Animals, study protocol
Female 6–8 week old, specified-pathogen free BALB/c mice were purchased from SLAC Laboratory Animal Co., Ltd. Mice were kept under laboratory conditions (22°C, 50%-60% relative humidity, air circulation, 12h:12h light-dark cycle). Mice were randomized to two groups (10 mice/group) including normal control and HDM-induced asthma model groups. On day 0, 10μg/40μL HDM extract were administered intranasal after anesthetized with 2-2.5 % isoflurane in air. From days 7 to 11, challenge the mice daily intranasally by pipetting 40 μL of diluted HDM extract (20μg/40μL per mice) directly into the nostrils under mild anesthesia (Fig 1). For mice in normal control group, comparative volume of PBS was administered intranasally at the same time as mentioned above.
Methacholine (Mch) and pentobarbital sodium were purchased from Sigma-Aldrich. Isoflurane was purchased from Shenzhen Ruiwode Life Technology Co. Ltd. HDM extract (Dermatophagoides pteronyssinus) was purchased from Greer Laboratories. Mouse lung dissociation kit was purchased from Milteny Biotec. Live APC-Cy7 solution and anti-mouse CD45 eF506 antibody were purchased from Thermo Fisher Scientific. Anti-mouse CD11b BV711 and anti-mouse SiglecF BV421 antibodies were purchased from BD Pharmingen. Anti-mouse Ly6C PE antibody was purchased from BioLegend. Anti-GPX4 and anti-15 Lipoxygenase 1 (15-LO1) antibodies were purchased from abcam, and anti-acyl-CoA synthetase long-chain family member 4 (ACSL4) and anti-catalytic subunit solute carrier family 7 member 11 (SLC7A11) antibodies were purchased from Novus. Monoclonal GPX4 antibody for immunohistochemistry assay was purchased from Novus. The GSH assay kit was purchased from Nanjing Jiancheng Bioengineering Institute and the reactive oxygen species (ROS) assay kit was purchased from Sigma-Aldrich. IgE, IL-5 and IL-13 Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Multisciences (LIANKE) biotech. RIPA lysis buffer was purchased from Beyotime Biotechnology. BCA protein test kit was purchased from Thermo Fisher Scientific. Immobilon Western Chemiluminescent HRP Substrate was purchased from Merck.
Measurement of AHR
Within 24h after the ﬁnal HDM challenge, airway hyperresponsiveness (AHR) presented as airway resistance (RL) and dynamic lung compliance (Cdyn) to Mch were measured according to the manufacturer’s instructions. Briefly, mouse was weighed, intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg), incised and tracheal intubated. Afterwards, mouse was placed in the chamber, and the tracheal intubation was connected to the ventilator. AHR was challenged with increasing doses of Mch (0, 6.25, 12.5, 50, 100mg/ml), and data was recorded and presented as changes in RL and Cdyn.
Hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining were performed to determine the inflammatory and mucus secretion changes. Briefly, the middle lobe of left lung from each mouse was removed, fixed in 4% paraformaldehyde, decalcified in EDTA, dehydrated in a graded series of ethanol solutions, and embedded in paraffin. Afterwards, lung tissue sections were sectioned to slices with thickness of 4μm. Lung slices were stained with H&E and PAS solution. A semi-quantitative grading method was used to evaluate the degree of peribronchial inflammation. PAS-positive cells in the airway were counted and their percentage was calculated in the airway epithelial cells. The data was collected by three independent blinded investigators.
Changes in mitochondrial structure were observed by transmission electron microscope (TEM). Mouse lung was cut into 1×1×3 mm3 and fixed in 2.5% glutaraldehyde phosphate buffer. Fixed samples were dehydrated with ethanol and acetone, embedded and dried. Afterward, the tissue was cut into 70 nm-thick sections, stained with uranyl acetate and citrate, and visualized with the JEM-1400 Plus transmission electron microscope (Japan Electron Optics Laboratory).
Leukocyte differential counts in BALF
Bronchalveolar lavage fluid (BALF) was collected by flushing the right lungs three times with 1 mL of PBS via a 1 mL syringe inserted in the cannula. BALF was centrifuged for 10 min at 500 × g and 4 °C, and the supernatant was collected for cytokines assay and the pellet containing the cells were resuspend in 100 μL PBS for leukocyte differential counts using BC-5000 Vet (MINDRAY, China).
Cytokines in BALF
IgE and T helper (Th) 2 cells played a pathogenic role in asthma. The levels of IgE, Th2 cytokines IL-5 and IL-13 in supernatants of BALF were investigated by using sandwich ELISA kits according to the manufacturer’s instructions.
Flow cytomerty of lung tissue
Eosinophil (Eos) has been implicated in the pathogenesis of asthma. Eos population in the lung tissue was detected by flow cytomerty. CD45+CD11b+Ly6C-SiglecF+ cells were identified as EOS population in our experiment. Mouse lung dissociation kit was used to dissociate the upper right lobe of each lung to single cells. Afterwards, the single cells were stained with the fluorescently labeled solutions and antibodies (Live APC-Cy7, CD45 eF506, CD11b BV711, SiglecF BV421, Ly6C PE) and detected by an Attune NxT instrument (Thermo Fisher Scientific).
ROS and GSH measurement
Ferroptosis is determined by the balance between iron accumulation-induced ROS production and the antioxidant system that avoids lipid peroxidation. In our study, the ROS and GSH levels were determined using commercial ROS and GSH assay kit according to the manufacturer's instructions.
Western blot assay
GPX4, ACSL4, 15-LO1 and SLC7A11 were mediators of lipid peroxidation and ferroptosis, and were reported to be involved in ferroptosis pathway. The expression levels of these proteins were assayed by western blot. Lung tissues were homogenated and sonicated in RIPA lysis buffer, and centrifuged at 12000 rpm for 10 min at 4C. Afterwards, the supernatants were collected to measure the total protein level using BCA protein test kit. A 12% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) separation gel was prepared for protein separation and transferred to PVDF membranes, followed by blockage in 5% non-fat milk at room temperature. Afterwards, the membranes were washed with Tris buffered saline tween (TBST) every 10 min for three times. After that, the PVDF membranes were incubated with GPX4(1:1000 dilution), ACSL4 (1:1000 dilution), 15-LO1 (1:1000 dilution) and SLC7A11 (1:1000 dilution) antibodies at 4℃ overnight. After three times of washing with TBST, the PVDF membranes were incubated with secondary antibodies for 1.5h at room temperature. Immobilon western chemiluminescent HRP substrate solution was used to exhibit protein band after three times of washing with TBST. β-actin was used as the internal control for the normalization of the data.
All data were analyzed and graphed by GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Data were presented as the mean ± standard deviation (mean ± SD). Student's t-test was used to analyze the difference between two samples. A P-value less than 0.05 was considered statistically signiﬁcant.