P 〈0. 05）. After stimulation of TGF-β1, FN protein expression increased in both normal HMCs and those transfected with AR （P 〈 0.05）. After preincubation with ARI, FN protein expression in HMCs transfected decreased significantly （P 〈 0. 05）. After stimulation of TGF-131,naive HMCs showed increased expression of phosphor-p38. In contrast, HMCs preincubated with ARIs showed reduced expression of phosphor-p38, and HMCs transfected with AR showed increased expression of phosphor-p38 （ P 〈 0. 05 ）. Conclusions AR regulates the expression of FN through the stimulation of TGF-β1, which may involve the activation of p38-MAPK signaling pathway. AR may play a role in the pathogenesis of glomerulosclerosis.