Abstract
Background: Macrophage-mediated inflammatory signaling drives pathological inflammation in diverse diseases. Zinc oxide nanoparticles (ZnO NPs) exhibit recognized anti-inflammatory activity, but their precise molecular mechanisms remain unclear. This study investigates the modulatory effects of ZnO NPs on Lipopolysaccharide (LPS)-induced inflammatory and underlying pathways in RAW264.7 macrophages. Methods: Scanning electron microscopy (SEM) was used to analyze the primary particle sizes of ZnO NPs. Their cytotoxicity on RAW264.7 cells was assessed using the CCK-8 assay. Cells were pretreated with ZnO NPs (0, 0.5, 1, 2, 5 μg/mL) for 1 h, then stimulated with LPS (1 μg/mL) for 24 h. Pro-inflammatory factors (TNF-α, IL-1β, IL-6, iNOS, and COX-2), as well as the anti-inflammatory factor Arg-1, were detected using qRT-PCR, ELISA, or Western blot. RNA-sequencing identified differentially expressed genes (DEGs). Nitric oxide (NO) production, Reactive Oxygen Species (ROS). Results: ZnO NPs reduced LPS-induced production of NO and pro-inflammatory factors but increased Arg-1 expression. RNA-sequencing identified 2638 DEGs (1822 upregulated, 816 downregulated) between LPS and LPS+ZnO NPs groups. Mechanistically, ZnO NPs exerted anti-inflammatory effects through: (1) blocking NF-κB activation by inhibiting IκB-α degradation and p65 nuclear translocation; (2) suppressing JAK1-mediated STAT1/3 activation and nuclear translocation. They also reduced LPS-induced ROS. Conclusion: ZnO NPs mitigate LPS-triggered inflammation by targeting NF-κB and JAK1-STAT1/3 pathways, and reducing ROS. These findings provide novel mechanistic insights into the anti-inflammatory effects of ZnO NPs, highlighting their therapeutic potential in macrophage-associated inflammatory disorders.