Study On Immunity Mechanism Of Intracellular Polysaccharide In Flammulina Rossica Redhead

1.Flammulina Rossica is a new record strain in China.Zhu Yan-yan and her colleagues have found that the extracellular polysaccharide of Flammulina rossica has immunity and anti-tumor activity.Its FREPs has a wide impact on the immune network of liver cancer mice,but the mechanism of its pharmacological effect is not clear.In order to enlarge the resources of edible fungi and enrich the edible fungus industry of our country,it is of great significance to develop and utilize the Flammulina rossica.This article examined the occurrence,development and therapeutic targets of immune inflammation from the point of view of cell signal,systematically collate the immune-related cytokines and signal pathways activated by cellular immunological inflammation,in order to determine the target of drug action.The mechanism of overcoming disease and the development and utilization of new drugs provides a theoretical basis.2.In order to increase the yield of FRPs,the fermentation conditions were optimized through single factor experiment and orthogonal optimization experiment in terms of liquid volume,inoculation volume,culture temperature and rotating speed of incubator.3 factors,such as extraction time,extraction temperature and liquid to material ratio,were selected,and the extraction rate of mycelia polysaccharides was taken as an index,and the best extraction process of polysaccharides was determined by orthogonal experimental design.The results showed that the suitable culture conditions for submerged fermentation of Flammulina rossica were: liquid volume 120 mL/250 mL,inoculation amount 10%,culture temperature 23?,and incubator speed 150 r/min.The optimum conditions of extraction of Polysaccharide from mycelium: extraction time 2 h,solid-liquid ratio 50:1,extraction temperature of 90?,the best process route is: 2h hot water extraction,filtering,merging 3 extracts,rotary evaporator concentrating fermentation liquid to the original volume of 1/10,4 times volume of 4 ?alcohol 24 h,3500 r/min,5 min after centrifugal precipitation collection,-80?,pre freezing overnight,vacuum freeze drying,weighing,re dissolved polysaccharides,centrifugation to precipitate,supernatant alcohol,again to freeze drying,mycelium polysaccharide(FRIPs),calculated the extraction rate of polysaccharide was 74.39%.3.Through the pharmacological experiments of normal BALB/c mice,the immune enhancement of FRPs was studied and the mechanism of its polysaccharide immunization was discussed.The lymphocyte proliferation test showed that FRPs and ConA synergistically stimulated mouse spleen lymphocyte proliferation and differentiation,and along with the increase of the dose,the OD value increased gradually,polysaccharides of 0.8 ug/mL and 1.6 ug/mL dose group compared with normal group,there was significant difference(p< 0.01).In vivo pharmacological assay of FRPs in different dosage group effects on spleen and thymus index of mice spleen lymphocyte subsets,detection of CD4+,CD8+,flow cytometry,serum ELISA detection kit in lymph and mononuclear factor IL-6 factor IL-1? and tumor necrosis factor TNF-? transforming growth factor TGF-? content,Western blot the experimental results show that the band optical density,influence the expression of FRPs at different doses on mouse spleen tissue I?B?,P-I?B?,P-NF-?Bp65 and NF-?Bp65,compared with the normal group,high dose group of FRIPs I?B? significant difference(p< 0.05),was significantly lower than the normal group,the expression of FRPs polysaccharide with different doses of P-I?B? was significantly increased compared with the normal group were significant difference(p< 0.01);compared with the normal group,FRIPs high dose group and low dose group of FREPs NF-?Bp65 showed significant difference(p< 0.01),the NF-?Bp65 ratio of FRIPs high dose group decreased and was lower than that of the normal group.There was a significant difference in P-NF-?Bp65 between different doses of FRPs polysaccharide group(p< 0.01),which was higher than that of the normal group.4.By establishing Immunosuppressive model on BALB/c mice,the thesis established in vivo pharmacological experiments,studied FRPs immunization and explored the mechanism of its polysaccharide's immune function.The thymus and spleen index of mice in different dose groups were determined.The results showed that compared with the model group,the FRPs dose group could significantly improve the thymus and spleen index of immunosuppressed mice.The contents of IL-6,mononuclear factor IL-1?,tumor necrosis factor TNF-? and transforming growth factor TGF-? in the serum of mice were detected by ELISA kit.To detect the changes of spleen lymphocyte subsets of mice for each group of cytokines CD4+,CD8+ and CD4+/CD8+ content by flow cytometry,FRIPs high dose group could significantly increase the content of CD4+(p< 0.01),significantly decreased the content of CD8+;By increasing the content of CD4+ and reduce the content of CD8+,the common high low dose of FRIPs increased CD4+/CD8+ ratio;the content of group CD8+,CD4+/CD8+ ratio decreased,the difference was extremely significant(p< 0.01);lower dose group CD4+ content in FRIPs,a significant difference(p< 0.05).Real-time PCR analysis of mouse mRNA cell factor expression in thymus tissue,ACTB,GAPDH,HPRT1,select the EEF1 A cross intron primers for reference,at the same time,extraction of mouse intestinal DNA,and at the same time reverse transcription products were amplified by PCR,agarose gel electrophoresis,genome sequencing,DNA interference elimination and calculate results.5.The effects of different doses of FRPs on the protein expression of thymus tissue in immunosuppressed mice were analyzed with Western blot strip light density.Compared with the normal group,the expression of I?B?,P-I?B?,P-NF-?Bp65 and NF-?Bp65 were significantly different between the 4 factors(p< 0.01),indicating that the BALB/c immunosuppressive mice model was successful.Compared with the model group,FRPs dose group showed significant difference(p< 0.01),proof of polysaccharides and positive drug CTX in immunosuppressive mice produce pharmacological effects;Model mice were stimulated by CTX,so their autoimmune destruction.I?B by IKK phosphorylation and degradation,resulting in activation of NF-?Bp65 the phosphorylation of NF-?B nuclear,I?B phosphorylation in the cytoplasm,I?B is inhibition of protein NF-?Bp65,up regulate the expression of FRPs I?B,thereby inhibiting NF-?Bp65 activity,the pharmacological effect.The expression of NF-?Bp65 in model group decreased NF-?Bp65 phosphate into the nucleus,I?B phosphorylation degradation;compared with the model group,NF-?Bp65 expression,and with the dose increase,the phosphorylation of NF-?Bp65 expression increased significantly(p< 0.01).Our results showed that FRPs played an immunoregulatory role by regulating I?B? and NF-?B phosphorylation,which provided a theoretical basis for the research and development of FRPs immunomodulation.It will also help to design specific tools for the treatment of specific human diseases.