Mitochondrial Damage and Epithelial-Mesenchymal Transition as Major Triggers of the Development of Idiopathic Pulmonary Fibrosis

2.1. The EMT Process of AECs Is a Crucial Feature of IPF TGF-β is a key fibrogenic cytokine often used as an in vitro model to induce pulmonary fibrosis, and TGF-β is also the target of the clinical anti-pulmonary fibrosis drug pirfenidone [13]. In RLE-6TN cells derived from the rat type II AECs line, a mesenchymal cell marker and fibroblast-like morphology were detected after six days of TGF-β1 treatment, with TNF-α enhancing this mesenchymal transition. A high number of cells were colocalized with thyroid transcription factor 1, pro-surfactant protein-B, and alpha smooth muscle actin (α-SMA). The mesenchymal marker proteins COL1A1 and vimentin are also increased by TGF-β1, with a simultaneous downregulation of aquaporin 5, zonula occludens-1, and cytokeratins in epithelial cells indicating EMT [14]. In patients with IPF and bleomycin (BLM) mouse models, the expression of Wnt-inducible signaling pathway protein 1, Twist, and sphingosine 1-phosphate in type II AECs was increased. The lung epithelial cell marker proteins tight junction protein, cadherin 1, and E-cadherin are down-regulated, while mesenchymal marker proteins α-SMA, Recombinant S100 calcium-binding protein A4, COL1A1, vimentin, and N-cadherin are up-regulated, promoting and maintaining the progressive development of fibrosis [15,16,17]. IPF patients and cell experiments showed that a transcription factor called snail family transcriptional repressors 1 and 2 up-regulated a type II AECs mesenchymal transition induced by TGF-β1, enhancing fibroblast differentiation [18]. In A549 cells derived from the human type II AECs line express fewer mRNAs for fibrosis-related proteins N-cadherin, α-SMA, and transcription factors slug, snail, and twist when treated with Sulforaphane in the TGF-β1-induced model; Sulforaphane treatment also reduces expression of TGF-β1 and collagen I in BLM mouse models [18]. The natural compound breviscapine inhibits EMT in BLM mouse models via the nuclear transcription kappa-B/NLR Family, Pyrin Domain Containing Protein 3 pathway [19] (Figure 1). 2.2. Mitochondrial Oxidative Stress Mediates Apoptosis of AECs Exogenous and endogenous factors that can cause oxidative stress include O₃, CO₂, SO₂, cigarette smoke, and ROS generated in the airways [20]. Both in vitro and animal experiments have confirmed that the cellular autoxidation inhibitor Lip-1 protects A549 cells and mice treated with bleomycin. Lip-1 can reduce lipid peroxidation markers malondialdehyde and ROS levels, interfere with the p53 signaling pathway, and attenuate apoptosis of AECs [21]. ROS regulator 1 (Romo1) mediates ROS production in mitochondria through the electron transport chain’s respiratory complex III. IPF patients express Romo1 mostly on alveolar and bronchial epithelial mitochondria, and TUNEL-positive cells are mainly in alveoli [22]. Therefore, Romo1 might be responsible for oxidative stress-induced apoptosis in the alveolar epithelium. Knockdown of Romo1 in vitro can inhibit the increase of ROS level and cell death induced by H₂O₂ treatment in A549 cells, playing a protective role [22]. In A549 cells, FMP-1, a morel polysaccharide exerts significant antioxidant activity in response to H₂O₂-induced oxidative damage, and FMP-1 can inhibit cytochrome C and caspase-3 release through the PI3K/AKT signaling pathway, reducing malondialdehyde and ROS levels and enhancing antioxidant enzymes superoxide dismutase and total antioxidant capacity [23]. A DNA splicing repair enzyme OGG1 attenuates PM2.5-induced PF by inhibiting signaling pathways related to NF-κB, type II AECs are protected from oxidative damage and death [24]. AECs apoptosis caused by PM 2.5 oxidative stress also involves MAPK signaling [25,26]. The H₂O₂-generating enzyme NOX4 is one of the specific pathological markers of IPF. type II AECs express it highly, and several studies have shown that type II AECs, one of the essential sources of H₂O₂, is associated with PF [27]. The anti-inflammatory factor IL-37 is significantly down-regulated in AECs of IPF patients [28]. Mouse AECs are resistant to oxidative stress when treated with IL-37, while knockout of IL-37 increases A549 cell death through inhibiting TGF-β1 receptor, phosphatidyl-inositol 3-kinase/serine-threonine kinase, Smad, and non-Smad (extracellular regulated protein kinases/mitogen-activated protein kinases) signaling pathways [29] (Figure 2). 2.3. Insufficient Autophagy of Type II AECs s Can Promote EMT Autophagy is a process in which lysosomes and autophagosomes work together to eliminate misfolded proteins and damaged organelles, such as mitochondria, from cells. Cells can survive on nutrients released by autophagy, such as fatty acids and amino acids released by the lysosomal tract; this is a crucial homeostatic mechanism [30,31]. Patients with IPF have significantly lower levels of lung autophagy, and adequate autophagy can lead to cellular senescence [32]. Decreased autophagy triggers EMT in AECs in the context of IPF [33], and depletion of autophagy-related gene 5 inhibits autophagy in type II AECs, increasing EMT through the p62/sequestosome 1-NF-κB-Snail2 pathway [34]. Lung tissue from patients with IPF shows phosphorylation activating the Janus kinase/signal transducer and activator of the transcription 3 signaling pathway [35]. In vitro experiments with primary type II AECs and A549 cells confirmed that JSI-124, an inhibitor of Janus kinase/signal transducer and activator of transcription 3, can prevent the EMT process caused by TGF-β and the release of IL-6/IL-13, while increasing the expression of autophagy markers Beclin-1 and LC-I/II expression [30,36]. Cells that are damaged or stressed show signs of the unfolded protein response (UPR), leading to the accumulation of UPR in endoplasmic reticulum stress (ERS) [37]. The ER can activate autophagy, and autophagosomes with double membrane sequester cytoplasm and organelles for lysosomal degradation, clearing the enlarged ER and promoting cell survival [37,38]. ERS can cause autophagy failure and lead to PF in mice [39]. Knockout of Atg4b results in reduced autophagy, impairing the pulmonary response to ERS [37,40]. The most common SP-C (I73T) mutation found in sporadic and familial cases of IPF is also mechanistically linked to insufficient autophagy in animal models [37,41], and the expression level of SP-C (I73T) is directly correlated with the severity of lung injury [41]. Type II AECs use autophagy to resolve ERS and other types of injuries (Figure 3). 2.4. Mitochondrial Injury Promotes Autophagy and Fibrosis Genetic susceptibility and repeated microinjury from environmental exposures such as cigarette smoke, inhalated dust, infection, and gastroesophageal reflux lead to damage in type II AECs mitochondria [42,43,44,45]. Damaged mitochondria release a plethora of bioactive molecules, namely damage-associated molecular patterns (DAMPs), which include mtDNA, ROS, and non-coding RNA, which activate fibroblasts/myofibroblasts, macrophages, and immune cell recruitment. These cells secrete inflammatory and growth factors that stimulate their growth and specialization to promote fibrosis [46]. Mitophagy, a selective process that degrades impaired mitochondria, occurs when autophagy targets mitochondria degraded by lysosomes [37,46]. At the mitochondrial outer membrane, the degradation machinery recognizes PTEN-induced putative kinase 1, which can recruit the E3 ubiquitin ligase PARK2 [37,47]. Substances such as B-cell lymphoma 2, mitofusin, and voltage-dependent anion channel, are ubiquitinated by p62/SQSTM1 and activate mitophagy through interaction with the LC3 protein [37,48]. In PF areas, type II AECs accumulate enlarged and misshapen mitochondria, correlating with markers of ERS [49]. In a mouse model, tunicamycin-induced ERS is sufficient to induce type II AECs mitochondrial enlargement, mediated by the UPR’s PERK arm. The production of activating transcription factor 3 represses the transcription of PINK1, resulting in higher mitochondrial amounts and spontaneous collagen deposition in alveolar walls in mice [37,50] (Figure 4). 2.5. The Development of PF Is Strongly Influenced by Ferroptosis Iron is essential for cellular functions, but excessive levels can be fatal. Iron overload leads to oxidative stress, lipid imbalances, and specific pathological manifestations in mitochondria, resulting in ferroptosis, a form of programmed cell death [51]. Ferroptotic cells display necrotic morphology, triggering inflammatory reactions and the release of DAMPs from the cells [52]. Eight ferroptosis-related gene signatures in bronchoalveolar lavage fluid samples from patients with IPF were associated with a poor prognosis in IPF [53]. In MLE-12 cells derived from the mouse lung epithelial cell line, a cell model of iron accumulation demonstrated that iron accumulation induced cytoplasmic oxidative stress, which subsequently triggers autophagy and the expression of profibrotic factors. In response to repetitive injury, lung epithelial cells undergo prolonged inflammation and sustained EMT [54]. The regulation of ferroptosis in EMT is significantly influenced by the maintenance of iron homeostasis and oxidation homeostasis [55]. The precise mechanism by which iron accumulation contributes to the development of IPF remains unknown [56]. Recent research has identified five ferroptosis-related genes, namely aconitase 1, NRAS proto-oncogene, ectonucleotide pyrophosphatase/phosphodiesterase 2, Mucin 1, and ZFP36 ring finger protein, as potential risk markers for IPF and predictors of unfavorable outcomes [56]. Additionally, the regulation of ferroptosis through caveolin 1, nitric oxide synthase 2, growth differentiation factor 15, and cyclin dependent kinase inhibitor 2A has been found to influence the progression of PF. These genes are involved in processes such as organic anion transport, hypoxia response, oxygen deficiency response, hypoxia-inducible factor 1 signaling pathway, and arachidonic acid metabolism [57]. The activation of N-methyl-d-aspartate receptors impacts iron homeostasis in type II AECs, leading to increased calcium influx. This, in turn, enhances the expression of neuronal nitric oxide synthase and iron regulator protein 1 genes related to iron metabolism, ultimately causing mitochondrial damage, dysfunction, and ferroptosis. This process ultimately contributes to the development of IPF [58]. Lip-1 and the iron chelator deferoxamine have been found to alleviate PF induced by bleomycin or LPS through iron chelation or reduction of lipid peroxidation in murine models [59,60]. Additionally, chloroquine, an autophagy inhibitor, effectively reduces iron-driven oxidative damage-induced EMT in AECs [54]. The regulation of EMT-induced PF through TGF-β/Smad, nuclear factor, erythroid derived 2, and Wnt signaling pathways is mediated by ferroptosis [55] (Figure 5). 2.6. In Summary: A Comprehensive Model of IPF from the Onset of Oxidative Stress to the End of EMT in AECs As previously mentioned, the EMT concept in the approach shed light on various characteristics of IPF lungs. The occurrence of oxidative stress and mitochondrial damage is significant, establishing AEC damage as a fundamental model for the development and progression of IPF. Both endogenous and exogenous oxidative stress factors contribute to inducing mitochondrial damage, releasing additional DAMPs and ROS, which worsens oxidative stress. Consequently, damaged mitochondria undergo autophagy for metabolic purposes while also contributing to ERS, which can activate autophagy as well. Inadequate autophagy can lead to EMT. Iron overload has become a prominent topic of investigation in current research, mainly due to excessive external supplements or internal metabolic dysregulation. This issue is especially common in individuals aged 65 and older. Iron, acting as a powerful catalyst, helps produce oxygen free radicals, which can lead to the peroxidation of the phospholipid membrane in lung epithelial cells. This process notably causes oxidative damage to the mitochondrial membrane and triggers ferroptosis. After apoptosis, AECs release DAMPs, which worsen the progression of EMT (Figure 6).