Research On The Effect And Mechanism Of Sarcodon Aspratus Polysaccharides In Improving NAFLD Based On Micro-ecological Balance Of Gut Microbiota And Endoplasmic Reticulum Stress

Non-alcoholic fatty liver(NAFLD)is a liver disease mainly characterized by diffuse hepatic bullous steatosis.Continuous development will lead to steatohepatitis and liver fibrosis.It is one of the major metabolic syndromes caused by obesity.The incidence of NAFLD is global,and it has surpassed viral hepatitis to become the largest liver disease in China.Therefore,controlling and improving the occurrence and development of NAFLD has become an urgent problem.So far,many researchers have found that polysaccharides from medicinal or edible fungal have unique advantages in improving metabolic syndrome,and their role in regulating gut microbiota has also received widespread attention and recognition.Sarcodon aspratus,a rare and valuable wild edible fungus,the activity studies of its polysaccharides(SATP)are mainly on immune,anti-oxidation and anti-tumor.However,it remained unknown whether SATP produce any effect on anti-obesity and NAFLD.In this paper,we systematically research the effect of SATP on gut microbiota dysbiosis and endoplasmic reticulum(ER)stress-induced inflammation in improving NAFLD by establishing obese mouse models,NAFLD mouse models,and oleic acid(OA)-induced LO2 liver cell injury models,which will lay the foundation for the development and utilization of Sarcodon aspratus for functional food or medicine.The main contents include the following sections: 1.Sarcodon aspratus polysaccharides(SATP)was prepared,and its total sugar content and protein content were analyzed,and the IR absorption spectrum and monosaccharide composition of SATP were detected.It was found that the total sugar content of SATP was 84.28%,and its infrared absorption spectrum conformed to the infrared absorption characteristics of polysaccharides;SATP was mainly composed of galactose,arabinose,mannose and glucose,and their molar ratio was 24.2: 8.39:3.29: 1.00.2.In this chapter,we evaluated the anti-obesity activity of SATP by detecting the body weight gain,plasma lipid profiles,adipose tissue inflammation,and expressions of key enzymes of fat metabolism in a high glucose and fat diet(HFD)-induced obese mice.The results indicated that the mice in the model group showed obvious obesity characteristics.After intervention with SATP(400,200,100 mg/kg),the excessive weight gain and fat accumulation of obese mice were controlled.SATP could improve the glucose tolerance in HFD-fed obese mice,down-regulate TC,TG,NEFA content,and up-regulate HDL-C levels.SATP could also significantly improve the infiltration of macrophages in adipose tissue in obese mice,and exert anti-obesity effects by inhibiting the expressions of key enzymes(ACC-1,PGC-1?)for fat synthesis and adipocyte differentiation.3.In this chapter,we further study the protective effect of SATP on hepatic steatosis in a HFD induced NAFLD mice by pathological examination such as HE staining,oil red O staining,Masson staining and other biochemical indicators such as transaminase,blood lipid,proinflammatory factor,and oxidative stress.The results showed that SATP could significantly improve the characteristics of HFD-induced liver steatosis,such as balloon-like lesions,cell necrosis,and inflammatory cell infiltration;it can effectively reduce fat accumulation and progressive fibrosis in the liver.SATP could also down-regulate the abnormal expressions of amino transferases ALT and AST,and could significantly reduce the levels of lipid per-oxidation products MDA and LPO in the liver,improve the activity of antioxidant enzymes SOD and GSH-Px.In addition,SATP could effectively reduce the over-expressed pro-inflammatory cytokines(IL-1?,TNF-?)in the serum of NAFLD mice,and protect liver tissue damage caused by steatosis.4.In this chapter,we researched the protective mechanism of SATP in improving hepatic steatosis injury based on the micro-ecological balance of gut microbiota in NAFLD mice.First,HE staining,WGA-FITC,and endotoxin detection were used to evaluate the protective effect of SATP on the intestinal barrier in NAFLD mice.Then,16 S rRNA high-throughput sequencing technology was used to analyze the diversity of gut microbiota in each group of mice.Then,the relationship between hepatic steatosis and changes in intestinal short-chain fatty acids(SCFAs)was explored.Finally,the effect of SATP on gut microbiota dysbiosis was analyzed using fecal microbiota transplantation(FMT)experiments.The results showed that the intestinal barrier of NAFLD mice was damaged,the levels of endotoxin in the serum of mice were significantly increased,and the intestinal mechanical barrier function was impaired.SATP intervention could maintain normal intestinal mucosal barrier function in mice.Different diets and interventions can lead to different development of the intestinal microbial community.The gut microbial diversity was reduced in NAFLD mice,and the ratio of Firmicutes/Bacteroidetes(F/B)in NAFLD mice was significantly higher than normal mice.SATP could effectively increase the diversity of gut microbiota and down-regulate F/ B.SATP also could significantly up-regulate the relative abundance of Lactobacillus,Bacteroides and Akkermansia.In addition,the content of acetic acid,propionic acid,butyric acid,and total SCFAs in the intestine of NAFLD mice were decreased significantly.The intervention of SATP could significantly increase the concentration of SCFAs in the colon,which also explained the regulation of the disordered and unbalanced gut microbiota.The gut microbiota from NAFLD mice could aggravate lipid accumulation in the liver,increase blood lipid levels,cause abnormal liver function,and increase serum levels of proinflammatory factors.Moreover,it could cause damage to the intestinal barrier in mice.Transplanting SATP-regulated gut microbiota could significantly improve the degree of fatty liver damage and protect the intestinal barrier function.5.In this chapter,we explored the molecular regulatory mechanism of SATP on fatty liver cell injury in OA-induced LO2 cells.The expression levels of ER stress-related factors in mice from liver and LO2 cells were measured.The production of ROS,the changes of ER ultrastructure and expressions of inflammatory factors were analyzed in OA-induced LO2 cells.The results showed that ER stress markers such as GRP78 / Bip and CHOP in liver of NAFLD mice and LO2 cells were significantly expressed.OA could induce excessive production of ROS in LO2 cells,cause changes in the ultrastructure of ER and increase proinflammatory cytokines(IL-1?,TNF-?).SATP intervention could significantly reduce the expressions of ER stress markers in liver and LO2 cells,inhibit the production of ROS and activation of inflammatory response.In addition,SATP could down-regulate the levels of p-IRE1?/IRE1? and p-NF-?B p65/ NF-?B p65,which might regulate the IRE1?/NF-?B pathway to improve fatty liver cell injury.