Anti-inflammatory effects in a mouse osteoarthritis model of a mixture of glucosamine and chitooligosaccharides produced by bi-enzyme single-step hydrolysis.

We developed a novel technique of bi-enzyme single-step hydrolysis, using recombinant chitosanase (McChoA) and exo-β-D-glucosaminidase (AorCsxA) constructed previously in our lab, to degrade chitosan. The hydrolysis product was shown by HPLC, FTIR, and chemical analyses to be a mixture (termed "GC") composed primarily of glucosamine (80.00%) and chitooligosaccharides (9.80%). We performed experiments with a mouse osteoarthritis (OA) model to evaluate the anti-inflammatory effects of GC against OA. The three "GC groups" (which underwent knee joint damage followed by oral administration of GC at concentrations 40, 80, and 160 mg/kg·bw·d for 15 days) showed significantly downregulated serum expression of pre-inflammatory cytokines (IL-1β, IL-6, TNF-α), and significant, dose-dependent enhancement of anti-inflammatory cytokine IL-2, in comparison with Model group. Levels of C-reactive protein, which typically rise in response to inflammatory processes, were significantly lower in the GC groups than in Model group. Thymus index and levels of immunoglobulins (IgG, IgA, IgM) were higher in the GC groups. Knee joint swelling was relieved and typical OA symptoms were partially ameliorated in the GC-treated groups. Our findings indicate that GC has strong anti-inflammatory effects and potential as a therapeutic agent against OA and other inflammatory diseases.