【摘要】 目的探讨醛糖还原酶(AR)对转化生长因子β1(TGFβ1)诱导的大鼠系膜细胞(MsC)细胞外基质成分纤连蛋白(FN)和IV型胶原(ColIV)蛋白表达的影响及其可能涉及的信号传导通路。方法经亚克隆构建AR真核表达质粒pCDNA3AR,经脂质体介导及G418筛选后,将pCDNA3AR稳定转染至大鼠MsC中。应用逆转录聚合酶链反应(RTPCR)、Western印迹法和免疫荧光法检测转染MsC表达AR情况;以Western印迹法检测正常MsC、AR转基因MsC,应用醛糖还原酶抑制剂(ARI)Sorbinil和Zopolrestat分别孵育的MsC:在TGFβ1刺激前后的FN、ColIV以及MAPK/phosphoMAPK表达变化。结果与正常MsC相比,两种ARI———Sorbinil和Zopolrestat均可下调MsC的FN和ColIV蛋白表达,而AR转基因MsC中FN和ColIV表达均升高(均P<0.05);TGFβ1作用后,MsC的FN和ColIV蛋白表达升高,预先使用Sorbinil或Zopolrestat孵育后,二者表达均下降,而AR转基因MsC中二者表达进一步增高(均P<0.05)。各组细胞在TGFβ1刺激前后总ERK、JNK和p38表达均无明显变化。正常MsC在TGFβ1刺激后phosphoERK、phosphoJNK、phosphop38表达升高,与之相比,若预先使用Sorbinil或Zopolrestat孵育后,则phosphoJNK、phosphop38表达下降,AR转基因MsC中phosphoJNK表达更为升高(P<0.05)。结论AR基因作为TGFβ1反应性基因之一,可能参与TGFβ1诱导的FN和ColIV表达的调控,且AR的作用有可能与TGFβ1刺激后的JNKMAPK和p38MAPK信号通路的活化有关。AR可能参与了非糖尿病导致的肾小球纤维化、硬化的病理发展过程。更多还原

【Abstract】 Objective To study the effects of aldose reductase (AR) on the transforming growth factor (TGF)-β1-induced expression of fibronectin (FN) and collagen IV.Methods Restriction endonucleases digestion and ligation were used to reconstruct the eukaryotic expression plasmid pCDNA3-AR. Rat mesangial cells (MsCs) were isolated, cultured, and transfected with pCDNA3-AR, or blank vector. The AR expression in the MsCs was examined by immunofluorescence analysis. RT-PCR was performed to detect the mRNA expression of AR in the MsCs and Western blotting was used to detect the protein expression of AR. AR inhibitors (ARIs), Sorbinil and Zopolrestat were added and co-incubated, then TGF)-β1 was added and Western blotting was used to analyze the protein expression of FN, collagen IV (Col IV), and mitogen-activated protein kinases (MAPKs) in the MsCs. Results Immunofluorescence analysis showed stronger expression of AR in the MsCs transfected with AR then in the normal MsCs and the MsCs transfected with blank vector. In comparison with the normal MsCs and those transfected with blank vector, the MsCs transfected with AR showed stronger protein expression of FN and Col IV (all P<0.05). After incubation of ARIs the protein expression of FN and Col IV decreased by 1.8 and 2.0 times respectively in the MsCs transfected with AR (all P<0.05). After stimulation of TGF-β1, the protein expression of FN and Col IV increased in both the normal MsCs and those transfected with AR (P<0.05 for the latter). After preincubaton with ARIs the protein expression of FN and Col IV in the MsCs transfected decreased significantly (both P<0.05). After stimulation of TGF-β1, the normal MsCs showed increased expression of phospho-ERK, phospho-JNK, and phospho-p38; the MsCs preincubated with ARIs showed reduced expression of phospho-JNK and phospho-p38; and the MsCs transfected with AR showed increased expression of phosphor-JNK (P<0.05). Conclusions AR can regulates the expression of FN and Col IV with the stimulation of TGF-β1 as AR gene is one of the responsive genes of TGF-β1, which may have relations with the activation of JNK-MAPK and p38-MAPK signaling pathways induced by TGF-β1. AR may play a role in the pathogenesis of glomerulosclerosis.更多还原