| Object: To assess the immune activity of polysaccharide from Saccharum Alhagi(SAP) in vitro and in vivo, and explore the preliminary study about mechanism of immune activity. Methods:(1) The polysaccharides were extracted with water, and precipitated with ethanol from Saccharum Alhagi. SAP-1 were precipitated by 50% ethanol, SAP-2 were precipitated by 80% ethanol.(2) To establish the model of immunosuppression mice by cyclophosphamidum, they were intragastrically administered with SAP. Nonspecificimmune function was observed by the method of carbon granular clearance test, antibody level was observed by hemolytic test and to determine cell factors(IL-2, IFN-γ, IL-10 and IL-6) from serum. Through these indexes to evaluate the immune activity of SAP.(3) Spleen lymphocyte could be extracted from mice, to intervene with SAP-1 and SAP-2 respectively, to assess the drugs on proliferation of spleen lymphocyte by CCK-8 assay, and to detect the concentration of cytokines(IL-2, IL-6, IL-10, IFN-γ and IL-12) by ELISA assay.(4) To intervene the macrophages RAW264.7 by SAP-1 and SAP-2, to investigate its proliferation by MTT assay, to assess the phagocytosis by neutral red, to detect the concentration of NO and cytokines(IL-2, TNF-α and IL-1β) by Griess and ELISA.(5) To assess the regulation of gene expression in Spleen lymphocyte and RAW264.7 by RT-(q)PCR.(6) To evaluate the scavenging rate of SAP on hydroxyl radicals(·OH) and superoxide anion radical(O2-·). Results:(1) Through the test of carbon granular clearance and hemolytic test, the results showed that SAP with high concentration can restore the phagocytic function and antibody level of immunosuppression mice. It also could enhance the concentration of IL-2 and IL-6 in serum of immunosuppression mice.(2) According to CCK-8 test, the results showed that SAP-1 and SAP-2 with high concentration can promote the proliferation of spleen lymphocyte, it also could enhance the concentration of IL-2, IL-6 and IL-10.(3) According to MTT assay in RAW264.7, the results showed that SAP-1 and SAP-2 with high concentration can promote the proliferation of RAW264.7 and the concentration of cytokines(IL-2, TNF-α and IL-1β), it also could promote the proliferation and NO of RAW264.7.(4) According to RT-(q)PCR assay, the results showed that SAP-1 and SAP-2 could up-regulate expression of IL-6, TNF-α, NF-κB, IL-1β and i NOS.(5) Through the determination of hydroxyl radicals(·OH) and superoxide anion radical(O2-·), the results showed that it has the same ability on scavenging of hydroxyl radicals as reference when the concentration of SAP was 15 mg·m L-1. But it had week effect on scavenging of superoxide anion radical. Conclusion:(1) SAP could enhance the immune activities of immunosuppression mice in vivo.(2) SAP-1 and SAP-2 could promote the proliferation and immune activities of spleen lymphocyte and RAW264.7, SAP-1 showed better activity.(3) Through the speculation of gene expression and regulation, SAP-1 and SAP-2 could regulate gene expression of immune factors by NF-κB signal transduction.(4) SAP-1 and SAP-2 showed certain antioxidant activities.(5) SAP showed better immune activities, especially the large molecular weight SAP-1. |
