| 摘要: |
| 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pTRC-shSIRT1并进行鉴定。将RMC分为高糖组（用高糖培养液培养）、白藜芦醇 高糖组（用含1μmol/L SIRT1激活剂白藜芦醇的低糖培养液培养24 h后，换用高糖培养液培养）、SIRT1 RNAi组（添加干扰病毒pTRC-shSIRT1感染4 h后，换用低糖培养液培养）、SIRT1 RNAi 高糖组（添加干扰病毒pTRC-shSIRT1感染4 h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。高糖刺激使RMC SIRT1基因表达降低，NF-κB p65蛋白乙酰化增强，MCP-1 mRNA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-1 mRNA和蛋白表达。结论 SIRT1可抑制高糖诱导的RMC MCP-1 mRNA和蛋白的表达，其机制可能与NF-κB P56去乙酰化有关。 |
| 关键词: RNA干扰 沉默信息调节因子1 NF-κB 单核细胞趋化蛋白1 糖尿病肾病 |
| DOI：10.3724/SP.J.1008.2014.00722 |
| 投稿时间：2013-08-09修订日期：2014-05-18 |
| 基金项目:浙江省自然基金 |
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| The Effect of SIRT1 Gene Silencing on Acetylation of NF-κB p65 Subunit and the Expression of MCP-1 in Rat Mesangial Cells Induced by High Glucose |
| DuYue-guang,Chai Ke-fu,Wang Li-pei |
| () |
| Abstract: |
| Objective To explore the effect of silent information regulator 1（SIRT1） on acetylation of nuclear factor-κB (NF-κB) p65 subunit and the expression of monocyte chemoattractant protein 1 (MCP-1) in rat mesangial cells (RMC) induced by high glucose. Methods Construct the lentiviral shRNA plasmid pTRC-shSIRT1 to interference SIRT1 gene and identification . The RMCs was classified into high glucose group (high glucose culture medium used), resveratrol high glucose group (with low glucose culture medium containing 1μmol / L SIRT1 activator resveratrol for 24 h and then switching to high glucose culture medium), SIRT1 RNAi group (4h after viral pTRC-shSIRT1 infection, and then switch to low-glucose culture medium), SIRT1 RNAi high glucose group (4h after viral pTRC-shSIRT1 infection, and then switch to high glucose culture medium) , While negative control group and the control group (hypertonic mannitol). The mRNA expression of SIRT1 and MCP-1 gene was analyzed by real-time quantitative PCR. The protein expression of SIRT1 and the acetylation of NF-κB p65 subunit were observed by Western Blot. The protein expression of MCP-1 was detected by ELISA. Results DNA sequencing demonstrated that the plasmid pTRC-shSIRT1 was successfully constructed. Viral produced by pTRC-shSIRT1 infected RMCs and knocked down SIRT1 expression both at mRNA and protein levels. The expression of SIRT1 was decreased and acetylation of NF-κB p65 subunit was significantly increased by high glucose. Resveratrol, as an activator of SIRT1 can decreases high glucose-induced acetylation of NF-κB p65, which results in the reduction of MCP-1 secretion. Conversely, Gene silencing of SIRT1 shows increased the acetylation of cellular p65 protein and secretion of MCP-1 when challenged with high glucose. Conclusion The plasmid pTRC-shSIRT1 was successfully constructed and can effectively down regulate SIRT1 mRNA and protein expression. SIRT1 activation can significantly inhibited high glucose mediated upregulation of MCP-1, This is probably attributable to the increasing SIRT1-mediated NF-κB p65 deacetylation. |
| Key words: RNA interference silent information regulator 1 NF-κB monocyte chemoattractant protein 1 diabetic Nephropathy |
