脾酪氨酸激酶对氧化应激诱导的气道上皮细胞黏液高分泌的影响

Effects of spleen tyrosine kinase on oxidative stress-induced mucous hypersecretion of airway epithelial cells

目的研究脾酪氨酸激酶(Syk)在氧化应激诱导的气道黏液高分泌中的作用。方法体外培养人气道上皮细胞(16HBE),用H_2O_2刺激细胞构建黏液高分泌模型,以Syk siRNA、丝裂原活化蛋白激酶(MAPK)信号通路中的细胞外信号调节激酶(ERK1/2)特异性抑制剂U0126及P38特异性抑制剂SB203580为干预因素,将细胞随机分为对照组、H_2O_2刺激组、H_2O_2+Syk siRNA组、H_2O_2+阴性siRNA组、H_2O_2+U0126组及H_2O_2+SB203580组。四甲基偶氮唑盐法(MTT)测试细胞活力;RT-PCR和ELISA法分别检测黏蛋白(MUC)5AC的转录及分泌水平;Western blotting法检测Syk、磷酸化ERK1/2(p-ERK1/2)和磷酸化P38(p-P38)蛋白表达水平;激光共聚焦技术检测MUC5AC蛋白的含量和分布。结果 H_2O_2刺激后Syk、p-ERK1/2、p-P38、MUC5AC表达水平显著高于对照组(均P=0.000),而利用Syk siRNA、SB203580和U0126处理后,H_2O_2导致的上述效应明显得到抑制(均P=0.000)。结论 Syk通过ERK1/2、P38 MAPK信号通路,参与氧化应激诱导的气道黏液高分泌。

Objective To explore the effects of spleen tyrosine kinase（Syk） on oxidative stress-induced airway mucous hypersecretion.Methods Human airway epithelial cells 16 HBE were cultured in vitro.The mucous hypersecretion model was established by stimulating cells by H2O2.Intervention factors were Syk siRNA and extracellular signal regulated kinase（ERK1/2） specific inhibitor U0126 and P38 specific inhibitor SB203580 of mitogen-activated protein kinase（MAPK） signaling pathway.The cells were randomly divided into the control group,H2O2 group,H2O2 ＋Syk siRNA group,H2O2 ＋ negative siRNA group,H2O2 ＋ U0126 group,and H2O2 ＋ SB203580 group.The cell viability was detected by MTT assay.Transcription and protein expression of mucin（MUC） 5AC were detected by RT-PCR and ELISA respectively.The protein expression of Syk,phosphorylated ERK1/2（p-ERKl/2）,and phosphorylated P38（p-P38） were detected by Western blotting.The content and distribution of MUC5 AC protein were detected by confocal laser technology.Results Compared with the control group,expressions of Syk,p-ERKl/2,p-P38,and MUC5 AC significantly increased after being stimulated by H2O2（P = 0.000）.Above effects of H2O2 were significantly inhibited after being treated by Syk siRNA,U0126,and SB203580（P = 0.000）.Conclusion Syk involves the oxidative stress induced airway mucous hypersecretion via ERK1/2 and P38 MAPK signaling pathways.