Effect Of Ginsenoside Rh2 On Glucose Metabolism In HepG2 Cells Based On P-AKT/FoxO1 Pathway And Its Mechanism

Objective:To investigate the relationship between ginsenoside Rh2(G-Rh2)and arsenic trioxide transactivator protein 3(AsTP3),and its effects and mechanisms on the glucose metabolism of HepG2 cells.Methods:1.Observe the effect of G-Rh2 on glucose metabolism:(1)Dissolve G-Rh2 in methanol,set 5 different concentration gradients of 0 ?M,5 ?M,10 ?M,20 ?M,40 ?M,and intervene in HepG2 cells for 24 h,respectively.G-Rh2 with different concentration gradients was detected by CCK-8.The cellular activity was then screened for the optimal concentration of G-Rh2 in subsequent experiments;(2)G-Rh2 with different concentration gradients were applied to HepG2 cells in the optimal concentration range of G-Rh2,through glucose production experiments,reactive oxygen species(ROS)detection,RT-PCR,western-blot to detect intracellular glucose level,ROS level and the expression of glucose metabolism-related genes phosphoenolpyruvate carboxykinase1(PEPCK1)and glucose-6-phosphate 1-dehydrogenase(G6PD),so as to explore the effect of G-Rh2 on glucose metabolism and its mechanism;2.Observe the effect of AsTP3 on glucose metabolism:The AsTP3 gene overexpression vector(pAsTP3),small interfering RNA(siAsTP3)and their respective negative controls NC and siNC were transiently transfected into HepG2 cells,and the RNA and total protein were extracted after 24 h and 48 h,respectively.The overexpression and interference of AsTP3 gene were verified by RT-PCR and western-blot.The effect of AsTP3 on the glucose metabolism of HepG2 cells was investigated by detecting the expression of glucose,ROS and glucose metabolism in the cell;3.Observe the effect of G-Rh2 on AsTP3:In the optimal concentration range,RNA and total protein were extracted from HepG2 cells by G-Rh2 with different concentration gradient after 24 h and 48 h,respectively,and the expression levels of AsTP3 gene mRNA and protein were detected after the intervention of G-Rh2,to explore the relationship between AsTP3 and G-Rh2.Meanwhile,G-Rh2 intervention was conducted under the condition of successful interference of AsTP3 expression,and glucose level,ROS level and glucose metabolism related gene expression in HepG2 cells were detected to further verify the relationship between AsTP3 and GRh2.Result:1.The effect of G-Rh2 on glucose metabolism:(1)The results of CCK-8 showed that compared with G-Rh2 concentration of 0 ? M,the G-Rh2 concentration of 20 ? M showed a slight decrease in cell activity,and the difference was statistically significant(P < 0.05),and the G-Rh2 concentration of 40 ?M showed a significant decrease(P < 0.0001);(2)G-Rh2 can reduce the levels of glucose and ROS in HepG2 cells in a concentration-dependent manner.At the optimal concentration of G-Rh2,G-Rh2 can down-regulate the expression of forkhead box protein O1(FoxO1)protein and up-regulate the expression of phosphoprotein kinase B(p-PKB,also known as p-AKT),thus decreasing the expression of PEPCK1 mRNA and protein levels,increasing the expression of G6 PD mRNA and protein expression levels.2.The effect of AsTP3 on glucose metabolism:After transfection,AsTP3 overexpression and interference were verified by mRNA and protein levels.At the same time,under the premise of overexpression and interference of AsTP3,the level of glucose in HepG2 cells decreased,the level of ROS decreased,the expression of FoxO1 protein decreased,the expression of p-AKT protein increased,and the expression of PEPCK1 mRNA and protein decreased,G6 PD mRNA and protein levels increased,while interference with AsTP3 expression was reversed;3.The effect of G-Rh2 on AsTP3: G-Rh2 up-regulated AsTP3 gene expression at mRNA level and protein level in a concentration-dependent manner,and the up-regulation effect was most significant at G-Rh2 concentration of 20 ?M(P < 0.001).Intervention with G-Rh2 in the presence of interference with AsTP3 expression,G-Rh2 can attenuate the role of AsTP3 in promoting glucose production in HepG2 cells.Conclusion: 1.Both g-rh2 and AsTP3 can reduce hepatic gluconeogenesis,possibly by regulating the p-akt /FoxO1 signaling pathway to regulate the expression of glucose metabolism-related genes in HepG2 cells;2.G-Rh2 can up-regulate AsTP3 expression,and G-Rh2 intervention in the case of interfering with the expression of AsTP3 can reduce the increased glucose metabolism of HepG2 cells to a certain extent when simply interfering with the expression of AsTP3,indicating that AsTP3 may be a new target for the regulation of glucose metabolism in the liver by G-Rh2.