1-磷酸鞘氨醇对人胎儿肺成纤维细胞中α-平滑肌肌动蛋白表达的作用

Effect of sphingosine 1-phosphate onα-smooth muscle actin expression in human fetal lung fibroblasts

目的探讨1-磷酸鞘氨醇(S1P)通过1-磷酸鞘氨醇受体2(S1P2)及RhoA/Rho激酶通路对人胎儿肺成纤维细胞(HFL-1)中α-平滑肌肌动蛋白(α-SMA)表达的作用。方法本研究为实验研究。培养14~19代HFL-1。应用蛋白质印迹法分别检测不同浓度(10-7 mol/L、10^(-6)mol/L、10-5 mol/L)S1P刺激下HFL-1中α-SMA的表达,10^(-6)mol/L S1P及10^(-6)mol/L S1P2拮抗剂JTE-013刺激下HFL-1中α-SMA、RhoA的表达,10μg/L RhoA抑制剂C3胞外酶、10-5 mol/L Rho激酶抑制剂Y27632、10^(-6)mol/L S1P刺激下HIF-1中α-SMA的表达。结果S1P 10^(-6)mol/L组、S1P 10-5 mol/L组α-SMA的相对表达量0.641(0.591,0.747)、0.662(0.633,0.810)较空白对照组0.116(0.089,0.456)升高,差异均有统计学意义(均P<0.05)。S1P组α-SMA的相对表达量0.907(0.466,1.629)较空白对照组0.540(0.210,0.807)、JTE-013组0.296(0.182,0.598)升高,差异均有统计学意义(均P<0.05)。S1P10^(-6)mol/L 5 min组RhoA的相对表达量0.931(0.660,0.977)较S1P 10^(-6)mol/L 0 min组0.060(0.023,0.061)升高;S1P 10^(-6)mol/L 10 min组RhoA的相对表达量0.086(0.016,0.097)较S1P 10^(-6)mol/L 5 min组RhoA的相对表达量0.931(0.660,0.977)降低;S1P 10^(-6)mol/L组RhoA的相对表达量2.221(2.063,3.061)较空白对照组0.833(0.092,1.393)、JTE-01310^(-6)mol/L组0.619(0.145,0.967)、JTE-01310^(-6)mol/L+S1P 10^(-6)mol/L组0.984(0.354,1.442)升高,差异均有统计学意义(均P<0.05)。S1P组α-SMA的相对表达量0.738(0.389,0.774)较空白对照组0.120(0.057,0.120)、C3胞外酶组0.073(0.027,0.093)高,差异均有统计学意义(均P<0.01)。Y27632+S1P组α-SMA的相对表达量0.142(0.014,0.430)较空白对照组0.544(0.489,0.632)低,差异有统计学意义(P<0.05)。结论S1P在毫摩尔水平浓度通过S1P2及其下游信号RhoA/Rho激酶通路调控HFL-1中α-SMA的表达。

Objective To investigate the effect of sphingosine 1-phosphate(S1P)onα-smooth muscle actin expression in human fetal lung fibroblasts(HFL-1)through S1P2 and RhoA/Rho kinase pathways.Methods This was a laboratory study.HFL-1 cells of 14-19 generations were cultured for the experiments.Western blot method was used to detect the expression ofα-SMA in HFL-1 stimulated with different concentrations of S1P(10-7 mol/L,10^(-6)mol/L,and 10-5 mol/L),the expressions ofα-SMA and RhoA stimulated by 10^(-6)mol/L S1P2 antagonist JTE-013,the expression ofα-SMA stimulated by 10μg/L RhoA inhibitor C3 extracellular enzyme,by 10-5 mol/L Rho Kinase inhibitor Y27632 and 10^(-6)mol/L S1P in HIF-1.Results The relative expression levels ofα-SMA in S1P 10^(-6)mol/L and S1P 10-5 mol/L groups were 0.641(0.591,0.747)and 0.662(0.633,0.810),higher than those in blank control group 0.116(0.089,0.456),and the differences were statistically significant(all P<0.05).The relative expression ofα-SMA in S1P group was 0.907(0.466,1.629),higher than that in blank control group 0.540(0.210,0.807)and JTE-013 group 0.296(0.182,0.598),and the differences were statistically significant(all P<0.05).The relative expression level of RhoA in S1P 10^(-6)mol/L 5 min group was higher at 0.931(0.660,0.977)than that in S1P 10^(-6)mol/L 0 min group at 0.060(0.023,0.061).The relative expression of RhoA in S1P 10^(-6)mol/L 10 min group was 0.086(0.016,0.097),which was lower than that in S1P 10^(-6)mol/L 5 min group 0.931(0.660,0.977).The relative expression of RhoA in S1P 10^(-6)mol/L group was 2.221(2.063,3.061),which was higher than that in blank control group 0.833(0.092,1.393),JTE-01310^(-6)mol/L group 0.619(0.145,0.967),and JTE-01310^(-6)mol/L+S1P 10^(-6)mol/L group 0.984(0.354,1.442),with statistical significance(all P<0.05).The relative expression level ofα-SMA in S1P group was 0.738(0.389,0.774),higher than that in blank control group 0.120(0.057,0.120)and C3 extracellular enzyme group 0.073(0.027,0.093),and the differences were statistically significant(all P<0.01).The relative expression level ofα-SMA in Y27632+S1P group was 0.142(0.014,0.430),lower than that in blank control group 0.544(0.489,0.632),and the difference was statistically significant(P<0.05).Conclusions S1P stimulates the expression ofα-SMA in lung fibroblasts through S1P2 and its downstream signaling RhoA/Rho kinase pathway.