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

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

目的 探讨PKC信号通路与醛糖还原酶在非高糖条件下对转化生长因子（TGF）-β1诱导人肾系膜细胞（HMC）细胞外基质成分纤连蛋白表达的影响.方法 应用醛糖还原酶抑制剂、PKC信号通路抑制剂G（O）6983、转染pcDNA3-醛糖还原酶及siRNA分别作用于人系膜细胞后,再用TGF-β1刺激,观察刺激前后人系膜细胞表达纤连蛋白的情况.Western blot检测系膜细胞内纤连蛋白和醛糖还原酶的变化,即时RT-PCR鉴定转染和干扰效果.结果 系膜细胞在TGF-β1作用后,醛糖还原酶和纤连蛋白表达升高;单独使用醛糖还原酶抑制剂并不能下调纤连蛋白的表达;但预先使用醛糖还原酶抑制剂孵育后,再用TGF-β1刺激,纤连蛋白表达减少为对照组的34%（P〈0.05）.单独使用PKC信号通路抑制剂并不能下调纤连蛋白的表达;用PKC信号通路抑制剂后,再用TGF-β1刺激,纤连蛋白表达下降为对照组的42%（P〈0.05）.预先使用醛糖还原酶抑制剂、PKC抑制剂孵育细胞后,再用TGF-β1刺激,纤连蛋白表达下降;转染pcDNA3-醛糖还原酶后,系膜细胞中醛糖还原酶mRNA表达增多超过10倍,纤连蛋白表达增加了2.5倍（P〈0.05）,再用TGF-β1刺激,纤连蛋白表达增加了3.6倍（P〈0.05）;转染siRNA后,系膜细胞中醛糖还原酶mRNA表达减少为对照组的20%,纤连蛋白表达减少为对照组的6%（P〈0.01）,即使再用TGF-β1刺激,纤连蛋白表达仍然下降,为对照组的12%（P〈0.01）.结论 醛糖还原酶基因参与了系膜细胞中TGF-β1诱导纤连蛋白表达过程的调控,这一过程可能与PKC信号通路的活化有关.

Objective To study the effect of PKC signalling pathway and aldose reductase （AR） on the expression of fibronectin （FN） induced by transforming growth factor-β1 （TGF-β1）. Methods Human mesangial cells （HMCs） were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA （siRNA） and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed （34%） in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl （P 〈0. 05） , and the induction effect on FN expression was suppressed by G（O）6983 （42%） in HMCs （P 〈 0. 05） . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs（P 〈0. 01） , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA （12%） in HMCs （P 〈0. 01）. Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.