The Effects Of S1P Induced Autophagy Regulation On Cardiomyocytes Hypoxia/Reoxygenation Injury

Objective:To observe the effect of S1P(sphingosine-1-phosphate)postconditioning on autophagy in H9c2 cells cardiomyocytes hypoxia/reoxygenation injury,explore the relationship between S1 P induced autophagy modulation and antimyocardial ischemia reperfusion injury effects.And study the correlation of autophagy modulation of S1 P and AMPK/mTOR pathway.Methods:1.The cells culture of H9c2 cells and model establishement of cells hypoxia reoxygenation injuryH9c2 cells were taken out from liquid nitrogen container and then were placed with DMEM medium containing 10% FBS by using convention adherent cell culture methods.They were cultured in an atmosphere of 5% CO2 and the temperature were keeping 37 oC.The culture medium was replaced and cells subculture every 2-3 days.24 hours later,the cells were divided into seven groups randomly and then six groups were subjected to 16 hours hypoxia in a 95% N2 and 5% CO2 gas mixture except the normal group.The culture medium was replaced by hypoxia buffer for 16 hours then retrieved again for 4 hours reoxyenation.2.Divide cells into seven groups randomly The H9c2 cells were divided into seven groups randomly and numbered in turn:(1)the normal group: cells were cultured with DMEM medium for 4 h;(2)hypoxia/reoxyenation group(H/R): cells were cultured with hypoxia buffer for 16 h and reoxyenation for 4 h;(3)S1P group: 4 ?M S1 P was added into the medium after the 16 h hypoxia and then reoxyenation for 4 h;(4)S1P+3-methyladenine group(S1P+3-MA): after the 16 h hypoxia,cells were treated with 10 mM 3-MA(inhibitor of autophagy)and 4 ?M S1 P for 4h with reoxygenation(5)3-methyladenine group(3-MA): 10 m M 3-MA was added into the medium after the 16 hypoxia and then reoxyenation for 4 h;(6)S1P+Rapamycin group(S1P+RAPA): After the 16 h hypoxia,cells were treated with 500 nM RAPA(inducer of autophagy)and 4 ?M S1 P for 4 h with reoxygenation;(7)Rapamycin group(RAPA): 500 nM RAPA was added into the medium after the16 hypoxia and then reoxyenation for 4 h;3.To explore the influence of S1 P,3-MA and RAPA on the cell viability in the H/R modelThe cell viability in 7 groups was measured by using the MTT assay.Insure the protective effect of S1 P postconditioning.4.To explore the influence of S1 P,3-MA and RAPA on the LDH levels in cell culture medium.The LDH levels in cell culture medium was measured to reflex the level of cell injury.5.To explore the relationship between autophagy and the protective effect of S1 P postconditioning on reducing the hypoxia/reoxygenation injury.The level of autophagy-related protein(LC3,Beclin-1,LAMP-2)were assayed by Western Blot to study the influence of S1 P on autophagy process by hypoxia/reoxygenation.6.To explore the relationship between the S1 P induced autophagy regulation and AMPK/mTOR pathway.The phosphorylation of AMPK and mTOR were measured by western blot to determine whether S1 P induced autophagy regulation was connected with AMPK/mTOR pathway.7.To explore the influence of S1 P on the cell apoptosis.The cell apoptosis was measured by using FITC Annexin V Apoptosis Detection Kit I via flow cytometry.Results:1.Cell viabilityThe viability of cells was decreased significantly in the H/R group than normal group.In the S1 P group,the viability of cells was increased significantly than H/R group.In the S1P+RAPA group,the viability of cells was increased than S1 P group.RAPA could counteract the effect of S1 P inducing cardiomyocytes H/R injury.2.The level of LDH in the culture mediumThe level of LDH in the normal group was low.Compared with the normal group,the LDH levels in the H/R group was increased significantly.S1 P decreased markedly the activity of LDH in the S1 P group compared with H/R group.In the3-MA group,the level of LDH was decreased.Compared with S1 P group,the level of LDH in S1P+3-MA group was decreased markedly.RAPA could increase the level of LDH.When the cells were treated with S1 P and RAPA,the level of LDH was increased significantly.This result revealed that the effect of S1 P reduing the level of LDH was suppressed by RAPA.3.The influence of S1 P on the expression of LC3,Beclin-1 and LAMP-2 protein.The results of western blot reveal that the expression of LC3-? protein in the normal group was high,but the expression of LC3-? protein was low.In with the H/R group,the expression of LC3-? protein and the specific value of LC3-? and LC3-? was increased markedly.Compared with the H/R group,the expression of LC3-? protein and the was decreased.RAPA could rise the specific value of LC3-?and LC3-?mildly and surpress the effect of S1 P reducing the specific value of LC3-? and LC3-?.The results of western blot reveal that the expression of Beclin-1 in the normal group was low.The expression of Beclin-1 in the H/R group was increased significantly than the normal group.In the S1 P group,the expression of Beclin-1protein was decreased than the H/R group.In the S1P+RAPA group,the expression of Beclin-1 protein was increased than the S1 P group.This phenomenon revealed that the effect of S1 P decreasing the expression of Beclin-1 protein was depressed.The results of western blot reveal that the expression of LAMP-2 in the normal group was high.The expression of LAMP-2 in the H/R group was significantly decreased than the normal group.In the S1 P group,the expression of LAMP-2 was increased than the H/R group.In the S1P+RAPA group,the expression of LAMP-2was significantly decreased than the S1 P group.This phenomenon revealed that the effect of S1 P increasing the expression of LAMP-2 was depressed.4.The influence of S1 P on the phosphorylation expression of AMPK and m TOR protein.The result of the Western blot revealed that the expression of t-AMPK protein had no obvious difference in groups.In the normal group,the expression of p-AMPK protein was low.Compared with the normal group,the expression of p-AMPK protein in the H/R group was increased markedly.S1 P and RAPA had no effect on the increase of the expression of p-AMPK protein result from H/R.The expression of t-mTOR protein had no obvious difference in groups.Compared with the normal group,the expression of p-mTOR protein in the H/R group was increased significantly.S1 P could raise the expression of p-mTOR protein markedly.The expression of p-mTOR protein in the RAPA group was decreased significantly.RAPA could suppress the effect of S1 P reduing the expression of p-mTOR protein.5.Cell apoptosisThe rate of cell apoptosis in the normal group was low.Compared with normal group,the rate of cell apoptosis in the H/R group was enhanced significantly.The rate of apoptotic cells in S1 P group was decreased markedly compared with H/R group.RAPA had no effect on the cell apoptosis and could suppress the impact of S1 P decreasing cell apoptosis.Conclusion:1.S1 P can induce autophagy regulation,which further antagonize myocardial hypoxia reoxygenation injury,inhibit myocardial apoptosis.2.S1 P inhibits autophagy via promoting the phosphorylation of m TOR,not the phosphorylation of AMPK.