| Type II diabetes mellitus Diabetes is a metabolic disease caused by glucose metabolism disorder caused by insulin resistance.Ganoderma lucidum is a traditional Chinese medicine for the treatment of diabetes.It is rich in polysaccharides,triterpenoids,alkaloids and other bioactive components.Among them,Ganoderma lucidum polysaccharides have multiple physiological effects,such as regulating glycolipids,lowering cholesterol and regulating immunity.However,because it is a polymer composed of several monosaccharides linked by glycosidic bonds,it cannot be digested and absorbed by the human stomach.The intestine can use its own microbial flora to ferment the macromolecular polysaccharides that cannot be digested by gastric digestive enzymes and produce various beneficial metabolites.However,the specific mechanism of improving insulin resistance based on the metabolites of Ganoderma lucidum polysaccharide intestinal flora has not been deeply studied at present.Therefore,based on the above reasons,this project takes Ganoderma lucidum polysaccharide and its flora metabolite（GLP/GLP-F）as the research object and HepG2 cell as the action medium,and evaluates the changes of composition of Ganoderma lucidum polysaccharide metabolites through physicochemical characterization analysis;On the basis of evaluation of cell phenotype and function,we evaluated the effect of Ganoderma lucidum polysaccharides on reducing blood glucose,and verified the improvement of insulin resistance after the metabolism of intestinal microflora,so as to provide a theoretical basis for the mechanism of Ganoderma lucidum traditional Chinese medicine in alleviating type II diabetes.This study is mainly carried out from the following aspects,and the results are as follows:1.firstly,the physicochemical properties of Ganoderma lucidum polysaccharide and its intestinal flora metabolism were detected.Ganoderma lucidum polysaccharide was extracted and purified by water extraction and alcohol precipitation and Sevage deproteinization.The yield was 0.857%.The contents of total sugar,reducing sugar,protein and triterpenoids in Ganoderma lucidum polysaccharides were detected by phenol sulfuric acid method,DNS method,Coomassie brilliant blue method and vanillin glacial acetic acid method.The contents of short chain fatty acids in the metabolites of Ganoderma lucidum polysaccharides after 48hours of intervention on intestinal flora were analyzed by gas chromatography.The results showed that the total sugar content of Ganoderma lucidum polysaccharides metabolized by intestinal flora decreased from 64.84±1.96%to 15.48±0.03%;The content of reducing sugar decreased from 6.14±0.004%to 1.43±0.03%;The contents of triterpenoids were0.53±0.02%and 0.55±0.03%,respectively.The contents of protein were 5.51±0.04%and 5.56±0.26%,respectively.The content of SCFAs in intestinal microflora metabolites of Ganoderma lucidum polysaccharide treated group for 48 hours was significantly higher than that of the blank control group,in which acetic acid,propionic acid and butyric acid were mainly produced.2.HepG2 cells were induced by insulin and dexamethasone to establish insulin resistance model.Insulin were used Concentration gradient of 10-1,10-2 and 10-3μM/L and dexamethasone the concentration gradient of 1,5 and 10μM/L was used to induce HepG2cells for 48h by orthogonal test,and the insulin resistance model（IR-HepG2 cells）was constructed.CCK-8 cell viability test kit and glucose oxidase method kit were used to test whether the model was successfully constructed.The results show that when the concentration is 10-3+10μM/L Glucose consumption decreased significantly（p<0.01）,and had no significant effect on cell viability.3.To explore the effect of GLP/GLP-F on IR-HepG2 cells.Use 0,250,500,750 and1000μg/ml Ganoderma lucidum polysaccharide and its flora metabolites intervened in IR-HepG2 cells.The optimal concentration range and action concentration of drug intervention were detected by CCK-8 cell viability detection kit,glucose oxidase method kit and glycogen synthesis kit.The results showed that when the concentration of GLP/GLP-F was 500μg/ml,the cell viability began to decrease significantly（p<0.05）;Use 50,100,200 and 400μg/ml concentration gradient intervened in IR-HepG2 cells,the cell viability had no significant effect,and showed a certain concentration dependence in this range.When the concentration is 400μg/ml,glucose consumption increased significantly（p<0.01）,which were 15.80±0.77%and17.66±0.51%respectively,and glycogen synthesis increased significantly（p<0.01）,which were 0.1089±0.0068%and 0.1141±0.0004%,respectively.4.To investigate the effect of GLP/GLP-F on the key proteins of glucose metabolism pathway in IR HepG2 cells.The IRS-1/PI3K/Akt glucose metabolism signal pathway of IR-HepG2 cells after GLP/GLP-F intervention was detected by Wstern blot.Mainly include the expression of key proteins IRS-1,P-Akt/Akt,the expression of GLUT2,a key protein in glucose uptake pathway,Glycogen synthesis pathway key protein P-GSK3β/GSK3βExpression and the expression of PEPCK,a key protein in gluconeogenesis pathway.The results showed that GLP/GLP-F activated the IRS/Akt glucose metabolism pathway of IR-HepG2 cells,increased the expression of IRS-1 protein and promoted the phosphorylation expression of Akt;Increased the transport of GLUT2 protein from glucose uptake pathway to cell membrane;Increased the phosphorylation expression of GSK3β,a key protein in glycogen synthesis pathway;The expression of PEPCK key protein in gluconeogenesis signaling pathway was reduced.Conclusion:The polysaccharide and reducing sugar components in Ganoderma lucidum polysaccharide are utilized by intestinal flora as the carbon source,and its main metabolite is short chain fatty acids,can promote glucose consumption,glycogen synthesis,gluconeogenesis,and type II diabetes caused by insulin resistance by activating the IRS-1/PI3K/AKT glucose metabolism signaling pathway in HepG2 cells. |
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