The Research On Structural Identification,Immune Activity,Anti-Tumor Activity And Its Mechanism Of Polysaccharide From Ramaria Flaccida(Fr.) Quél.(RF-1)

Ramaria flaccida(Fr.)Quél.also known as broom fungi,belongs to aphyllophorales,ramariaceae,ramaria.But up to now,the research on Ramaria flaccida(Fr.)Quél.is limited to resource investigation,identification and classification,and chemical composition.However,there is no report on the complete polysaccharide structure analysis of Ramaria flaccida(Fr.)Quél.,and immunoregulation and anti-tumor activity of the polysaccharide from Ramaria flaccida(Fr.)Quél..In this study,crude polysaccharides were obtained by solid-liquid extraction and protein removal.Then crude polysaccharides were purified by DEAE-52 cellulose chromatography,and then pure polysaccharides from Ramaria flaccida(Fr.)Quél.(RF-1)were obtained.By using HPGPC,FT-IR,HPLC,GC-MS and NMR methods,the structure of RF-1 was identified.On this basis,cell models were established in vivo and in vitro,and S180 tumor models were established in vivo to investigate the immunoregulatory activity and anti-tumor activity of RF-1 in vivo and in vitro.Finally,high throughput sequencing technology was used to sequence the tumor tissues of S180 model group and RF-1 medicine group.The differentially expressed genes were analyzed,and all differentially expressed genes were further annotated by GO function and enriched by KEGG pathway.The results showed that the weight-average molar mass of RF-1 was 17,093 Da,and it was a homogeneous pure polysaccharide.The monosaccharide composition of RF-1 was glucose and galactose,and the ratio was 2:1.The main chain of RF-1 consisted of(1?6,2)-?-D-galactopyranose and(1?6,4)-?-D-glucopyranose.A branched chain was connected by(1?6)-?-D-glucopyranose to the 4-O of the main chain(1?6,4)-?-D-glucopyranose.A terminal glucose(4-?-D-glucopyranose)was attached to 1-O of the branched chain.The other two terminal glucoses were linked to the 2-O of the main chain(1?6,2)-?-D-galactopyranose,respectively.By using CCK-8 method,it was found that with the increase of mass concentration of RF-1,the proliferation of macrophages,T and B lymphocytes was significantly promoted(P<0.01),and showed a dose-dependent relationship.When the concentration of RF-1 was 5 ?g/mL,macrophages could significantly promote the phagocytosis of neutral red and fluorescent microspheres(P<0.01).And RF-1 could extremely significantly stimulate macrophages to secrete NO,IL-2,IL-10 and TNF ?(P<0.01).It also had a significant effect on stimulating the secretion of IL-6 in macrophages(P<0.05).This indicates that RF-1 has good immunoregulatory activity.With the increase of the mass concentration of RF-1,the anti-cancer rate also showed an upward trend.The anti-cancer rate also showed a dose-dependent relationship with the concentration of RF-1.When the concentration of RF-1 was 5 ?g/mL,it could significantly inhibit the proliferation of CT26.WT,MFC and S180 cells(P<0.01),and the inhibition rates for these three cells were 19.5 %,36 % and 21 %,respectively.However,it did not inhibit MDCK,indicating that RF-1 had no toxicity to normal cells.RF-1 could inhibit the growth of S180 tumors in vivo through the experimental analysis of mice S180 tumors model.When the concentration of RF-1 was 20 mg/kg,the inhibition rate of S180 tumors in mice was 40 %.Compared S180 tumor model group with RF-1 group,1,971 differentially expressed genes were obtained.Among them,818 genes were up-regulated and 1,153 genes were down-regulated,which may be related to the anti-tumor activity of RF-1 in vivo.A Gene Ontology(GO)enrichment analysis generated 47,091 assignments to biological processes,5,250 assignments to cellular components,and 6,466 assignments to molecular functions.KEGG pathway enrichment analysis showed that Wnt signaling pathway and MAPK signaling pathway were significantly enriched.The number of differentially annotated genes in these two pathways was 19 and 33,respectively.In conclusion,RF-1 has good anti-tumor activity.The key mechanism may be through Wnt signaling pathway and MAPK signaling pathway.Finally,it was found that the protein levels of IL-1?,IL-6,VEGF and VEGFR in RF-1 group were significantly lower than those in S180 group(P<0.01).The results of this study provide a theoretical basis for the follow-up research and development of Ramaria flaccida(Fr.)Quél.,and provide a theoretical reference for the further study of the immune regulation mechanism and anti-tumor mechanism of polysaccharides from Ramaria flaccida(Fr.)Quél..