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