| Type 2 diabetes mellitus(T2DM)is a chronic metabolic disease characterized by hyperglycemia resulting from progressive loss of ?-cell insulin secretion frequently on the background of insulin resistance.T2DM,also known as non-insulin-dependent diabetes,accounts for more than 90%of all cases of diabetes.Individuals with T2DM usually suffer serious complications from hyperglycemia.In a sense,the deterioration of complications is the most direct cause of death in diabetic patients.Accumulating studies have shown that polysaccharides,as an important bioactive component of plants,exert visible antidiabetic effects with few or no side effects.Polysaccharide is one of the major active ingredients of legume,and has various biological activities,including antioxidant and immunoregulatory activities.In this paper,four legume polysaccharides were extracted from azuki bean(Vigna angular is),mung bean(Vigna radiata),cowpea(Vigna unguiculate)and pea(Pisum sativum Linn),and its hypoglycemic activity was compared in T2DM mice.Then,the polysaccharide from azuki bean(ABP)with the strongest hypoglycemic activity was selected to study its ameliorative effects on glucose and lipid metabolism and insulin resistance,protective effects on liver and kidney damage,and mechanism of antidiabetic activity in T2DM rats.Finally,the metabolic markers of T2DM rats were identified by UPLC-Triple-TOF/MS metabolomics analysis,and the effects of ABP on metabolic markers and involved metabolic pathways were investigated.The main research contents.and results were summarized as follows:(1)Comparison of hypoglycemic effects of polysaccharides from four legume species.The polysaccharides from azuki bean,mung bean,cowpea and pea were extracted and named CPP,PP,MBP and ABP respectively.The molecular weight and monosaccharide composition of ABP,MBP,CPP and PP were determined.The T2DM mice model was induced by high-fat diet and intraperitoneal injection of streptozotocin(STZ).These four kinds of legume polysaccharides(400 mg/kg·bw/d)were given orally to study their effects on fasting blood glucose(FBG),serum insulin(FINS),hepatic glycogen content,serum triglyceride(TG),serum total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),islet hormone sensitive index(ISI)and pancreatic histopathology in T2DM mice.The results showed that there were significant differences in the molecular weight and monosaccharide composition of these four kinds of legume polysaccharides;ABP can significantly reduce FBG,TG and LDL-C,increase hepatic glycogen reserve and ISI,and resist apoptosis of islet ? cells in T2DM mice;CPP can significantly reduce FBG,improve ISI and protect islet ? cells in T2DM mice.Based on our results,the observed hypoglycemic effects of four legume polysaccharides in STZ-induced diabetic mice decreased in the order of ABP>CPP>PP>MBP.Therefore,ABP was selected for further antidiabetic research.(2)The effects of ABP on glucose and lipid metabolism in type 2 diabetic rats were studied.The model of T2DM rats was induced by high-fat diet and tail vein injection of STZ.The effects of low(100 mg/kg·bw/d),medium(200 mg/kg·bw/d)and high(400 mg/kg·bw/d)doses of ABP on glucose and lipid metabolism in T2DM rats were studied.The main indexes were body weight,FBG,TG,TC,LDL-C,HDL-C,hepatic glycogen,glycosylated serum protein(GSP),glucose tolerance,serum free fatty acids and fecal short-chain fatty acids(SCFAs).It was found that low,medium and high doses of ABP can reduce the FBG,GSP and serum free fatty acid content in T2DM rats.Medium and high doses of ABP can effectively improve the glycogen reserve and reduce the serum TG content in T2DM rats.High dose of ABP can improve the glucose tolerance of diabetic rats,decrease the content of short chain fatty acids,increase the total amount of short chain fatty acids of feces in diabetic rats.The above results indicated that ABP could significantly improve glucose and lipid metabolism disorder in T2DM rats caused by hyperglycemia.(3)The effect of ABP on insulin resistance in type 2 diabetic rats was studied.Insulin resistance(IR)is the main cause of T2DM.The effects of ABP on the improvement of IR and the protection of pancreas were evaluated by measuring serum FINS,calculating HOMA-IRI,histopathological observation of pancreas and immunohistochemical analysis of Bax and Bcl-2 protein expression.The results showed that ABP could significantly reduce the compensatory increase of blood insulin caused by hyperglycemia in T2DM rats,improve the HOMA-IRI,and increase the hypoglycemic potency of unit insulin.In addition,ABP could maintain the integrity of islet structure and resist the apoptosis of ? cell in islet by up regulating the expression of Bcl-2 protein and down regulating the expression of Bax protein in pancreas of diabetic rats.(4)The protective effects of ABP on the liver and kidney of type 2 diabetic rats were observed.Diabetes often causes multiple organ damage,among which liver damage and kidney damage are common.In this study,the organ index,alanine aminotransferase(ALT),alanine aminotransferase(AST),ALT/AST,total protein(TP),albumin(ALB),globulin(GLO),urea nitrogen(BUN)and creatinine(SCr)of T2DM rats were measured to evaluate the liver and kidney function,and the damage of liver and kidney were observed by HE and oil red O staining.The results showed that the liver index and kidney index of diabetic rats treated with ABP were significantly decreased,the activity of ALT and AST was significantly decreased,the content of serum BUN,SCr,TP,ALB and GLO was increased,indicating that the liver function and renal function were significantly improved.Histopathological sections showed that ABP could repair liver and kidney damage and reduce the accumulation of renal fat in diabetic rats.Therefore,ABP had a good protective effect on the liver and kidney of diabetic rats.(5)The mechanism of antidiabetic effects of ABP was discussed in terms of oxidative stress and insulin signal transduction.After ABP treatment,the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)increased,and the content of malondialdehyde(MDA)decreased in T2DM rats,indicating that ABP can promote the clearance of active oxygen and reduce insulin resistance by decreasing the level of oxidative stress.Real-time Quantitative PCR(RT-PCR)and western blotting analysis showed that ABP significantly increased the expression of insulin receptor(INSR),insulin receptor substrate-1(IRS-1),phosphoinositide 3-kinase(PI3K),protein kinase B(AKT)and glucose transporter-2(GLUT-2),suggesting that the antidiabetic effect of ABP is related to the activation of insulin/PI3K/Akt signaling pathway and the regulation of glycogen synthesis and glucose metabolism related enzyme activity.(6)The effect of ABP on serum metabolites in type 2 diabetic rats was studied by metabolomics analysis.Through UPLC-Triple-TOF/MS metabolomics analysis,15 metabolic markers in serum of T2DM rats were identified,including 1-methylhistidine,5-hydroxy-6-methoxyindole glucuronide,citric acid,cysteine-S-sulfate,deoxycytidin,formyl-5-hydroxykynurenamine,acetoacetic acid,indolelactic acid,lysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)),tetradecanedioic acid and thromboxane B2 were significantly up-regulated in T2DM rats,while the concentrations of hydroxybutyrylcarnine,L-lysine,lysophone(20:0/0:0)and stearic acid were lower than those in normal rats.The metabolic pathway analysis showed that ABP improved the metabolism of T2DM rats mainly through the synthesis and degradation of ketone bodies,pentose and glucuronide conversions,butyrate metabolism and tricarboxylic acid cycle. |
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