| Background Chronic Obstructive Pulmonary Disease(COPD)is the most common chronic airway disease worldwide,characterized by incomplete reversible airway obstruction and high mortality and disability rates.Risk factors for COPD include genetic factors,smoking,and airway inflammation,with smoking being the most significant factor.Meanwhile,studies have shown that iron and ferroptosis play a crucial role in the occurrence and development of COPD,but the specific mechanism is still not fully understood.Currently,the first-line treatment strategy for COPD is still bronchodilators,and all phenotypes of COPD do not significantly benefit from inhaled steroids.Therefore,finding new targets and new drugs for the treatment of COPD airway inflammation is a core problem to be solved.Many natural products derived from plants have shown significant anti-inflammatory activity in respiratory system diseases in vitro and in vivo.Isorhamnetin(ISO)is a natural flavonoid compound isolated from sea buckthorn fruit and ginkgo biloba leaves.The main mechanism of action of ISO is related to antioxidant and inhibition of Reactive oxygen species(ROS)production,which is significantly correlated with ferroptosis.Therefore,exploring the therapeutic or preventive value of isorhamnetin in COPD by inhibiting the ferroptosis pathway is worth further study.Objective(1)Based on bioinformatics analysis,the ferroptosis regulatory network of COPD was revealed to elucidate the relationship between ferroptosis of airway epithelial and the occurrence and development of COPD.(2)Through network pharmacology analysis,the network pharmacological targets and mechanisms of isorhamnetin for preventing COPD were explored.(3)Through cellular and animal experiments,the efficacy of ISO in treating chronic obstructive pulmonary disease was explored,and the role of isorhamnetin in reducing COPD airway inflammation by inhibiting the ferroptosis pathway was elucidated.MethodsPart 1:(1)Based on the Gene Expression Omnibus(GEO)public database,datasets related to "COPD" and "airway epithelial" were screened out.(2)Three COPD airway epithelial datasets were used to identify the differentially expressed genes(DEGs)in COPD airway epithelium.The genes that were differentially expressed in all three datasets were selected as target genes,and the cytohubba plugin in Cytoscape was used to screen 5% of the genes as critical genes.(4)The ferroptosis genes set was obtained from the Ferr Db database.DEGs were compared with the critical genes,and the ferroptosis-related hub genes(FRHGs)of COPD were identified.(5)Bioinformatics methods were used to analyze the FRHGs,including protein-protein interaction network(PPI),mi RNA-m RNA regulatory network,Gene Ontology,and Kyoto Encyclopedia of Genes and Genomes analysis,and genome enrichment analysis.(6)The diagnostic value of FRHGs was validated using an independent validation dataset.(7)By analyzing two independent datasets,it was investigated whether smoking affected the expression of FRHGs and whether smoking cessation could reverse smoking-induced differential expression of FRHGs.Part 2:(1)The two-dimensional molecular structure of isorhamnetin was downloaded from the Pub Chem database and uploaded to the Swiss Target Prediction database and Similarity Ensemble Approach database to collect the isorhamnetin-target gene set.(2)The Venn diagram was used to analyze the overlap between the isorhamnetin-target gene set and the COPD airway epithelial DEGs obtained in Part 1,and the overlapped genes were selected as potential targets for isorhamnetin to counter COPD.(3)Network pharmacology analysis was performed on the potential targets of isorhamnetin to counter COPD,including protein-protein interaction network,Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis,and genome enrichment analysis.(4)Molecular docking experiments were performed to investigate the interaction between isorhamnetin and the potential targets for isorhamnetin to counter COPD obtained in Part 1.Part 3:(1)Cellular experiments: 1)Using BEAS-2B cells to study the harmful effects of CSE,the potential benefits of ISO,and their mechanisms.2)The CCK-8and Annexin V/PI double staining experiments were performed to detect cell viability and apoptosis.3)The changes in MDA and GSH/GSSH contents were determined using a kit.4)Transmission electron microscopy was used to observe the mitochondria of control and experimental cells.5)Immunofluorescence was performed to observe the expression of NRF2,SLC7A11,and GPX4 in the control and experimental groups.(2)Animal experiments: 1)A COPD mouse smoking model was established,and the mice were randomly divided into the CON group,CS group,and CS+ISO group.2)After exposure to cigarette smoke(CS)for 90 days,lung function tests were performed on the mice.3)After the lung function test,blood was collected from the mice for red blood cell counting and hematocrit analysis.4)The m RNA dataset of lung tissue from the smoker mouse model was downloaded from the GEO database,and differential expression genes and their enriched pathways were analyzed.5)Pathological examination and morphological analysis of lung tissue from different groups of mice were performed.6)Western blotting was used to detect the expression levels of GPX4,SLC7A11,and NRF2 in lung tissue.ResultsPart 1:(1)Transcriptomic analysis of three COPD airway epithelial datasets identified 220 DEGs,with 157 upregulated and 63 downregulated.(2)A PPI network with 190 nodes and 265 edges was constructed based on the 220 DEGs,with an average node degree of 2.79.(3)Thirteen key genes were identified using Cytoscape.(4)The Ferr Db database was used to compare key genes,resulting in the screening of7 potential COPD ferroptosis-related hub genes: NQO1,AKR1C3,AKR1C1,GPX2,TXNRD1,SRXN1,and SLC7A11.(5)GO and KEGG enrichment analysis and GSEA analysis revealed that these 7 genes were mainly involved in oxidative stress pathways and regulated by the NRF2 signaling pathway.(6)A mi RNA-m RNA regulation network containing 281 mi RNAs and 359 m RNA-mi RNA pairs was constructed based on the 7 genes.(7)These 7 genes have a significant diagnostic value for COPD smokers.(8)The expression of the 7 ferroptosis related key genes showed an increasing trend from non-smoking healthy individuals,smoking healthy individuals to smoking COPD patients.(9)Even short-term exposure to tobacco smoke can upregulate the expression of these 7 genes,but after smoking cessation,the expression of these genes can be reduced to normal levels.Part 2:(1)195 potential targets of ISO were obtained from the Swiss Target Prediction database and the Similarity ensemble approach.(2)Venn diagram analysis was performed on the 220 DEGs obtained in the first part of the study and the 195 potential targets of ISO,resulting in 11 overlapping targets,which were identified as pharmacological targets for COPD treatment with ISO.(3)The 11 pharmacological targets are primarily involved in biological processes such as oxidation-reduction.(4)A PPI network with 11 nodes and 24 edges,and an average node degree of 4.36,was constructed based on the 11 pharmacological targets.(5)Using Cytoscape,5 key targets were identified.(6)Molecular docking experiments showed that ISO can bind well to the protein structures of the 5 target genes,NRF2,SLC7A11,NQO1,AKR1C3,and AKR1C1.Part 3:(1)A concentration of 0.08% CSE and 40μM of ISO were determined for BEAS-2B cell experiments using CCK8 assays.(2)Under the microscope,the number of cells was significantly reduced in the CSE and Erastin groups,while the use of ISO and Fer-1 significantly improved cell death.(3)The CSE and Erastin groups significantly increased PI-positive cells in BEAS-2B cells,while the use of ISO and Fer-1 significantly reduced the number of PI-positive cells.(4)As compared to the control group,MDA levels were significantly increased in BEAS-2B cells treated with CSE and Erastin,while the use of ISO and Fer-1 significantly inhibited the increase in MDA induced by CSE.In addition,GSH and GSH/GSSG levels decreased while GSSG increased in BEAS-2B cells treated with CSE and Erastin,but both ISO and Fer-1 significantly alleviated CSE-induced changes in GSH,GSSG,and GSH/GSSG and restored them to normal levels.(5)Transmission electron microscopy revealed that mitochondrial morphology in BEAS-2B cells treated with CSE and Erastin changed significantly(mitochondrial shrinkage,increased membrane density,reduced Cristae,and even outer membrane rupture)compared to the control group.However,the use of ISO and Fer-1 significantly improved mitochondrial morphology,which was restored to the level of the control group.(6)Compared with the control group,CS-exposed mice had significantly reduced body weight,increased HCT,and peripheral red blood cell counts.After ISO intervention,the trend of these indicators was reversed.(7)Compared with the control group,mice exposed to CS exhibited significant airflow limitation,lung hyperinflation,and increased quasi-static compliance.After intervention with ISO,a significant decrease in FVC and RI increase and a reduction in the deterioration of FEV0.1/FVC and quasi-static compliance caused by CS exposure were observed.(8)Bioinformatics analysis of the m RNA dataset of lung tissues from smoking mice revealed significant enrichment of differentially expressed genes in the ferroptosis pathway.(9)Compared with the control group,pathological analysis of the lungs of CS-induced COPD mice showed pulmonary emphysema,damage to the alveolar wall,and fusion of alveolar walls.In addition,there were significant inflammatory changes in the lung tissues of the CS group compared with the control group,manifested by thickening of the bronchial wall,disorder of epithelial cells,and infiltration of numerous inflammatory cells.Compared with the CS group,MAA and MLI were increased in the ISO-treated group,which significantly reduced these histopathological injuries.In addition,compared with the CS group,ISO treatment significantly inhibited pulmonary emphysema and inflammatory cell infiltration induced by CS-induced COPD,effectively reducing the pathological score.(10)ISO treatment significantly reversed the decrease in NRF2,SLC7A11,and GPX4 protein expression induced by CS.(11)Compared with the control group,the MDA level in lung tissues of CS group mice was significantly increased,while GSH and GSH/GSSG were decreased and GSSG was increased.ISO treatment significantly reversed this change.(12)The expression of NRF2,SLC7A11,and GPX4 was significantly inhibited in the CSE group and the Erastin group,but increased after treatment with ISO and Fer-1.Conclusion1.Using bioinformatics analysis,220 differentially expressed genes in COPD airway epithelial were revealed.Seven potential COPD ferroptosis-related hub genes with good diagnostic characteristics were further screened,and a mi RNA-m RNA regulatory network was constructed.The seven COPD ferroptosis-related hub genes mainly involve biological processes such as oxidative stress and are regulated by the NRF2 pathway.Smoking can cause high expression of these seven genes,which can be reversed after smoking cessation.2.Network pharmacology was used to predict 11 genes that may be potential pharmacological targets of ISO for COPD treatment.Molecular docking experiments confirmed that ISO can target NRF2,SLC7A11,NQO1,AKR1C3,and AKR1C1.3.Through cell and animal experiments,it was found that ISO has pharmacological activity against airway epithelial ferroptosis,which can alleviate COPD airway inflammation caused by cigarette smoke and further explain the key role of the NRF2/SLC7A11/GPX4 pathway in ISO reducing COPD airway inflammation. |
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