The Mechanisms Of Dendrobium Mixture Inhibiting Gluconeogenesis Based On PI3K/Akt And GCGR/PKA Pathway

ObjectiveThe aim of this study is to explore the mechanism of Dendrobium mixture(DMix)in improving insulin resistance and inhibiting gluconeogenesis through regulating PI3K/AKT and GCGR/PKA signaling pathway in vivo and in vitro.Methods1.Detection of iTRAQ protein in rat liver tissue40 female Wistar rats were randomly divided into 10 rats in the normal group and 30 in the model group.The normal rats were fed with ordinary feed,and the model rats were induced by intraperitoneal injection of Streptozoein(25 mg/kg+m2,interval 72 h)after high glucose and high fat diet for 6 weeks.The diabetic rats were selected and randomly divided into 3 groups(model group,DMix group and metformin group).The rats in the DMix group were administered with DMixl(17.2 g/kg·d-1)and DMix2(12.4 g/kg·d-1);the rats in the Metformin group were administered with metformin(100 mg/kg ·d).and the model group and normal group were fed with normal saline.Continuous gavage of stomach for 60 days.All the animals were sacrificed 24 hours after the last treatment,then liver tissue and blood were collected.iTRAQ-based liquid chromatography-mass spectrometry was used to detect the liver proteomics of rat.The differential protein expression of the liver in each group was observed and the differential proteins and related signaling pathways between the groups were found,which lay the foundation for the follow-up study.2.Animal experiment50 healthy female Wistar rats were randomly selected for 11 as normal group,and the remaining 39 rats were modeled and grouped according to the above methods.Blood and liver tissue were taken after 12 weeks of continuous gavage,Detect fasting blood glucose,glycosylated serum protein,total cholesterol,triglycerides and other indicators.Serum insulin and glucagon were detected by ELISA;Observation of liver tissue morphology by HE staining;RT-PCR assay was used to detect the gene expression associated with Gluconeogenesis through PI3K/Akt and GCGR/PKA signaling pathways.The protein expression associated with Gluconeogenesis through PI3K/Akt and GCGR/PKA signaling pathways was detected by immunohistochemistry and Western-blotting method.3.Cell experimentAfter the establishment of insulin resistance hepatocyte model in vitro,the serum concentration of Dendrobium mixture,screened by MTT,was used to intervene.After the intervention,the cell culture supernatant was taken to measure the residual glucose level by Glucose oxidase-peroxidase method,And calculate the insulin sensitivity index.The total RNA and protein were extracted.RT-PCR and Western-blotting assay were performed to analyze gene and protein expression associated with Gluconeogenesis through PI3K/Akt and GCGR/PKA signaling pathways.Result1.The differential proteins identified by iTRAQ proteomics in liver tissues are involved in energy metabolism,carbohydrate metabolism,fat metabolism,amino acid metabolism and so on.The signaling pathways of gluconeogenesis,fatty acid metabolism,amino acid metabolism are directly or indirectly related to gluconeogenesis.2.The rising levels of FBG?GSP?Fins?HOMA-IR and Glucagon in model group decreased significantly(P<0.05 ?P<0.01)after the intervention of Dendrobium mixture.After the treatment of metformin,the levels of FBQ GSP,Fins and HOMA-IR decreased significantly(P<0.05?P<0.01),but the level of Glucagon did not decrease significantly(P>0.05).3.The rising levels of Tch?TG?LDL?AST and ALT in model group decreased significantly(P<0.05 ?P<0.01)after the intervention of Dendrobium mixture.After the treatment of metformin,the levels of Tch,LDL?AST and ALT decreased significantly(P<0.05 ?P<0.01),but the level of TG did not decrease significantly(P>0.05).4.HE staining in liver tissue showed that the shape of hepatocytes in the model group was changed,the cell contour was not clear,the lobule of hepatic lobules was arranged,and the hepatic sinusoids were enlarged.Dendrobium mixture can significantly improve the liver morphology and the effect is better than metformin group.5.Dendrobium mixture can significantly(P<0.05 ?P<0.01)increase the mRNA expression of InsR,beta-catenin and TCF7L2,and down regulate the mRNA expression of GCGR,PKA,FoxO1,PEPCK and G6Pase.Dendrobium mixture also increased the protein expression of InsR,p-PI3K,p-Akt,beta-catenin,TCF7L2 and lowered the protein expression of GCGR,PKA,FoxOl,PEPCK and G6Pase(P<0.05 ?P<0.01)to inhibit gluconeogenesis in diabetic rats.6.The serum containing Dendrobium mixture can significantly increase the glucose uptake ability(P<0.05)of IR hepatocytes and improve insulin resistance.7.The serum containing Dendrobium mixture can significantly(P<0.05 ? P<0.01)increase the mRNA expression of InsR,and down regulate the mRNA expression of FoxO1,PEPCK and G6Pase.It also significantly(P<0.05 ?P<0.01)increased the protein expression of InsR?PI3K?p-Akt,p-FoxO1 and lowered the protein expression of Fox0O1?PEPCK and G6Pase to inhibit gluconeogenesis in IR hepatocytes.Conclusion1.The differential proteins identified by iTRAQ proteomics in liver tissues involved these signaling pathways,such as gluconeogenesis pathway,fatty acid metabolism,amino acid metabolism and energy metabolism.These pathways are associated with gluconeogenesis related directly or indirectly.2.The DMix can regulate the metabolism of glycolipid in diabetic rats,improve insulin resistance,improve the liver morphology and promote the recovery of liver function.3.The DMix can regulate gene and protein expression associated with Gluconeogenesis through PI3K/Akt and GCGR/PKA signaling pathways to inhibit gluconeogenesis in diabetic rats.4.The serum concentration of DMix could improve the glucose uptake of IR hepatocytes,thus improve insulin resistance.5.The serum concentration of DMix can regulate gene and protein expression through PI3K/AKT signaling pathways to inhibit gluconeogenesis in IR hepatocytes.