鞘氨醇-1-磷酸对肺成纤维细胞活性的作用及其机制

Effect of sphingosine⁃1⁃phosphate on activity of lung fibroblasts and its mechanism

目的 探讨鞘氨醇-1-磷酸(sphingosine-1-phosphate, S1P)对肺成纤维细胞活性的影响及其可能机制。方法 以0,0.01,0.03,0.1,0.3,1,3μmol/L S1P干预肺成纤维细胞24 h,筛选最佳干预浓度;随后以确定的S1P干预浓度刺激细胞0,24,48,72 h,CCK-8法检测细胞增殖。将肺成纤维细胞分为对照组、S1P组、JTE013+S1P组、CAY10444+S1P组、control siRNA+S1P组和YAP siRNA+S1P组。S1P组以1μmol/L S1P干预肺成纤维细胞24 h;JTE013+S1P组和CAY10444+S1P组细胞先分别用10μmol/L选择性S1P受体2(sphingosine-1-phosphate receptor 2, S1PR2)拮抗剂JTE013或10μmol/L选择性S1PR3拮抗剂CAY10444预处理1 h后再给予1μmol/L S1P干预24 h;control siRNA+S1P组和YAP siRNA+S1P组细胞先分别转染control siRNA或YAP siRNA,再给予1μmol/L S1P干预24 h。CCK-8法检测细胞增殖;免疫印迹法检测t-Yes相关蛋白(Yes-associated protein, YAP)、p-YAP、α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)、纤维粘连蛋白(Fibronectin)水平。结果 S1P干预后,肺成纤维细胞增殖随浓度和时间依赖性增加(P<0.05),筛选最佳干预浓度为1μmol/L。与对照组相比,S1P组细胞p-YAP水平减少(P<0.001),α-SMA、CollagenⅠ和Fibronectin蛋白水平增多(P<0.05)。与S1P组相比,JTE013+S1P组和CAY10444+S1P组细胞p-YAP水平均增多(P<0.05),α-SMA、CollagenⅠ和Fibronectin蛋白水平均减少(P<0.05),细胞增殖均受到抑制(P<0.05);细胞增殖均受到抑制(P<0.05);且JTE013的作用效果较CAY10444显著(P<0.05);YAP siRNA+S1P组细胞α-SMA、CollagenⅠ和Fibronectin蛋白水平,以及细胞增殖均降低(P<0.05)。结论 S1P通过与S1PR2/3结合激活YAP,诱导肺成纤维细胞活化。

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(0,0.01,0.03,0.1,0.3,1,3μmol/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 1μmol/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(10μmol/L JTE013)or a S1PR3 antagonist(10μmol/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 1μmol/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),CollagenⅠand 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),andα-SMA,CollagenⅠand Fibronectin were upregulated(P<0.05).Compared with S1P group,the protein levels of p-YAP were upregulated(P<0.05),andα-SMA,CollagenⅠand 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,CollagenⅠand 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.