Study On The Mechanism Of Immune Activity Of Thorn Sugar Polysaccharide On Mouse TLR4 Target

Objective: To study the immunological activity of polysaccharide from Saccharum Alhagi in vitro and in vivo,and to explore its immunological activity on TLR4 target.Methods:1.Establish an animal model of intraperitoneal injection of cyclophosphamide immunosuppressive mice.The mice were intragastrically administered to AP1-1,and the cytokines IL-1?,IL-2,IL-4,IL-6 in the serum of the mice were determined.The production levels of IL-12,TNF-?,LgG,and LgM were used to evaluate the immunological activity of AP1-1.The expression of TLR4 receptor in mouse tissues was detected by RT-PCR and Western Blot.3.Western Blot assay was used to detect the blocking expression of macrophage RAW264.7 by TLR4 receptor inhibitors.The proliferation activity of thorn polysaccharides AP1-1 and AP1-2 on macrophage RAW264.7 immunodeficiency model was investigated by MTT assay.5.Determination of cytokines IL-1?,IL-2,IL-12,TNF-? and NF-?B in macrophage RAW264.7immunodeficiency model after serotonin AP1-1 intervention by ELISA kit Level.The expression of MyD88,TRAF6,TRAM and TRIF in macrophage RAW264.7immunodeficiency model was detected by RT-PCR and Western Blot.Results:1.According to the results of ELISA kit,IL-2,IL-12,TNF-a and LgG cytokines were significantly higher than the positive drug group,the difference was statistically significant(P<0.05).The expression of TLR4 receptor in mouse tissues was detected by RT-PCR and Western Blot.The results were consistent and dose-related.3.Western Blot results showed that the optimal time for the administration of thorn polysaccharide was 1to 3 days.The MTT assay for the proliferation of macrophage RAW264.7immunodeficiency model was better than AP1-2.The mass concentration of thorn polysaccharide AP1-1 was 25~100?g/mL for macrophages RAW264.The survival of the immunodeficiency model showed a significant promotion(P<0.05).5.ELISA kit can be used to determine the secretion of IL-1?,IL-6,IL-12 and NF-?B by macrophage RAW264.7 in each dose group.1 dose increased and the difference was statistically significant(P <0.05).Western blot analysis showed that different concentrations of thornpolysaccharide AP1-1 interfered with macrophage RAW264.7 immunodeficiency model for 24 h,and then regulated by MyD88-dependent pathway MyD88 and TRAF6 of TLR4 receptor,at the concentration of 50,75?g / mL was significantly up-regulated(P<0.05).Conclusion:1.Polysaccharide AP1-1 can enhance the immune function of mice in immunosuppressive mice.2.Polysaccharide AP1-1 can promote the proliferation of macrophage RAW264.7 immunodeficiency model and its immune activity.3.Polysaccharide AP1-1 promoted the secretion of cytokines IL-1?,IL-6,IL-12 and NF-?B in macrophage RAW264.7 immunodeficiency model.Western blot results showed that the polysaccharide polysaccharide activates the immune response of the TLR4 receptor through the MyD88-dependent pathway.