| Diabetes(DM)is an endocrine disease characterized by hyperglycemia and accompanied by metabolic disorders of glucose,lipids and proteins.With the rapid increase in the incidence of DM,it has become the third disease that poses a serious threat to human health after cardiovascular and neoplastic diseases.Although many drugs help slow the progression of type 2 diabetes,long-term use can have toxic side effects on the body.Therefore,it is very important to find and develop highly effective and non-toxic natural hypoglycemic drugs.Shiitake is known as the "king of the mountains" and is a high-protein,low-fat nutritional health food.Lentinan polysaccharide is the main active ingredient of Lentinula edodes,which has the functions of lowering blood sugar,antiviral,antitumor,regulating immune function and stimulating the formation of interferon.However,the main mechanism of anti-diabetic pharmacological activity of Lentinan polysaccharide is unclear.Therefore,this subject extracts,isolates,and purifies lentinan to obtain lentinan,and analyzes the structure and in vitro activity of lentinan.Based on the in vitro activity results,a T2DM mouse model is established,and DM mice are protected at different doses of lentinan.The protective effect of polysaccharides on type 2 diabetes mellitus and its possible protective mechanisms,in order to understand the anti-diabetic mechanism of lentinan polysaccharides,and provide new theoretical and experimental basis for the prevention and treatment of diabetes.The specific research results are as follows:(1)Extraction,Purification and Structural Identification of Polysaccharide from Lentinusedodesa.Lentinusedodes was extracted by boiling water,precipitated by ethanol,and deproteinized to obtain crude polysaccharide(LNT).Three kinds of purified polysaccharides were obtained from DEAE cellulose separation and purification of the crude polysaccharides from Lentinula edodes,which were water-washed polysaccharide LNT-1,0.05mol/L salt-washed polysaccharide LNT-2,and 0.1mol/L salt-washed polysaccharide LNT-3.b.Structural identification of LNT,LNT-1,LNT-2,and LNT-3:①Visually observe a polysaccharide,LNT is a tan powder,LNT-1 is a light yellow powder,LNT-2 is a light yellow floc,LNT-3 For light brown flocculent.②Scanning electron microscope results showed that the four polysaccharide morphologies were significantly different.③The molecular weights of LNT-1,LNT-2,and LNT-3 by gel permeation chromatography were 6.408×103Da,1.956 × 105Da,and 6.870×103Da,respectively.④Infrared spectrum analysis and nuclear magnetic resonance analysis showed that all four polysaccharides were β-pyranose.⑤Congo red experiment proved that there is no triple-helix structure in the four polysaccharides.(2)in vitro activity analysis of lentinana.Antioxidation in vitro:In vitro antioxidant results show that LNT,LNT-1,LNT-2 and LNT-3 all have the ability to scavenge hydroxyl free radicals,DPPH free radicals and total reducing ability.And the clearance activity order is LNT-1>LNT>LNT-2>LNT-3.b.In vitro hypoglycemic results:In vitro hypoglycemic results show that LNT,LNT-1,LNT-2 and LNT-3 all have α-glucosidase inhibitory activity,and the order of inhibitory activity is LNT-1)LNT>LNT-2>LNT-3.LNT and LNT-1 were used to carry out the experiment on glucose consumption in IR-HEPG2 cells.Compared with the blank group,glucose consumption in the model group was reduced(*p<0.01),and compared with the model group,the LNT group was increased(#p<0.05).),LNT-1 group increased(#p<0.01).(3)Protective effect of LNT and LNT-1 on T2DM micea.Construction of T2DM model:A high-fat,high-sugar diet combined with STZ was used to establish the T2DM model.It was found that the model mice exhibited the typical "three more and one less" symptoms of polydipsia,polydipsia,polyuria and weight loss.Fasting blood glucose,serum insulin,TG,TC,LDL-C levels increased(#p<0.001,#p<0.001,#p<0.01,#p<0.01,#p<0.001),and HDL-C levels decreased(#p<0.01),indicating that the glucose and lipid metabolism of the mice in the model group is abnormal.H&E staining of liver tissue samples from mice revealed that the DM group mice had an irregularly arranged liver plate structure,constricted hepatocyte nuclei,marked congestion of the central vein of the hepatic lobules,and hepatocytes vacuolated.H&E staining sections of pancreatic tissue samples found that the number of pancreatic islets in the model group was significantly reduced,the morphology was irregular,and the pancreas showed serious pathological changes,indicating that the mouse liver and pancreas were damaged during the modeling process.Comprehensive physiological and biochemical indexes and pathological results of mice in the DM group showed that the model was successfully established.b.Protective effect of LNT and LNT-1 on T2DM mice:Compared with the model group,4 weeks after gavage of LNT and LNT-1 solution,the symptoms of "three more and one less" were alleviated,and fasting blood glucose,serum insulin,TG,TC and LDL-C levels decreased(#p<0.01,#p<0.01,#p<0.05,#p<0.01,#p<0.001),and HDL-C levels increased(#p<0.05,#p<0.01)It shows that LNT and LNT-1 can improve the abnormal glucose and lipid metabolism in T2DM mice.Microscopic observation of the liver tissue revealed that the cells had a complete morphology,a clear structure,a regular arrangement,a significant reduction in vacuolation,and no congestion in the central vein.And LNT-1 is more effective than LNT,which is the same as the order of in vitro activity.This indicates that LNT and LNT-1 have protective effects on liver injury in T2DM mice.Microscopic observation of mouse pancreatic tissue revealed that the number of islets increased,the structure and organization of pancreatic cells were normal,the cells in the islets were neatly arranged,the cytoplasm was full,and the boundaries between pancreatic acinar and islet cells were clear.This shows that LNT and LNT-1 have a stimulating effect on ductal stem cells by restoring cells and increasing mitotic activity to differentiate and regenerate islet cells and have a protective effect on damaged islet cells.(4)Protective mechanism of LNT and LNT-1 on T2DM micea.UPLC-Q-TOF analysis of the serum of each group of mice found that the potential serum metabolites were mainly lipids,and the metabolic pathways were mainly arachidonic acid metabolism and glycerophospholipid metabolism,indicating that the mice produced lipid peroxidation and oxidation.Stress response.b.Based on the Nrf2/HO-1 pathway,study the protective effects of LNT and LNT-1 on T2DM mice:After four weeks of intragastric administration of LNT and LNT-1 solutions,the content of Nrf2 protein and mRNA in liver tissues of T2DM mice increased,indicating that lentinan can Effectively promote the nuclear translocation of Nrf2.The content of HO-1 protein and mRNA in liver tissue of mice in the protection group were higher than those in the model group.It is shown that lentinan can induce Nrf2 activation,leading to an increase in downstream antioxidant enzyme levels.This shows that lentinan has significant antioxidant activity in vitro and has a significant effect on diabetes.Its antioxidant activity in the body may enhance the body’s antioxidant capacity,relieve oxidative stress and reduce body damage by activating the Nrf2/HO-1 pathway. |
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