Study On The Mechanism Of Yuye Decoction In Preventing And Treating Pancreatic Islet β-cell Dysfunction And Apoptosis Based On Network Pharmacology And Molecular Docking

Objective:(1)In this study,the optimal extraction process of Yuye Decoction was determined through orthogonal experiments,and the quality control and comprehensive scoring of Yuye Decoction water extracts were carried out by combining one test with multiple evaluations and information entropy methods.(2)The key targets and core compounds of Yuye Decoction in the prevention and treatment of type 2 diabetes mellitus(T2DM)were predicted by network pharmacology and molecular docking technology.(3)The mouse insulinoma cells(Min6)were induced by high glucose palmitic acid to establish an in vitro model of isletβ-cell dysfunction and apoptosis,and to verify the protective effect and mechanism of Yuye Decoction on isletβ-cell dysfunction.Methods:(1)Investigation of extraction process:In this study,the ratio of solid to liquid,extraction time and extraction times were used as the investigation factors,and the yield of dry paste,total saponin content,puerarin content,mangiferin content and verbascoside glucoside content were used as evaluation indicators.Combined with the information entropy method,the water extract of Yuye Decoction was comprehensively scored,and the L9(3~3)orthogonal test table was established to optimize the optimal extraction process of Yuye Decoction.(2)Predicting the core targets and core compounds of Yuye Decoction in the prevention and treatment of T2DM based on network pharmacology and molecular docking technology:The principal components of Yuye Decoction water extracts were identified by UPLC-MS/MS technology;the Pharmmapper database was used to predict the chemical constituents.Targets of action;CTD and Genecards databases were used to search the gene targets of T2DM,and finally the core targets of Yuye Decoction for prevention and treatment of T2DM were obtained;KEGG and GO enrichment analysis was performed,and the protein interaction network was drawn using String data and Cytoscape software;Cytoscape software draws component-target and component-target-network diagrams respectively;the selected core components are molecularly docked with the core targets,and finally the main active compounds of Fufang Yuye Decoction in the prevention and treatment of T2DM and their targets are obtained.(3)High glucose and palmitic acid were used to induce mouse islet tumor cells(Min6)to establish an in vitro model of pancreatic isletβ-cell dysfunction in T2DM.Cell morphology was observed and cell viability was detected by CCK8;TUNEL staining was used to detect the positive rate of cell apoptosis;Western-blot assay was used to detect cells Bax,Bcl-2,p53,caspase 3,PI3K,AKT,p-AKT,Foxo1,p-The protein expression level of Foxo1;q RT-PCR detected the expression of metastasis associated in lung denocarcinoma transcript 1(MALAT1)at the cellular m RNA level.Results:(1)The optimal extraction process of Yuye Decoction is to soak in 10 times the amount of water for 90 minutes,and extract twice for 2.5 hours each time.(2)The results of network pharmacology indicated that Yuye Decoction exerted its preventive effect on T2DM through 350 potential targets,which were enriched in 152 signaling pathways and involved in multiple biological functions.The top 10 core targets are SRC,MAPK1,PIK3R1,AKT1,HRAS,HSP90AA1,GRB2,PTPN11,RXRA and RHOA,and the core compounds are gomisin G,luteolin,γ-Linolenic acid,schisandrin B and isoliquiritigenin have a high degree of connection with the core target,which may play an important role in the prevention and treatment of T2DM.In addition,this study performed molecular docking analysis on the four potential targets of PIK3R1,AKT1,Foxo1 and PPARG,and the prediction results showed that the highest binding scores to PIK3R1 were monolinolein,glycerol linolenic acid,andα-linolenic acid;the binding scores to AKT1The highest ones are pelargonidin-3-O-glucoside,acacetin,andα-linolenic acid;the highest binding scores to Foxo1 are mangiferin,timosaponin A-Ⅲ,isovitexin;the highest binding scores to PPARG The ones are pelargonidin-3-O-glucoside,monolinolein,glyceryl linolenic acid.(3)In vitro cell experiments showed that:Yuye Decoction can significantly inhibit the apoptosis of isletβcells induced by high glucose palmitic acid,reduce the protein expressions of Bax caspase3,p53 and Foxo,and up-regulate Bcl-2,PI3K,AKT,p-AKT and p-Foxo protein expression,and significantly decreased lnc MALAT1 m RNA expression.Conclusion:Yuye decoction water extract can exert its protective effect on isletβ-cell apoptosis by regulating PI3K/AKT/Foxo signaling pathway and inhibiting the expression of lnc MALAT1,and the core components of this effect may be gomisin G,luteolin,gamma-linolenic acid,schisandrin B and isoliquiritigenin.