Studies On The Hypoglycemic And Mechanism Of Ginsenosides Rk3/Rh4

Type 2 diabetes mellitus(T2DM)is a characterized by persistent hyperglycemia.Persistent hyperglycemia have an increased risk of developing a number of serious life-threatening health complications resulting in high medical care costs,decreased quality of life and increased mortality.There are more evidence approves that ginsenosides can reduce blood glucose level,improve T2 DM,and almost no toxic side effects.However,the mechanism of different ginsenosides on T2 DM remains unclear.In this study,we obtained the ginsenosides Rk3 and Rh4 by extracting,transforming,and separating and purifying from total ginsenosides,then a model of T2 DM mice and in vitro cell model were applied in this research.Next,the effect and mechanism of ginsenoside Rk3 and Rh4 on T2 DM were investigated,respectively.The mainly studies of this thesis are as below:(1)Preparation of ginsenoside Rk3 and Rh4This research takes panax notoginseng root as the object,by optimizing the extraction conditions,the content of total ginsenosides in the extract was 44.41%.Next,panaxatriol saponins were isolated from total ginsenosides by D101 macroporous resin.Single factor and response surface experiments were used to optimize acid hydrolysis conditions to convert the panaxatriol saponins into rare ginsenosides.When the concentration of citric acid is 8 mmol/L,the reaction temperature is 110?,and the reaction time is 4 h,the content of rare ginsenosides is the highest.Next,through the optimization of the resin conditions,it was found that D101 resin can obtain more than 70% mixture of ginsenosides Rk3 and Rh4.It was separated and purified by HPLC,and the purity of the final prepared ginsenoside Rk3 and Rh4 greater than 98%,respectively.(2)The effect and mechanism of ginsenoside Rk3 on T2DMIn this study,the T2 DM mice model was induced by HFD/STZ,and Rk3(30 mg/kg bw)and(60 mg/kg bw)were given orally for 4 consecutive weeks.The serum levels of mice in each group were detected.It was found that ginsenoside Rk3 could reduce the FBG level,improve glucose tolerance and insulin resistance.Serum level and Oil Red O staining were used to verify that Rk3 inhibit the lipid accumulation of T2 DM mice.Kit detection,q PCR and western blot were used to verify that ginsenoside Rk3 inhibit the inflammation levels of T2 DM mice.Flow cytometry,western blot and q PCR were used to verify that ginsenoside Rk3 improve insulin sensitivity,inhibit the hepatic gluconeogenesis and hepatic lipolysis of T2 DM mice.We further studied the effect of ginsenoside Rk3 on the upstream signaling events of hepatic gluconeogenesis and lipid accumulation.Our result indicated that ginsenoside Rk3 can activate the AMPK/Akt pathway in IR-Hep G2 cells,thereby improves the glucose and lipid metabolism disorders.These studies suggest that ginsenoside Rk3 ameliorate T2 DM via activating the AMPK/Akt signaling pathway.It may be a new type of hypoglycemic drug and provide an important reference for clinical application.(3)The effect and mechanism of ginsenoside Rh4 on T2DMTo evaluate the anti-T2 DM effect of ginsenoside Rh4 and verify its potential mechanism,HFD/STZ induced model of T2 DM was applied in this research.T2 DM mice were treated orally for 4 weeks with 20 and 40 mg/kg body weight of ginsenoside Rh4.The serum levels of mice in each group were detected.It was found that ginsenoside Rh4 could reduce the FBG level and improve serum lipid in T2 DM mice.High-throughput sequencing,q PCR,transmission electron microscopy,immunofluorescence staining and western blot were used to verify that ginsenoside Rh4 can significantly increase the antioxidant capacity of pancreatic tissue in T2 DM mice,improve pancreatic ?-cells dysfunction and promote insulin secretion.To further confirm the mechanism of ginsenoside Rh4 in the improving of T2 DM from the aspects of oxidative stress and insulin signal transduction,alloxan induced model of INS-1 cells was applied in this research.Immunohistochemistry,GSIS and western blot were used to verify that ginsenoside Rh4 promoted Nrf2 nucleus translocation as well as up-regulated the expression of HO-1,NQO1 and GCLC.Furthermore,we also found that ginsenoside Rh4 increased insulin secretion by activating the signal pathway of PDX-1,Glut2 and GCK.More importantly,the protective effects of ginsenoside Rh4 on alloxan-induced by upregulation of Nrf2 target gene and insulin secretion were abolished by Nrf2 knock down.Finally,we explored the upstream pathway of Nrf2 by ginsenoside Rh4 and found that the Akt deficiency inhibited ginsenoside Rh4-mediated Nrf2 nuclear translocation.Therefore,we demonstrate that pancreatic ?-cells protective effect of ginsenoside Rh4 activited Nrf2 signaling pathway might be through regulating the levels of p-Akt.In conclusion,our study illustrates that Rk3 improves T2 DM via activating the AMPK/Akt pathway.Ginsenoside Rh4 improves pancreatic ?-cells dysfunction by activating Akt mediated Nrf2 and its target gene.This study provides an effective theoretical basis for ginsenoside Rk3 and Rh4 in the treatment of T2 DM.