| Background: Insulin resistance is the main pathological mechanism of type 2diabetes mellitus.More than 90% of patients with type 2 diabetes mellitus are associated with insulin resistance.Therefore,relieving insulin resistance is the key to delaying and treating type 2 diabetes mellitus.Studies have confirmed that Yuye Decoction,a classic prescription,has pharmacological effects such as falling blood sugar,inhibiting inflammation and improving insulin resistance.It has definite clinical efficacy in the treatment of diabetes and its complications,but its mechanism of action is still lacking in in-depth research.Objective: The mechanism of action of Yuye Decoction in the treatment of type 2diabetes mellitus and its related signal pathways were predicted by network pharmacology and molecular docking technology.The combination of internal and external experiments were conducted to observe the improving effect of Yuye Decoction on insulin resistance of T2 DM rats induced by high fat diet combined with STZ and IR-HepG2 cells induced by palmitic acid.Then,Then,the PI3K/AKT signaling pathway predicted by network pharmacology was used as the entry point to explore the mechanism of Yuye Decoction in improving liver insulin resistance of type 2 diabetes mellitus.Methods: 1.To explore the mechanism of action of Yuye Decoction in the treatment of type 2 diabetes t mellitus hrough network pharmacology and molecular docking technology:Based on TCMSP,Swiss target prediction and other databases,the effective compound components and corresponding target proteins of Yuye Decoction were investigated.Gene Cards database was used to screen the differential genes of type 2 diabetes mellitus,and the GO enrichment analysis and KEGG pathway analysis were performed on the common targets of the two.The String platform and Cytoscape 3.7.2 software were used to construct the protein interaction network.Cytoscape 3.7.2 software was used to construct TCM-component-target-disease network diagram,and target-enrichment pathway network diagram to screen core compounds,targets and pathways.Finally,molecular docking technology was used to further simulate the binding of core targets and compounds.2.In vivo experiment: The model of T2 DM IR in SD rats was established by high fat feeding for 4 weeks combined with STZ(35mg/kg)injection.Sixty T2 DM rats successfully constructed were randomly divided into model group,metformin group,Yuye Decoction high-dose,medium-dose and low-dose groups with 12 rats in each group and 10 rats in normal group.The rats in each group were given corresponding drug intervention,and the changes of body weight and fasting blood glucose in each group were detected regularly.Oral glucose tolerance and fasting insulin tolerance were measured at the end of week 7 and 8,respectively,and the area under the curve was evaluated.After 8 weeks of intervention,samples were collected from the abdominal aorta and serum was separated.Serum Hb A1 c and fasting insulin levels were detected,insulin resistance index was assessed,TC,TG,LDL,HDL、ALT,AST,ALP,TP levels were detected.The liver was weighed,the organ coefficient was calculated,the glycogen content of liver tissue and the pathomorphological changes of liver were detected.By observing the above indexes,it is clear that Yuye Decoction can regulate glucose and lipid metabolism disorder,reduce insulin resistance and protect liver injury in T2 DM IR rats.In order to further clarify the mechanism of Yuye Decoction in improving insulin resistance in liver,the expression levels of TNF-α and IL-6 in liver tissue were detected by ELISA.The expressions of IRS-1,GSK3β,AKT,PI3 K p110a and their phosphorylated proteins in liver PI3K/AKT signaling pathway were detected by Western blot,and the relative expressions of IRS-1,PI3 K,AKT and GSK3β mRNA were detected by qRT-PCR.3.In vitro experiment: HepG2 cells were induced with 0.25 mM palmitic acid for 24 h to establish IR-HepG2 cell model.Cell morphology was observed by inverted microscope,cell viability was detected by CCK-8,and cell glucose consumption was measured by glucose oxidase method.Finally,100μg/ m L,200μg/ m L and 400μg/ m L were determined as the low,medium and high concentration groups of Yuye Decoction for subsequent experiments.The glucose consumption and glycogen content of IR-HepG2 were determined by glucose oxidase assay and anthrone assay,respectively.The expression levels of PI3K/ AKT signaling pathway related proteins IRS-1,p-IRS-1,PI3 K p110a,p-PI3 K,AKT,p-AKT,GSK3β and p-GSK3β were detected by Western blot.The mRNA expression levels of G6 Pase,GSK3β,AKT,IRS-1 and PI3 K were detected by qRT-PCR,and the network pharmacological and animal experimental results were further verified by in vivo experiments.Results:1.Results of network pharmacology and molecular docking: After network pharmacology analysis,108 core compounds,117 drug-disease common targets and 152 enrichment signal pathways of Yuye Decoction were finally screened.The results of molecular docking showed that the core compounds in Yuye Decoction had good binding ability to Akt1 and GSK3β in PI3K/ AKT signaling pathway.2.In vivo results : In vivo results:(1)Compared with model group,body weight of T2 DM rats in Yuye Decoction groups was significantly increased,FBG,Hb A1 c,FINS,HOMA-IR were significantly decreased(P < 0.05),ISI level was significantly increased(P< 0.05),serum levels of ALT,TC,TG were decreased(P < 0.05).Yuye Decoction can regulate glucose and lipid metabolism and increase insulin sensitivity.(2)Yuye Decoction improved liver tissue morphology,reduced liver coefficient,and increased liver glycogen synthesis content.(3)ELISA results showed that the expression levels of TNF-α and IL-6 in liver of T2 DM rats in Yuye decoction high-dose group and metformin group were significantly decreased(P < 0.01).(4)Western blot results showed that both Yuye Decoction and metformin groups could up-regulate the protein expressions of IRS-1,p-IRS-1,PI3 K,p-PI3 K,AKT,p-AKT,and down-regulate the protein expressions of GSK3β and p-GSK3β in liver of T2 DM rats(P < 0.05 or P < 0.01).Among them,the protein expressions of IRS-1,PI3 K and p-PI3 K in the high-dose group of Yuye Tang were significantly up-regulated(P < 0.01),and the expression of p-GSK3β was down-regulated(P < 0.01).The expression of p-IRS-1 in the medium-dose group of Yuye Tang was significantly up-regulated,and the expression of p-AKT was enhanced(P < 0.01).(5)The qRT-PCR results showed that the mRNA expression of PI3 Kand the mRNA expression of IRS-1 and GSK3β in the Yuye Decoction group and the metformin group could be up-regulated to varying degrees,and the mRNA expression of IRS-1 and GSK3β could be decreased(P < 0.01).Among them,the high dose group of Yuye Decoction significantly down-regulated IRS-1 mRNA and up-regulated the expression of PI3 K mRNA(P < 0.01),and the low dose group of Yuye Decoction significantly down-regulated the expression of GSK3β mRNA(P < 0.01).3.HepG2 cells treated with 0.25 mM palmitic acid for 24 h significantly increased glucose consumption and significantly decreased glycogen content(P < 0.05),suggesting that IR-HepG2 cell model was successfully established.Different concentrations of Yuye Decoction were used to intervene IR-HepG2 cells.According to glucose consumption and glycogen content,400μg/ m L,200μg/ m L and 100μg/ m L of Yuye Decoction were selected as high,medium and low concentrations for molecular mechanism study.The qRT-PCR results showed that the expression levels of G6 pase mRNA and GSK3β mRNA in IR-HepG2 cells were down-regulated in Yuye Decoction high-concentration group(P <0.05),and the expression levels of IRS-1mRNA and PI3 K mRNA were up-regulated(P <0.05).Western blot showed that: Compared with model group,the protein expressions of AKT,IRS-1 and p-IRS-1 in Yuye Decoction groups were up-regulated(P < 0.05),and the protein expressions of GSK3β and p-GSK3β were down-regulated(P < 0.05),and the protein expressions of GSK3β and p-GSK3β in Yuye Decoction high-concentration group were significantly inhibited(P < 0.01).In addition,the expression of PI3 K and its phosphorylated protein could be up-regulated in the low concentration group of Yuye Decoction(P < 0.05),and the results were similar to the metformin group.Conclusion:1.Yuye Decoction may play a therapeutic role in the treatment of T2 DM by regulating PI3K/AKT signaling pathway.Molecular docking results showed that the core compounds in Yuye Decoction had good binding ability with AKT1 and GSK3β in PI3K/ AKT pathway.2.Yuye Decoction can regulate glucose and lipid metabolism disorder in T2 DM rats,improve insulin sensitivity,improve insulin resistance and protect liver injury.3.Yuye Decoction improved the level of insulin signaling in liver tissue,reduced liver inflammatory response and insulin resistance,and improved T2 DM.The mechanism was achieved by up-regulating the expression of IRS-1,p-PI3 K,p-AKT,AKT protein,and down-regulating the levels of GSK3β protein and mRNA.4.Yuye Decoction can regulate key enzymes in IR-HepG2 cells to promote glycogen synthesis and reduce gluconeogenesis,and improve insulin resistance of PA-induced IR-HepG2 cells by activating PI3K/AKT pathway. |
PDF Full Text Request