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.