| ObjectiveDiabetes is a kind of multifactorial and complex metabolic disorder,with high morbidity and mortality.And it is called one of the world three big disease with cancer and cardiovascular disease.Western medicine therapy can effectively control blood sugar quickly,but the deficiency is that they are still lack of effective measures for the prevention and treatment of chronic complications.Besides,treatment failure,insulin resistance and hyperinsulinemia would appear as a result of continuous use of oral hypoglycemic drugs.Traditional Chinese medicine(TCM)can produce one or more significant overall pharmacological effects because of its multicomponent,multiple target points and a variety of mechanisms,and it conform to the complicated pathological features of diabetes.And it has stable curative effects,low toxicity and adverse reactions.So it can be used for a long time.In recent years,TCM is very active in the prevention and treatment of diabetes.Since ancient times,the ancient medical books have the symptoms of diabetes treatment leaves records,such as Compendium of Materia Medica load Mulberry is Hand Foot Yangming medicine...Only the diabetes",Japan ancient book<>also recorded the Mulberry leaf tea health in mind improve water" disease "(Modern Medicine Diabetes)role.It has been proved that miRNA can directly or indirectly participate in the occurrence and development of many diseases by regulating the complex network of cellular signaling pathways and the biological behavior of cells.In view of this,this paper from the whole,cell and gene post transcriptional regulation in three aspects,to study the protective effects of Mulberry leaf on type 2 diabetes islet beta cell function,and to further explore the relationship between microRNA mediated apoptosis,and clarify the Mulberry leaf active site protection of islet cells,improve the mechanism of type 2 diabetes.Methods1.The total flavonoids from Mulberry leaves by orthogonal design method,the total alkaloid and total polysaccharide extraction process of screening,in order to improve the extraction rate of various components.Among them,the polar alkaloids and flavonoids of Mulberry leaves were similar,the extraction conditions of two kinds of components in the same experiment.In the purification process,the separation method of different principle to improve the purity of each active site.Based on the total flavonoids from Mulberry leaves by macroporous resin and polyamide column chromatography separation series,Mulberry leaf alkaloid by acid-base solution separation method with cation exchange resin combination of Mulberry polysaccharide with three trichloroacetic acid precipitation,water extraction and alcohol precipitation method and the dialysis separation purification.2.The high-fat diet combined with low dose of STZ,induced by intraperitoneal injection of type 2 diabetic rat model,were given 1000 mg/kg.d-1 Mulberry leaf total polysaccharide,total flavonoids from Mulberry leaves and leaves 350 mg/kg.d-1 30 mg/kg.d-1 total alkaloids were treated,continuous regular administration of 6 weeks.The level of blood glucose and insulin secretion was measured by large fasting,detection of islet beta cell apoptosis by TUNEL,and the related tissues(pancreas,liver,kidney,lung and muscle)on the morphological observation,the comprehensive evaluation of the 2 active sites of Mulberry leaves The effect of tissue and organ in type 2 diabetic rats.3.The activity of INS-1 cell CCK8 assay,flow cytometry,detection of Caspase3,RT-PCR Bax,Bcl-2 changes in the level of gene,to investigate the protective effect of Mulberry leaves on active site and wither INS-1 cell injury death;on this basis,further using miRNA gene chip were used to detect the difference between the expression levels of miRNAs,and the use of miRanda,targetscan miRBase,microRNA and other bioinformatics databases of candidate miRNAs target gene crossover,enrichment analysis,the introduction of KEGG and GO software to dig the biological function and biological pathway information of target gene enrichment analysis,the construction,of miRNA-mRNA gene regulatory network,regulation of transcription level of miRNA mediated apoptosis in INS-1 cells.Results1.Extraction of total flavonoids from Mulberry leaves is determined for A3B1C1D3,namely,reflux extraction 3 times,5 times the amount of solvent,extraction time was 1H,the ethanol concentration was 70%,the average content of purified total flavonoids from Mulberry leaves for the extraction of total alkaloids from Mulberry leaves 81.42%.eventually identified as A3B4C1D1,namely,reflux extraction 3 time,solvent amount was 8 times,the extraction time was 1H,the ethanol concentration was 50%,the average content of total alkaloids after purification process conditions of 16.80%.Mulberry leaf total polysaccharide extraction is confirmed to be A1B3C3,which is 6 times the amount of water,extraction time For 2h,leaching times was 3,the average content of purified Mulberry leaf total polysaccharide was 67.55%.2.Under an electron microscope,compared with T2DM model group,Mulberry leaf polysaccharide group,total flavonoids in the intervention group,intervention group total alkaloids large islet volume catachromasis,a more regular oval,clear boundary,dense secretory cells;liver cells compared with normal liver,muscle fiber bundle cable structure repair;basic and close neat;glomerular structure is normal,the cavity ratio is basically normal,renal tubular epithelial cell expansion and swelling decreased;alveolar sac and alveolar expansion was not obvious.The preliminary infer that Mulberry leaf total polysaccharide,total flavonoids and total alkaloids of the pancreas in diabetic rat liver,kidney,muscle tissue and lung tissue,in the form of all There are different degrees of improvement.3.Mulberry leaf polysaccharide group,total flavonoids in the intervention group,intervention group reduced the total alkaloids of INS-1 cell injury and apoptosis induced by high glucose(P<0.05 or P<0.01),down-regulation of the pro apoptotic factor Caspase3,Bax expression in mRNA and protein levels(P<0.05),expression of anti apoptotic protein Bcl-2 in mRNA and protein levels(P<0.05;miRNAs)from differential expression analysis:(1)compared with the negative control group,high glucose treatment 24h group miRNAs a total of 283,including Increase of 202,down 81;high glucose treatment 48h group miRNAs a total of 293,which increased 174,down 119;(2)compared with high glucose treatment 24h,Mulberry leaf polysaccharide intervention group significantly miRNAs a total of 351,which increased 171,down there are 180;Total Flavonoids from Mulberry leaves in intervention group significantly miRNAs a total of 239,which increased 107,down 132;Total alkaloids from Mulberry leaves in intervention group significantly miRNAs total of 373,The increase of 169,down 194;(3)compared with high glucose treatment 48h,Mulberry leaf polysaccharide intervention group significantly miRNAs a total of 96,which increased 46,down 50;Total Flavonoids from Mulberry leaves in intervention group significantly miRNAs a total of 236,which increased 122.By 114;Total alkaloids from Mulberry leaves in intervention group significantly miRNAs a total of 187,which increased 48,down 138;in vivo,in vitro quantitative detection technology of PCR,the main gene rno-The expression levels of miR-30d-5p,rno-miR-126a-3p and so on were consistent with the microarray results.Conclusions1.The establishment of Mulberry leaf total flavonoids,extraction of total alkaloids and total polysaccharide purification process is simple,stable and reproducible.The purity of three active sites were relatively early research group increased significantly,the content of total flavonoids and total polysaccharides of more than 50%.2.Mulberry leaf total polysaccharide,total flavonoids and total alkaloids of the pancreas in type 2 diabetic rats liver,kidney,muscle and lung tissue injury in morphology have certain effect;and Mulberry total polysaccharide and total flavonoids in normal rats showed different degrees of reduction of blood sugar and promote the role of insulin secretion.3.Mulberry leaf total polysaccharide,total flavonoids and total alkaloids by inhibiting the mitochondrial apoptotic pathway,damage and apoptosis of islet INS-1 cells induced by high glucose protection;in vivo,in vitro quantitative detection technology of PCR,which demonstrate the main gene rno-miR-30d-5p,the expression level of gene chips and rno-miR-126a-3p agreement;in the pathogenesis of type 2 diabetes mellitus in rno-miR-30d-5p,can be directly targeted by Atgl2 autophagy factor mediated autophagy,apoptosis and regulation of islet beta cells,can be targeted to IRS1 Protein mediated insulin resistance,but also relates to the biological process of multiple signaling pathways involved in indirect type 2 diabetes;and another has proven rno-miR-126a-3p mainly targeting plk2 factor mediated cell cycle,and apoptosis of beta cells,also targeting IRS 1 protein mediated insulin resistance and multiple signaling pathways. |
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