The Effects Of Dendrobium Officinale Polysaccharide On Hyperglycemia In Type ? Diabetic Mice And Its Potential Mechanisms

Dendrobium officinale(the dry stems of Dendrobium officinale Kimura et Migo),is mainly distributed in Zhejiang,Guangzhou and Yunnan.As a traditional and precious Chinese medicine,it has the functions of invigorating stomach,nourishing Yin,clearing heat,nourishing lung and nourishing kidney.Dendrobium officinale polysaccharide(DOP),one of the main bioactive components of Dendrobium officinale,exhibits significant pharmacological activities,including antioxidant,antiviral,anti-inflammatory,anti-aging,hypoglycemic and immune regulation.In recent years,our research group has been devoted to the extraction,separation and purification of DOP and the related biological activities research.Our previous studies showed that DOP could effectively alleviate the glucose metabolic disorders of type?diabetes mellitus(T2DM)in mice and the hypoglycemic mechanism might be associated with the insulin-mediated PI3K/Akt signaling pathway.However,many researches on the hypoglycemic mechanism of DOP mainly focuses on the insulin signaling pathway,which is too simple to be fully studied.Therefore,it is particularly important to explore the hypoglycemic mechanism of DOP comprehensively and deeply.In this study,Dendrobium officinale polysaccharide was extracted,separated and purified from the dry stems of Dendrobium officinale from Leqing,Zhejiang province using the established extraction technology.In vivo experiments,T2DM mice model was established by high-fat diet(HFD)combined with streptozotocin(STZ).In vitro experiments,insulin resistant HepG2 cells were performed.We aimed to study the effects of Dendrobium officinale polysaccharide on hyperglycemia in type?diabetic mice and its potential mechanisms,improving its molecular network mechanism and providing new way for the further development and utilization of DOP.Part One:The extraction,purification and identification of DOPDendrobium officinale polysaccharide was obtained from the dry stems of Dendrobium officinale in Leqing,Zhejiang province by water extraction combined with alcohol precipitation method.The refined DOP was isolated after repeated freeze-thawing removal and ultrafiltration.The results showed that the total sugar content of DOP was more than 99.36%,and the weight average molecular weight of DOP was1.90×10~5 Da.The UV results showed that there was almost no nucleic acid and protein.The monosaccharide compositions of DOP comprised D-Glcp and D-Manp in the proportions of 1.00:4.41.In this part,DOP extracted and purified from Dendrobium officinale has the advantages of stable yield,uniform molecular weight and high purity,which meeting the requirements of subsequent pharmacological studies.Part Two:The effects of DOP on ameliorating hyperglycemia in type?diabetic miceC57BL/6 mice were fed with HFD for 8 weeks and successfully induced to establish a T2DM model(FBG?11.1mmol/L as the standard)by combining with intraperitoneal injection of STZ.Then the diabetic mice were administrated with low,medium and high dose of DOP for 4 weeks,respectively.During the experiment,the activity status of mice in each group was observed regularly,and the body weight and FBG of mice in each group were recorded;Oral glucose tolerance test(OGTT)was performed before mice were sacrificed;The serum glucagon content in each group were measured;H&E staining sections of liver and pancreas were prepared.The experimental results showed that elevated FBG in the diabetic mice significantly decreased after DOP treatment.At the same time,the FBG in the prevention group is significantly lower than that in diabetic control group,showing that the DOP had certain prevention effect on hyperglycemia in diabetic mice.OGTT experiment results showed that the DOP could obviously improve the glucose tolerance and repair the impaired glucose tolerance in diabetic mice.Moreover,the serum glucagon level in DOP-treated group significantly decreased compared with that in diabetic control group,which suggested that DOP could inhibit the glucagon secretion and then relieve hyperglycemia in diabetic mice.In addition,H&E staining also showed that DOP could significantly improve fatty degeneration in livers and pancreas damage in diabetic mice.The experimental results in this part confirmed that DOP could reduce the FBG level,improve glucose tolerance,reduce serum glucagon level and relieve liver steatosis and pancreas injury,which could alleviate hyperglycemia in type?diabetic mice.Part Three:The mechanisms of DOP on ameliorating hyperglycemia in type? diabetic miceAfter DOP treatment,the livers were isolated and stained with PAS to observe the distribution of hepatic glycogen.The content of hepatic glycogen was also determined by biochemical method.The experimental results showed that the DOP could significantly improve the content of hepatic glycogen in diabetic mice.The molecular mechanisms of DOP to ameliorate hyperglycemia in type?diabetic mice were discussed from two aspects:hepatic glycogen metabolism and hepatic gluconeogenesis.The expression of the hepatic glycogen-related metabolism enzymes including GS,p-GS,PYGL and GBE,and the related protein in cAMP-PKA signaling pathway including glucagon,GCGR,AC,PKA-C and p-PKA were detected by western blots;The results showed that DOP could downregulate the related protein expression in cAMP-PKA signaling pathway to improve the hepatic glycogen metabolism,finally contributing to ameliorate hyperglycemia in type?diabetic mice.In addition,the expression of the hepatic gluconeogenesis enzymes including PEPCK and G6Pase,and the related protein in Akt/FoxO1 signaling pathway including Akt?p-Akt?FoxO1 and p-FoxO1 were also detected by western blots;The results showed that DOP could upregulate the expressions of p-Akt and p-FoxO1 to inhibit the hepatic gluconeogenesis,finally contributing to ameliorate hyperglycemia in type?diabetic mice.The experimental results in this part indicated that DOP could influence the glucagon-mediated cAMP-PKA and Akt/FoxO1 signaling pathways,promote hepatic glycogen synthesis and inhibit glycogenolysis and gluconeogenesis,which eventually improved hepatic glucose metabolism to ameliorate hyperglycemia in type?diabetic mice.Part Four:Research of DOP on the regulation of cAMP-PKA signaling pathway to improve hepatic glycogen metabolism in vitroIn order to verify the metabolism that DOP could inhibit cAMP-PKA signaling pathway to regulate hepatic glycogen metabolism and alleviate hyperglycemia in type?diabetic mice,an insulin resistance HepG2 cell model was established in this part and intervened by DOP for 24 h.The protein expressions of GS,p-GS,PYGL and GBE,and the related proteins in the cAMP-PKA signaling pathway including glucagon,GCGR,AC,PKA-C and p-PKA were detected by western blots in insulin resistance HepG2 cells,and the results were consistent with that of in vivo experiments.It suggested that DOP could inhibit the cAMP-PKA signaling pathway to regulate the hepatic glycogen metabolism in insulin resistance HepG2 cells.To further verify the inhibitory effect of DOP on the cAMP-PKA signaling pathway,we added adenylate cyclase(AC)activator in the cell experiments.The results showed that after DOP intervention for 24 h,the expressions of related proteins in the cAMP-PKA signaling pathway were significantly down-regulated in HepG2 cells,and AC activator could compensate for this effect of DOP,which again confirmed that DOP could inhibit the cAMP-PKA signaling pathway.Part Five:The effects of DOP on the structure of the liver glycogen in type? diabetic miceLiver glycogen was extracted from the liver tissues of mice in each group by the mild extraction method,and then purified and freeze-dried.Finally,white water-soluble cotton flocculent liver glycogen was obtained.Size exclusion chromatography(SEC)was used to analyze the structure of liver glycogen.The results showed that the structure of liver glycogen in normal control mice was stable,and there was no significant change before and after DMSO treatment.However,the liver glycogen structure of type 2diabetic mice was not stable and?particles were fragile,which could be easily degraded into?particles in DMSO.After 4 weeks'DOP treatment,the liver glycogen structure in DOP-treated groups was stable compared with in diabetic mice,which suggested that DOP could improve the instability of liver glycogen structure in diabetic mice and resist the effect of DMSO on the degradation of?particles,thus improving hyperglycemia in type 2 diabetic mice.