摘要
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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2014年第6期451-455,共5页
Chinese Journal of Nephrology
基金
安徽省自然科学基金(11040606M161)
安徽省高校自然科学基金(KJ2011A157)