ZnO NPs Attenuate LPS-Induced Inflammation in RAW264.7 Macrophages by Inhibiting NF-κB and JAK1-STAT1/STAT3 Pathways and Reducing ROS

Hui Tang,1,2 Li Zhang,1,2 Chengchen Deng,2,3 Yuechuan Li,2,3 Guiying Ren,2,4 Ziru Sun,2,4 Lebing Chang,2,4 Meihong Xu,2,4 Duanqiang Xiao,2,4 Haijun Zhang1,2 1Department of Interventional and Vascular Surgery, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, 200072, People’s Republic of China; 2National United Engineering Laboratory for Biomedical Material Modification, Branden Industrial Park, Dezhou, Shandong, 251100, People’s Republic of China; 3Key Laboratory for Biorheological Science and Technology of Ministry of Education, National Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, People’s Republic of China; 4School of Materials Science and Engineering, Shandong University of Technology, Zibo, Shandong, 255000, People’s Republic of ChinaCorrespondence: Haijun Zhang, Department of Interventional and Vascular Surgery, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, No. 301, Middle Yanchang Road, Shanghai, 200072, People’s Republic of China, Email [email protected]: 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.Keywords: macrophages, ZnO NPs, inflammatory cytokine, NF-κB signaling pathway, JAK-STAT signaling pathway, ROS