Effect of SIRT1 on high glucose-induced NF-κB p65 subunit acetylation and MCP-1 expression in rat mesangial cells / 第二军医大学学报

ABSTRACT

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-shSIRT1 was 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 1 μmol/L resveratrol for 24 h, and then with high glucose culture medium), SIRT1 RNAi group (4 h after viral pTRC-shSIRT1 infection, and then treated with low-glucose culture medium), SIRT1 RNAi + high glucose group (4 h 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-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 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(P<0.01). High glucose decreased SIRT1 expression and promoted acetylation of NF-κB p65 subunit, and increased MCP-1 mRNA and protein expression. Resveratrol, an activator of 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(P<0.01 or P<0.05). Conclusion: SIRT1 can inhibit high glucose-induced MCP-1 mRNA and protein expression in RMCs, which may involve NF-κB p65 deacetylation.