Roles And Mechanisms Of Ferroptosis In Asthmatic Airway Remodeling

BackgroundAsthma is a chronic disease of the respiratory system associated with varying degrees of airway inflammation,airway hyper-responsiveness,reversible airflow limitation and airway remodeling.As one of the major pathological features of asthma,airway remodeling may contribute to irreversible airflow limitation and progressive lung function decline,making asthma symptoms poorly controlled,which ultimately exacerbates the condition.Transforming growth factor β1(TGF-β1)is an important initiator and process factor in airway remodeling of asthma.Ferroptosis is a newly identified iron-dependent form of cell death.Intracellular disorders of iron homeostasis and imbalances in the antioxidant system involving regulatory factors such as glutathione peroxidase 4(GPX4)contribute to the accumulation of reactive oxygen species(ROS)and the lipid per-oxidation metabolite malondialdehyde(MDA),which ultimately triggers ferroptosis.In recent years,a growing body of evidence has demonstrated the intimate association of ferroptosis with the pathogenesis of a variety of clinical conditions,however,the contribution of ferroptosis to airway remodeling in asthma has rarely been reported.Increased quantities of iron-loaded cells in bronchoalveolar lavage fluid in patients with mild to moderate or severe asthma have been shown to be correlated with lower one-second rates(FEV1/FVC),suggesting that fixed airflow limitation in asthmatic individuals might be tightly linked to high iron content reserves and ferroptosis.Thus,this research will provide additional insight into the connection between ferroptosis and asthma in the perspective of airway remodeling and explore the possible therapeutic value of ferroptosis in asthma airway remodeling.ObjectivesTo investigate whether iron death is engaged in the process of airway remodeling in asthma and the underlying mechanisms of the links between them,and to provide new directions in the treatment of airway remodeling.Methods1.Transcriptomic data from the GSE6858 dataset were used to enrich the KEGG pathway for differentially expressed genes between the asthma and control groups,and to analysis the expression levels of the ferroptosis-related gene GPX4 between the two groups.2.Ovalbumin sensitization and aerosol excitation were performed to establish a chronic asthma mouse model.The iron concentration was measured by iron detection kit and Prussian blue staining,and the protein expression of GPX4 was evaluated by western blot and IHC.MDA assay kit for assessing lipid per-oxidation levels and TEM to observe ultra-structural changes in the airway epithelium.MASSON staining was used to observe the level of airway remodeling.3.Following intraperitoneal administration of Ferrostatin-1,an ferroptosis inhibitor,in the mouse model,lung tissue was stained with HE,PAS and MASSON to evaluate airway inflammation,mucus hyper-secretion and collagen deposition,as well as ELISA to detect serum IgE levels,and tissue IF with Western Blot to detect GPX4 protein expression levels and indicators of airway remodeling.4.The levels of GPX4 were measured by Western Blot and qRT-PCR after stimulation with TGF-β1 at different time and concentration gradients in human bronchial cell line BEAS-2B.Under different concentrations of TGF-β1,intracellular ROS levels were measured by flow cytometry(FCM),intracellular MDA levels were determined by MDA assay kit,cell viability was detected by CCK8,cell proliferation capacity was tested by EdU proliferation assay,and indicators of airway remodeling were examined by qRT-PCR.5.After Ferrostatin-1 was applied to BEAS-2B,ROS,MDA,cell viability and cell proliferation were examined,and the protein expression levels of indicators of airway remodeling were measured by Western Blot.6.Ferroptosis PCR arrays were conducted to examine changes in ferroptosis-related mRNA levels under TGF-β1 stimulation,and Western Blot to examine the expression levels of ferritinophagy-related proteins in response to TGF-β1 activation.Results1.Bioinformatics analysis indicated that the ferroptosis pathway was enriched in the asthma group and the GPX4 expression level was significantly lower in the asthma group than in the normal one,suggesting that ferroptosis may be involved in asthma illness.2.In the chronic asthma model,collagen deposition was accompanied by a series of ferroptosis features such as increased iron concentration,decreased GPX4 expression,MDA accumulation and ferroptosis-like ultra-structural changes in the airway epithelium,suggesting that asthma airway remodeling is intimately linked with ferroptosis.3.Treatment with Ferrostatin-1 in asthma models showed a reduction in asthma airway inflammation,mucus hyper-secretion and collagen deposition,as well as a decrease in serum IgE levels and an elevation in GPX4 expression,with changes in N-cadherin,E-cadherin,α-SMA and Fibronectin airway remodeling indicators being suppressed.These results suggest that inhibition of ferroptosis may alleviate asthma airway inflammation and airway remodeling.4.In TGF-β1-activated BEAS-2B cells,both mRNA or protein expression levels of GPX4 were decreased,with increased intracellular ROS,higher MDA concentrations and reduced cell viability and proliferation capacity,accompanied by changes in airway remodeling indicators.It is suggested that the pathway by which ferroptosis plays a role in asthma airway remodeling may be through TGF-β1.5.After treatment with Ferrostatin-1 in BEAS-2B cells,TGF-β1-induced ferroptosis-related alterations were inhibited and the protein expression levels of N-cadherin,Fibronectin and Collagen I,all indicators of airway remodeling,were reduced.This demonstrates that Ferrostatin-1 inhibits airway remodeling in asthma by suppressing TGF-β1-induced ferroptosis.6.Following the application of TGF-β1 on bronchial epithelial cells,the mRNA levels of transferrin(TF),transferrin receptor 2(TFR2)and nuclear receptor coactivator 4(NCOA4)were increased,and the ferritin expression level decreased progressively after a feedback increase,indicating that TGF-β1 induced ferroptosis in epithelial cells by promoting iron uptake and ferritinophagy.Conclusion1.Ferroptosis participates in the pathogenesis of asthma airway remodeling,inhibiting ferroptosis attenuates airway remodeling in both in vivo and in vitro asthma models.2.TGF-β1 triggers ferroptosis in bronchial epithelial cells by facilitating iron uptake and ferritinophagy to participate in the airway remodeling process.