Research On The Mechanisms Of Macrophage Activation By Chito-oligosaccharides Mediated By TLR4Receptor

Background: Chito-oligosaccharides, the degration product of chitosan, hasmore valuable biological activities than Chitosan such as enhance immunity,anti-tumor, antioxidant, antimicrobial, promoting healing and so on, which is due toits feature of low molecular weight, non-toxic and good water solubility. There’sstudy showed that chito-oligosaccharides can regulate immune function of theorganism and inhibit tumor cell from growing and metastasis. Our work provides afoundation for further research of immunoregulation mechanism of oligochitosanwith macrophage.Methods: Chitosanase was used to prepare chito-oligosaccharides through theenzyme hydrolysis method in this project,and HPLC method was used to identifythe composition of COS. The effects of different concentrations of COS on theproliferation, neutral red phagocytosis ability, and tumor necrosis factor (TNF-alpha)secretion of mouse peritoneal macrophages were tested in vitro studies. Mice wereadministrated with different doses of COS to elucidate the impact of COS on serumIgG and IgM levels and on the thymus, spleen index of mouse. COS was thenlabeled with fluorescein isothiocyanate (FITC) to get FITC-COS. Phagocytosis ofCOS by mouse peritoneal macrophages’ and its relationship with TOLL-like receptor4(TLR4) was investigated.Results: HPLC analysis results showed that COS, prepared by chitosanasehydrolysis, mainly consist of3-7degree of polymerization of monosaccharide.Chitosan has preferably immunomodulatory functions.Chitosan oligosaccharide significantly enhanced the phagocytic function of macrophages and stimulatemacrophages to secrete TNF-α after being phagocytosis by macrophages.The resultsof in vivo studies showed that administration of mice with COS could significantlyincrease the spleen index and serum IgG levels. While the thymus index and serumIgM levels were not significantly changed.Fluorescence microscopy results showedthat FITC-COS could be phagocytized by mouse peritoneal macrophages in atime-dependent manner,reaching the peak in15min or so. Pretreating ofmacrophages with TLR4monoclonal antibody for1hr, phagocytosis of COS wasalmost completely inhibited. Chito-oligosaccharides can promote the proliferation ofRAW264.7cells.And it can also increase the cell’s ability of the phagocytosis ofneutral red and the capacity of secreting NO in which the effect of hexose is thebest.The effect of anti-TLR4pretreatment on cells is that it can dramatically reducethe cell’s ability of secreting NO.The chito-oligosaccharide is able to induce thegene transcription in RAW264.7cell,such as TNF-a、iNOS、TLR4and so on.Conclusions: COS with3-7polymerization degree can be phagocytized bymacrophage and then activate macrophages. COS has well in vitro and in vivoimmunological function. The activation of macrophages by COS is mediated by theTLR4receptor on cell surface.