Ferrostatin-1 and 3-Methyladenine Ameliorate Ferroptosis in OVA-Induced Asthma Model and in IL-13-Challenged BEAS-2B Cells

Ferroptosis was reported to be involved in the occurrence and development of asthma. However, the potential mechanism underlying the role of ferroptosis in asthma remains unclear. In this study, we established the mouse asthma model following the ovalbumin (OVA) method in C57BL/6 mice and the cell model with IL-13 induction in bronchial epithelial cells (BEAS-2B cells). Treatment of ferrostatin-1 (Ferr-1) and 3-methyladenine (3-MA) decreased iron deposition in IL-13-induced BEAS-2B cells and lung tissues of asthma mice, opposite to that in bronchoalveolar lavage fluid (BALF). Meanwhile, excessive lipid peroxidation asthma model in vivo and in vitro was alleviated by Ferr-1 or 3-MA treatment. In addition, Ferr-1 and 3-MA inhibited the expression of LC-3 in these cells and lung tissues of mice. Moreover, Ferr-1 and 3-MA also suppressed the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and oxidative stress factors (ROS and MDA), while promoting the level of SOD, in vivo and in vitro. Furthermore, application of Ferr-1 exhibited a greater inhibitory effect on iron release and lipid peroxidation in IL-13-induced BEAS-2B cells and asthma mice than 3-MA, accompanied with a weaker effect on ferritinophagy than 3-MA. Collectively, Ferr-1 and 3-MA ameliorated asthma in vivo and in vitro through inhibiting ferroptosis, providing a new strategy for the clinical treatment of asthma.


Introduction
Asthma is a chronic airway inflammation which is associated with multiple cells and cellular components [1]. This inflammation often induces adverse respiratory reactions and symptoms such as shortness of breath, chest tightness, and cough [2]. Furthermore, asthma is a common chronic respiratory disease with a high incidence in all age groups [3]. In recent years, the incidence of asthma has been increasing around the world. Several overlapping factors such as inhaled allergens, air pollution, respiratory virus infections, and genetics could trigger the initiation of asthma [4]. In addition, the ferroptotic and autophagic molecular mechanisms of the early pathophysiology of asthma are still unclear to develop new clinical therapies.
Ferroptosis is an iron-dependent form of cell death [5]. The occurrence of ferroptosis is mainly due to the excessive accumulation of reactive oxygen species (ROS) related to iron ions in the cells and the attenuation scavenging effect of glutathione peroxidase 4 (GPX-4). These can lead to the formation of lipid oxides [6], and the homeostasis of the production and degradation of lipid oxide is also imbalanced, resulting in the disability of clearing the excessive accumulation of lipid oxides in time, which will finally cause cell death [7]. Iron in cells is often in the form of ferritin, such as ferritin light chain (FTL) and ferritin heavy chain (FTH1), and iron response element binding protein 2 (IREB2) plays a critical role in the formation of ferritin [8]. There is a study revealing that airway inflammation, lipid peroxidation and ROS levels were elevated in asthma, accompanied with the occurrence of ferroptosis, indicating that ferroptosis was involved in the pathogenesis of allerigic asthma [9].
The occurrence of inflammation of respiratory epithelial cells was a common pathogenesis of the asthma [10]. The study suggested that the expression of interleukin-(IL-) 13 could activate the expression of members of the lipoxygenase family (ALOX15 and ALOX5) and therefore aggravated the oxidation of polyunsaturated fatty acid phosphatidylerhanolamine (PUFA-PE), thereby inducing the occurrence of ferroptosis [11]. However, the production of GPX-4 could induce the production of reduced glutathione (GSH), and the releasing of GSH relieved the peroxidate of these cells, so as to avoid ferroptosis [12]. However, whether GPX-4 and GSH could alleviate asthma by relieving ferroptosis is unclear.
In this study, we established an asthma model in vivo and in vitro and used ferrostatin-1 (Ferr-1), a synthetic antioxidant that has been recognized as an inhibitor of ferroptosis [13,14], and 3-methyladenine (3-MA), an inhibitor of phosphatidylinositol 3-kinases (PI3K) that is commonly used as autophagy inhibitor [15,16], for treatment, to explore the molecular mechanism of ferroptosis during asthma. This study may provide a novel therapeutic strategy for the treatment of asthma.

Materials and Methods
2.1. Animal Assays. First, male C57BL/6 mice were obtained from Shanghai Institute of Zoology, Chinese Academy of Sciences, and housed in a temperature-and humiditycontrolled room under a 12/12 h light/dark cycle with free access to water and food. All animal studies were approved by the ethics review committee of Shengjing Hospital of China Medical University.
The in vivo experiments consist of two parts (Figures 1(a) and 1(b)). For part 1, 10 mice were stochastically divided into 2 groups: the control group and model group. The animal model of asthma was established following the ovalbumin (OVA) method. In brief, the mice of the model group were sensitized by intraperitoneal injection of 1 mg OVA/40 mg Al(OH) 3 gel in 0.2 ml phosphate buffer saline (PBS; once a week) for 3 times. On the next day, after the last time of OVA injection, the mice were then challenged by intranasal inhalations with 5% OVA for a week. Mice in the control group received the same schedule for sensitization and were administrated with an equivalent amount of 0.9% sterile saline instead of OVA.
For part 2, 25 mice were randomly divided into 5 groups: the control group, model group, Ferr-1 group, 3-MA group, and Ferr-1+3-MA group. The mice in the control and model groups were proceeded as described above. The mice of the Ferr-1 group were treated with the Ferr-1 (2.5 μmol/kg, dissolved in dimethylsulfoxide (DMSO); Selleck, USA) by the intraperitoneal injection 2 h prior to OVA challenge. The dose of Ferr-1 was selected according to previous studies [17,18]. The mice of the 3-MA group were intraperitoneally injected with the 3-MA (15 mg/kg, dissolved in DMSO; Sigma-Aldrich, USA) 2 h prior to OVA challenge [19,20]. The mice of Ferr-1+3-MA group were treated with both Ferr-1 and 3-MA. Finally, these mice were executed to euthanasia. Subsequently, PBS was used for the lavage of the bronchus. Then, the PBS was collected as the bronchoalveolar lavage fluid (BALF) for the next research. In addition, a part of lung tissues were fixed in 4% formaldehyde, and the remaining lung tissues were stored at -80°C for further experiments.
2.2. Cell Culture and Treatment. Bronchial epithelial cell line (BEAS-2B cells) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured with RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA). These cells were cultured in the 37°C humid atmosphere supplemented with 5% CO 2 . These cells were cultured in air-liquid interface (ALI) as previously described [12] and treated with the IL-13 (2 ng/ml, 10 ng/ml, and 50 ng/ml,   2.5. Immunohistochemical Staining. We used paraffin to embed the lung tissue and cut the tissue into thin slices. Next, xylene was used for the dewaxing of these tissues. After that, these tissues were incubated with H 2 O 2 and stewed in the citrate buffer solution to expose the antigen sites. Then, these tissues were blocked with BSA (Beyotime, China) and incubated with the primary antibody against LC-3 (ab48394, Abcam) at 4°C overnight. On the second day, the tissues were incubated with the secondary antibody (Goat Anti-Rabbit IgG H&L, ab205718, Abcam) at room temperature for 2 hours. Then, these tissues were stained with the hematoxylin solution (Beyotime, China). Finally, the expression of LC-3 in these tissues was observed under the microscope (Olympus, Japan).
2.6. Immunofluorescence. BEAS-2B cells were plated on the glass slide. These cells were fixed with 4% paraformaldehyde    7 Oxidative Medicine and Cellular Longevity and incubated with 0.5% Triton X-100 (Beyotime, China) to enhance the permeability of the cell membranes. Then, 5% skim milk powder solution was used for the block of these cells. And these cells were incubated with the primary antibody against LC-3 (ab48394, Abcam) at 4°C overnight. After that, these cells were incubated with the secondary antibody (Goat Anti-Rabbit IgG, ab150077, Abcam) in the dark room. Finally, DAPI was used for the mark of the cell nucleus and the fluorescence was observed under the confocal microscope (Olympus, Japan).

ELISA Assays.
Human IL-6 ELISA kit (ab178013, Abcam), human TNF-α ELISA kit (ab181421, Abcam), and human IL-1β ELISA kit (ab229384, Abcam) were used for the detection of these factors in culture medium of BEAS-2B cells. In addition, mouse IL-1β ELISA kit (ab197742, Abcam), mouse IL-6 ELISA kit (ab222503, Abcam), and mouse TNF-α ELISA kit (ab208348, Abcam) were applied for the detection of the expression of these factors in BALF and lung tissues of mice. In addition, mouse FTL ELISA kit (ab157713, Abcam) and mouse FTH1 ELISA kit (ab65080, Abcam) were applied for the detection of the expression of these proteins in BALF and lung tissues of mice. The operation was following the instruction.

The Detection of Reduced Glutathione (GSH) and
Oxidized Glutathione (GSSG). The commercial kits (Beyotime, China) were used for the detection of GSH and GSSG in lung tissues and BEAS-2B cells. The operation was following the instruction.
2.9. Detection of ROS, MDA, and SOD. The production of SOD and MDA in lung tissues and BEAS-2B cells were determined with their commercial kits from Beyotime (S0088 and C0017, Shanghai, China). All operations of this assay were following the instruction. The ROS level was detected using its commercial kit from Shanghai Jianglai Biological Technology Co., Ltd. (Shanghai, China).

Cell
Counting Kit-8 (CCK-8) Assay. BEAS-2B cells were plated into 96 plates. After the incubation for 24, 48, and 72 h, respectively, CCK-8 (Dojindo, Japan) was diluted and added into 96 plates. Next, these cells were incubated in the incubator for 2 hours. Finally, the absorbance at 450 nm of these cells was determined with the spectrophotometer.

Statistical Analysis.
All data in this research were analyzed with the GraphPad Prism 8.0 (GraphPad Software, Inc.). The experiments of this study were repeated for three times, and the data in this research were displayed as mean ± SD. The comparison between diverse groups was performed with Student's t-test or one-way ANOVA test. The difference was considered as statistical significance until the value of p was less than 0.05.

The Ferroptosis Occurred in the Lung
Tissues of Mice during the Development of Asthma. In this part, we established the asthma model mice and collected the BALF and lung tissues after the sacrifice of these mice. As shown in Figure 2(a), a severe pathological injury of lung tissues was observed in asthma mice. The results (Figures 2(b)-2(e)) of Prussian blue staining and iron ion detection revealed an iron deposition in the lung tissues of asthma mice, compared to the mice of the control group. In addition, we found that the expression of FTL and FTH1 was suppressed in the lung tissues of asthma model mice (Figures 2(f) and 2(g)). All these results suggested that the ferroptosis was activated during the development of asthma.

Treatment of Ferr-1 and/or 3-MA Relieved Asthma-
Induced Damage of Lung Tissues of Mice. Next, Ferr-1 and 3-MA were used for the treatment of asthma mice. Results (Figure 3(a)) of H&E staining showed that the application of Ferr-1 and 3-MA alleviated the pathological injury of lung tissues of asthma mice. Meanwhile, a high expression of LC3, the marked protein of autophagy, was identified in lung tissues of asthma mice, which was then partly abolished by Ferr-1 and/or 3-MA treatment (Figure 3(b)). In addition, the concentrations of IL-4, IL-5, and IL-13, as well as IL-1β, IL-6, and TNF-α, were greatly elevated in BALF and lung tissues, especially in lung tissues, of asthma mice, which were suppressed after the treatment of Ferr-1 and/or 3-MA (Figures 3(c) and 3(d)). The commercial kits were also used for the detection of ROS, SOD, and MDA in BALF and lung tissues of these mice. What is more, the downregulated SOD and upregulated ROS and MDA in BALF of asthma mice were obviously reversed by Ferr-1 and/or 3-MA treatment. The levels of ROS, MDA, and SOD in the lung tissue had the same trendline of change with those in BALF (Figure 3(e)).

Treatment of Ferr-1 and/or 3-MA Relieved Iron
Accumulation in Lung Tissues in Asthma Mice. Next, we explored the specific regulatory role of Ferr-1 and 3-MA in asthma. As shown in Figures 4(a) and 4(b), the levels of FTL and FTL1 in BALF were increased after the occurrence of asthma while decreased after the Ferr-1 and/or 3-MA treatment. On the contrary, the levels of FTL and FTL1 in lung tissues were decreased in asthma mice but increased after Ferr-1 and/or 3-MA treatment. Meanwhile, as shown in Figures 4(c)-4(e), the levels of free iron in BALF were decreased during the development of asthma and increased after the treatment of Ferr-1 and/or 3-MA, which was opposite to those in lung tissues. Similarly, the results of Perls staining also revealed an accumulation of iron in lung tissue of asthma mice, while Ferr-1 and 3-MA treatment relieved this accumulation (Figures 4(f) and 4(g)). These results suggested that Ferr-1 and/or 3-MA treatment reduced iron deposition in lung tissues of asthma mice.

Treatment of Ferr-1 and/or 3-MA Alleviated Lipid Peroxidation and Ferritinophagy of Lung Tissues in Asthma
Mice. In addition, we also explored the lipid peroxidation and ferritinophagy, which were critical regulatory mechanisms underlying the initiation of ferroptosis, in Ferr-1-and 3-MAtreated asthma mice. As exhibited in Figures 5(a)-5(c), the treatment of Ferr-1 and 3-MA promoted the production of GSH and restricted the production of GSSG in the lung tissues of asthma mice. Meanwhile, we found a downregulated

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Oxidative Medicine and Cellular Longevity expression of antioxidant proteins (SLC7A11, SLC3A2, and GPX-4) and an upregulated expression of ALOX5, which was related to lipid peroxides-induced ferroptosis, in the lung tissues of asthma mice; however, the expression of these antioxidant proteins was enhanced, while the levels of ALOX5 were suppressed after the treatment of Ferr-1 and 3-MA ( Figure 5(d)). Moreover, a previous report pointed out that the increased TFR1 and DMT1 expression were linked with elevated ferroptosis and severe asthma [21], which was also verified in the present study, but Ferr-1 and/or 3-MA treatment could greatly reduce the protein expression of TFR1 and DMT1 ( Figure 5(e)). Furthermore, the expression of ferritinophagy-related proteins (NCOA4, ATG-5, and ATG-7) in the lung tissues was elevated upon the occurrence of asthma but was then repressed after the treatment of Ferr-1 and 3-MA ( Figure 5(f)).

IL-13 Induced Inflammatory and Oxidative Damage and
Induced Ferroptosis of Bronchial Epithelial Cells. To further concentrate on the regulatory mechanism of Ferr-1 and 3-MA in asthma, bronchial epithelial cells (BEAS-2B cells) were applied and stimulated with IL-13 to mimic asthma in vitro. Firstly, stimulation of IL-13 induced the decreasing of the viability of BEAS-2B cells dose dependently (Figure 6(a)). The production of IL-1β, IL-6, and TNF-α was also hugely elevated after the stimulation of IL-13 at 10 ng/ml and 50 ng/ml (Figures 6(b)-6(d)). Moreover, stimulation of IL-13 also enhanced the production of ROS and MDA, while repressing the activity of SOD in BEAS-2B cells (Figures 6(e)-6(g)). In addition, the results ( Figure 6(h)) of immunofluorescence also showed that stimulation of IL-13 enhanced the expression of LC-3. Next, the reduced GSH/ GSSG ratio and the elevated free iron upon IL-13 stimulation revealed that IL-13 induced oxidative damage and iron deposition in BEAS-2B cells (Figures 7(a)-7(f)), which was also evidenced by the downregulated protein expression of GPX4, SCL7A11, and SLC3A2 and the upregulated expression of ALOX5 upon IL-13 stimulation (Figure 7(g)), suggesting that IL-13 triggered the initiation of ferroptosis in BEAS-2B cells. Consistently, IL-13 also promoted the expression of ferroptosis-related proteins (TFR1, DMT1) and ferritinophagy-related proteins (NCOA4, ATG-5, and ATG-7) in BEAS-2B cells (Figures 7(h) and 7(i)).
3.6. Ferr-1 or 3-MA Relieved the IL-13-Induced Inflammation, Oxidative Damage, and Ferroptosis of Bronchial Epithelial Cells. Finally, due to a severe damage to cell viability of IL-13 at 50 ng/ml, IL-13 at 10 ng/ml was selected for the subsequent experiments. In addition, Ferr-1 and 3-MA were applied for treatment in vitro. As shown in Figure 8

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Oxidative Medicine and Cellular Longevity 8(g)). Moreover, we also found that treatment of Ferr-1 and 3-MA inhibited the expression of LC-3 in these cells (Figure 8(h)). In addition, the results also showed that the free iron ion was decreased after the treatment of Ferr-1 or 3-MA (Figures 9(a)-9(c)). The treatment of Ferr-1 also rescued the production of GSH while inhibiting the releasing of GSSG in IL-13-stimulated BEAS-2B cells, but 3-MA did not affect the level of GSH and GSSG in IL-13-stimulated BEAS-2B cells (Figures 9(d)-9(f)). Moreover, Ferr-1 treatment remarkably rescued the expression of SLC7A11, SLC3A2, GPX-4, and ALOX5, but 3-MA only rescued the expression of SLC7A11 and SLC3A2 in IL-13-stimulated BEAS-2B cells (Figure 9(g)). The levels of TFR1 and DMT-1 in these cells were further inhibited after the treatment of Ferr-1 or 3-MA (Figure 9(h)). Furthermore, in terms of ferritinophagy-related proteins (NCOA4, ATG-5, and ATG-7), 3-MA exhibited a significantly inhibitory effect on IL-13-mediated ferritinophagy, but Ferr-1 did not affect it well (Figure 9(i)).

Discussion
Asthma is a widespread respiratory disease. The occurrence and development of asthma affect the health of many people. At present, the main clinical therapy is the use of glucocorticoids or leukotriene receptor antagonists to relieve the symptoms of asthma patients [22]. While traditional antiinflammatory drugs could induce many side effects and damage human health, new treatment options were needed to further improve safety and effectiveness [23]. Therefore, we need to further study the molecular mechanisms of the occurrence and development of asthma to develop the latest treatment options. This study evaluated the initiation of ferroptosis in the pathogenesis of asthma in vivo and in vitro, as well as the potential regulatory mechanism.
Ferroptosis usually refers to the process of excessive lipid peroxide caused by the accumulation of a large amount of free iron in the cell, which eventually induces cell death [24]. As aforementioned, GSH, a major antioxidant that acts as a reducing substrate of GPX4 to mitigate the accumulation of ROS, can reduce and alleviate the accumulation of lipid peroxides in cells [25]. Heterodimeric cystine/glutamate antiporter system Xc − (mainly consisting of SLC7A11 and SLC3A2) mainly focuses on taking cystine into cells and reducing it to cysteine to synthesize GSH, thereby protecting cells from oxidative damage. Therefore, regardless of the abnormal function of the Xc --system, the abnormal GSH synthesis, or the decreased GPX4 activity caused by various factors, it could induce the hindering of the reduction of lipid peroxides and finally led to the ferroptosis of cells [26]. Currently, ferroptosis has been reported to be closely involved in and participated into human biological processes and multiple human diseases, including cancer, lung injury, and kidney diseases [13,27,28]. Nevertheless, there has been very few evidence regarding ferroptosis in