Study On The Protective Effect And Mechanism Of Puerarin On Diabetes Based On Network Pharmacology And Metabolomics

Diabetes(DM)is a chronic metabolic disease mainly characterized by hyperglycemia.At present,the number of patients with DM is increasing year by year,which has caused widespread concern around the world.Therefore,finding early diagnostic markers and therapeutic targets for diseases is of great significance for the prevention and treatment of DM.Pueraria is the dried root of wild kudzu or dried kudzu.It is often used clinically to treat symptoms such as fever,yin deficiency and thirst,spleen deficiency and diarrhea.Puerarin is the active ingredient with the highest content in kudzu root.Puerarin has better effects in improving insulin resistance and eliminating inflammation.Therefore,this study intends to use the network pharmacology technology to construct a "drug-disease-target" network relationship diagram to obtain the relevant targets and pathways of puerarin on DM,followed by the injection of STZ to induce a DM mouse model,with metformin as a positive In contrast,different doses of puerarin were used as protective drugs,and the model was verified based on physiological and biochemical indicators combined with histomorphology,and the protective effect of puerarin on DM was discussed.Using metabolomics technology to analyze metabolites in the serum of the control group,the diabetes group(DM),the metformin group(Met)and the puerarin medium-dose group(Pue-M)to find potential biomarkers that trigger DM To explore the possible mechanism of puerarin protection of DM.Combined with Western blot and RT-PCR technology to verify the possible mechanism of puerarin protection of DM.The IR-HepG2 cell model was constructed to further verify the mechanism of puerarin protection of DM at the cellular level.To provide data reference for clinical diagnosis and treatment of DM.The main research contents are as follows:1.Mechanism of Puerarin in the Treatment of Diabetes Based on NetworkPharmacologyA total of 132 potential action targets for puerarin and 4,545 targets for DM were screened through the database.Network visualization and PPI node analysis of puerarin and DM interaction targets were found and found that AKT1 and VEGFA are core nodes connecting other nodes.Perform GO function annotation and KEGG pathway enrichment analysis on the involved nodes,and obtain high-scoring signal pathways such as PI3K-AKT,MAPK,TNF,etc.Among the relevant protein genes involved are PI3K,AKT,NF-?B,mTOR,etc.2.Construction of a Mouse Model of Diabetes and the Protective Effect of PuerarinOne week after the adaptive feeding of 60 SPF male mice,the DM mouse model was induced by intraperitoneal injection of STZ,and the physiological and biochemical indicators and histomorphologically verified models were successfully constructed and the protective effects of puerarin were explored.The results showed that compared with the Control group,the blood glucose of the DM group was significantly increased(p<0.01),the drinking water,diet and urine increased,and the body weight continued to decrease,which was in line with the clinical characteristics of diabetes.Mice's tolerance to glucose decreased significantly(p<0.001),and insulin content and insulin resistance coefficient increased significantly(p<0.01).Biochemical index results showed that compared with the Control group,TG,TC,and LDL in the DM group were significantly increased(p<0.01 or p<0.001),and HDL was significantly decreased(p<0.01);the oxidation index results showed that the DM group mice MDA content increased(p<0.01),SOD activity decreased.After oral administration of metformin and puerarin,the symptoms of DM mice improved,body weight became normal,blood glucose decreased,glucose tolerance improved,insulin resistance coefficient decreased significantly(p<0.001),serum tetralipid significantly improved compared with DM group,and MDA in liver The content decreased significantly(p<0.05),and SOD activity increased.Histomorphological results showed that liver tissues of the control group were intact,cells were arranged neatly,and no inflammation infiltration was observed.In the pancreas,the number of islets was large,the edges were smooth,and the cytoplasm was rich.Fat cells in the liver of mice in the DM group appeared fatty vacuoles and were mostly infiltrated with inflammation;in the pancreas,the islets were irregular in shape and the edges were blurred,the number of islet cells was small,and the cytoplasm was lost.After metformin and puerarin protection,the inflammation and infiltration of hepatocytes decreased;the islet morphology in the pancreas tended to be normal,the number of islets increased,and vacuoles were rare.The above results show that STZ-induced DM mice have glucose and lipid metabolism disorders,produce oxidative stress,and cause damage to the liver and pancreas.Metformin and puerarin have a certain protective effect on DM mice.optimal.3.Protective effect of puerarin on diabetes based on metabonomicsUPLC-Q-TOF was used to analyze the metabolites in the serum of the control group,DM group,Met group,and Pue-M group.The results showed that the serum samples of each group were completely separated,and the degree of aggregation in the group was better..Analysis of metabolic pathways for differential metabolites.The main metabolic pathways involved are the glycerophospholipid metabolic pathway and the mutual conversion of pentose and glucuronic acid.The analysis of the main metabolites indicates that the occurrence of DM and the protective effect of puerarin on DM may be related to Glycolipid metabolism is closely related to inflammatory factors.4.Protective effect of puerarin on diabetes based on AMPK/mTOR and PPARy/NF-?B signaling pathwayCompared with the Control group,the DMK mouse AMPK and PPARy protein and mRNA expressions were down-regulated(p<0.01 or p<0.001),the mTOR and NF-?B protein and mRNA expressions were up-regulated(p<0.01 or p<0.001),metformin and kudzu root After the protection of serotonin,the expression levels of the above proteins tended to be normal.It is suggested that puerarin can inhibit mTOR expression by activating AMPK,improve insulin signal transduction and glucose and lipid metabolism;in addition,puerarin can inhibit NF-?B activity by activating PPARy,and inhibit inflammatory response in DM mice,thereby exerting its effect on DM mice.Protective effect of DM mice.5.Protective effect of puerarin on diabetes by constructing IR-HepG2 cell modelIn order to further evaluate the effects and mechanisms of puerarin on DM and insulin resistance,in vitro studies were conducted to induce IR-HepG2 cells with high concentration of insulin,to observe the effects of puerarin on IR-HepG2 cells,and to explore its molecular mechanism.The results showed that compared with the Control group,high concentration of insulin would affect the consumption of glucose in HepG2 cells,causing the insulin,TG,ROS and MDA content in the cells to increase(p<0.01 or p<0.001),and the SOD activity decreased;the cells were destroyed Structure that accelerates apoptosis.After metformin and puerarin protection,IR-HepG2 cells increased glucose consumption,decreased insulin and TG content,improved oxidation indexes,improved cell morphology,and decreased apoptosis rate.In addition,compared with the Control group,the AMPK and PPARy protein and mRNA expressions were down-regulated(p<0.05 or p<0.01)and the mTOR and NF-?B protein and mRNA expressions were up-regulated(p<0.05 or p<0.01 or p<0.001);Compared with IR group,protein and mRNA expressions in Met group and puerarin dose groups tended to be normal.The above results suggest that puerarin can improve insulin resistance in IR-HepG2 cells at physiological and biochemical levels;and further verified at the cellular level that puerarin can improve AMPK/mTOR and PPAR?/NF-?B signaling pathways,thereby improving liver glucose and lipid metabolism Abnormalities and insulin resistance reduce inflammation damage.