The Effect And Mechanism Of Apelin-13in The Atherosclerosis Formation

AIM:In vitro cell model of Human aortic smooth muscle cells (HASMC) and human umbilical vein endothelial cells (HUVEC), study the effect of apelin-13on Human aortic smooth muscle cell proliferation and migration,and the effect of apelin-13on the expression of intercellular adhesion molecule(ICAM-1) and vascular cell adhesion molecule (VCAM-1) in Human umbilical vein endothelial cells, The aim is to detect the effect of apelin-13in atherosclerosis formation process, and corresponding cell signaling pathway.Content:Through culturing human arotic smooth cells (HASMC), total RNA extraction, reverse transcription and PCR to detect the expression of APJ(putative receptor protein related to the angiotensin receptor ATI, APJ) in HASMC cells;through stimulating the cells with different concentrations of apelin-13(0.001μM,0.01μM,0.1μM,1μM,10μM) for24,48,72hours respectively.Apply MTT colorimetric to detect the effect of apelin--13on human aortic smooth muscle cell proliferation; through Stimulating the cells with different concentrations of apelin-13(0.001μM,0.01μM,0.1μM,1μM,10μM) for4hours, to detecte the effect of apelin-13on human aortic smooth muscle cell migration through cell count.Through culturing human umbilical vein endothelial cells (HUVEC), total RNA extraction, reverse transcription and PCR to detect the expression of APJ in HUVEC cells; through stimulating the cells with different concentrations of apelin-13(0.001μM,0.01μM,0.1μM,1μM,2μM) for0,3,5,8,12,24hours respectively, to looking for the optimum concentration and the optimum time of apelin-13promoting expression of VCAM-1and ICAM-lin human umbilical vein endothelial cell; With LPS as a positive control, to detect the type of receptor coupled G-protein related in apelin-13promoting expression of VCAM-1and ICAM-1through Gi protein inhibitor PTX, Gq protein inhibitor YM254890and apelin costimulating human umbilical vein endothelial cells.Through the Nuclear factor NF-κB inhibitor BAY11-7082and apelin costimulating human umbilical vein endothelial cells to detect the effect of NF-κB on apelin-13promoting the expression of VCAM-1and ICAM-1. Through Gi protein inhibitor PTX, Gq protein inhibitor YM254890and apelin costimulating human umbilical vein endothelial cells to test the effect of apelin-13on NF-κB phosphory--lation, further testing the type of receptor coupled G-protein.Result:Sequencing results showed that the APJ receptor expression both in human aortic smooth muscle cells and human umbilical vein endothelial cells; MTT assay results showed that apelin-13does not promote the proliferation of human aortic smooth muscle cells; traswell cell migration results showed that apelin-13does not promote human aortic smooth muscle cell migration; western blot results showed that apelin-13can promote the expression of VCAM-1, ICAM-1expression in human umbilical vein endothelial cells,and the expression of VCAM-1and ICAM-1are maximum at0.1μM apelin, and are maximum at8h; during the process of apelin-13promoting VCAM-1、ICAM-1expression, detected the phosphorylation of nuclear factor NF-kB; when apelin-13stimulate PTX pretreatment cells, the role of apelin-13promoting the expression of VCAM-1, ICAM-1is inhibited. And PTX can inhibite the nuclear the factor NF-kB phosphorylation when apelin-13stimulated the HUVEC.Conclusion and significance:(1) APJ expression both in human aortic smooth muscle cells and human umbilical vein endothelial cells.(2) apelin-13did not affect the proliferation and migration of human aortic smooth muscle cells.(3) apelin-13can promote the expression of VCAM-1, ICAM-1in human umbilical vein endothelial cells, apelin-13possibly through apelin-13receptor APJ-G; protein-NF-κB signaling pathway to promote ICAM-1expression;apelin-13possibly through apelin-13receptor APJ-Gi protein-NF-KB signaling pathway to promote VCAM-1expression; apelin-13can promote atherosclerosis formation in the early process through promoting the expression of VCAM-1, ICAM-1in endothelial cells. The study of the effect of apelin in the artery atherosclerosis form provide a theoretical basis for finding a new target for the treatment of arterial atherosclerosis and open up a new areas for the atherosclerosis treatment, and there is also the argument for obesity may lead to atherosclerosis.