Regulatory Effect Of Proton-sensing Receptor GPR4 On Vascular Endothelial Cells Apoptosis And Its Mechanism

Objective:To investigate the regulatory effects of LPC and acidic microenvironment on vascular endothelial cell apoptosis induced by GPR4.Methods:Normal HUVECs cells and HUVECs with the knockdown or overexpression of GPR4 were used as cell models.When the cells were stimulated by LPC and acid medium(p H 6.8)alone or together,apoptosis and the expression of inflammatory factors were detected.The expression of GPR4 m RNA level was detected by RT-q PCR method;The effect of LPC or acidic environment on the proliferation of HUVEC cells was detected by CCK8method;The effect of acidic medium of p H 6.8 or stimulation of LPC on apoptosis of HUVEC cells was detected by flow cytometry;GPR4 knockdown HUVEC cell line was constructed by sh RNA technique,and GPR4 overexpression cell line was constructed by a lentiviral vector with overexpressing GPR4.ELisa was used to detect the expression of TNF-αand IL-6 in cells.Results:In HUVEC cells,GPR4 is highly expressed,while other receptors are hardly expressed.Compared with the normal control group,10μM LPC inhibited the proliferation of HUVEC cells in a dose-dependent manner,and the cell proliferation decreased gradually with the decrease of p H value of the medium.Both LPC and acid medium induced apoptosis,especially in an acid environment;however,it is interesting that when cells were treated with LPC and acid medium at the same time,apoptosis did not simply increase.LPC inhibits apoptosis induced by an acid environment.The expression of GPR4 m RNA was significantly up-regulated by 5-fold and 25-fold due to the stimulation of LPC and p H6.8 acid medium,respectively,but the expression of GPR4 was significantly lower than that of cells stimulated by acid medium alone when LPC and acid medium were stimulated together.The knockdown of the GPR4 gene attenuated apoptosis in the LPC and acid environment,while the overexpression of the GPR4 gene further confirmed that LPC inhibited apoptosis induced by the acid environment to some extent when cells were treated with LPC and acid medium at the same time.The results of ELISA showed that the LPC and the acidic medium of p H 6.8could promote the levels of TNFαand IL-6 either alone or simultaneously.Both acidic medium and LPC upregulated the m RNA expression of P53.Conclusion:Both LPC and proton(H~+)can activate GPR4 and induce two different signal pathways to induce apoptosis in HUVEC cells,which have feedback regulation between each other.LPC and proton(H~+)promote apoptosis of HUVEC cells through GPR4-induced expression of inflammatory factors and the p53 pathway.