SIRT1对高糖诱导的系膜细胞NF-κBp65乙酰化及MCP-1表达的影响

Effect of SIRT1 on high glucose-induced NF-κB p65 subunit acetylation and MCP-1 expression in rat mesangial cells

目的探讨沉默信息调节因子1(SIRT1)对高糖诱导的大鼠肾小球系膜细胞(RMC)核因子κB(NF-κB)p65蛋白乙酰化及单核细胞趋化蛋白1(MCP-1)表达的影响。方法构建干扰SIRT1基因的shRNA慢病毒质粒pTRCshSIRT1并进行鉴定。将RMC分为高糖组(用高糖培养液培养)、白藜芦醇(SIRT1激活剂)+高糖组(用含1μmol/L白藜芦醇的低糖培养液培养24h后,换用高糖培养液培养)、SIRT1RNAi组(添加干扰病毒pTRC-shSIRT1感染4h后,换用低糖培养液培养)、SIRT1RNAi+高糖组(添加干扰病毒pTRC-shSIRT1感染4h后,换用高糖培养液培养),同时设正常对照组和甘露醇高渗对照组。以实时荧光定量PCR检测SIRT1、MCP-1的mRNA表达,蛋白质印迹法检测SIRT1和NF-κB p65乙酰化蛋白的表达,ELISA技术检测MCP-1蛋白含量。结果质粒测序证实干扰SIRT1基因的shRNA慢病毒载体构建成功,且能抑制RMC中SIRT1基因表达(P<0.01)。高糖刺激使RMC SIRT1基因表达降低,NF-κB p65蛋白乙酰化增强,MCP-1mRNA和蛋白水平增高;SIRT1激活剂白藜芦醇可逆转高糖引起的变化;而沉默SIRT1可促进高糖诱导的RMC NF-κB p56乙酰化及MCP-1mRNA和蛋白表达(P<0.05或0.01)。结论 SIRT1可抑制高糖诱导的RMC MCP-1表达,其机制可能与NF-κB p56去乙酰化有关。

Objective To explore the effect of silent information regulator 1（SIRT1）on high glucose-induced nuclear factor-κB（NF-κB）p65 subunit acetylation and monocyte chemoattractant protein 1（MCP-1）expression in rat mesangial cells（RMCs）.Methods The lentiviral shRNA plasmid pTRC-shSIRT1was constructed for interference of SIRT1 gene and was identified.The RMCs were divided into high glucose group（treated with high glucose culture medium）,resveratrol（SIRT1 activator）＋high glucose group（treated with low glucose culture medium containing 1μmol/L resveratrol for 24h,and then with high glucose culture medium）,SIRT1 RNAi group（4h after viral pTRC-shSIRT1 infection,and then treated with low-glucose culture medium）,SIRT1 RNAi＋ high glucose group（4h after viral pTRC-shSIRT1 infection,and then treated with high glucose culture medium）;we also established normal control group and hypertonic mannitol control group.The mRNA expression of SIRT1 and MCP-1gene was analyzed by real-time quantitative PCR;the protein expression of SIRT1 and the acetylation of NF-κB p65 subunit were observed by Western blotting analysis.The protein level of MCP-1 in the supernatants was detected by ELISA.Results DNA sequencing confirmed the successful construction of the plasmid pTRC-shSIRT1,which could knocked down SIRT1 mRNA expression（P0.01）.High glucose decreased SIRT1 expression and promoted acetylation of NF-κB p65 subunit,and increased MCP-1 mRNA and protein expression.Resveratrol,an activatorof SIRT1,could reverse the above changes induced by high glucose.Conversely,silencing SIRT1 gene significantly accelerated the high glucose-induced acetylation of NF-κB p65 subunit and MCP-1 expression at both mRNA and protein levels（P0.01or P0.05）.Conclusion SIRT1 can inhibit high glucose-induced MCP-1 mRNA and protein expression in RMCs,which may involve NF-κB p65 deacetylation.