【摘要】 目的:研究沉默信息调节因子1(SIRT1)对高糖诱导的大鼠肾小球系膜细胞NF-κB p65蛋白乙酰化影响及其保护作用。方法:培养大鼠肾小球系膜细胞,实验分为5组:正常对照组、甘露醇组、高糖组、白藜芦醇组和SIRT1 RNAi组。MTT比色法检测细胞活性;以实时荧光定量PCR检测SIRT1、单核细胞趋化蛋白1(MCP-1)、血管黏附分子1(VCAM-1)、肿瘤坏死因子α(TNF-α)和转化生长因子β1(TGF-β1)mRNA水平;Western blotting检测SIRT1和NF-κB p65乙酰化蛋白的表达水平,ELISA检测MCP-1、VCAM-1、TNF-α、TGF-β1和丙二醛(MDA)的含量。结果:高糖刺激使肾小球系膜细胞的活力降低,超氧化物岐化酶(SOD)活性降低,MDA含量增加;SIRT1 mRNA及蛋白表达降低,NF-κB p65蛋白乙酰化水平增高,MCP-1、VCAM-1、TNF-α、TGF-β1mRNA和蛋白水平增高。白藜芦醇可逆转高糖引起的变化,而沉默SIRT1基因使高糖诱导的系膜细胞NF-κB p56乙酰化水平、MCP-1、VCAM-1、TNF-α、TGF-β1mRNA和蛋白水平升高。结论:高糖可降低SIRT1水平,增加炎症因子的表达。SIRT1的激活可使NF-κB的亚单位RelA/p65去乙酰化,从而抑制炎症因子的产生。SIRT1可作为治疗DN的潜在靶点。更多还原

【Abstract】 AIM:To investigate the effects of silent information regulator 1(SIRT1) on high glucose-induced acetylation of NF-κB p65 subunit and its protective role in rat mesangial cells.METHODS:Rat mesangial cells were cultured in DMEM supplemented with 10% FBS and were divided into control group,mannitol group,high glucose group,resveratrol group and SIRT1 RNAi group.The cell viability was determined by MTT assay.The mRNA expression of SIRT1,monocyte chemoattratant protein 1(MCP-1),vascular cell adhesion molecule 1(VCAM-1-1),tumor necrosis factor α(TNF-α),transforming growth factor β1(TGF-β1) was analyzed by real-time quantitative PCR.The protein expression of SIRT1 and the acetylation of NF-κB p65 subunit were determined by Western blotting.The protein concentrations of MCP-1,VCAM-1,TNF-α,TGF-β1and malondialdehyde(MDA) were detected by ELISA.RESULTS:The cell viability,superoxide dismutase(SOD) activity,and the expression of SIRT1 at mRNA and protein levels were decreased by high glucose treatment as compared with control group.The acetylation of NF-κB p65 subunit was significantly increased after interfered with high glucose,resulting in the increase in the secretion of MCP-1,VCAM-1,TNF-α and TGF-β1.Resveratrol decreased high glucose-induced acetylation of NF-κB p65 subunit.However,silencing SIRT1 significantly enhanced the acetylation of NF-κB p65 subunit and the expression of MCP-1,VCAM-1,TNF-α and TGF-β1.CONCLUSION:SIRT1 remarkably inhibits the inflammatory reactions by deacetylating NF-κB p65,suggesting that SIRT1 is a possible target for preventing diabetic nephropathy.更多还原