| ObjectiveAnimal experiments and cell experiments were separately carried out to observe the regulation of total flavonoids from Ampelopsis grossedentata on glucose metabolism in spontaneous diabetic ZDF rats,and to explore the molecular mechanism of skeletal muscle insulin resistance through IRS-1/PI3K p85/AKT/GLUT4 signaling pathway in skeletal muscle cells.The molecular mechanism of skeletal muscle glucose metabolism provides more data basis for the clinical hypoglycemic application of Ampelopsis grossedentata.Methods1.Animal experiments 40 male ZDF rats(fa/fa),an animal model of spontaneous type 2 diabetic mellitus,were adaptively fed for 1 week and then fed with pumina#5008 high-fat feed for 4 weeks.Successful diabetic models were screened according to the tail vein blood glucose level and included in the experiment.There were randomly divided into model group(DM),metformin group(MET),high-dose group of total flavonoids from Ampelopsis grossedentata(AGH),medium-dose group of total flavonoids from Ampelopsis grossedentata(AGM).and low-dose group of total flavonoids from Ampelopsis grossedentata(AGL)according to random number table,8 rats in each group.At the same time,8 male(Zucker lean)ZL(fa/+)rats of the same age as the normal control group(NC)were fed with ordinary feed.The positive drug control group(MET):0.158 g/Kg·d-1.The high,medium and low dose groups of total flavonoids from Ampelopsis grossedentata were 400 mg/Kg·d-1,200 mg/Kg·d-1,100 mg/Kg·d-1,respectively.Drugs were dissolved in normal saline and then administered by gavage for 6 consecutive weeks.Observe the general state of the rats,and monitor the fasting blood glucose and body weight of the rats every week.After the intervention,the rats were anesthetized to take pancreas and gastrocnemius.The pancreatic tissue homogenate was used to detect insulin levels.Gastrocnemius muscle tissue was used to detect skeletal muscle glycogen content and histomorphological detection.Formalin-fixed paraffin-embedded gastrocnemius muscle section were stained with hematoxylin and eosin to show morphological changes,and stained with periodic acid-schiff to show muscle glycogen changes.The image processing software Avizo was used to analyze muscle glycogen deposition area.Western Blot and Real-time PCR methods were used to detect the protein expression and the transcription level of target genes of the skeletal muscle insulin signal transduction pathway.2.cell experiments The CCK-8 method was used to detect the effects of different concentrations of total flavonoids from Ampelopsis grossedentata(5 μg·ml-1,10 μg·ml-1,20μg·ml-1,30 μg·ml-1,50 μg·ml-1,80 μg·ml-1)on cell proliferation of C2C12 cells.Calculate the cell survival rate and determine the high,medium and low concentrations of in subsequent experiments.0.5 mmol·1-1 palmitic acid modeling solution was used to establish a cellular insulin resistance model.The myotube cells induced to mature by C2C12 cells were divided into normal group(NC),high glucose model group(DM),and total flavonoids from Ampelopsis grossedentata in high,medium,and low dose groups(AGH,AGM,AGL).Their concentrations are 80 μg·ml-1,50 μg·ml-1,30 μg·ml-1).The glucose consumption of each group of cells was detected,and the protein expression of insulin signaling pathway in C2C12 cells was detected by Western Blot method.Results1.Animal experiments(1)Body weight and serum detection of glucose metabolism:The body weight of rats in the model group,metformin group and total flavonoids from Ampelopsis grossedentata group after 6 weeks of intervention was significantly higher than that of the normal group at the time of each week of the intervention for 6 weeks(P<0.01)The body weight of rats in the high and medium dose groups of total flavonoids from Ampelopsis grossedentata and the metformin group was significantly lower than that of the model group at the 4th and 5th weeks respectively(P<0.05).and the body weight showed a continuous downward trend after the intervention for 6 weeks.Compared with the model group,the high-dose group of total flavonoids from Ampelopsis grossedentata,the metformin group and the middle-dose group of total flavonoids from Ampelopsis grossedentata showed a significant reduction in fasting blood glucose at the 4th,5th,and 6th week(P<0.05或 P<0.01).OGTT and ITT peak delayed(P<0.01).The insulin level of rat pancreatic tissue homogenate in the model group was significantly higher than that in the normal group(P<0.01),the insulin level of each dose group of total flavonoids from Ampelopsis grossedentata and metformin group was significantly lower than that of the model group.Estimating the trend of HOMA-IR,the model group HOMA-IR was significantly higher than that of the normal group(P<0.01),the HOMA-IR of the total flavonoids from Ampelopsis grossedentata in high and medium dose groups and the metformin group was significantly lower than that of the model group(P<0.01).The skeletal muscle glycogen content of rats in the model group was significantly reduced after 6 weeks of intervention(P<0.01),the skeletal muscle glycogen in high-dose group of the total flavonoids from Ampelopsis grossedentata and the metformin group were significantly higher than that of the model group(P<0.05).(2)Skeletal muscle morphology detection:it is shown that from Hematoxylin-Eosin staining each dose group of total flavonoids from Ampelopsis grossedentata and metformin group improved the atrophy of skeletal muscle fibers in rats.It is indicated that from the Periodic Acid-Schiff stain each dose group of total flavonoids from Ampelopsis grossedentata and metformin group obviously improved the staining of muscle fiber glycogen compared with the model group.The skeletal muscle glycogen deposition area of total flavonoids from Ampelopsis grossedentata in high and medium dose groups and metformin group was significantly higher than that of the model group(P<0.01).(3)The high-dose group of total flavonoids from Ampelopsis grossedentata and the metformin group promoted the IRS-1/PI3K p85/AKT/GLUT4 signaling pathway and significantly increased the protein expression of IRS-1,PI3K p85,AKT and GLUT4(P<0.01 or P<0.05).Each dose group of total flavonoids from Ampelopsis grossedentata and metformin group significantly increased the transcription levels of IRS-1,PI3K p85,AKT,and GLUT4 genes(P<0.01 or P<0.05).2.cell experiments According to the detection of C2C12 cell viability,the high,medium,and low concentrations of total flavonoids from Ampelopsis grossedentata used in cell experiments were 80 μg·ml-1,50 μg·ml-1,and 30 μg·ml-1,respectively.Compared with the high glucose model group,the glucose consumption of each concentration group of total flavonoids from Ampelopsis grossedentata increased significantly(P<0.01).The high and medium dose groups of total flavonoids from Ampelopsis grossedentata significantly increased the protein expression of IRS-1/PI3K p85/AKT/GLUT4 signaling pathway(P<0.01).Conclusions1.Total flavonoids from Ampelopsis grossedentata significantly reduced the body weight and improved glucose metabolism,hyperinsulinemia and IR of ZDF rats.2.Total flavonoids from Ampelopsis grossedentata improve skeletal muscle glycogen content,muscle glycogen area of ZDF rats and skeletal muscle IR.3.Total flavonoids from Ampelopsis grossedentata up-regulate the protein and gene expression of IRS-1/PI3K p85/AKT/GLUT4 in skeletal muscle cells,and up-regulate the protein expression of IRS-1/PI3K p85/AKT/GLUT4 insulin signaling pathway in C2C12 cells.which may be one of the mechanisms of action to improve skeletal muscle IR. |
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