HDM Induces Airway Epithelial Cell Ferroptosis And Promotes Inflammation By Activating Ferritinophagy In Asthma

Background and purposeAsthma is a disease characterized by airway epithelial barrier destruction,chronic airway inflammation and airway remodeling.Repeated damage to airway epithelial cells by allergens in the environment plays an important role in the pathophysiology of asthma.At present,the clinical treatment methods and means for asthma are limited,and finding new treatment options has always been a difficult clinical challenge.Ferroptosis is a novel form of regulated cell death(RCD)mediated by lipid peroxidation in association with free iron-mediated Fenton reactions.Studies have shown that ferroptosis is involved in the occurrence and development of various diseases,but the relationship between ferroptosis and asthma has been rarely reported,and whether inhibiting ferroptosis will affect the occurrence and development of asthma remains to be studied.Therefore,in this study,we explored whether ferroptosis occurs in airway epithelial cells in a house dust mite(HDM)-induced asthma model through in vitro and in vivo experiments,and if so,what is the mechanism of this process,and whether inhibition of ferroptosis improves asthma symptoms.Experimental Approach1.In vitro experiment:Human bronchial epithelial cell line was used and cultured with KM medium.The cells were treated with different reagents according to the experimental requirements,and the expression of ferroptosis-related proteins such as GPX4,FTH,HO-1,and Nrf2 was detected by western blotting,and the cell-related indicators were detected by functional experiments such as fluorescent probes and CCK8.2.In vivo experiment:BALB/c mice were randomly divided into 4 groups:(1)control group;(2)HDM group;(3)HDM+DFO group;(4)HDM+Fer-1 group.The HDM group,HDM+DFO group,HDM+Fer-1 group were sensitized on days 0 and 7 by intraperitoneal injection of 0.2 ml of normal saline solution containing HDM.Then,the mice were intranasally challenged with 0.02 ml of normal saline solution containing HDM every other day for 3 weeks,and the control group received the same volume of normal saline.The HDM+DFO group and HDM+Fer-1 group were intraperitoneally injected with ferroptosis inhibitors DFO(100 mg/kg)and Fer-1(20 mg/kg)1 hour before each HDM challenge.All mice were sacrificed on day 28,and lung tissue and bronchoalveolar lavage fluid were collected.The infiltration of airway inflammatory cells was detected by H&E staining,the expression of GPX4,TFH and other proteins was detected by western blot,and the inflammation was analyzed by cell count in bronchoalveolar lavage fluid and Elisa assay.Result1.In vivo and in vitro experiments,HDM induces ferroptosis in airway epithelial cells.In the cell model,the use of HDM to stimulate cells can reduce the expression of GPX4,cause lipid peroxidation in the cells,and change the morphology of mitochondria,which is aggravated by knockdown of GPX4.These changes were improved after administration of the ferroptosis inhibitors DFO and Fer-1.In the HDMinduced mouse asthma model,the reduction of GPX4 expression in airway epithelial cells was also detected,and the death of airway epithelial cells in HDM mice could be alleviated by DFO and Fer-1.2.DFO and Fer-1 inhibited the inflammatory response induced by HDM.We detected HDM by H&E staining,cell count,and Elisa detection of related inflammatory factors.The airway inflammation of the mice in the group was severe,and the specific manifestations were a large number of inflammatory cells infiltration around the airway,an increase in the number of total cells and eosinophils in the lavage fluid,and the type 2 inflammatory factors IL-4,IL-5,IL-13 levels increased,while these changes were alleviated in the HDM+DFO and HDM+Fer-1 groups.These results indicate that inhibition of ferroptosis can alleviate HDM-induced T2-type inflammatory response to a certain extent.3.HDM increases cellular iron levels and aggravates oxidative stress.We found higher iron levels and more severe lipid peroxidation in lung tissue of HDM mice.In cell experiments,HDM can increase the level of cellular iron ions and aggravate oxidative stress.After administration of ferrous ammonium sulfate to cells,the experimental results are consistent with HDM,indicating that HDM induces an increase in the level of cellular free iron ions and promotes oxidative stress.4.HDM activates NCOA4-mediated ferritinophagy.Through co-immunoprecipitation experiments,we found that HDM can make the cell NCOA4 have a higher degree of binding to FTH,and degrade FTH to release iron ions.Knocking down NCOA4 can restore the iron ion level to normal.Likewise,inhibition of autophagy resulted in reduced FTH degradation and reduced iron levels.It was shown that HDM can activate NCOA4-mediated ferritinophagy to degrade ferritin and release iron ions.