Study On The Effect And Mechanism Of Polysaccharide SAFP From Sarcodon Imbricatus In Ameliorating Gastric Ulcer

Gastric ulcer is a kind of digestive disease with a high incidence rate,which is widespread in the world.However,the existing chemical drugs can not completely repair gastric mucosa damage,with certain side effects and a high recurrence rate.Therefore,it is important to find a natural drug that have non-toxic side effect,effectively prevent and treat gastric ulcer.Sarcodon imbricatus?L.?P.Karst.)is a rare wild edible fungus with unique flavor.Polysaccharide is an important active component.The current studies mainly focus on the extraction and isolation of polysaccharides,immunoregulation and anti-tumor,but there are no reports on the prevention and treatment of gastrointestinal ulcer.In this study,the polysaccharide SAFP was first extracted and isolated from Sarcodon imbricatus and its physicochemical properties were characterized.Secondly,the protective effect of SAFP on cellular damage of gastric mucosal epithelial cells?GES-1cells?induced by aspirin was investigated.Finally,the mechanism of SAFP in ameliorating ulcerative bleeding in stress gastric ulcer rats by modulating the dysbiosis of gut microbiota was studied.The main research contents and conclusions are as follows:?1?Isolation and purification of SAFP and physicochemical properties:The polysaccharide SAFP was extracted by water extraction and alcohol precipitation combined with protein removal by Sevage method.The preliminary physical and chemical properties showed that SAFP was soluble in water,with the total sugar content was 82.8%,the total protein content was 4.34%.The results of monosaccharide composition experiment showed that SAFP was composed of four monosaccharides?D-mannose,D-glucose,D-galactose,D-fucose?,and the molar ratio was 8.14:7.42:1.02:11.41.D-mannose and D-fucose were the main components.Infrared spectrum analysis showed that SAFP was pyranose with obvious characteristic absorption peaks of polysaccharides.?2?Protective effect of SAFP on GES-1 cellular damage:Aspirin was used to induce human gastric mucosal epithelial cells?GES-1 cells?to establish the cell damage model.In the GES-1cell damage model,SAFP(100?g m L^-1,200?g m L^-1,400?g m L^-1)was used for early intervention.The results showed that SAFP had no toxic side effect on GES-1 cells,could reduce the cell apoptosis rate,reverse the alteration of nuclear morphology,effectively improved the level of SOD and GSH-p X in cells,reduced the level of MDA and ROS,and reduced the damage of oxidative stress on cells.Western Blot indicated that SAFP could reduce oxidative stress response and protect GES-1 cells from aspirin induced damage by activating Keap1/Nrf2 signaling pathway.?3?Study on the improvement of SAFP on stress gastric ulcer in rats and the related mechanism:60 SD rats aged 8 weeks?200±20g body weight?were selected and the stress gastric ulcer model was established by water-immersion and restraint stress.After SAFP(100mg kg^-1,200 mg kg^-1,400 mg kg^-1)preventive treatment,HE staining showed that the ulcerative bleeding in rats was improved to varying degrees,with only a small amount of inflammatory cell infiltration.The effect of the high-dose SAFP group was similar to that of the ranititin?RNTD?positive drug group.SAFP could reduce the incidence of gastric ulcer in rats,protect the integrity of gastric mucosa and prevent the occurrence of gastric ulcer.SAFP could significantly increase the level of SOD and GSH-PX,reduce the level of MDA,and decrease the level of inflammatory factors?,IL-?,IL-6?.Immunohistochemistry,Western Blot and q RT-PCR analysis showed that SAFP can up-regulate the m RNA and protein expression of Nrf2,Ho-1 and NQO1 genes,inhibit the m RNA and protein expression of Keap1 and NOX4 genes,and inhibit the protein expression of TLR4,NF-?Bp65,My D88.The results indicated that SAFP can reduce oxidative stress and inflammation by activating Keap1/Nrf2 signaling pathway and inhibiting TLR4/NF-?Bp65 signaling pathway,thus protecting gastric mucosa and improving stress gastric ulcer.?4?Study on the regulation of SAFP on the gut microbiota of stress gastric ulcer rats.High-throughput pyrosequencing of 16S r RNA suggested that the gut microbiota of stress gastric ulcer rats was seriously dysfunctional,while the preventive treatment of SAFP could improve the diversity and richness of the gut microbiota.SAFP supplementation could significantly downregulate the abundances of Proteobacteria while upregulating the abundances and multiplication capacity of Firmicutes,Verrucomicrobia and Actinobacteria at the phylum level,which returned to normal levels.Furthermore,pretreatment with SAFP led to a significant reduction in the relative abundance of Bacteroides,Helicobacter,Escherichia and Parabacteroides when compared with the SGUM group.Detailed analysis showed that the relative abundance of Lactobacillus,Oscillospira,Akkermansia and Bifidobacterium were dramatically upregulated by SAFP supplementation.When the dose of SAFP was 400 mg kg^-1,the effect was equivalent to or even better than RNTD treatment.The results indicated that SAFP could protect the gut microbiota in rats against damage,modulate the dysbiosis of gut microbiota,and maintain the homeostasis of gut microbiota by enhancing the relative abundance of probiotics,decreasing WIRS-triggered bacteria proliferation.The results of this paper show that SAFP has a good prevention and improvement effect on gastric ulcer,and SAFP can regulate gut microbiota and maintain the homeostasis of gut microbiota,which has potential value in the development and application of natural polysaccharide gastric mucosa protectants and nutritional supplements targeting intestinal flora.