Effect of sphingosine-1-phosphate on activity of lung fibroblasts and its mechanism
Objective To investigate the effect of sphingosine-1-phosphate(S1P)on the activity of lung fibroblasts and its possible mechanism.Methods Lung fibroblasts were incubated with different concentrations of S1P(0,0.01,0.03,0.1,0.3,1,3 μmol/L)for 24 h to determine the optimal intervention concentration,and the cells were pretreated with the optimal intervention concentration of S1P for different times(24,48,72 h),and then the cell proliferation was measured by CCK-8 assay.Lung fibroblasts were divided into six groups:control group,S1P group,JTE013+S1P group,CAY10444+S1P group,control siRNA+S1P group and YAP siRNA+S1P group.Lung fibroblasts were treated with 1 μmol/L S1P for 24 h in S1P group.In JTE013+S1P group and CAY10444+S1P group,lung fibroblasts were pretreated with a selective sphingosine-1-phosphate receptor(S1PR)2 antagonist(10 μmol/L JTE013)or a S1PR3 antagonist(10 μmol/L CAY10444)for 1 h before stimulation with S1P.In control siRNA+S1P group and YAP siRNA+S1P group,lung fibroblasts were respectively transfected with control siRNA or YAP siRNA,and then treated with 1 μmol/L S1P for 24 h.The cell proliferation was measured by CCK-8 assay,the protein levels of t-Yes-associated protein(YAP),p-YAP,α-smooth muscle actin(α-SMA),Collagen Ⅰ and Fibronectin were determined using immunoblotting.Results S1P dose-and time-dependently induced the proliferation of lung fibroblasts(P<0.05).Compared with control group,the protein level of p-YAP was downregulated in S1P group(P<0.001),and α-SMA,Collagen Ⅰ and Fibronectin were upregulated(P<0.05).Compared with S1P group,the protein levels of p-YAP were upregulated(P<0.05),and α-SMA,Collagen Ⅰ and Fibronectin levels and the cell proliferation were downregu-lated in both JTE013+S1P group and CAY10444+S1P group(P<0.05).Moreover,JTE013 was more effective than CAY10444(P<0.05).Compared with S1P group,α-SMA,Collagen Ⅰ and Fibronectin levels and the proliferation of lung fibroblasts were inhibited in YAP siRNA+S1P group(P<0.05).Conclusion S1P can induce the activation of lung fibroblasts,which is related with the activa-tion of YAP by binding to S1PR2/3.
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