二甲双胍对大鼠肾小球系膜细胞腺苷酸活化蛋白激酶表达的影响

Effects of metformin on expression of AMP- activated protein kinase in rat glomerular mesangial cells

目的观察二甲双胍对大鼠肾小球系膜细胞（MCs）腺苷酸活化蛋白激酶（AMPK）、核因子κB（NF—κB）和转化生长因子β1（TGF-β1）表达的影响，探讨其肾脏保护机制。方法体外培养MCs，随机分为正常糖浓度（NG）组、高糖（HG）组及高糖＋不同浓度二甲双胍（M1、M2、M3）组，48h后收获各组细胞及上清液。采用荧光实时定量PCR法检测细胞NF—κB、TGF-β1的mRNA含量；Western印迹法检测细胞磷酸化AMPK（p-AMPK）、总AMPK、NF—κBp65及TGF—β1的蛋白表达水平。结果肾小球系膜细胞可表达AMPK，NF-κB和TGF-β1。与NG组相比，HG组MCs的NF—κB、TGF-β1mRNA和蛋白表达水平升高（均P〈0．05）；二甲双胍干预组MCs的NF-κB、TGF-β1的mRNA和蛋白表达水平降低（均P〈0．05），且呈浓度依赖性。与NG组相比，HG组MCs的p-AMPK蛋白表达水平降低（P〈0．05）；二甲双胍组MCs的p-AMPK蛋白表达水平升高（P〈0．05），且呈浓度依赖性；各组总AMPK表达量的差异无统计学意义（均P〉0．05）。结论二甲双胍可激活。肾小球系膜细胞的腺苷酸活化蛋白激酶，抑制NF—κB及其下游产物TGF-β1的合成，此作用可能与其肾脏保护机制相关。

Objective To obselve the effects of metformin on expression of Adenosine 5＇ - monophosphate （AMP）-activated protein kinase （AMPK）, nuclear factor-κB （NF-κB） and transforming growth factor β1 （TGF- β1） in cultured rat glomerular mesangial cells （MCs）, and explore its reno- protective mechanisms. Methods MCs were cultured in the medium with normal glucose （group NG, 5.6 mmol/L）, high glucose （group HG, 25mmol/L） and different concentrations of mefformin （group M1, M2, M3）. After 48 h exposure, the supernatants and MCs were collected. The expression of NF-κB and TGF-β1 mRNA was analyzed by real time-PCR. Total-AMPK, phospho-Thr-172 AMPK （p-AMPK）, NF -κB p65 and TGF-β1 were visualized by Western blot. Results The real time-PCR and Western blot result showed MCs could express AMPK, NF-κB and TGF-β1 mRNA and protein. After stimulated by HG, the levels of intracellular NF- κB and TGF- β1 expressions were significantly increased compared with group NG （P 〈 0.05）; The levels of NF-κB and TGF-β1 were significantly decreased in group M1, M2 and group M3 compared with group HG in a dose-dependent manner. After stimulated by HG, the level of intracellular p-AMPK were down-regulated compared with group NG（all P 〈 0.05）; The expression of p- AMPK increased with the rising of metformin concentration, presenting the opposite trend （P 〈 0.05）, while the level of total-AMPK protein was unchanged with exposure to HG or different concentrations of metformin（P 〉 0.05）. Conclusion Metformin can suppress the expression of NF- κB and TGF-β1 of glomemlar MCs induced by HG via AMPK activation, which may partly contribute to its reno-protection.