Study On The Effect Of Curcumin On The Proliferation Of Vascular Smooth Muscle Cells (VSMC) In Acidified Environment

Background:In the pathologic conditions of ischemia,hypoxia,inflammation and injury,local cell glucose metabolism disorder,the increase of glycolysis resulted in local accumulation of acidic substances such as lactic acid,resulting in lower pH value of the internal environment and acidification of extracellular environment,thus affecting the function of various cells.Vascular smooth muscle cells(VSMC)will go to the differentiation to obtain strong ability of proliferation and migration in pathological factors,and this is the pathophysiological basis of cardiovascular diseases such as hypertension,atherosclerosis,and restenosis after angioplasty.Recently,it is a hot area of research that the activation of acid-sensitive ion channels(ASICs)resulted from the low p H and H~+in the internal environment,the activation of ASICs mediated cationic flow,and then activated downstream MAPK,ERK1/2 and other signaling pathways,finally affected the activity of vascular smooth muscle cells.In this study,the rat thoracic aorta vascular smooth muscle cells were cultured in vitro under different the pH stimulations.To observe relationship between the functional changes of vascular smooth muscle cells and the activation of acid-sensitive ion channels,in order to explore the mechanism of curcumin inhibitory proliferation of VSMC and to broaden the clinical application prospect of curcumin.Objective:To observe the relationship between the proliferation of VSMCs and the ASIC 1a under different pH in vitro,in order to explore the pathway and mechanism of curcumin inbitory proliferation of VSMC..Methods:1.Culture and identification of VSMCs:adopt cell patch method.By separating the aortic vessels of the rats,the outer membrane of the blood vessels was removed,and the inner membrane was scraped into small tissue pieces,and the tissue was soaked in a culture bottle for 5-7d.Cell purity was identified by flow cytometry and cell anti-a-sma antibody.The vascular smooth muscle cells used in the experiment were all cells from the 4th to 6th generation.2.Detection of acidic conditions the expression of vascular smooth muscle cells ASCIS 1a:groups:pH6.9+control,pH6.9+curcumin(20mmo L/L),pH6.9+PCTX1,pH6.9+PCTX1+curcumin(20 mmoL/L),pH7.4+control.The expression of ASICS 1a protein was detected by western-blot method.3.Test the expression of phosphorylated ERK1/2 protein of cells in acidicenvironment:groups:pH7.4+control;pH6.9+control;p H6.9+curcumin;p H6.9+PCTX1;pH6.9+PD98059;pH6.9+curcumin+PD98059;pH6.9+PCTX-1+PD98059.ERK1/2 protein expression was detected by Western-Blot method.4.CCK-8 method for the determination of proliferation of vascular smooth muscle cells:groups:pH7.4+control;pH6.9+control;pH6.9+curcumin(20mmol/L);pH6.9+PCTX1;pH6.9+PCTX1+curcumin(20 moL/L);Cell activity and proliferation rate were detected by CCK-8.Results:1.Compared with control group,the acidification of the vascular smooth muscle cells under the stimulate of express ASIC 1a increased(P<0.05),respectively in specific inhibitors and curcumin PCTX-1,ASIC 1a expression of different degrees were inhibited(P<0.05).There was no significant difference between the expression of ASIC 1a and the control group(P<0.05).2.Compared with the control group,the phosphorylation ERK1/2 protein expression in VSMC cells increased significantly(P<0.05).However,the expression of the cell phosphorylation ERK1/2 protein was inhibited by the three factors of curcumin,PCTX-1 and ERK1/2 pathway inhibitor PD98059,and the inhibitory degree of each group was different(P<0.05).The experimental group of curcumin+PD98059 and the group of PCTX-1+PD98059 were significantly enhanced in the original inhibitory degree,and the inhibitory degree of the two groups was different(P<0.05),but there were still differences between the two groups(P<0.05).3.Compared with the control group,the OD value of vascular smooth muscle(VSMC)decreased(P<0.05)in the acidification environment,considering the possibility of cell damage mediated by calcium ions.However,the OD values of curcumin,PCTX-1 and the both were significantly decreased(P<0.05),and there were differences in each group.In terms of proliferation activity,the experimental group was significantly higher than the other groups,and the difference was significant.While,the group which combine curcumin and PCTX-1,the proliferation activity of cells was inhibited,and the degree of inhibition was different(P<0.05).Combined with PCTX-1and curcumin,thegroup has the greatest inhibitory effect on cell proliferation.Conclusion:In the acidification environment caused by ischemia and hypoxia,the expression of ASIC 1a in the vascular smooth muscle of the thoracic aorta in rats increase,and regulate the proliferation of vascular smooth muscle,through the phosphorylation of ERK1/2 in the cell.It is suggested that curcumin inhibit the proliferation of VSMC through ASIC 1a and phosphorylation of ERK1/2 in acidification environment.