Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by the loss of respiratory lobules caused by fibrosis and the remodeling of alveolar units, which leads to a decline in pulmonary function. Many factors, including susceptible genetic factors, viral infection, radiation, and smoking, may cause IPF (Cong et al., 2017). The only two drugs approved by the FDA for IPF, pirfenidone and ninedanib, slow disease progression in some IPF patients but have little impact on the survival rate of IPF patients and have some side effects (King et al., 2014; Richeldi et al., 2014; Rangarajan et al., 2016). At present, lung transplantation is the only therapeutic strategy that significantly prolongs the survival of severe IPF patients, but the probability of pulmonary fibrosis (PF) caused by allograft rejection is also high. Therefore, it is urgent to identify effective therapeutic strategies for PF.

In recent years, it has been reported that pulmonary iron accumulation and dysregulated iron homeostasis are associated with or promote the progression of many lung diseases, such as IPF, chronic obstructive pulmonary disease, cystic fibrosis, alveolar proteinosis, lung cancer, asthma and acute lung injury (Ghio et al., 2008; Ali et al., 2017; Cloonan et al., 2017; Neves et al., 2019; Zhang et al., 2019; Zhu et al., 2021). Accumulated iron generates excessive ROS through the Fenton reaction, which causes functional changes in cells, such as inflammation or ferroptosis mediated by lipid peroxidation (Kajarabille and Latunde-Dada, 2019; Battaglia et al., 2020). Because the lung contacts the external environment directly, maintaining pulmonary iron homeostasis is a major challenge. The regulation of pulmonary iron homeostasis mainly depends on iron regulatory molecules, such as ferritin, transferrin receptor (TFRC), divalent metal ion transporter (DMT1), and ferroportin 1 (FPN1). Cytosol iron is either wrapped by ferritin, thus reducing iron toxicity to cells, forms a liable iron pool (LIP) or is relocated to mitochondria to form iron sulfur clusters and heme. TFRC mediates the endocytosis of Fe³⁺ bound to transferrin (TF). DMT1 transports Fe²⁺ from the extracellular space to the cytosol, and FPN1 pumps iron out of the cell. It has been reported that pulmonary levels of iron and ferritin are elevated in bleomycin (BLM)-induced fibrotic mouse lungs, and the levels of iron regulatory proteins are also altered (Zhu et al., 2021).

Aging is one of the predisposing factors of IPF. With aging, mitochondria show abnormal morphology and structure, such as swelling, the disappearance of cristae, and the rupture of the intima (Mora et al., 2017). Importantly, oxidative stress dysregulates mitochondrial dynamics, such as mitochondrial fission and fusion imbalance, thus affecting the shape, size and number of mitochondria, mitochondrial dysfunction and subcellular transport, further affecting a variety of cell functions and even causing cell death and a variety of human diseases (Yu et al., 2020). In the lungs of IPF patients and BLM-PF lungs, mitochondrial morphology and number were dysregulated, and mitochondrial dysfunction occurred (Bueno et al., 2015; Rangarajan et al., 2018; Yu et al., 2018).

Vitamin E (VE) is a group of antioxidant liposoluble vitamins, including α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol; α-tocopherol is the most biologically active form of VE (Abdala-Valencia et al., 2013). VE has been reported to attenuate BLM-induced PF in experimental animals in only a few reports, and its explored functional mechanisms are restricted to antioxidation and the inhibition of inflammation (Deger et al., 2007; Shi et al., 2013; Ramezani et al., 2021). In addition, VE supplementation immediately after irradiation was reported to protect against radiation-induced PF in rats (Bese et al., 2007). In recent years, iron metabolism and mitochondrial metabolism have received much attention and become the focus of pathogenesis studies on PF. However, the effects of VE on iron metabolism and mitochondrial structure and function in fibrotic lungs have not been explored. Therefore, in this study, we administered VE to experimental mice with PF or cultured pulmonary cells and investigated the unknown aspects of its functional mechanisms, especially whether the improvement of iron and mitochondrial metabolism plays a role in the inhibition of PF by VE.

VE possesses anti-inflammatory, anticancer and antimutagenic properties (Jin et al., 2014; Tang et al., 2016). There has been only a few reports about the attenuation of experimental PF caused by different inducers besides BLM, such as amiodarone, radiation, melphalan, and hexavalent chromium, however, all of the identified molecular mechanisms are limited to antioxidation and the inhibition of inflammation (Card et al., 2003; Bese et al., 2007; Hemmati et al., 2008; Wigenstam et al., 2009). This study observed the improvement in BLM-induced lung pathology and survival decrease of experimental animals by VE, confirming a potent mitigation effect of VE in severe PF. For the first time, we uncovered and evidenced the novel mechanisms of VE against PF include suppressing the pathological activation and fibrotic differentiation of lung fibroblasts and epithelial-mesenchymal transition, and importantly, the improvement of metabolisms of iron and mitochondria, and apoptosis in BLM-induced fibrotic lungs. We also investigated the molecular functional mechanisms of VE in vitro. These results deepen our understanding of the protective effects of VE against PF, which may be underestimated before.

Oxidative stress and inflammation play important roles in IPF and experimental PF models (Bringardner et al., 2008). VE concentrations in the hamster lungs increase significantly after intratracheal BLM administration, and diet-deficiency of VE increases levels of lipid peroxide in the hamster lungs at a very early stage post BLM treatment (Kato et al., 1990) Administration of α-tocopherol increases glutathione level and catalase activity, and decreased malondialdehyde level in bleomycin-induced fibrotic lungs of rats (Deger et al., 2007). It has been reported that VE reduces the release of proinflammatory cytokines by limiting the production of ROS and ROS-mediated signal transduction, preventing chronic inflammation, such as liver fibrosis (Singh et al., 2005; Zingg, 2007; Calder et al., 2009). Consistently, VE deficiency enhances pulmonary inflammatory response and oxidative stress induced by single-walled carbon nanotubes in mice (Shvedova et al., 2007). Tocopherol diet normalizes belomycin-induced increase in reactive oxygen and nitrogen species, and mitigated inflammation progression, demonstrated by reduced macrophage activation and function, and limited enzymes and cytokines involved in pro-inflammation in mouse lungs (Shi et al., 2013). In a clinical trial, the supplementation with a combination of vitamins D, C and E remarkably reduced the serum levels of protein carbonyl, high-sensitivity C-Reactive Protein and TGF-β in IPF patients (Yavari et al., 2022). Consistently, this study confirmed that VE significantly upregulated Gpx4 expression and suppressed the transcript levels of inflammatory cytokines in BLM-induced fibrotic lungs and MLE 12 cells in vitro. Thus, inhibition of the oxidative stress and inflammatory response contribute to the protective effects of VE against PF.

Iron is an essential trace element for multicellular organisms and most microorganisms. It is a catalytic component of some enzymes that mediate many redox reactions that are critical for energy production and intermediate metabolism (Ganz and Nemeth, 2015). Iron also plays an important role in the regulation of the cell cycle, cell division and cell metabolism (Heath et al., 2013). Therefore, the maintenance of iron homeostasis in organs is crucial for their normal function in the organism. Iron catalyzes electron exchange and disturbs iron homeostasis through oxidative stress in the lung, resulting in lung tissue damage. BLM-induced PF and decreased lung function in experimental mice are related to pulmonary iron accumulation (Ali et al., 2020). Alpha-tocopherol decreased BLM-triggered pulmonary iron concentrations in rats (Ertekin et al., 2007). In this study, VE treatment significantly reduced BLM-induced iron aggregation and normalized the expression of ferritin and iron-regulatory proteins, indicating that VE improves pulmonary iron level as well as metabolism. It has been reported that iron promotes the proliferation of pulmonary fibroblasts in BLM-induced fibrotic lungs and in vitro (Zhu et al., 2021). Thus, VE reduces the total number of fibroblasts and the number of proliferating fibroblasts in BLM-induced lungs, which may be mediated by reducing pulmonary iron levels. However, in vitro, VE had no significant effect on FAC-induced expression of ferritin, cellular iron, or cell supernatant iron levels or on the proliferation level in TGF-β1-induced myofibroblasts. These results indicate that VE does not directly chelate iron or directly affect fibroblast proliferation under physiological conditions, but VE reduces the pathological increase in pulmonary iron levels in vivo, which is likely dependent on less inflammatory microenvironment in the fibrotic lung, thereby reducing the proliferation of pulmonary fibroblasts in vivo. In addition, VE decreased the number of myofibroblasts and the expression of the main extracellular matrix component collagen I in BLM-PF lungs, and it also reduced TGF-β1-induced fibrotic differentiation in lung fibroblasts and EMT in epithelial cells in vitro. These effects do not seem to be dependent on iron levels, and they may be related to VE-reduced cellular ROS production or secretion of proinflammatory factors.

Mitochondrial dysfunction has been found in pulmonary AECIIs, fibroblasts, and macrophages in IPF patients, which increases sensitivity to fibrosis (Bueno et al., 2020). Mitochondrial dysfunction results from mitochondrial damage, which is caused by adverse environmental conditions, including excessive mitochondrial reactive oxygen species (mtROS), changes in mitochondrial morphology, disturbed oxidative phosphorylation, impaired energy production, alterations in metabolic pathways (i.e., fatty acid β-oxidation) (Nunnari and Suomalainen, 2012; Cloonan and Choi, 2016; Bueno et al., 2020). Alpha-tocopherol is crucial for mitochondrial integrity, as profound deficiency of α-tocopherol causes mitochondrial degeneration and leads to necrosis of skeletal muscle in rats (Van Vleet et al., 1968). VE has been reported to play an important role in reducing mitochondrial damage and mitochondrial oxidative dysfunction mediated by clearing ROS (Napolitano et al., 2019), or prevent the reduction in the electron transfer rate in aged mice (Navarro et al., 2005; Polyak et al., 2018). For instance, VE restored the activity and expression of the third subunit of cytochrome-c oxidase in lung mitochondria and bronchial epithelia, and improved mitochondrial ultrastructure of bronchial epithelia in an ovalbumin-challenged allergic asthma murine model (Mabalirajan et al., 2009). VE fully prevented the brain death of zebrafish in the rotenone-induced mitochondrial complex I disease model (Polyak et al., 2018). However, VE did not prevent mitochondrial dysfunction in amiodarone-induced pulmonary fibrosis in hamster (Card et al., 2003). The potential reasons for the divergent conclusions include different action mechanisms of the drugs, outcome measures, species, and dietary conditions. In this study, AECIIs in BLM-induced lungs showed mitochondrial swelling, which was inhibited by VE. Importantly, the total mitochondrial numbers in AECIIs were increased in BLM-induced lungs, and VE treatment significantly reduced mitochondrial numbers, suggesting that VE suppressed the accumulation of mitochondria with impaired structure and function. Notably, we found that the expression of mitochondrial structure- and function-related proteins (Ampkα2, Ucp1, Nd5, and Sirt3) was downregulated by BLM exposure, and this change was restored by VE treatment significantly or in a trend, strongly suggesting that VE improves the structure and function of BLM-damaged mitochondria.

Mitochondrial damage and dysfunction can lead to cell senescence and even cell death. BLM-induced fibrotic mouse lungs exhibited higher levels of apoptosis than the controls (Sul et al., 2022) and ferroptosis of epithelial cells was included (Pei et al., 2022). In this study, altered expression levels of the apoptosis-related proteins Parp1, p53, and Bax in BLM-PF lungs indicated increased cell apoptosis, which was restored by VE significantly or in a trend. Consistently, Trolox mitigated BLM-induced apoptosis in pulmonary epithelial cells and inhibits fibroblast death from BLM injury in vitro. As VE compensates for Gpx4 loss by protecting hepatocytes against deleterious lipid peroxidation (Carlson et al., 2016), the normalization of BLM-reduced Gpx4 levels by VE is suggested to contribute to the inhibition of pulmonary cell death in our study. Similarly, VE protects against cigarette smoke extract-induced cell apoptosis in a dose- and time-dependent manner in mouse embryonic cells (Chen et al., 2011). The inhibitory effect of VE on apoptosis in fibrotic lungs and pulmonary epithelial cells may involve improvements in mitochondrial structure damage and dysfunction.

In conclusion, in addition to confirming the strong inhibitory effect of VE on PF, this study demonstrated novel mechanisms by which VE protects against PF, including stabilizing iron metabolism in an experimental mouse model, improving mitochondrial structure, and suppressing the activation and fibrotic differentiation of fibroblasts and EMT, besides the known weakening of inflammatory response and strong antioxidant ability. Considering the safety and efficacy of VE, it is a potential therapeutic for treating PF and is worth further clinical trials to determine its clinical application. The regulation in metabolisms of iron and mitochondria by VE that were delineated in this work also sheds light on the therapeutics of other diseases with similar metabolic disorders.