| Pleurotus eryngii is a fungus with both food and medicine value that is widely cultivated in our country.It has rich nutritional value and a variety of active substances,such as polysaccharides,polyphenols,proteins,minerals,vitamins,etc.Pleurotus eryngii polysaccharides are reported to have many effects such as anti-tumor,antibacterial,antiviral,preventing diabetes and improving immunity,but the research on the function mechanism are not deep enough,especially the research on the lipid-lowering mechanism.In this paper,Pleurotus eryngii fruiting bodies were used as raw materials to extract polysaccharides(PEP)with traditional water extraction and alcohol precipitation.The three-component polysaccharides PEP1-A,PEP2-A and PEP3-A were obtained by purification,and their structures were characterized.In view of the preliminary in vitro antioxidant activity screening results,the polysaccharide PEP with better antioxidant activity was selected for anti-fatigue and lipid-lowering activity researches,through the detection of conventional biochemical indicators,liver and adipose tissue morphological observations,it was found that PEP has anti-fatigue and lipid-lowering effect.Through LC-MS technology,Western blotting and qRT-PCR technology,the lipid-lowering mechanism of PEP was studied from metabolomics,AMPK and ACC protein and gene expression levels.It was found that PEP lowers lipids by regulating the changes of amino acids,fatty acids and other endogenous compounds,and the various metabolic pathways they participate in.The mechanism of PEP lowering lipids in the AMPK-ACC pathway was through the regulation of AMPK(Adenylate Activated Protein Kinase),ACC(acetyl-coenzyme A carboxylase)protein and gene expression levels.The specific research results were as follows:(1)Optimizing the optimal process parameters for extracting PEP,measuring the hydration properties of PEP,and obtaining three polysaccharide components of PEP1-A,PEP2-A and PEP3-A through separation and purification,and measuring and comparing the content of polysaccharides,protein,uronic acid,and sulfuric acid group.It was found that the content of protein,uronic acid and sulfuric acid group decreased while polysaccharide content increased after purification.Through single factor and response surface experiments.The highest yield(7.52%)of Pleurotus eryngii polysaccharide was obtained when the extraction temperature was 80℃,the extraction time was 3h,and the extraction material-liquid ratio was 1:50(g/mL).The total sugar,protein and uronic acid contents of PEP were detected to be 67.84%,4.42%,and 0.28%,respectively.The water solubility of PEP was 45.8%,the water holding capacity was 19.7 g/g,and the expansion force was 24.00 mL/g.(2)Preliminary analysis of the structure of PEP1-A,PEP2-A and PEP3-A.The monosaccharide composition of PEP1-A,PEP2-A and PEP3-A had four monosaccharides with different content of fucose,galactose,glucose and mannose.Among them,the glucose content was the highest and the fucose content was the least.In addition,PEP3-A contained 1.39%arabinose.Ultraviolet and infrared results showed that PEP 1-A,PEP2-A and PEP3-A all contain the characteristic absorption peaks of polysaccharides.NMR results showed that PEP1-A,PEP2-A and PEP3-A all have pyranose rings,the sugar residues of PEP1-A were connected by β-glycosidic bonds,and the sugar residues of PEP2-A and PEP3-A were connected by α-Glycosidic bond connection.The methylation results showed that the main chain of PEP 1-A was connected by 1,3-Glc(p),1,4-Glc(p),1,6-Gal(p),and 1,6-Glc(p).The branches were 1,3,4-Glc(p),1,2,6-Man(p).The backbone of PEP2-A was connected by 1,3-Glc(p),1,4-Man(p),and 1,6-Gal(p),and the branch was 1,2,6-Glc(p).The backbone of PEP3-A was composed of 1,3-Glc(p),1,6-Glc(p),1,4-Man(p),1,6-Gal(p),1,6-Ara(p),branching was the connection of 1,3,6-Glc(p)and 1,2,6-Glc(p).Scanning electron microscopy showed that PEP1-A,PEP2-A and PEP3-A have similar strip-like structures.Due to self-charge,intramolecular forces and multi-branched chains,irregular lumps,islands and grain pile of PEP1-A,PEP2-A,and PEP3-A were observed under the atomic force microscope.(3)The anti-oxidation experiment of Pleurotus eryngii polysaccharide in vitro showed that PEP,PEP1-A,PEP2-A,PEP3-A all have a certain reducing power,superoxide radical scavenging ability,hydroxyl radical scavenging ability,DPPH Free radical scavenging ability,ABTS+radical scavenging ability.Especially,PEP’s anti-oxidation ability in vitro was best,so PEP was selected for further research on anti-fatigue and Lipid lowering activities in mice.(4)In the anti-fatigue activity,PEP had no significant effect on the weight and liver index changes of mice.Compared with normal control group the effect of PEP on the exhausted swimming time of mice was significantly increase(p<0.05).The increasing rates of exhausted swimming time in the high,medium and low dose PEP groups were 89.70%,75.12%and 53.11%,respectively,and the effect was positively related to the dose within the experimental range.The anti-fatigue biochemical index showed that compared with the control group,the PEP intervention group reduced the levels of BUN and LD and increased the activity of LDH.(5)Different doses of PEP gavaged high-fat mice,mice body weight,biochemical indicators and histopathological sections all showed that PEP had the effect of lowering lipids.Simvastatin group and high-dose PEP group could significantly reduce the weight of mice,while the weight loss of middle and low-dose PEP group was not significant,indicating that gavage concentration had an effect on weight change.The biochemical indicators and enzymatic test results of mice showed that after simvastatin and different doses of PEP gavage,the content of TC,TG,LDL-C,ALT,AST decreased significantly,while the content of HDL-C increased significantly,and the degree of decrease and increase were dose-dependent with PEP.It showed that PEP had a lipid-lowering effect and could prevent food-borne obesity,and high-dose PEP had the best effect on lowering lipids,followed by medium-dose PEP,and low-dose PEP had the lowest effect.Morphological observation of mouse liver and epididymal adipose tissue showed that after different doses of PEP gavage treatment,the fat vacuoles in the liver tissue of the mice were smaller compared with the high-fat model group.The size and number of adipocytes reduced compared with the high-fat model group,and the high-dose PEP group had the best effect.(6)By LC-MS technology,the serum samples of each group of mice were analyzed by metabolomics to clarify the lipid-lowering effect and mechanism of PEP.The results showed that the high-fat model group and the PEP group with different doses of serum metabolites had a good degree of separation,indicating that PEP had a certain lipid-lowering effect on high-fat modle mice,and could be used for the prevention and treatment of high-fat mice.After comparative analysis of each group,the metabolic differences in the serum of high-fat mice after PEP were found to include amino acids,fatty acids,choline,lipid variants,as well as other endogenous compounds and changes in the metabolic pathways involved.It was found that multiple pathways such as glycerophospholipid metabolism,tricarboxylic acid cycle,fatty acid metabolism have changed,indicating that the effect of different doses of PEP on hyperlipidemia caused by obesity may be related to energy metabolism.(7)Preliminary analysis of the lipid-lowering effect of the AMPK-ACC signal pathway mediated by PEP.Western blot and qRT-PCR were used to analyze the expression levels of key proteins and genes in the AMPK-ACC pathway.It was found that PEP can up-regulate the protein expression of pAMPK and pACC levels and up-regulate the gene expression levels of AMPK and ACC,thus having hypolipidemic effects,Thereby inhibiting the occurrence of metabolic disorders such as hyperlipidemia caused by obesity.Through this study,it is discovered that the polysaccharide of Pleurotus eryngii body had anti-fatigue and lipid-lowering biological activities,and the structure of PEP1-A,PEP2-A and PEP3-A was analyzed.From the metabonomics and the protein and gene expression levels of AMPK and ACC,the lipid-lowering mechanism of PEP was clarified.It laid a theoretical foundation for the in-depth research of Pleurotus eryngii polysaccharides and the development and utilization of health products. |
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