Yujun Sung, Siriporn Namwongsa, Sineenart Songkoomkrong, Supawadee Duangprom, Napamanee Kornthong
Oxidative stress-induced macrophage activation and vascular injury are major contributors to atherosclerosis, a chronic inflammatory disease associated with approximately 20 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 proteins, with iNOS and COX-2 exhibiting the most favorable docking scores and extensive hydrogen-bonding and polar interactions. 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) and Ptgs2 (COX-2) 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 relevant pathways. These findings suggest that COS may modulate macrophage inflammatory responses through coordinated regulation of oxidative stress and inflammation-related signaling pathways.