The Regulatory Role Of GPR4in Cell Inflammatory Molecules Gene Expression In HUVEC Cells

In the development and progression of atherosclerosis,inflammation was accompanied. Endothelial cells contain and release inflammatory molecules to promote tissue damage.In inflammatory environments, anaerobic Glycolysis of hypoxic environment has led to the accumulation of lactic acid, cause a decrease of in lesion area pH,This heralds acidic conditions may have a material effect on arteriosclerosis;Lysophos-phatidylcholine(LPC)is derivative of the phosphatidycholine, it generated from phospholipase A2(PLA2) catalyticing the hydrolysis of phosphatidylcholine (PC), LPC is the main ingredient of oxidative low density lipoprotein(ox-LDL),so LPC is high concentration in atherosclerosis.Studies have shown that GPR4is an important dualligand receptor in endothelial cells, it plays an important role in the acidic environment, such as the promotion of adhesion molecules expression and inflammatory factors. The molecular mechanism that GPR4as dualligand receptor in endothelial cells involved in inducing of regulation of the expression of inflammatory factors in endothelial cells is still unknown. This study first described a new phenomenon of proton sensing receptor GPR4in the process of expression of inflammatory factors in endothelial cell,may also provide a better understand the pathogenesis of atherosclerosis and potential targets for the treatment of atherosclerosis.Aims:Explore regulatory effects of proton-sensing recepor GPR4on the expression of IL-6、TNF-α in HUVEC cells, and to clarity the cell signaling pathways induced by LPC and protons.Methods:Reverse transcription PCR was used to cloned GPR4gene,GPR4gene was subclone to the pCR2.1vector and through the sequencing,digestion,ligated into the eukaryotic expression vector pIRES-EGFP.GPR4expression vector transient transfected to HUVEC cell;shRNA-GPR4interference vector was screened and transient transfected to HUVEC cells. The expression of GPR4was detected by RT-PCR in HUVEC cells, GPR4overexpression cells and GPR4interferenced cells. Expression of IL-6and TNF-a was detected after stimulated by LPC and proton (H+) in these cells.,and PTX, the specifity of Gi protein inhibitor was to confirm G protein involved in these process. Deal with transgenic cells with NF-κB inhibitors BAY, to determine NF-κB signaling pathway is involved in GPR4-induced inflammatory cytokines process.Results:Restriction enzyme digestion and sequencing results indicated that GPR4gene has cloned and pIRES-EGFP-GPR4eukaryotic expression vector was constructed; we also got GPR4gene over-expressing and GPR4interferenced shRNA-GPR4HUVEC cells. In the transfected GPR4recombinant cells, acid stimulation induced increased expression of IL-6and TNF-a; LPC can inhibit the increased expression of IL-6, TNF-a induced by proton; In the shRNA-GPR4interference cells, acid stimulation reduced the expression of IL-6and TNF-a, and LPC can reduce the amount of their expression induced by proton. PTX not affect the expression of IL-6and TNF-a; BAY,were significantly decreased the expression of IL-6and TNF-a.Conclusion:GPR4is one of the important proton-sensing receptors in endothelial cells,related to the expression of IL-6and TNF-a, is closely related to the inflammatory response. LPC has been shown to antagonize the proton-induced IL-6and TNF-a expression. Protons caused the expression of IL-6and TNF-a independent of PTX. Proton caused the expression of IL-6and TNF-a depends on the NF-κB signaling pathway.Results show that the proton-sensing receptor GPR4plays an important role in regulating the development of atherosclerosis.