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Abstract (The First Hospital of Qinhuangdao a. Pediatric ICU; b. College of Pharmacy, Qinhuangdao 066000, China)
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-α in 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-JAK2 and 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.
Key words: azithromycin; Janus kinase 2; signal transduction and transcription promoter 3; alveolar epithelial cells; inflammatory lung injury
（Acta Laser Biology Sinica, 2024, 33(3): 267-274）
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