高糖及核因子κB通路诱导足细胞上皮间充质转分化的研究

High glucose induces podocyte epithelial-mesenchymal transition and cell migration through NF-κB pathway

目的:探讨高糖对小鼠足细胞上皮间充质转分化(EMT)与迁移的影响及其分子机制。方法:以永生化小鼠足细胞株(足细胞)为研究对象,设置正常糖组(5 mmol/L葡萄糖)、甘露醇组(5 mmol/L葡萄糖+25mmol/L甘露醇)、高糖组(30 mmol/L葡萄糖)、核因子κB(NF-κB)特异性抑制剂小白菊内酯(PTL)+高糖组,作用时间24h。采用Transwell检测细胞迁移能力,Western Blot检测蛋白表达,q PCR检测mRNA表达情况,免疫荧光检测NF-κB入核情况。结果:与正常糖及甘露醇组比较,高糖刺激足细胞24h后细胞的迁移能力明显增强(P<0.05),足细胞特征性蛋白nephrin明显下调(P<0.05),纤连蛋白(FN)及mRNA表达显著增加(P<0.05),而E钙黏蛋白(E-cadherin)及其mRNA表达明显下降(P<0.05)。同时,与上述对照组比较,高糖明显上调p-IκBα(P<0.05)下调IκBα(P<0.05)促使足细胞胞质中的NF-κB入核同时上调p-p65的表达;干预细胞PTL后,足细胞的EMT和迁移得到明显抑制(P<0.05),nephrin蛋白表达明显上升(P<0.05)。结论:高糖通过激活NF-κB通路诱导足细胞发生EMT及迁移。

Objective： To explore the possible molecular mechanism of epithelial-mesenchymal transition（ EMT）and cell migration in high glucose（ HG）-induced podocytes. Methodology： Immortalized conditional mouse podocytes（ MPC5） worked as the research object. Podocytes were divided into HG group（ stimulated with 30 mmol/L glucose for 24hrs）,the normal glucose group（ 5 mmol / L glucose） and the mannitol group were set as control groups. Cell migration was examined by Transwell. The expression of protein was detected by Western blot. The mRNA level of protein was determined by q PCR. The nuclear translocation of NF-κB was observed by immunofluorescence. Results： Compared with the control groups,the cell migration was significantly enhanced（ P〈0. 05）,and the specific protein of podocytes＇ Nephrin was significantly down-regulated（ P〈0. 05）. FN was significantly up-regulated（ P〈0. 05） while E-cadherin was markedly down-regulated（ P〈0. 05） after stimulated by HG for 24 hrs. Meanwhile,HG increased p-IκBα（ P〈0. 05） and decreased IκBα（ P〈0. 05） greatly,then promoted the nuclear translocation of NF-κB and up-regulated phosphorylation level of p65. Moreover,after treatment with the specific inhibitor Parthenolide（ PTL）,EMT and migration in podocytes were significantly inhibited and the protein level of Nephrin was up-regulated（ P〈0. 05）. Conclusion： HG induces EMT and cell migration in podocytes through NF-κB pathway.