1. Introduction
Pulmonary fibrosis is a mostly irreversible lung disease resulted from acute lung injury, including severe infections such as COVID-19, radiation, chemotherapy, toxins, COPD, etc(George, Wells & Jenkins, 2020; Meziani et al., 2018; Wijsenbeek, Suzuki & Maher, 2022; Wilson & Wynn, 2009). The incidence of PF is not easy to estimate. Whereas, idiopathic pulmonary fibrosis (IPF), a specific chronic and progressive PF, was once classed as a rare disease but with an incidence rate continuously increased these years(Hutchinson, Fogarty, Hubbard & McKeever, 2015; Navaratnam, Fogarty, Glendening, McKeever & Hubbard, 2013). Importantly, the average survival year of IPF is around 3-5 years after diagnosis(Vancheri, Failla, Crimi & Raghu, 2010). However, the approved treatments for PF are limited or with low efficacy, including pirfenidone and nintedanib(Johannson, Chaudhuri, Adegunsoye & Wolters, 2021). Therefore, it is still required efforts to elucidate important mechanisms for developing efficient strategies to treat PF progression.
With years’ efforts, it has been now well recognized that the progression of PF shares three common phases, including injury, inflammation, and tissue repair and contraction(Wilson & Wynn, 2009). In brief, the lung injury caused by multiple factors mentioned above would lead to inflammation for replacing the damaged epithelial or endothelial cells to maintain barrier function and integrity, from which several cytokines are produced, leading to EMT in AT II cells for tissue repair but also initiates fibrosis by synthesizing ECM components like fibronectin and collagen type 1, etc.(Kim et al., 2006; Kim et al., 2009). EMT has been reported to be an important process for fibrosis in multiple lung diseases, except IPF with controversial conclusions(Gauldie, 2002; Strieter, 2002). Recently, a single cell-RNA Seq analysis identified that EMT process in AT II cells does require for IPF progression(Xu et al., 2016). These studies together emphasize the importance of EMT in promotion of pulmonary fibrosis. Till now, multiple molecular targets modulating EMT have been reported to be effective in fibrotic lung diseases in mice model, however, no relevant effective clinic strategies so far have been reported(Bartis, Mise, Mahida, Eickelberg & Thickett, 2014).
Several animal models have been established to understand the PF progression, from which PQ poisoning-induced acute lung injury is widely recognized to induce PF in a short period. Other models, like bleomycin-induced PF, require a relatively long period (O’Dwyer & Moore, 2018), which contains EMT, endothelial-to-mesenchymal transition, fibroblast-to-myofibroblast transition (FMT), etc(Moss, Ryter & Rosas, 2022). Considering others and our previous studies all identify EMT as a key process in PQ-driven PF(Chowdhury, Zielonka, Kalyanaraman, Hartley, Murphy & Avadhani, 2020; Su, Cong, Bi & Gao, 2018; Yamada, Aki, Unuma, Funakoshi & Uemura, 2015), PQ-induced acute lung injury would be an ideal model to identify potential strategies targeting EMT for treating PF. PQ has been well recognized to produce ROS that leads to severe cellular damage, EMT, and thus pulmonary fibrosis(Zheng, Goncalves, Abiko, Li, Kumagai & Aschner, 2020), which oxidative stress has been well characterized as a central signal for multiple factors-raised PF. Interestingly, clinical treatments to eradicate ROS production are not sufficient to treat PQ-induced PF and following high mortality (Dinis-Oliveira, Duarte, Sanchez-Navarro, Remiao, Bastos & Carvalho, 2008; Subbiah & Tiwari, 2021), indicating that other important mechanisms are still required to be elucidated. Recent studies have identified several other potential mechanisms and relevant new strategies for treating PQ poisoning, however, the efficacy is limited, yet exhibiting attenuation of the symptom (Subbiah & Tiwari, 2021), including metformin treatment only slightly reducing PQ induced pulmonary fibrosis(Wu et al., 2019) and maintaining an around 30% survival rate in PQ-poisoned mice model (Algire et al., 2012), rapamycin treatment exhibiting an increased survival rate on day 2 but almost losing the efficacy on day 3 in PQ-poisoned zebrafish model (Feng, Bian, Zhang, Wang & Chen, 2019). Similarly, rapamycin attenuates but not reverses PQ-induced pulmonary fibrosis in animal models(Tai et al., 2020; Xu et al., 2017). These observations raised us to identify the molecular target for PQ-induced EMT. Our previous work has successfully revealed that STIM1 is another important molecular target of PQ for EMT in AT II cells(Yang et al., 2022). STIM1 is an essential component for extracellular calcium entry by association with either ORAI1 or TRP family members(Soboloff, Rothberg, Madesh & Gill, 2012). We found that PQ targeting STIM1 promotes the exposure of the poly-lysine (K) region in the C-terminal of STIM1, which facilitates the association between STIM1 and TRPC1 for extracellular calcium entry and intracellular calcium increase(Yang et al., 2022). Note, it has been reported that intracellular calcium burden is required for EMT in multiple cancer cells for tumor metastasis. Therefore, we speculated that intracellular calcium increase is an important signal for PF, targeting intracellular calcium burden would be a potential strategy to treat PF(Wei et al., 2021). Herein, we utilized PQ-induced EMT and PF model with combination of information from mice model, cynomolgus model, and PQ poisoned patients, to examine the precise restriction of intracellular calcium burden as a potential strategy to treat PF.