N-acetyl-chitooligosaccharides attenuate LPS-induced inflammation in macrophage by MR/NOD2/NF-κB signaling

Chitooligosaccharides (COS) and fully N-acetylated chitooligosaccharides (NACOS) differ in degree of acetylation (DA), yet their anti-inflammatory activities remain unclear. This study evaluated COS and NACOS with defined degree of polymerization (DP; 2 and 6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Both oligosaccharides reduced the levels of proinflammatory mediators, with NACOS6 exhibiting superior activity. Transcriptome profiling revealed NACOS6 primarily modulated pathways involved in immunity and inflammation, comprising C-type lectin receptor signaling, NOD-like receptor signaling, NF-κB signaling and antigen processing. RT-qPCR and Western blotting demonstrate that COS and NACOS upregulated mannose receptor (MR) expression, modulated the NOD2-RIPK2 axis, and attenuated NF-κB activation via reducing IκBα and p65 phosphorylation. Molecular docking suggested potential interactions of COS and NACOS with both MR and NOD2, with acetylation altering binding residue preferences. Receptor blocking assays revealed that MR inhibition had no effect on anti-inflammatory activity, whereas NOD2 inhibition abolished the suppressive effect of COS2, NACOS2, and NACOS6 on NF-κB activation and nitric oxide production, while COS6 was less affected. Collectively, NACOS, especially NACOS6, exhibited stronger anti-inflammatory activity than COS in LPS-stimulated macrophages, and this effect is primarily mediated through NOD2-dependent regulation of inflammatory signaling. These findings suggested NACOS as a potential functional ingredient for the dietary management of inflammation-related disorders.