Silicosis is a progressive, irreversible, and often fatal occupational disease caused by the inhalation, deposition, and retention of silica particles. The pathogenesis of silicosis has not been fully elucidated and therapeutic interventions are still limited due to inadequate basic knowledge [2]. Few effective drugs for silicosis are currently in clinical development. BIC derived from the traditional Chinese medicine (TCM) Schisandra chinensis (Wuweizi) of North, is an approved hepatoprotective drug with well-characterized safety [9]. Recent studies on different fibrotic animal models for example, bile duct ligation or dimethylnitrosamine-induced liver fibrosis [31, 32], renal interstitial fibrosis induced by unilateral ureteral obstruction [33] and bleomycin-induced idiopathic pulmonary fibrosis (IPF) [34] have demonstrated the efficacy of BIC against fibrosis. As expected, BIC exerted a significant therapeutic effect on SiO2-induced pulmonary fibrosis in the aspects of improvement of pulmonary function, attenuation of inflammation and slowing down progression of fibrosis in the current study, supporting its efficacy as a potent therapeutic agent for silicosis.
Impairment of pulmonary function increases with progression of silicosis, even after the patient is no longer exposed to silica [35]. In the early stages of silicosis, patients may display no abnormalities in pulmonary function. With disease progression, architectural distortion leads to deterioration of pulmonary function [36]. Silicosis patients with poor pulmonary function have a lower quality of life and higher risk of mortality than the patient groups with better pulmonary function [37]. Thus, PFTs were employed as a means to assess the efficacy of BIC in addition to pathological staining. Items indicating lung compliance, such as Cst, or expiratory capacity, such as FEV/FVC, FEF and TPEF, were abnormal in the SiO2 group, compared with the control group, suggestive of reduced pulmonary function in silicosis rats. All the parameters were recovered to varying extents in silicosis rats after BIC treatment, associated with reduction of fibrosis in lungs, as detected with pathological staining. While abnormalities of pulmonary function parameters in silicosis could be either obstructive, restrictive or mixed and not specific for assessment of the therapeutic effects of BIC, improvements in pulmonary function could still be effectively used as an indicator of the efficacy of BIC.
Inflammation is considered the first stage of silicosis. Silica particles enter the lungs through the respiratory tract, where they continuously stimulate AMs to release substantial amounts of inflammatory factors (IL-1β) and induce apoptosis [20]. Damaged AMs and particles that are not eliminated in a timely manner accumulate and provoke an immune response, which triggers proliferation of neutrophils, macrophages and lymphocytes, as well as generation of TGF-β1, TNF-α, and IL-6 [4]. These cells and cytokines further aggravate the inflammatory response and promote fibrotic progression. Hence, it is possible to delay silicosis development through suppression of inflammation. Our data showed that BIC exerted anti-inflammatory effects in response to silica particles in many respects. Specifically, BIC protected macrophages against polarization and apoptosis and reduced inflammatory cell aggregation and infiltration into lungs. On the other hand, BIC suppressed the expression of IL-1β, IL-6, TGF-β1, and TNF-α, which contribute to amplification of the inflammation reaction and activation of myofibroblasts. Suppression of inflammation by BIC ultimately slowed down fibrosis progression.
TGF-β1 has been identified as a key inflammatory cytokine. The predominant role of TGF-β1 in silicosis is the promotion of fibroblast proliferation, myofibroblast differentiation, and collagen synthesis through autocrine and paracrine mechanisms [38]. BIC is reported to inhibit the expression of TGF-β1 in liver to reduce fibrosis [32, 39]. Coincident with earlier findings, BIC suppressed TGF-β1 expression in BALF, serum, and lung tissues in our silicosis rat model, while the three other cytokines examined (IL-1β, IL-6, and TNF-α) were only decreased in BALF and lung tissue and not in serum. Moreover, TGF-β1 was the only inflammatory cytokine that induced both EMT and FMT processes in vitro, similar to that observed with the supernatant of SiO2-stimulated RAW264.7 cells. Notably, in our study, TGF-β1 failed to stimulate inactive macrophages directly as observed with SiO2 particles, indicating that TGF-β1 promotes pulmonary fibrosis induced by SiO2 rather than initiating inflammatory progression. Thus, other initiation mechanisms that are stimulated by SiO2 to induce macrophage production of TGF-β1 may exist. Importantly, BIC could block both EMT and FMT processes induced by either direct addition of TGF-β1 or the supernatant containing TGF-β1 in a dose-dependent manner, suggesting that TGF-β1 could serve as a therapeutic target of BIC against silicosis.
TGF-β1 promotes phosphorylation of the signal transducer proteins SMAD2/3, following which phosphorylated SMAD2/3 forms a heterotrimeric complex with SMAD4 and translocates into the nucleus. The complex binds a consensus sequence and regulates fibrotic gene transcription, which is described as a canonical (SMAD-dependent) pathway [29]. Activation of canonical signaling induces loss of phenotype of epithelial cells and EMT, which is characterized by low expression of epithelial markers (E-cadherin) and high expression of mesenchymal markers (α-SMA) [40], leading to significantly increased production of ECM components [41]. In our experiments, BIC blocked phosphorylation or nuclear translocation of SMAD2/3 in TC-1 cells co-cultured with SiO2-stimulated macrophages or TGF-β1, indicating that inhibition of the canonical TGF-β1 signaling pathway in lung epithelial cells to suppress EMT may be a key strategy of BIC to reduce fibrosis.
In addition to the canonical signaling pathway, TGF-β1 activates a variety of SMAD-independent pathways (known as non-canonical signaling) to modify cell function. These non-SMAD pathways include MAPK, PI3K/AKT and Rho-like GTPase signaling [6, 42]. In fibroblasts and fibrotic diseases, such as systemic sclerosis and CCl4-induced liver fibrosis, another SMAD-independent TGF-β1 activation pathway has been described that activates JAK2 and STAT3 [43–46]. Abnormalities of the JAK/STAT signaling pathway play important roles in both cancer progression and inflammatory and autoimmune diseases [47–49]. Inhibition of both p-JAK2 and p-STAT3 protected lung fibroblasts from undergoing FMT in IPF [43, 50]. Similarly, in our silicosis model, both JAK2 and STAT3 were activated by TGF-β1 in fibroblasts, but not the other three cytokines, leading to promotion of the FMT process and wound healing. Our findings indicate that FMT in fibroblasts driven by JAK2/STAT3 signaling could present another pathway for accumulation of ECM. Consistently, upon BIC-mediated down-regulation of phosphorylated JAK2 and STAT3 in silicosis rat lung and NIH-3T3 cells via suppression of TGF-β1 secretion, ECM deposition was markedly decreased in both tissues and cultured cells, indicating that fibroblast activation and FMT via TGF-β1/JAK2/STAT3 signaling may serve as another mechanism by which BIC targets TGF-β1 to exert therapeutic effects against silicosis.
Other than driving FMT, the JAK2/STAT3 signaling pathway is significantly activated under conditions of interactions of a range of profibrotic/pro-inflammatory cytokines during physiological and pathological processes to regulate the inflammatory response [23, 43, 51]. In our study, significant BIC-induced inhibition of the phosphorylation levels of both JAK2 and STAT3 was observed in lung tissues of the silicosis rat model and macrophages in vitro, leading to down-regulation of SOCS3 in a cascade of diminished secretion of inflammatory cytokines, such as TNF-α, IL-1β and IL-6 [33, 34], as well as negative feedback regulation of JAK2 phosphorylation [32]. However, single cytokines, including TGF-β1, failed to activate this signaling pathway in non-stimulated macrophages as observed in fibroblasts in vitro, indicating that activation of JAK2/STAT3 signaling in macrophages presents a means to amplify rather than initiate the inflammatory response. Hence, inactivation of JAK2/STAT3 signaling in macrophages may not be the only mechanism by which BIC reduces inflammation during silicosis.
Two types of cellular transformation processes, EMT and FMT, are characteristic of fibrotic diseases, including silicosis. The two main components of lung tissues, epithelial cells and fibroblasts, undergo EMT and FMT, respectively, in response to inflammatory stimulation by silica particles at the fibrosis stage of silicosis. Consequently, alveolar epithelial cells lose their epithelial phenotype. Meanwhile, fibroblasts activate and proliferate abnormally, and subsequently lose their differentiation ability and acquire the mesenchymal phenotype of myofibroblasts, ultimately exacerbating the synthesis and abnormal deposition of ECM in damaged lungs. Therefore, suppression of abnormal ECM is another goal of silicosis therapy in addition to reducing the inflammatory response. In our study, BIC down-regulated α-SMA in epithelial cells and fibroblasts and up-regulated E-cadherin in both in vivo and in vitro silicosis models, leading to reduced synthesis of ECM components, indicating that inhibition of EMT and FMT processes by BIC could present other modes of action against silicosis, rather than the simple subsequent response of inflammation blockade.
In summary, our findings highlight the significant therapeutic effects of BIC against silicosis and provide valuable insights into the underlying molecular mechanisms. BIC attenuated SiO2-induced inflammation via acting on macrophages, leading to reduced secretion of inflammatory cytokines, in particular, TGF-β1. During the fibrotic stage, BIC targeted TGF-β1 to block both canonical and non-canonical signaling pathways in epithelial cells and fibroblasts and inhibited the EMT and FMT processes, followed by suppression of the synthesis and deposition of ECM components, ultimately resulting in delayed fibrosis progression (Fig. 10). Our collective findings provide an experimental basis for the clinical application of BIC in silicosis.