【摘要】 为了探究阿奇霉素对脂多糖（LPS）诱导的人肺泡上皮细胞增殖、凋亡及Janus激酶2(JAK2)/信号转导和转录启动因子3(STAT3)通路的调控作用，体外培养人肺泡上皮细胞A549，分为空白组（不做干预）、LPS组（10μg/m L LPS处理24 h）、低/中/高浓度试验组（10μg/mL LPS+1、2、4μg/mL阿奇霉素）、阿奇霉素组（10μg/mL LPS+4μg/mL阿奇霉素）、抑制剂组（10μg/mL LPS+50μmol/L JAK2/STAT3通路抑制剂AG490）、阿奇霉素+抑制剂组（10μg/mL LPS+4μg/m L阿奇霉素+50μmol/L AG490）、阿奇霉素+激活剂组（10μg/m L LPS+4μg/m L阿奇霉素+0.5μmol/L JAK2/STAT3通路激活剂Colivelin）。干预24 h后，采用酶联免疫吸附试验（ELISA）、细胞计数试剂盒-8(CCK-8)、5-乙炔基-2’脱氧尿嘧啶核苷（EdU）、Hoechst 33258染色法、蛋白免疫印迹法检测炎症因子白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的表达水平、细胞活力、增殖率、凋亡率、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、细胞周期素D1(Cyclin D1)及JAK2/STAT3信号通路相关蛋白表达水平。结果显示：LPS组细胞炎症因子IL-6、IL-8和TNF-α表达水平高于空白组，细胞活力低于空白组；与LPS组相比，高浓度试验组炎症因子IL-6、IL-8、TNF-α表达水平显著降低，细胞活力显著升高。选择在LPS组基础上有差异的4μg/m L阿奇霉素作为阿奇霉素组进行后续试验。LPS组细胞增殖率、Cyclin D1蛋白表达水平低于空白组，细胞凋亡率、Caspase-3、p-JAK2、p-STAT3蛋白表达水平高于空白组。阿奇霉素组和抑制剂组显著扭转了LPS组上述指标的变化。与阿奇霉素组相比，阿奇霉素+抑制剂组细胞增殖率、Cyclin D1蛋白表达水平进一步显著升高，细胞凋亡率、IL-6、IL-8、TNF-α、Caspase-3、p-JAK2、p-STAT3蛋白表达水平进一步显著降低，阿奇霉素+激活剂组则显著逆转了上述指标的变化。本研究表明，阿奇霉素可通过抑制JAK2/STAT3信号通路减轻LPS诱导的A549细胞炎症损伤，促进细胞增殖并抑制凋亡。更多还原

【Abstract】 To explore the regulatory effects of azithromycin on lipopolysaccharide(LPS)-induced proliferation, apoptosis and Janus kinase 2(JAK2)/signal transduction and transcription promoter 3(STAT3) signaling pathways in human alveolar epithelial cells, in vitro culture of human alveolar epithelial cells A549, were divided into blank group(no intervention), LPS group(10 μg/mL LPS treated for 24 h), low/medium/high concentration experimental group(10 μg/mL LPS+1, 2, 4 μg/mL azithromycin) and azithromycin group(10 μg/mL LPS+4 μg/mL azithromycin), inhibitor group(10 μg/mL LPS+50 μmol/L JAK2/STAT3 pathway inhibitor AG490), azithromycin+inhibitor group(10 μg/mL LPS+4 μg/mL azithromycin+50 μmol/L AG490) and azithromycin+activator group(10 μg/mL LPS+4 μg/mL azithromycin+0.5 μmol/L JAK2/STAT3 pathway activator Colivelin). After 24 hours of intervention, enzyme-linked immunosorption assay(ELISA), cell counting kit-8(CCK-8),5-acetyney-2’ deoxyuracil nucleoside(EdU), Hoechst 33258 staining and Western blotting(WB) were used for the expression levels of inflammatory factors interleukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-α(TNF-α), cell viability, proliferation rate, apoptosis rate, Caspase-3 and Cyclin D1 and the expression levels of JAK2/STAT3 signaling pathway related proteins. The results show that: compared with blank group, the expression levels of inflammatory cytokines IL-6, IL-8 and TNF-αin LPS group significantly increased, and cell viability significantly decreased. Compared with LPS group, the expression levels of inflammatory cytokines IL-6, IL-8 and TNF-α in high-concentration experimental group significantly decreased, and cell viability significantly increased. In this study, 4 μg/mL azithromycin with significant difference from LPS group was selected as azithromycin group for follow-up experiments. Compared with blank group, the cell proliferation rate and Cyclin D1 protein expression levels in LPS group significantly decreased, while the apoptosis rate, Caspase-3, p-JAK2 and p-STAT3 protein expression levels significantly increased. Compared with LPS group, azithromycin group and inhibitor group significantly reversed the changes of the above indexes. Compared with azithromycin group, cell proliferation rate and Cyclin D1 protein expression levels in azithromycin+inhibitor group further significantly increased, while apoptosis rate, IL-6, IL-8, TNF-α, Caspase-3, p-JAK2and p-STAT3 protein expression levels further significantly decreased. Azithromycin+activator group significantly reversed the changes of the above indexes. Azithromycin can reduce LPS-induced inflammatory damage of A549 cells by inhibiting JAK2/STAT3 signaling pathway, promote cell proliferation and inhibit apoptosis, and provide evidence for exploring azithromycin treatment of LPS induced acute lung injury.更多还原