调控铁死亡防治心肌缺血再灌注损伤的关键基因及潜在中药化合物筛选OA

Objective To screen key genes and potential herbal compounds regulating ferroptosis for the prevention and treatment of myocardial ischemia-reperfusion injury(MIRI)based on network pharmacology,molecular docking technology,and in vitro cellular experiments.Methods(1)Ferroptosis-related genes were downloaded from the FerrDb V2 database,and MIRI-related targets were obtained from the GeneCards and OMIM databases.The intersection of these gene sets was taken to identify potential targets regulating ferroptosis for ameliorating MIRI.The intersecting genes were imported into the STRING platform to construct a protein-protein interaction(PPI)network of potential targets and screen core targets.The HERB database was used for reverse screening of potential herbal compounds regulating ferroptosis for MIRI prevention and treatment.A target-compound network was established using Cytoscape 3.9.1 software to screen core compounds.Molecular docking simulations between core targets and core compounds were performed using AutoDock software to identify potential effective compounds.(2)HL-1 cardiomyocytes were injured by hypoxia/reoxygenation(H/R).Cells were treated with different concentrations of celastrol(0.25,0.5,1.0 μmol·L-1),different concentrations of dioscin(0.25,0.5,1.0 μmol·L-1),and different concentrations of ursolic acid(20,40,80 μmol·L-1).After 6 hours of hypoxia and 2 hours of reoxygenation,relevant indicators were assessed.Cell viability was detected using the CCK-8 method.Lactate dehydrogenase(LDH)content in the cell culture supernatant was measured by ELISA.Reactive oxygen species(ROS)content and mitochondrial membrane potential(∆Ψm)were detected using a live-cell imaging system.The expression levels of glutathione peroxidase 4(GPX4)and transferrin(TF)were detected by immunofluorescence staining.Results(1)A total of 128 common targets were identified as potential targets regulating ferroptosis for ameliorating MIRI.Core targets included TP53,IL6,HIF1A,IL1B,STAT3,ALB,JUN,mTOR,PTEN,and SRC.Through reverse screening using the HERB database,470 potential herbal compounds regulating ferroptosis for MIRI prevention and treatment were identified.Core compounds included resveratrol,curcumin,dioscin,ursolic acid,honokiol,and celastrol.Ursolic acid,celastrol,withaferin A,triptolide,and dioscin showed stable binding to the core targets.Based on the molecular docking results,three herbal compounds with high docking activity—celastrol,dioscin,and ursolic acid—were selected for in vitro experimental validation.(2)Compared with the normal group,the model group showed significantly decreased viability of HL-1 cardiomyocytes(P＜0.01),significantly increased LDH leakage(P＜0.01),significantly increased cellular ROS level(P＜0.01),significantly decreased ΔΨm(P＜0.01),significantly downregulated GPX4 protein expression(P＜0.01),and significantly upregulated TF protein expression(P＜0.01).Compared with the model group,the 0.25 μmol·L-1 celastrol group,the 0.25-1.0 μmol·L-1 dioscin groups,and the 80 μmol·L-1 ursolic acid group showed significantly increased viability of HL-1 cardiomyocytes(P＜0.05,P＜0.01);the 0.25-1.0 μmol·L-1 dioscin groups showed significantly decreased LDH leakage(P＜0.05,P＜0.01);the 1.0 μmol·L-1 dioscin group showed significantly decreased ROS level(P＜0.01),significantly increased ΔΨm(P＜0.01),significantly upregulated GPX4 protein expression(P＜0.01),and significantly downregulated TF protein expression(P＜0.01).Conclusion Celastrol,dioscin,and ursolic acid may serve as potential herbal compounds regulating ferroptosis for the prevention and treatment of MIRI.Dioscin ameliorates H/R-induced injury in HL-1 cardiomyocytes by regulating the expression of ferroptosis-related proteins GPX4 and TF,alleviating oxidative stress and mitochondrial damage,and inhibiting ferroptosis.