Study On Sulfated Modification,Antioxidant And Immunomodulatory Activities Of Cyclocarya Paliurus Polysaccharides

In this study,a crude polysaccharide from Cyclocarya paliurus(CPP)was extracted by the hot water extraction-ultrasonic method,and was purified from CPP with a further step by DEAE-Cellulose(CPP_0.05).CPP and CPP_0.05 were performed with chlorosulfonic acid-pyridine(CSA-Pyr)method to obtain sulfated products(S-CPP,S-CPP_0.05).Through the determination of physicochemical properties and structural characterization,the primary structure of S-CPP_0.05 was inferred.Based on the determination of free radical scavenging ability,and the H₂O₂-induced oxidative damage model of mouse bone marrow-derived dendritic cells(BM-DCs)was established to explore the protective effects of CPP_0.05 and S-CPP_0.05 and the molecular mechanism on DCs with oxidative stress.DCs was cultured with polysaccharides and the immunosuppressive mice model was established to evaluate its immunomodulatory activity and explore its mechanism.The main results were as follows:(1)The results of structure characterization showed that polysaccharides were successfully modified.The backbone of S-CPP_0.05 was composed of?3)-?-D-Galp-(1?4)-?-D-Glcp-(1?substituted by?-L-Araf-(1?,?4,6)-?-D-Galp-(1?and?-D-Glcp-(1?at O-4/5/6 position,terminated by?-L-Araf-(1?and occasionally by?-D-Glcp-(1?.Sulfate groups were substituted at C6 of?4)-?-D-Glcp-(1?.(2)Improved antioxidant activity:Polysaccharides were used to culture DCs induced by H₂O₂.The antioxidant properties and molecular mechanism of CPP_0.05 and S-CPP_0.05 on DCs were comparatively investigated by DPPH,ABTS,hydroxyl radical,reducing power assay,the determination of SOD,CAT,MDA levels and Nrf2,Keap1proteins levels.Results indicated that both of them had good antioxidant activity,and S-CPP_0.05 was stronger.Meanwhile,it found that Nrf2 proteins were significantly increased by stimulus of polysaccharides with Keap1 decreasing which proved that antioxidant effect was possibly performed via Keap1-Nrf2 pathway.(3)Improved the immunomodulatory activity in vitro:Polysaccharides were used to culture DCs to evaluate their immunomodulatory activity and explore mechanism.Although the purity of the purified polysaccharide was significantly improved,the functional activity of CPP was significantly stronger than that of the CPP_0.05.Meanwhile,S-CPP showed stronger immunomodulatory activity than CPP through determination of cytokine expression levels.It found that p-JNK,p-p38MAPK and NF-?B p65 proteins were significantly increased by stimulus of CPP and S-CPP,blocking TLR2/4 could significantly decreased proteins above which proved that immune regulation effect of CPP and S-CPP on DCs was performed via MAPK and NF-?B signaling pathways by triggering TLR2/4.(4)Improved the immunomodulatory activity in vivo:Through the establishment of the Cy-induced immunosuppression model in mice,the immunoregulatory activities and the repair effect on intestinal damage of CPP and S-CPP in vivo were explored.As a result,CPP and S-CPP could alleviate intestinal villus damage and improve the diversity and composition of intestinal microbiota,the expression levels of cytokines in serum and small intestine tissues were improved,the gene and protein expression levels of tight junction proteins were increased which promoted the repair of intestinal mechanical barrier and enhanced the immune ability.And the immunomodulatory activity of S-CPP was superior to CPP.Sulfated modification played a promoting role in antioxidant activity of Cyclocarya paliurus polysaccharides in vitro and intestinal immunomodulatory function in vivo,which related to the substitution of sulfate groups,especially to the position and degree of substitution.The study proved that the substitution at the C6position of glucose in the main chain could effectively improve the antioxidant and immunomodulatory activities of Cyclocarya paliurus polysaccharides.