CAJ | 학술논문

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,48 h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-κB p65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-β1 mRNA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.
목적 관찰이갑쌍고대대서신소구계막세포(MCs)선감산활화단백격매(AMPK)、핵인자κB (NF-κB)화전화생장인자β1(TGF-β1)표체적영향,탐토기신장보호궤제.방법 체외배양MCs,수궤분위정상당농도(NG)조、고당(HG)조급고당+불동농도이갑쌍고(M1、M2、M3)조,48 h후수획각조세포급상청액.채용형광실시정량PCR법검측세포NF-κB、TGF-β1적mRNA함량;Western인적법검측세포린산화AMPK (p-AMPK)、총AMPK、NF-κB p65급TGF-β1적단백표체수평.결과 신소구계막세포가표체AMPK,NF-κB화TGF-β1.여NG조상비,HG조MCs적NF-κB、TGF-β1 mRNA화단백표체수평승고(균P＜0.05);이갑쌍고간예조MCs적NF-κB、TGF-β1적mRNA화단백표체수평강저(균P＜0.05),차정농도의뢰성.여NG조상비,HG조MCs적p-AMPK단백표체수평강저(P＜0.05);이갑쌍고조MCs적p-AMPK단백표체수평승고(P＜0.05),차정농도의뢰성;각조총AMPK표체량적차이무통계학의의(균P＞ 0.05).결론 이갑쌍고가격활신소구계막세포적선감산활화단백격매,억제NF-κB급기하유산물TGF-β1적합성,차작용가능여기신장보호궤제상관.
Objective To observe 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 renoprotective 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 metformin (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 mefformin(P ＞ 0.05).Conclusion Metformin can suppress the expression of NF-κB and TGF-β1 of glomerular MCs induced by HG via AMPK activation,which may partly contribute to its reno-protection.