Study On The Structure Of Ulva Pertusa Polysaccharide And The Immunomodulatory Effect Of Its Iron(?)Complex

The immune system can exert immunomodulatory effects including immune defense,surveillance and self-stabilization through specific immunity and non-specific immunity.Once the immunity ability declines or the immune system becomes abnormal,the immunomodulatory effect will be disordered.In severe cases,a variety of immune related diseases,such as immunodeficiency,hepersensitivity and autoimmunity,can be caused.Thus,maintaining the stability of immune system is of great significance to human health.The ocean is ri ch in resources and many kinds of ocean plants can be used as food or drugs to enhance immunity.Ulva Pertusa,a green algae,mainly distributed along the coasts of the East and South Sea of China,contains a large amount of biological substances,among which the content of polysaccharides is relatively high.Currently,the detailed structure of the immunologically active Ulva Pertusa polysaccharide and its regulation mechanism are not yet clear.Thus,the research on the molecular structure and the related regulation mechanisms of the immunologically active polysaccharides from Ulva Pertusa is particularly important.In this study,Ulva Pertusa polysaccharides were extracted by the method of water extraction and alcohol precipitation,and purified by DEAE-52 cellulose gel column and Sephacryl S400 column.The polysaccharide fragment UPP with the best immunological activity was screened by comparing the effects on the proliferation and phacocytic capacity of RAW264.7 macrophages,and its chemical composition and molecular structure were finally determined.In addition,the immunomodulatory effects of UPP on the Cyclophosphamide(Cy)-induced immunosuppressive mice were studied,and the immune enhancement mechanisms were explored through itraq technology.On the other hand,UPP-iron(?)complex was prepared and its in vivo immunomodulatory activity and the detailed mechanisms were also investigated in depth.Firstly,the crude Ulva Pertusa polysaccharides were purified by DEAE-52 and Sephacryl S400,and the polysaccharide fragments with immunomolatory activity were screened through RAW264.7 macrophages.The molecular structure of UPP was identified by UV spectroscopy,infrared spectroscopy,HPGPC,GC-MS,1D-NMR and 2D-NMR,and the results showed that UPP,with an average molecular weight of 235.62 k Da,was relatively homogeneous polysaccharide.And,UPP was mainly composed of rhamnose,arabinose,xylose,glucose and galactose,and the percentage of each monosaccharide is 51.94%,6.39%,35.95%,2.79%and2.93%.The body structure of UPP is identified to be?4)-?-D-Xylp-(1?4)-?-L-Rhap3S(1?3,4)-?-L-Arap(1?3)-?-L-Rhap(1?2,4)-?-L-Rhap(1?4)-?-D-Galp(1?,and it contains two main branches,?3,4)-?-L-Arap(1?4)-?-D-Glcp3Me(1?4)-?-D-Glcp A(1?connected to the C₃position of1,3,4-?-L-Arap,and?4)-?-D-Glcp A(1?4)-?-D-Xylp(1?linked to the C₂ position of 1,2,4-?-L-Rhap,respectively.The morphological characteristics of UPP were analyzed by Congo red test,scanning electron microscopy(SEM),atomic force microscope(AFM)and X-ray diffraction(XRD).The results showed that UPP,without a triple helix structure,is a sheet with smooth surface and belong to a semi-crystalline polymer.The in vitro experiment showed that UPP can significantly improve the proliferation activity,phagocytic capacity,NO secretion and cytokine expression of RAW264.7 macrophages.Through the Cy-induced immunosuppressive mice model,UPP was found to enhance the proliferation of splenic lymphocytes and the bioactivity of peritoneal macrophages,restore the serum hemolysin and cytokine levels,and improve the ability of haematogenesis.Furthermore,the itraq analysis determined that UPP can significantly change seven biological processes related to the immune system,and improve lipid metabolism pathways and immune system-related pathways,and can markedly influence eight target proteins related to immunity.And improving the complement and coagulation cascade immune pathways was found to play an important role in immune regulation mechanism.Furthermore,the immunomodulatory activity of UPP-iron(?)was studied and the results showed that compared with UPP,UPP-iron(?)can more effectively increase the proliferation of RAW264.7 macrophages,enhance the phagocytic capacity and promote the secretion of NO and cytokine.The in vivo experiment results suggested that compared with UPP,UPP-iron(?)can significantly enhance the hematopoietic ability of Cy-induced immunodeficient mice without affecting the immunomodulatory activity of UPP.And the itraq analysis indicated that UPP-iron(?)can significantly change both the biological processes related to the immune system and lipid metabolism,and the complement and coagulation cascades and phagocytosis,thereby exerting an immune regulation.In this study,UPP,a polysaccharide component with immunomodulatory activity,was isolated and purified from Ulva pertusa,and its molecular structure was characterized and analyzed.In addition,UPP-iron(?)was prepared and the experiment results showed that both UPP and UPP-iron(?)exert immunomodulatory activity on RAW264.7 macrophages in vitro and Cy-induced immunosuppressive mice in vivo.The itraq technology also helped reveal the immunomodulation mechanisms of UPP and UPP-iron(?).