Differential Modulation of Macrophage Polarization and NF-κB Signaling by Distinct Antibiotics in an in vitro Acute Inflammatory Model

Chunmin Chen, Jingdi Diao, Yuan Chen, Yi BoEmergency Department, Lianyungang Hospital of Jiangsu Provincial Hospital of Traditional Chinese Medicine, Lianyungang, Jiangsu, 222004, People’s Republic of ChinaCorrespondence: Yi Bo, Email [email protected]: This study aimed to compare the regulatory effects of cefotaxime (CTX), levofloxacin (LVFX), and azithromycin (AZM) on macrophage polarization and NF-κB signaling in an in vitro LPS-induced acute inflammation model.Methods: RAW264.7 murine macrophages were pretreated with CTX (25 μg/mL), LVFX (25 μg/mL), or AZM (10 μg/mL) for 1 h and subsequently stimulated with LPS (100 ng/mL). Cell viability was assessed using CCK-8 assays. TNF-α, IL-1β, and IL-10 levels were quantified by ELISA. M1-associated markers (iNOS and CD86) and M2-associated markers (Arg-1 and CD206) were evaluated using qRT-PCR and immunofluorescence. NF-κB pathway activation was examined by Western blot analysis of p65 nuclear translocation, IκBα expression, and IKKα/β and p65 phosphorylation.Results: All three antibiotics attenuated LPS-induced proinflammatory responses, as shown by reduced TNF-α and IL-1β levels and increased IL-10 secretion. CTX, LVFX, and particularly AZM suppressed M1-associated marker expression while increasing M2-associated marker expression. Western blot analysis showed that antibiotic pretreatment inhibited NF-κB activation by reducing IKKα/β and p65 phosphorylation, limiting p65 nuclear translocation, and restoring IκBα expression, with AZM exerting the strongest effect among the three antibiotics.Conclusion: CTX, LVFX, and AZM exerted differential immunomodulatory effects in LPS-stimulated RAW264.7 macrophages. AZM showed the most pronounced ability to attenuate inflammatory activation, promote an M2-like macrophage phenotype, and suppress NF-κB signaling. These findings provide preliminary in vitro evidence that antibiotics may differ in their immunomodulatory properties, although further validation in primary macrophages, pharmacologically relevant exposure ranges, and in vivo inflammatory models is required.Keywords: macrophage polarization, acute inflammation, NF-κB signaling, azithromycin, LPS-stimulated RAW264.7 cells