The Role And Mechanism Of PPAR γ In The Vascular Endothelial Injury Induced By Intermittent Hypoxia

Objective Obstructive sleep apnea(OSA)is a common chronic respiratory disease.Chronic intermittent hypoxia(CIH)is the most important pathophysiological feature of OSA.Increasing medical researches showed that endothelial dysfunction was one of the important causes of various cardiovascular diseases related to CIH.It was indispensable to find the effective means to prevent and treat OSA-related cardiovascular diseases.The objective of this study:1.To identify the target proteins in CIH-related vascular dysfunction by comparative proteomics analysis.2.To explore the role of peroxisome proliferator-activated receptor gamma(PPARγ)on vascular endothelial injury under intermittent hypoxia(IH)mode.3.To explore the interaction between mi R-130a-3p and PPARγ,and to explore the potential mechanism of mi R-130a-3p in IH-induced vascular endothelial injury.4.To explore the role of PPARγ on mitochondrial function in IH-treated vascular endothelial injury.Methods1.A comparative proteomics analysis was conducted in aortic samples of rats treated with CIH and those with normoxia.Bioinformatics analyses were performed to determine the potential roles of major proteins.The expressions of target proteins were measured by western blotting.2.Rat aortic endothelial cells and human umbilical vein endothelial cells were used as the research objects.The experimental groups were divided into normoxia control group(Control group),intermittent hypoxia group(IH group)and intermittent hypoxia + rosiglitazone group(IH + Rosiglitazone group).The m RNA levels of PPARγ were detected by q RT-PCR.The protein levels of PPARγ and apoptosis-related proteins were detected by western blot.Cell apoptotic ratio was detected by Flow cytometer.Cell viability were detected by Cell Counting Kit-8(CCK-8).Cell proliferation were detected by Ed U(5-ethynyl-2’-deoxyuridine)-594.3.The target mi RNA of PPARγ were predicted and verified by bioinformatics analysis and luciferase assays.The effect and potential mechanism of mi R-130a-3p in IH-treated vascular endothelial injury were detected by rescue experiment.4.The mitochondrial function injury in IH treated HUVECs were detected by reactive oxygen species(ROS)kit,JC-1 kit and the protein levels of DRP1 and Mitofusin.In IH treated HUVECs,after mitochondrial-specific antioxidant mito-Tempo was given and PPARγ was knocked down,the changes of mitochondrial function,cell viability,cell proliferation ability,apoptosis rate and apoptotic protein were observed.Results1.3593 proteins in aortic tissues of rats were quantified.92 upregulated proteins and468 downregulated proteins were identified when the cut-off of fold change was set at1.5(CIH vs.normoxia).The results of bioinformatics analysis revealed that the differentially expressed proteins were enriched in the processes of energy metabolism and lipid metabolism.Reduced expression level of PPARγ protein was identified in thoracic aortic tissues of rats with CIH by proteomics analysis and western blotting(p<0.05).2.In intermittent hypoxia-treated RAOECs and HUVECs,PPARγ protein levels were reduced,and the cell viability,cell proliferation were markedly decreased(p<0.05);The apoptosis rate and the protein levels of caspase-3 and Bax were markedly elevated(p<0.05).Importantly,forced expression of PPARγ by rosiglitazone in intermittent hypoxia-treated rat aortic endothelial cells not only attenuated caspase-3and Bax protein levels,cell viability and cell proliferation,but also reduced the rate of apoptosis(p<0.05).3.Bioinformatics revealed that the seed sequence of mi R-130a-3p is complementary to the 3’UTR site of PPARγ,which suggested that PPARγ may be a target gene of mi R-130a-3p.Dual-luciferase reporter also confirmed that PPARγ was the direct downstream target of mi R-130-3p.In rescue experiment,HUVECs were transfected with Si-PPARγ,mi R-130-3p inhibitor,the effects of mi R-130-3p silence on cell viability,cell proliferation,the levels of apoptosis-related proteins and the cell apoptotic rate were all reversed by PPARγ knockdown(all p<0.05).4.Compared with the control group,accumulated ROS,decreased the mitochondrial membrane potential(JC-1),and increased the expression of mitochondrial division protein were detected in IH group(all p<0.05).The mitochondrial-specific antioxidants and PPARγ inhibitors were transfected into HUVECs.The results showed that inhibiting the expression of PPARγ could regulate mitochondrial function and affect the cell viability,cell proliferation,the levels of apoptosis-related proteins and the cell apoptotic rate(all p<0.05).Conculsions1.PPARγ is critical in endothelial dysfunction of rats with CIH.Additionally,further studies on these differentially expressed proteins associated with IH-related endothelial dysfunction are necessary.2.Rosiglitazone,the PPARγ agonist,reduced IH-induced vascular endothelial cell injury.3.mi R-130-3p mediated IH-induced vascular cell injury by target PPARγ.4.The role of PPARγ on IH-related vascular endothelial injury was related to mitochondrial function.