The Immune Regulation Effects Of N-acetyl Chitooligosaccharides On Mouse RAW264.7 Macrophages

N-acetyl chitooligosaccharides(NACOS)are the hydrolytic products of chitin.Compared with chitosan oligosaccharides(COS),which are the hydrolytic products of chitosan,the bioactivity of NACOS differs from that of COS,as they have more acetyl groups than COS.Given the lack of efficient chitinase,the traditional preparation of NACOS requires chemical or enzymatic acetylation of COS,which is complicated and inefficient as well as not easy to obtain.Therefore,compared with COS,the biological activity and the underlying mechanisms of NACOS are rarely reported,and the immunomodulatory effects and regulatory mechanisms of NACOS are still unclear.In previous study,a recombinant chitinase with high activity was obtained by our research group,which can degrade shrimp shells or chitin to prepare NACOS,making the commercial production of NACOS possible.In this study,we used two enzymatically produced NACOS with different molecular compositions(LNACOS and HNACOS)and six NACOS(NACOS1–6)with a single degree of polymerization,to verify their immunomodulatory effects on the RAW264.7 macrophage cell line.We aimed to identify any differences between COS and various NACOS with a single degree of polymerization.The research results were shown as follows:(1)NACOS had similar immune enhancement effects on RAW264.7 cells as COS,all NACOS could significantly improve the generation of reactive oxygen species(ROS),phagocytotic activity,and production of pro-inflammation cytokines(IL-1?,IL-6,and TNF-?).However,unlike COS and lipopolysaccharide(LPS),NACOS1 and NACOS6 significantly inhibited nitric oxide(NO)production.In addition,The phosphorylation levels of NF-?B signaling pathway-related protein(I?B?,p65)were detected by Western blot and found that NACOS significantly promoted the phosphorylation levels of these two proteins.These results indicated that NACOS could promote the non-specific immune response of macrophages and the production of pro-inflammatory cytokines through the NF-?B signaling pathway.(2)NACOS inhibited the LPS-induced RAW264.7 inflammatory response as COS,but there were some differences in the anti-inflammatory effects of NACOS with different degrees of polymerization.HNACOS,NACOS1 and NACOS6 could significantly inhibit the production of NO and proinflammatory cytokines(IL-6 and TNF-?)induced by LPS in macrophages.All NACOS could significantly inhibit the phagocytic activity of LPS-induced macrophages and the production of pro-inflammatory cytokine IL-1?,especially HNACOS,NACOS1 and NACOS6 had a stronger inhibitory effect.Regarding the production of ROS,it was found that LNACOS,NACOS1,NACOS2 and NACOS3 could significantly reduce LPS-induced ROS production.The phosphorylation levels of NF-?B signaling pathway-related protein(I?B?,p65)were detected by Western blot and found that NACOS significantly inhibited the phosphorylation levels of I?B? and p65 stimulated by LPS,especially NACOS6 had the strongest inhibitory effect.These results indicated that NACOS could inhibit the nonspecific immune response of LPS-induced macrophages and the production of pro-inflammatory cytokines through the NF-?B signaling pathway.In summary,we confirmed that the NF-?B pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells.This study could serve inform the application of NACOS with varying different degrees of polymerization in human health.