基于JAK2/STAT3通路探讨阿奇霉素对脂多糖诱导肺泡上皮细胞的保护作用

Investigation of the Protective Effect of Azithromycin on Lipopolysaccharide-Induced Alveolar Epithelial Cells Based on JAK2/STAT3 Pathway

目的探究阿奇霉素(AZI)对脂多糖(LPS)诱导的人肺泡上皮细胞增殖生长、迁移的作用及相关机制。方法体外培养人肺泡上皮细胞A549,分为对照组(不做干预)、LPS组(10μg/ml LPS处理24 h)、低/中/高剂量实验组(10μg/ml LPS+2、4、8μg/ml AZI)、AZI组(10μg/ml LPS+4μg/ml AZI)、抑制剂组(10μg/ml LPS+50μmol/L JAK2/STAT3通路抑制剂AG490)、AZI+抑制剂组(10μg/ml LPS+4μg/ml AZI+50μmol/L AG490)、AZI+激活剂组(10μg/ml LPS+4μg/ml AZI+0.5μmol/L JAK2/STAT3通路激活剂Colivelin)。干预24 h后,采用细胞计数试剂盒-8(CCK-8)、酶联免疫吸附试验(ELISA)、倒置显微镜、划痕法、蛋白免疫印迹(WB)法检测炎症因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6表达水平、细胞生长、迁移率、上皮间质转化(EMT)及JAK2/STAT3信号通路相关蛋白表达水平。结果LPS组细胞活力较对照组下降(P<0.05)。中/高剂量实验组细胞活力较LPS组上升(P<0.05)。因此选择有显著差异的较低浓度(4μg/ml AZI)作为AZI组进行后续实验。与对照组相比,LPS组细胞生长受抑制,TNF-α、IL-1β、IL-6、E-钙黏蛋白(E-cadherin)表达降低(P<0.05),细胞迁移率、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维粘连蛋白(FN)、p-JAK2、p-STAT3蛋白表达升高(P<0.05)。与LPS组相比,AZI组和抑制剂组显著扭转了上述指标的变化(P<0.05)。与AZI组相比,AZI+抑制剂组细胞生长状态较好,E-cadherin蛋白表达进一步升高(P<0.05),细胞迁移率、TNF-α、IL-1β、IL-6、N-cadherin、Vimentin、FN、p-JAK2、p-STAT3蛋白表达进一步降低(P<0.05),AZI+激活剂组则显著逆转了上述指标的变化(P<0.05)。结论阿奇霉素能够通过抑制JAK2/STAT3信号通路减轻对A549细胞的炎症损伤,促进细胞生长,并抑制其迁移与上皮间质转化(EMT)进程。

Objective To explore the effects of azithromycin(AZI)on the proliferation,growth and migration of human alveolar epithelial cells induced by lipopolysaccharide(LPS)and the related mechanisms.Methods In vitro culture of human alveolar epithelial cells A549 were divided into blank group(no intervention),LPS group(10μg/ml LPS treated for 24 h),low-dose/medium-dose/high-dose experimental group(10μg/ml LPS+2,4,8μg/ml AZI)and AZI group(10μg/ml LPS+4μg/ml AZI),inhibitor group(10μg/ml LPS+50μmol/L JAK2/STAT3 pathway inhibitor AG490),AZI+inhibitor group(10μg/ml LPS+4μg/ml AZI+50μmol/L AG490)and AZI+activator group(10μg/ml LPS+4μg/ml AZI+0.5μmol/L JAK2/STAT3 pathway activator Colivelin).After 24 hours of intervention,The expression levels of inflammatory factors tumor necrosis factor(TNF-α),interleukin(IL-1β)and IL-6,cell growth,mobility,epithelial mesenchymal transformation(EMT)and J AK2/STAT3 signaling pathway related protein expression levels were detected by cell counting Kit 8(CCK-8),enzyme-linked immunosorption assay(ELISA),inverted microscopy,scratching method,and protein western blotting(WB).Results The cell viability of LPS group was lower than that of control group(P<0.05).The cell viability of medium/high dose experimental group was higher than that of LPS group(P<0.05).Therefore,a different lower concentration(4μg/ml AZI)was selected as AZI group for follow-up experiments.Compared with the control group,the growth of cells in LPS group was inhibited,and the expressions of TNF-α,IL-1β,IL-6 and E-cadherin were decreased(P<0.05).The expressions of cell mobility,N-cadherin,Vimentin,fibronectin(FN),p-JAK2 and p-STAT3 were increased(P<0.05).Compared with LPS group,AZI group and inhibitor group significantly reversed the changes of the above indexes(P<0.05).Compared with AZI group,AZI+inhibitor group showed better cell growth,and E-cadherin protein expression was further increased(P<0.05).Cell mobility,TNF-α,IL-1β,IL-6,N-cadherin,Vimentin,FN,p-JAK2 and P-Stat3 protein expressions were further decreased(P<0.05),while those of AZI+activator group were significantly reversed(P<0.05).Conclusion AZI can reduce inflammatory damage to A549 cells,promote cell growth,and inhibit migration and epithelial mesenchymal transition(EMT)process by inhibiting JAK2/STAT3 signaling pathway.