In total, 20 female (6-8 week old, 20±2 g), specified-pathogen free BALB/c mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were kept under laboratory conditions (22˚C; 50-60% relative humidity; air circulation; 12-h light-dark cycle with free access to water and food). The experimental procedures were approved by the Ethics Committee of Animal Experiments of Fudan University (authorization no. 2020-10-HSYY-WY-01; Shanghai, China). Mice were randomized to two groups (10 mice/group): The normal control and HDM-induced asthma model groups. The asthma model was prepared as previously described (19,20) and was modified according to pre-experimental results. On day 0, HDM extract (10 µg/40 µl) was administered intranasally after anesthetization with 2-2.5% isoflurane in air. From days 7-11, the mice were challenged daily by pipetting 40 µl diluted HDM extract (20 µg/40 µl per mouse) directly into the nostrils under mild anesthesia (Fig. 1A). For mice in the normal control group, the same volume of PBS was administered intranasally at the exact time as aforementioned.

In the present study, AHR levels, leukocyte differential counts in bronchoalveolar lavage fluid (BALF), inflammatory cytokines and histology were assessed to determine whether the asthma model was successfully established. Mice with outliers (One mouse in the control group was removed and two mice in the model group were removed) (21,22), including abnormal values of airway resistance (R_L), hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining and leukocyte differential counts in BALF, were considered to have failed the modeling process, and all the values were removed from the subsequent statistical analysis.

Methacholine (Mch) and pentobarbital sodium were purchased from Sigma-Aldrich (Merck KGaA). HDM extract (Dermatophagoides pteronyssinus) was purchased from Greer Laboratories, Inc. The mouse lung dissociation kit was purchased from Miltenyi Biotec, Inc. Live eFluor780 (cat. no. 65-0865-14; 1:200 dilution) solution and anti-mouse CD45 eFluor 506 (cat. no. 69-0451-82; 1:100 dilution) antibodies were purchased from Thermo Fisher Scientific, Inc. Anti-mouse CD11b BV711 (cat. no. 563168, 1:100 dilution) and anti-mouse SiglecF BV421 (cat. no. 562681, 1:100 dilution) antibodies were purchased from BD Pharmingen (BD Biosciences). Anti-mouse Ly6C PE (cat. no. 128007, 1:200 dilution) antibodies were purchased from BioLegend, Inc. Anti-GPX4 (cat. no. Ab125066; 1:1,000 dilution) and anti-15 Lipoxygenase 1 (15-LO1; cat. no. Ab244205; 1:1,000 dilution) antibodies were purchased from Abcam, and anti-acyl-CoA synthetase long-chain family member 4 (ACSL4; cat. no. NBP2-16401; 1:1,000 dilution) and anti-catalytic subunit solute carrier family 7 member 11 (SLC7A11; cat. no. NB300-318; 1:1,000 dilution) antibodies were purchased from Bio-Techne. The GSH assay kit (cat. no. A006-2) was purchased from Nanjing Jiancheng Bioengineering Institute and ROS was measured using dihydroethydium (DHE; Sigma-Aldrich; Merck KGaA) oxidation. IgE [cat. no. 70-EK275-96; Multi Sciences (Lianke) Biotech, Co., Ltd.], IL-5 [cat. no. 70-EK205-96; Multi Sciences (Lianke) Biotech, Co., Ltd.] and IL-13 ELISA kits [cat. no. 70-EK213/2-96; Multi Sciences (Lianke) Biotech, Co., Ltd.] were purchased from Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd. RIPA lysis buffer was purchased from Beyotime Institute of Biotechnology. The BCA protein test kit was purchased from Thermo Fisher Scientific, Inc. and the Immobilon Western Chemiluminescent HRP Substrate was purchased from Merck KGaA.

Within 24 h after the final HDM challenge, AHR presented as R_L and dynamic lung compliance (Cdyn) to Mch were measured according to the manufacturer's protocols (DSI Buxco^® FinePointe Resistance and Compliance system; Data Sciences International; Harvard Bioscience, Inc.). Mice were weighed, intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg), incised and intubated via the trachea. Afterwards, mice were placed into the chamber of the Resistance and Compliance system at room temperature and the tracheal intubation was connected to a ventilator. AHR was challenged with increasing doses of Mch (0, 6.25, 12.5, 50 or 100 mg/ml; Each dose has a 3-min response time and there was a 1-min interval between each dose) and data was recorded and presented as changes in R_L and Cdyn.

Anesthetized mice were sacrificed by cervical dislocation following AHR detection. Intact left and both upper and lower lobes of the right lung were snap frozen in liquid nitrogen (-196˚C) and then stored at -80˚C until further use. The middle lobe of the right lung was fixed in 4% paraformaldehyde at room temperature for 24 h and used for subsequent histological analysis. H&E and PAS staining were performed to determine the inflammatory and mucus secretion changes. The middle lobe of right lung from each mouse was removed, fixed in 4% paraformaldehyde at room temperature for 24 h, decalcified in EDTA, dehydrated in a graded series of ethanol solutions and embedded in paraffin. Afterwards, lung tissue sections were obtained at a thickness of 4-µm. Lung slices were stained with H&E (hematoxylin staining for 10 min at room temperature; eosin staining for 3 min at room temperature) and PAS solution and imaged with light microscopy (x100 or x200, magnifications; Nikon ECLiPSE Ni; Nikon Corporation). A semi-quantitative grading method was used to evaluate the degree of peribronchial inflammation as described by Myou et al (23). The severity of the inflammation was graded in five categories: 0, Normal; 1, few inflammatory cells; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells of four cells deep. PAS-positive cells in each airway were counted, divided by the circumference of the basement membrane and multiplied by 100(24). The data was collected by three independent blinded investigators (FZT, JC, JJQ).

Changes in mitochondrial structure were observed using TEM. Lower lobe of right lung was cut into 1x1x3 mm³ sections and fixed in 2.5% glutaraldehyde phosphate buffer at 4˚C for 24 h. Fixed samples were dehydrated with ethanol and acetone, embedded (Epon812; cat. no. 45345; Sigma-Aldrich; Merck KGaA) and dried. Afterward, the tissue was cut into 70-nm thick sections, stained with uranyl acetate and citrate at room temperature for 30 min and visualized using the JEM-1400 Plus transmission electron microscope (magnification, x20,000; JEOL, Ltd.).

BALF was collected by flushing the right lungs three times with 1 ml PBS using a 1-ml syringe inserted into a cannula. BALF was centrifuged for 10 min at 500 x g and 4˚C, after which the supernatant was collected for cytokines analysis. The pellet containing the cells was resuspend in 100 µl PBS to determine the leukocyte differential count using the BC-5000 Vet auto hematology analyzer (MINDRAY Medical International Co., Ltd.).

IgE and T helper (Th) 2 cells play a pathogenic role in asthma (25). The levels of IgE, along with Th2 cytokines IL-5 and IL-13, were therefore investigated in the supernatants of BALF samples using sandwich ELISA kits according to the manufacturer's instructions.

Eosinophil (Eos) has been implicated in the pathogenesis of asthma (26). Eos populations in lung tissue were detected using flow cytometry. CD45⁺CD11b⁺Ly6C-SiglecF⁺cells were identified as the EOS population as described by a previous study (27). The mouse lung dissociation kit was used to dissociate the upper right lobe of each lung to single cells. Afterwards, single cells were stained with the fluorescently labeled dyes or antibodies (Live eFluor780, CD45 eFluor 506, anti-mouse CD11b BV711, anti-mouse SiglecF BV421, anti-mouse Ly6C PE) at 1x10⁶ cells/100 µl at 4˚C for 30 min detected using an Attune NxT instrument (Thermo Fisher Scientific, Inc.). Flow analysis was done using the FlowJo version 10 software (FlowJo, LLC).

For ROS, the tissue samples were embedded with Tissue-Tek^® OCT (cat. no. 4583; Sakura Finetek USA, Inc.) and then prepared into frozen sections. The embedded samples were cut into 10-µm thick slices using a cryostat (CM1860; Leica Microsystems GmbH) and transferred onto glass slide and stored at -20˚C. They were then stained according to the protocols of the ROS kit. For GSH sample preparation, the tissue sample was prepared into a 10% tissue homogenate [tissue weights (g): Volume of normal saline (ml)=1:9], and the subsequent determination was performed according to the protocols of the GSH kit. Ferroptosis is determined by the balance between iron accumulation-induced ROS production and the antioxidant system that avoids lipid peroxidation (16). In the present study, the upper lobe of the left lung is used for ROS and part of the lower right lung is used for GSH detection, ROS and GSH levels were determined using commercial ROS and GSH assay kits according to the manufacturer's instructions.

GPX4, ACSL4, 15-LO1 and SLC7A11 are mediators of lipid peroxidation and ferroptosis, and have been reported to be involved in the ferroptosis pathway (16). The expression levels of these proteins were assayed using western blotting. Lung tissues were homogenized and sonicated in RIPA lysis buffer, and then centrifuged at 13,201 x g for 10 min at 4˚C. Afterwards, the supernatants were collected and the total protein level was measured using a BCA protein test kit. A 12% SDS-PAGE separation gel (30 µg per lane) was prepared for protein separation and transferred to PVDF membranes, followed by blockage in 5% non-fat milk at room temperature for 1 h. Afterwards, the membranes were washed three times with Tris buffered saline 0.1% Tween-20 every 10 min. After that, the PVDF membranes were incubated with GPX4 (1:1,000), ACSL4 (1:1,000), 15-LO1 (1:1,000) and SLC7A11 (1:1,000) antibodies at 4˚C overnight. After three washes with TBST, the PVDF membranes were incubated with secondary antibodies for 1.5 h at room temperature (goat anti-rabbit IgG HRP-conjugated secondary antibody; cat. no. L3012; Signalway Antibody LLC; 1:10,000 dilution). Immobilon western chemiluminescent HRP substrate solution was used to exhibit protein band after three washes with TBST. β-actin (1:10,000; dilution; cat. no. HRP-60008; Proteintech Group, Inc.) was used as the internal control for the normalization of the data using ImageJ (v152p; National Institutes of Health).

All the experiments were repeated ≥ three times. All data were analyzed and graphed using GraphPad Prism 8 (GraphPad Software, Inc.). Data are presented as the mean ± standard deviation. Unpaired Student's t-test or Mann-Whitney U test were used to analyze the difference between two samples. P<0.05 was considered to indicate a statistically significant difference.

Changes in AHR were investigated in the current study. Compared with mice in the normal control group, when the Mch dose increased, the R_L increased significantly (P<0.05 or P<0.001; Fig. 1B), whereas Cdyn decreased significantly (P<0.05 or P<0.01; Fig. 1C). There was no obvious increase in R_L between the two groups at Mch doses <50 mg/ml (Fig. 1B). However, Mch challenge at 50 and 100 ng/ml resulted in the prominent augmentation of R_L. At Mch doses of 12.5 (P<0.05), 50 (P<0.01) and 100 (P<0.01) mg/ml, HDM inhalation induced a significant decrease in Cdyn compared with the control group (Fig. 1C). These data indicated that HDM exposure could aggravate AHR.

H&E staining was performed to evaluate the inflammatory changes within the lung. The results revealed that inflammatory cell infiltration around the bronchus (Fig. 2A and B) was observed in mice receiving HDM inhalation, and that HDM exposure significantly aggravated the pulmonary inflammatory response compared with the control (P<0.05; Fig. 2B). Mucus hypersecretion is a notable feature of allergic asthma (28). Therefore, PAS staining was conducted to evaluate the degree of goblet cell metaplasia and mucus secretion. As presented in Fig. 2A and C, mucus secretion increased significantly compared with mice in the normal control group. In addition, inflammatory cells in BALF were also detected, and the results demonstrated that the total leukocytes, lymphocytes (Lym), Eos and monocytes (Mon) increased significantly after HDM exposure compared with the control (P<0.05; Fig. 2D). These aforementioned data suggest that HDM exposure promoted lung inflammation and goblet cell proliferation.

CD45⁺CD11b⁺SiglecF⁺Ly6C in Eos populations of the lung tissue were detected using flow cytometry. Mice in the HDM-induced asthma group demonstrated significantly increased profiles of CD45⁺CD11b⁺SiglecF⁺Ly6C in the Eos proportion compared with mice in the control group (P<0.01; Fig. 3A and B). Furthermore, IgE, IL-5 and IL-13 levels in BALF were also investigated using ELISA. Elevated IgE, IL-5 and IL-13 expression levels were detected compared with control group mice (P<0.05, P<0.01 or P<0.001; Fig. 3C-E). Therefore, HDM-induced airway inflammation was characterized by Th2-mediated eosinophilic inflammation in the lungs.

Imbalance between ROS production and the antioxidant system is associated with ferroptosis (16). The present study revealed that HDM exposure resulted in significantly elevated ROS (P<0.05; Fig. 4A and B) and reduced GSH levels (P<0.05; Fig. 4C) in the lung tissue compared with those in the control mice. It has been reported that ferroptosis is associated with mitochondrial fragmentation and cristae decrease (29). To explore the role of HDM inhalation in ferroptosis, the morphological changes of mitochondria were observed using TEM. Consistent with literature reports (15,27,30), dysmorphic small mitochondria were observed in the lung cells after HDM inhalation. Representative results are presented in Fig. 4D. Decreased mitochondria crista, condensed and ruptured outer membranes were also discovered in the lung cells of mice in HDM-induced asthma group. These results provided evidence that HDM exposure could induce ferroptosis in the lungs.

To investigate the underlying mechanisms associated with ferroptosis, key proteins involved in ferroptosis were measured. The protein expression levels of GPX4, ACSL4, 15-LO1 and SLC7A11 were detected. The quantified western blotting results revealed that mice that received HDM inhalation had significantly downregulated activities of GPX4 compared with the normal control group (P<0.01; Fig. 4E and G). Furthermore, a significant upregulation of ACSL4 and 15-LO1 as well as downregulation of SLC7A11 were detected in HDM-induced asthmatic mice compared with the controls (P<0.05; Fig. 4E, F, H and I). These results demonstrated that GPX4, ACSL4, 15-LO1 and SLC7A11 were involved in ferroptosis in the lungs of HDM-induced asthmatic mice.