Structurally Defined Chitosan Oligosaccharides (COS) Suppress Macrophage Inflammatory Signaling and Enzymes in Atherosclerosis Relevant Pathways

Oxidative stress-induced macrophage activation and vascular injury are major contributors to atherosclerosis, a chronic inflammatory disease associated with approximately twenty million deaths worldwide. This study investigated the antioxidant and anti-inflammatory activities of structurally characterized low-molecular-weight chitosan oligosaccharides (COS) derived from mud crab (Scylla olivacea) shell waste by hydrochloric acid hydrolysis and explored their molecular interactions with inflammation-related targets. Structural characterization by ¹³C-NMR and MALDI-TOF confirmed the identity of COS, while DPPH and ABTS analysis demonstrated concentration-dependent antioxidant activity. In LPS-induced RAW 264.7 macrophages, COS showed no cytotoxicity and significantly reduced nitric oxide production at 80 and 160 µg/mL. Molecular docking predicted favorable interactions of COS with several inflammation-associated receptors, including VEGFA, FGF1, and HPSE, showing stronger binding affinity than the reference drug diclofenac, and identified stable interactions with iNOS and COX-2 through extensive hydrogen-bond and polar contact networks. Transcriptomic profiling further revealed broad transcriptional remodeling and enrichment of pathways related to inflammation and atherosclerosis, including TNF, NF-κB, MAPK, Toll-like receptor, and lipid-and-atherosclerosis signaling. COS markedly suppressed inflammatory mediators, particularly Nos2 (iNOS; log₂FC = −7.92) and Ptgs2 (COX-2; log₂FC = −0.84), together with multiple cytokines and chemokines, consistent with reduced nitric oxide production and modulation of macrophage activation and foam cell-associated processes. Integration of docking and transcriptomic analyses identified iNOS and COX-2 as convergent candidate anti-inflammatory targets of COS, supporting its ability to attenuate inflammatory signaling through multi-target regulation rather than single-pathway inhibition. These findings suggest that COS may serve as a promising preventive strategy for atherosclerosis through coordinated regulation of oxidative stress, inflammatory responses, and vascular remodeling.