| ObjectiveIn this study,network pharmacology and molecular docking methods were used to investigate the main mechanism and regulatory targets of guava leaf,a traditional Chinese medicine used for treating type 2 diabetes mellitus(T2DM).To further elucidate the regulation of guava leaf on the hepatic PI3K/AKT insulin signaling pathway and its downstream gluconeogenesis pathway,utilized db/db mice with spontaneous T2DM were used as animal models to experiment in vivo.The purpose of this study was to comprehensively verify the hypoglycemic efficacy of guava leaf,and to provide experimental basis for its future clinical application.Methods1.Network pharmacology and molecular docking research.The main active ingredients contained in guava leaf raw medicinal materials were detected by high performance liquid chromatography,and their three-dimensional chemical structures were searched in the PubChem database and imported into the SwissTargetPrediction online analysis platform to predict their targets.Using "Type 2 diabetes mellitus" and "T2DM" as keywords to search for human disease-related genes in the GeneCards database,after removing duplicate values,the relevant target genes of human T2DM were obtained.Then the above results were standardized using the UniProt database.The intersection of the predicted targets of guava leaf and human T2DM disease-related genes was taken as the potential targets of guava leaf for treating T2DM,and the above potential targets were analyzed by using the String platform.Select the core target according to the Degree value in the network and visualize the"Tranditional Chinese Medicine-ingredient-target-disease" network using Cytoscape.Furthermore,performed enrichment analysis on potential targets of guava leaf using Gene Ontology(GO)and Kyoto Gene and Genome Encyclopedia(KEGG)through the Metascape online analysis platform,and conducts molecular docking verification on the core targets related to important metabolic pathways.2.Animal experiment research.A randomized controlled experiment was designed,a total of 24 6-week-old male db/db mice with spontaneous T2DM and 6 male normal db/m mice of the same age were included.After 1 week of adaptive feeding,6 db/m mice were set as the normal group(NC),and random blood glucose in different dates>11.1 mmol/L detected in 24 db/db mice was considered successful in T2DM modeling.db/db mice were randomly divided into model group(DM),guava leaf extracts high-dose group(GL-H),middle-dose group(GL-M)and low-dose group(GL-L)according to body weight and fasting blood glucose,with 6 mice in each group.After calculation,the high,medium,and low intervention doses of guava leaf extracts were respectively 1000 mg/kg/d(equivalent to 7.8 g/kg/d dose of the original medicinal materials),500 mg/kg/d(equivalent to 3.9 g/kg/d dose of the original medicinal materials),and 250 mg/kg/d(equivalent to 1.95 g/kg/d dose of the original medicinal materials),and mice in each intervention group were given 0.1ml/20g body weight of different doses of suspension liquid to gavage,and the NC group and DM group were gavaged with equal volume of deionized water.The experiment lasted for 4 weeks.During the experiment period,the fasting blood glucose and body weight of the mice were monitored every 2 weeks,and an oral glucose tolerance test(OGTT)was performed on the 4th week of drug intervention.After the end of the experiment,the blood of mice was taken on an empty stomach by removing eyes,then the blood was used to detect the biochemical indicators related to glucose and lipid metabolism,and the insulin resistance index was calculated.The expressions of PI3K/AKT signaling pathway related proteins IRS2,PI3Kp85α,AKT,p-AKT,and its downstream hepatic gluconeogenesisrelated proteins FOXO1,p-FOXO1,G6Pase and PEPCK were detected in liver tissues by Western Blot.Results1.Network pharmacology and molecular docking research results.(1)Six active components were detected in guava leaf by high performance liquid chromatography,namely:catechin,ursolic acid,gallic acid,guava glycosides,agaricin,and quercetin.(2)After sieved,a total of 20 potential core targets for the treatment of T2DM in guava leaf were obtained,ranked in descending order of degree value as TNF,AKT1,ALB,IL6,PPARG,EGFR,PTGS2,PPARA,ESR1,CAT,MMP9,KDR,IL2,PTPN1,CNR1,INSR,AKR1B1,SLC6A4,PRKCB,DPP4.(3)GO enrichment analysis showed that the potential targets of guava leaf may be involved in the activation of protein kinases,and may have a positive regulatory effect on phosphorylation.(4)According to KEGG enrichment analysis,in addition to cancer pathways,the pathway with the most enriched potential targets of guava leaf was the PI3K/AKT signaling pathway.(5)The results of molecular docking showed that the six active ingredients in guava leaf could freely bind to the core target proteins INSR,AKT1 and AKT2 on the PI3K/AKT pathway,and the binding affinity of ursolic acid to INSR and AKT1 proteins both are the highest among all ingredients,while quercetin has the strongest binding activity with AKT2.In addition,all components had higher binding affinity to AKT2 protein than AKT1.2.Animal experiment research results.(1)General situation:compared with the mice in the NC group,the mice in the DM group and each dose of guava leaf extracts intervention group were obese,dull in color,poor in spirit,unresponsive,slow in action,and smaller than those in the NC group,these mice obviously drink more,eat more,and urinate more.At 2 weeks of drug intervention,compared with the mice in the DM group,the body weight of the mice in the GLH and GL-L groups decreased(P<0.05);at the 4th week of intervention,compared with the DM group,the body weight of the mice in the GL-H group decreased(P<0.05).(2)Changes in blood glucose:after 2 weeks and 4 weeks of drug intervention,compared with the mice in the DM group,the fasting blood glucose of the mice in each dose of guava leaf extracts intervention group decreased(P<0.01).The area under the OGTT curve of mice in each dose of guava leaf intervention group were lower than that of DM group(P<0.05),and the area of mice in GL-L group was significantly lower(P<0.01).(3)Detection of serum biochemical indicators:compared with the DM group,TC,LDL,and FFA in the serum of mice in the GLH group were significantly reduced(P<0.01),and FFA in the mice in the GL-M group decreased(P<0.05),TC was significantly reduced(P<0.01),and the four detection results of TC,TG,LDL,and FFA in the serum of mice in the GL-L group were significantly reduced(P<0.01).(4)Evaluation of insulin sensitivity:compared with the DM group,the insulin levels and HOMA-IR index of the mice in each dose group of guava leaf extracts were significantly reduced(P<0.01).(5)Western Blot detection:compared with the DM group,the expression of IRS2 in the liver of mice in GL-H(P<0.05),GL-M(P<0.01)and GL-L(P<0.01)groups was increased to varying degrees,and the expression of p-AKT in the mice in each dose of guava leaf extracts intervention group increased(P<0.05);while there was no statistical difference in the expression of PI3Kp85α and AKT protein in each group(P>0.05),the pAKT/AKT ratio of mice in GL-L group was increased significantly(P<0.01).Compared with the DM group,the expressions of G6Pase in the liver of mice in the GL-H group decreased(P<0.05)while the expressions of p-FOXO1 increased(P<0.05).The expression level of pFOXO1 in the liver of mice in the GL-M group was not significantly different from that in the DM group(P>0.05),but the expression levels of FOXO1,G6Pase and PEPCK were significantly reduced(P<0.01);The expression levels of FOXO1,G6Pase and PEPCK in the mouse liver were significantly lower than those in the model group(P<0.01),but the expressions of p-FOXO1 increased(P<0.05),In addition,the ratio of p-FOXO1/FOXO1 in mice in each dose group of guava leaf extracts were significantly higher than that in DM group(P<0.01).Conclusion1.Guava leaves can reduce the body weight and blood glucose of db/db mice to a certain extent,and improve insulin resistance and lipid metabolism disorders.2.Guava leaves can activate the PI3K/AKT/FOXO1 signaling pathway in the liver,increase the phosphorylation level of key proteins AKT and FOXO1,and inhibit liver gluconeogenesis,thereby reducing blood glucose in T2DM. |
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