Study On Biologycal Activities And Mechanisms Of Zwitterionic Derivatives Of Glygosaminoglycans

Glycosaminoglycans(GAGs) are long, unbranched polysaccharides composed of repeating disaccharide units consisting of alternating uronic acids and amino sugars. Hyaluronic acid(HA) and chondroitin sulfate(CS), two common GAGs, are widely distributed throughout connective, epithelial and neural tissues. They are important regulator in inflammation and immunity in vivo. Along with the age growth, HA and CS will lose their acetyl groups under action of HA N-deacetylase in vivo. The structural modification of macromolecules including acetylation and deacetylation is an important means in the adjustment of their physiological function. However, the biological consequence of this physiological process remains largely unknown. Based on the above background, we conducted the following research:1. A decrease in moisture absorption–retention capacity of N-deacetylation of hyaluronic acid. Two highly N-deacetylated HAs(dHAs) were generated via the NH2NH2-HIO3 procedure. Their molecular weights were estimated to be 24 kDa and 16 kDa by high performance gel-permeation chromatography(HPGPC), and the N-deacetylation degrees were 79.4% and 93% respectively, as determined by 1H nuclear magnetic resonance(NMR). The study on moisture absorption(Ra) and retention(Rh) abilities demonstrated that the Ra values of dHAs under conditions of 81% or 43% relative humidity, as well as the Rh values of dHAs under dry condition or 43% relative humidity, were significantly smaller than that of their respective re-N-acetylated products. The decline of moisture absorption and retention capacity after HA N-deacetylation were consistent with the appearance of unsolvated amides remained in the N-deacetylated products, as indicated by circular dichroism(CD) spectroscopy. Our findings implied that HA N-deacetylation, in addition to the decrease of HA contents in the elderly persons, might account for manifestations of naturally aged skin, such as laxity, sagging, and wrinkling.2. Alteration in immune responses towards N-deacetylation of hyaluronic acid. Three highly N-deacetylated HAs(dHAs), d HA-5 kDa(Mw: 5 kDa, DD: 100%), dHA-16 kDa(Mw: 16 kDa, DD: 94%) and dHA-110 k Da(Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells(APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B(NF-κB) transcription factor in APCs and induced cytokine production through the Toll-like receptor(TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in ageinduced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.3. Effect of N-deacetylated hyaluronic acid on spleen lymphocyte and T, B cells proliferation. We tested the spleen cell proliferation and T and B cells activation activities of HA-120 kDa and dHA-110 kDa. Results showed that both HA and dHA with large molecular weight had a potential in promoting the proliferation of spleen lymphocytes. Also they elicited a collaborative effect on ConA-induced T-lymphocyte proliferation and LPS-induced B-lymphocyte proliferation. HA-120 kDa induced the spleen cell proliferation is mainly dependent on TLR4, however, the zwitterionic hyaluronic acid dHA-110 kDa is more dependent on TLR2. Flow cytometry results show that HA-120 kDa and dHA-110 kDa have the similar capacity in promoting the increment of T cells. And the effect on promoting CD4+ T cells and CD19+ B cells of dHA-110 kDa is much stronger than that of HA-120 kDa. These results indicate that the change of immune regulating of T, B cells may also be associated with the deacetylation phenomenon of hyaluronic acid in vivo.4. Mechanistic insights into cellular immunity of chondroitin sulfate A and its zwitterionic N-deacetylated derivatives. As a major component in extracellular matrix of the tissue, chondroitin sulfate A(CS-A) has been shown to exhibit either pro- or anti-inflammatory immune response which was largely dependent on its molecular size and cell types. In this study, we determined the signaling pathway involved in immune response of CS and its N-deacetylated derivative(dCS). Our data indicated that both CS and dCS could activate the NF-κB transcription factor in antigen presenting cells and induce TNF-α production through the TLR/MyD88 pathway. Further studies demonstrated that both CS and dCS had a potential in promoting the proliferation of spleen lymphocytes, and promoting the cytokines secretion by ovalbumin(OVA)-sensitized splenocytes. Thus, our finding provided a mechanistic insight into the understanding of cellular immunity of CS and dCS, which might be helpful to develop CS-based immune modulators against chronic inflammatory conditions, autoimmunity, infectious diseases, allergies and asthmatic conditions.Taken togother, the present work has provided an insight to uncover the role of immunological effect underlying the N-deacetylation of glycosaminoglycans in vivo. Also this proposal will enlarge our understanding of zwitterionic plosaccharide, and will give us an oportunity to find novel zwitterionic polysaccharides with immunomodulatory activities.