Role Of PPARγ In High Glucose-induced Insulin Resistance And Susceptibility In Vascular Endothelial Cells (HUVECs)

Objective:The aim of the study is to construct the recombinant plasmid for PPARy and transfect it into human umbilical vein endothelial cells (HUVECs) to research the role of PPARy in high glucose-induced insulin resistance (IR) and suceptibility in HUVECs.Methods:The nucleotide sequences of PPARy mRNA was retrieved from Genebank, and amplified by RT-PCR.The amplified sequence was cloned into the vector of pCMV5-flag plasmid and identified by restriction enzyme analysis, sequencing analysis and the efficiency of overexpression analysis. Four groups were individed, namely Control group, IR group, vehicle group, PPARy group. First, the model of high glucose-induced IR was established in HUVECs and then the combinant plasmid pCMV5-flag-PPARγ was transfected into HUVECs by lipofectamine 2000. After that, the expression levels of PPARy and eNOS, and the levels of NO and Ang II were detected to explore the role of PPARy in high glucose-induced IR in HUVECs. ultimately, the expression levels of IKKa/p, pS176-IKKa/p, IκBα, and the levels of TNF-a, IL-6, sICAM-1, sVCAM-1 were measured to study the mechanism of PPARy.Results:1.The restriction enzyme analysis and sequencing results demonstratedthat the recombinant vector was constructed successfully.2.The results of NO and Ang II levels also revealed that regardless of before modeling or after modeling, the overexpression of PPARy was able to induce the increasing levels of NO while the decreasing levels of Ang II, suggesting that the overexpression of PPARy significantly decreased the suceptibility of HUVECs to IR and improved the IR degree in HUVECs.3.The results of protein levels were demonstrated that the overexpression of PPARy was able to upregulate the expression levelsof eNOS and IκBα, but downregulate the levels of pS176-IKKα/β markedly.4.The results of TNF-a, IL-6, sICAM-1 and sVCAM-1 levels were unveiled that the overexpression of PPARy considerably decreased the levels of inflammatory factors.Conclusion:The overexpression of PPARy significantly decreased the suceptibility of HUVECs to IR induced by high glucose and improved the IR degree in HUVECs. The regulation mechanism of PPARy to IR and its susceptibility is dependent on the pathway of IKKα/β/IκBα/NF-κB.