来氟米特对高糖环境下足细胞表达的影响及其机制

Effects of leflunomide on podocytes exposed to high glucose condition and its mechanism

目的探讨高糖环境下来氟米特（A771726）对人足细胞Podocalyxin（PCX）、核因子（NF）-KB和基质金属蛋白酶9（MMP-9）表达的影响及其机制。方法在高糖环境下培养足细胞，Western印迹法检测不同时间点足细胞特征蛋白PCX表达的变化；再将足细胞分为正常糖组、来氟米特组、高糖组、高渗对照组进行培养，用Western印迹法检测各组足细胞PCX蛋白的表达。Western印迹法检测不同时间点高糖培养足细胞NF-KBp65及磷酸化NF-KBp65（P-NF．KBp65）活性；再将足细胞分为正常糖组、甘露醇高渗对照组、高糖组、来氟米特组和NF-KB阻断剂（PDTC）组，用Western印迹法检测各组足细胞MMP一9蛋白的表达。结果在高糖环境下，足细胞特征蛋白PCX的表达随时间持续下降；来氟米特组PCX蛋白表达明显高于高糖组（0．46±0．04比0．13±0．03，P〈0．05）。在高糖刺激足细胞在30min时，NF-KB的活化开始，P．NF-KBp65蛋白表达上升，60min达高峰，6h后接近基础水平；而在同一时点NF-KB阻断剂PDTC组和来氟米特组足细胞MMP-9蛋白表达明显低于高糖组，差异均有统计学意义（0．71±0．叭、0．64±0．03比1．64±0．03，均P〈0．05）。结论来氟米特（A771726）对高糖环境下的足细胞有保护作用，其机制可能是通过抑制NF-KB活化而减少足细胞表汰MMP-9。

Objective To explore the protective effects of leflunomide （A771726） on the expression of podocalyxin, NF-KB and matrix metalloproteinase-9 （ MMP-9 ） in podocytes exposed to high glucose environment and elucidate its mechanism. Methods Podocytes were cultured in high glucose. And the altered expressions of podocyte protein podocalyxin were detected by Western blotting at different timepoints. Then podocytes were divided into 4 groups of normal glucose control, leflunomide, high glucose and hypertonic control. The expression level of podocalyxin protein in each group was detected by Western blotting. And NF-KB 1o65 and phosphorylation of NF-KB 1365 （P-NF-KBp65） in podocytes cultured in high glucose were detected at different timepoints. And then the podocytes were divided into 5 groups of normal glucose, mannitol, hypertonic control, high glucose, leflunomide and PDTC （NF-KB blocker）. And the expressions of MMP-9 protein in these groups were also detected by Western blotting. Results In the high glucose environment, the expression of podocalyxin declined instantly. Compared with the high-glucose group, the podocalyxin expression of the leflunomide group was significantly higher than the high glucose group （0. 46 ± 0. 04 vs 0. 13 ± 0. 03, P 〈 0. 05 ）. After 30-minute stimulation by high glucose, the activation of NF-KB started and the expression of P-NF-KBp65 protein increased. Such activities peaked at 60 minutes and reverted to a basic level after 6 hours. Compared with the high glucose group, the expressions of MMP-9 in PDTC and leflunomide groups were significantly lower than the high glucose group. And the differences were statistically significant （0. 71 ± 0.01, 0. 64 ±O. 03 vs 1.64 ± O. 03, both P 〈 0. 05 ）. Conclusions Leflunomide has protective effects on podocytes in high glucose. And its mechanism is possibly due to alowered expression of MMP-9 through an inhibition of NF-KB activation.