| ObjectiveDiabetic retinopathy(DR)is a serious complication of diabetes mellitus,which nearly happens to all the diabetic sufferers.The aim of this study was to identify the preliminary molecular regulatory mechanisms involved in the efficacy of astragaloside Ⅳ(AS-Ⅳ)in the treatment of DR through a combination of bioinformatics,network pharmacology,molecular docking and experimentation.MethodsSPF male SD rats were injected intraperitoneally with streptozotocin(dose:60 mg/kg)to induce diabetes mellitus.Seventy-two hours after injection,blood samples were collected from the tails of the rats(fasted for 12 hours)to measure glucose levels.Fasting blood glucose levels above 16.7 mmol/l on three consecutive days were considered successful and were used as animal models for subsequent experiments.Optical Coherence Tomography(OCT)and hematoxylin-eosin staining(HE)were used to show the histopathological changes.The main targets of AS-Ⅳ were identified by searching public databases of herbal medicines(GeneCards,PharmMapper and Swiss Target Prediction).In addition,disease targets for DR were obtained by integrating data from the GEO dataset with predictions from public databases.The Protein-Protein Interaction Networks(PPI)network was constructed through Cytoscape and 10 core targets were selected and subjected to Gene Ontology(GO)and the Kyoto Encylopaedia of Genes and Genomes(KEG).Encylopaedia of Genes and Genomes(KEGG)for enrichment analysis.Interactions between AS-Ⅳ and these key genes were analysed using a molecular docking approach.Real time quantitative polymerase chains reaction(RT-q PCR)and Western blot(WB)were used to verify changes in expression of core targets.ResultsCompared to the model group,the AS-Ⅳ treated diabetic rats showed a significant reduction in blood glucose levels and a significant increase in body weight.HE staining showed that AS-Ⅳ administration significantly increased retinal thickness,neatly aligned cells in all layers,edema of nerve fibres,and reduced inflammatory cell infiltration and vacuole formation in diabetic rats.The retinal layer and fundus photographs were observed.Compared with the diabetic group,the total retinal thickness of AS-Ⅳ treated diabetic rats was significantly increased,vitreous opacity and corneal whitening in diabetic rats were alleviated,and the images of fundus vessels in the treated diabetic model rats were clear.Also,significantly reduced neovascularization in diabetic rats appeared disorderly in the fundus of diabetic rats.107 common targets of AS-Ⅳ and DR were determined after intersection.PPI network analysis filtered10 hub genes potentially targeted by AS-Ⅳ,including VEGFA,CASP3,HIF1α,STAT3,CTNNB1,SRC,AKT1,EGFR,IL1β and IL6.Enrichment analysis indicated that these genes were mainly enriched in biological processes like T cell activation,epithelial cell proliferation and protein kinase B signaling,and involved in oxidative stress,apoptosis and inflammation-related pathways.The molecular docking prediction suggested that AS-Ⅳ exhibited stable binding to these core targets.In addition,mRNA levels of core targets in diabetic rats were differentially expressed before and after AS-Ⅳ treatment.Western blot further revealed that AS-Ⅳ treatment elevated DR-depressed protein levels of PI3 K and AKT.ConclusionsOur study elucidated the effect of AS-Ⅳ in attenuating retinopathy induced by diabetes in rats and preliminarily unveiled the therapeutic efficacy of AS-Ⅳ in the treatment of DR might be attributed to activation of PI3K-AKT signaling pathway. |
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