Introduction
Pulmonary fibrosis diseases are fatal, chronic, progressive, and fibrotic interstitial lung diseases, which are characterized by immune cells recruitment, fibroblasts activation and proliferation, as well as crucially excessive accumulation of extracellular matrix (ECM). Typically, pulmonary fibrosis diseases are comprised of the most common idiopathic pulmonary fibrosis (IPF) with unknown origin and typical pneumoconiosis with definitive etiology. IPF assumes a paramount significance in idiopathic interstitial lung diseases accompanied by a very poor prognosis with a median survival of 2 to 3 years (King et al., 2011). Alternatively, pneumoconiosis, especially silicosis is the most pivotal occupational diseases worldwide, caused by long-term inhalation of dust particles during the working (Leung et al., 2012). Nowadays, despite an unclear mechanistic basis for this dogged advancement of pulmonary diseases, pirfenidone (PFD) and nintedanib (BIBF) (approved in Europe in 2011 and 2015 respectively) were invented to slow the progression of IPF in clinical trials (Noble et al., 2011; Richeldi et al., 2011). Regrettably, there exists no curative treatment for pneumoconiosis exclusive of lung transplantation. Consequently, the quest for pharmacological interventions capable of mitigating or even eradicating pneumoconiosis stands as the utmost critical and pressing imperative at present.
The emergence of PFD and BIBF has partly bridged the gap in anti-fibrotic medications. PFD was evaluated by three multi-national, randomized, placebo-controlled, phase III clinical trials (King et al., 2014; Noble et al., 2011), and identified as an effective agent that improved lung function decline, extended progression-free survival, and decreased death rates over 12 months (Collins & Raghu, 2019; Nathan et al., 2017; Nathan et al., 2019; Noble et al., 2016; Paterniti et al., 2017). Whereas initially applied to counteract inflammation through diminishing the production of cytokines and infiltration of immune cells (Bizargity et al., 2012; Gurujeyalakshmi et al., 1999; Hirano et al., 2006; Spond et al., 2003; Toda et al., 2018; Visner et al., 2009), PFD has been widely considered as an anti-fibrotic agent due to its role of suppressing fibrogenic growth factors to attenuate deposition of ECM (Conte et al., 2014; Ma et al., 2018; Molina-Molina et al., 2018; Qin et al., 2018). Additionally, safety and efficacy of BIBF were also assessed in the clinical trials, demonstrating the benefit of BIBF versus placebo in disease progression, time to first exacerbation, and treatment mortality (Richeldi et al., 2016; Richeldi et al., 2014). BIBF is an oral tyrosine kinase inhibitor mainly targeting fibroblast growth factor receptor (FGFR)-1, vascular endothelial growth factor receptor (VEFGR)-2, and platelet-derived growth factor receptor (PDGFR)-α and β(Wollin et al., 2015). Overall, PFD and BIBF exert anti-inflammatory and anti-fibrotic effects in the treatment of IPF, although they have different emphases and impact distinct signaling pathways. Given a host of congruencies are exhibited between IPF and pneumoconiosis at symptoms, etiology, and pathogenesis, PFD and BIBF may be also effective in treating pneumoconiosis. Meanwhile, the divergences prompted us to further explore the concrete molecular mechanisms specifically respective to pneumoconiosis. Moreover, further investigation is warranted to determine the feasibility of combined administration in pneumoconiosis treatment.
Recently, we have entered the era of multi-omics, wherein the integration of transcriptomics, proteomics, and metabolomics allows for a multi-level and multi-dimensional deciphering the overarching landscape of gene regulation. In this study, we established a murine model of advanced silicosis with severe fibrosis and administered different doses of monotherapy (PFD or BIBF) and combined therapy with PFD and BIBF regimens to evaluate the safety and efficacy, indicated by lung function, inflammation, and fibrosis. Subsequently, we employed transcriptomics and metabolomics to elucidate the commonalities and divergences in the mechanisms of actions of PFD and BIBF in the treatment of silicosis. Our study, on the whole, not only presents novel treatment strategies for pneumoconiosis but also contributes to a better understanding of the mechanisms underlying PFD and BIBF in treating pneumoconiosis, aiming to rectify the current clinical predicament of limited therapeutic options for this disease.
Methods and materials
Main reagents
The crystalline silica particles were purchased from Forsman Technology (China Beijing) Co., Ltd. (CAS7631-86-9; 99% purity), and the average particle size of them was 1.6 μm. The particulate endotoxin was removed by baking at 180°C for at least 2 h and then naturally cooled. Before use, the silica suspension was prepared by suspending in sterile phosphate buffer (PBS) at a concentration of 300 mg mL-1. Beijing Continental Pharmaceutical Co., Ltd. (Beijing, China) provided PFD for suspension in 1% sodium carboxymethylcellulose (CMC-Na, 419273, Sigma-Aldrich, St. Louis, Missouri, USA). BIBF was purchased from Boehringer Ingelheim, Germany and suspended in 1% CMC-Na. It should be noted that BIBF is difficult to dissolve, so it is best to slowly add BIBF powder and gradually mix it well. All the suspensions were sonicated for at least 30 min before use.