| Mulberry(Morus alba L.,family of Moraceae)leaf can be used not only as silkworm feed,but also in traditional Chinese medicine to treat diabetes certificate."Compendium of Materia Medica" records:mulberry fried juice and moxibustion replace of tea can prevent thirst quencher.In "Shen Nong's Herbal Classic",mulberry leaf is called "god leaf',which posses function of evacuating wind-heat,clearing the lung heat and moisten dryness,and liver eyesight.Modern pharmacological studies have shown that the mulberry leaf,inhibiting the rise of blood sugar,can prevent and treat diabetes.The chemical composition of mulberry leaves contain mainly flavonids;alkaloids,polysaccharides,and leaf protein,etc.Most domestic and foreign researches were focused on the,mulberry leaf hypoglycemic active ingredient of alkaloid 1-deoxynojirimycin(DNJ),which was used as template to develop the imno sugar drug-miglitol that has been approved in the market and widely applied in the treatment of diabetes.However,compared with alkaloid 1-DNJ,the hypoglycemic effect and mechanism of other chemical compositions are not studied in depth.Polysacchar:ides,another hypoglycemic active ingredient from mulberry leaves,has been shown having significant hypoglycemic effect,and are richer in content when compared with the alkaloid of 1-DNJ in mulberry leaves.However,current domestic and foreign researches were mainly conducted on the hypoglycemic activities of the crude polysaccharide extract,the exact hypoglycemic active component and its hypoglycemic mechanism has not deeply studied and was lack of experiment,which can not provide the basis for the pharmaceutical development of the polysaccharide from mulberry leaves.In view of this,a water-soluble polysaccharide ingredient(MLP?)that possessed hypoglycemic activity was purified from mulberry leaves.The preliminary structure of polysaccharide MLP? and its hypoglycemic effect and molecular mechanism on type 2 diabetic rats were also evaluated.The main results are as follows:(1)Purification and characterization of hypoglycemic active component-MLP ?.The mulberry leaf total polysaccharide was based on the water extraction and ethanol precipitation method,and then was fractionated on DEAE-52 Sepharose Fast Flow column and Sephadex G-100 column chromatography.In final,three homogeneous polysaccharide ingredients,named MLP I,MLP? and MLP?,were purified.Type 2 diabetic rat model was prepared using high-fat diet and low dose of STZ.The hypoglycemic effects of three polysaccharide ingredients on diabetic rats were determined,and the results showed that the hypoglycemic effect of MLP ? was the most obvious.The efficient steric exclusion chromatography,high performance liquid chromatography and infrared spectra were used to determine the molecular weight,monosaccharide composition and basic structure of hypoglycemic active components of MLP ?.The results showed that MLP ?,a water-soluble white powder with its molecular weight of 7.93 kDa,was made up of mannose(man),rhamnose(rha),glucose(glc),xylose(xyl)and arabinose(ara)with the molarity proportion of 8.73:1.04:6.53:2.13:1.00,and the monosaccharides mentioned above were linked with together by ?-glycosidic linkages.(2)The therapeutic effect of MLP ? on type 2 diabetic rats.MLP ? therapeutic intervention in type 2 diabetic rats were measured by 50 mg/kg,100 mg/kg and 150 mg/kg dose of MLP ? on the impact of diabetes glucose and lipid metabolism-related indicators,islet,and liver morphology,in order to investigate the therapeutic effect of the MLP ? on type 2 diabetic rats.The results showed that MLP ? could effectively control the levels of body weight and FBG levels,regulate glucose and fat metabolism and reduce islet ?-cells injury,promote the insulin synthesis and secretion in pancreatic ?-cells and improve hepatic lipid metabolism and insulin resistance.MLP ? showed a clear dose-dependent therapeutic effect on type 2 diabetic rats.In addition,the safety determination of MLP ? on rats showed that high doses of the MLP ?(1.5 g/kg)have no effect on liver,kidney,and other physiological indexes of rats,suggesting MLP ? has no obvious side effects on rats.(3)Effect of MLP ? on gene expression profiling of type 2 diabetic rats.The screening of differentially expressed genes in the islets and liver tissues of diabetic rats after intervention of MLP II were used by Roche-Nimblegen rat cDNA microarray.The results showed that,compared with control group,the up-regulated and down-regulated genes were respectively 902 and 1293 in the islet tissues of diabetic rats after intervention of 150 mg/kg MLP ?.The screening analysis was focused on islet cell function-related genes.Screened differentially expressed genes are mostly of cell apoptosis-related factors(eg.Bcl-2 family genes),oxidative stress-related factors(eg.Tgf-?1 gene),nuclear transcription-related factors(eg.Pdx-1 gene),and insulin secretion-related factors(eg.Glut2 gene),etc.In addition,we screened out 122 up-regulated genes and 138 down-regulated genes from the liver tissues of diabetic rats after intervention of MLP?.Insulin signal transduction-related screening analysis showed that the differentially expressed genes are mainly involved in the insulin receptor factors(eg.Insr gene),insulin receptor substrate factors(eg.Irs-2 gene),protein tyrosine kinase gene(eg.Ptp-1b gene),serine/threonine protein kinase gene(eg.Akt2 gene)and glycogen metabolism factors(eg.Gsk-3? gene),etc.(4)The molecular basis of protected effects of MLP ? on pancreatic ?-cell function in type 2 diabetic rats.On the basis of the results of gene microarray,immunofluorescence staining,western blotting and quantitative PCR were used to detect the effect of MLP ? on islet ?-cell apoptosis-related factors and insulin secretion factors of diabetic rats.The results showed that MLP ? can significantly inhibit the expression of pro-apoptotic factor BAX in islet ?-cell of diabetic rats,and increase the expression of the anti-apoptotic factor BCL-2,block cytochrome C(Cyt-C)released from the mitochondria to the cytoplasm,and then reduce the activation level of pro-apoptotic protein cysteine protease-3(CASP-3)in the cytoplasm,thus inhibiting the apoptosis of pancreatic ?-cells.In addition,MLP ? can inhibit the translocation from the nucleus to the cytoplasm for the transcription factor pancreatic islet cells in diabetic rats-duodenal homeobox factor(PDX-1),and improve the expression of PDX-1 protein in the nucleus and the expression of the downstream insulin secretion factor glucose transporter protein 2(GLUT2)and glucokinase(GCK),thus contributing to the sy nthesis and secretion of insulin.(5)The molecular basis of ameliorated effects of MLP ? on the hepatic insulin resistance in type 2 diabetic rats.On the basis of gene chip test results and PI-3K/AKT signal transduction pathway,immunofluorescence staining,western blotting and quantitative PCR were used to detect the effect of MLP ? on insulin signal transduction related factors in liver tissues of diabetic rats.The results showed that MLP ? can significantly inhibit the expression of tyrosine phosphatase 1B(PTP-1B)in the liver tissues of diabetic rats,and improve the phosphorylation level of insulin receptor substrate-2(IRS-2)and the expression of phosphatidylinositol-3-kinase(PI-3K),serine/threonine protein kinase 2(AKT2),and Glycogen synthase kinase 3?(GSK-3?),thus ameliorating insulin signal transduction barriers and reducing liver insulin resistance of type 2 diabetic rats.This paper systematically studied the therapeutic effects of MLP ? on type 2 diabetic rats,and its specific mechanism has also been discussed in depth from organization,protein and gene levels.It has provided a theoretical basis for the further development of the MLP ? that used in the prevention of type 2 diabetes. |
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