Pirfenidone inhibits epithelial-mesenchymal transition and pulmonary fibrosis in the rat silicosis model

Toxicology Letters 300 (2019) 59–66

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Pirfenidone inhibits epithelial-mesenchymal transition and pulmonary fibrosis in the rat silicosis model ⁎

Jingwen Guoa,b, Zhifeng Yangb, Qiang Jiab, Cunxiang Bob, Hua Shaob, , Zhenling Zhangb, a b

T ⁎

School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan Shandong 250062, China Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Science, Jinan Shandong 250062, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Pirfenidone EMT Pulmonary fibrosis Silica

To study the role of pirfenidone in rats exposed to silica dust, we established the rat silicosis model with 50 mg/ ml silica by intratracheal instillation. From the first day after silica instillation, rats were given pirfenidone (50, 100 mg/kg/day) and rats were sacrificed at 14 days and 28 days to observe the histopathology of lungs, to analyze the level of TNF-α, IL-1β, IL-6 in lung tissues and to measure the expression of TGF-β1, Smad2/3, vimentin, and E-cadherin in lung tissues. Results showed that pirfenidone (50, 100 mg/kg/day) reduced the silica-induced alveolar inflammation, the damage of alveolar structure and the blue areas of collagen fibers in the lungs of rats. At the same time, pirfenidone also reduced the level of TNF-α, IL-1β, IL-6 in lung tissues and the protein expression of collagen I. After pirfenidone intervention for 14 days and 28 days, the protein expression of vimentin was down-regulated and the protein expression of E-cadherin was up-regulated in lung tissues. In addition, the TGF-β1/smad2/3 pathway was activated at 14 days and 28 days after silica instillation, and pirfenidone reduced the expression of TGF-β1 and smad2/3 in the lungs. These results indicated that pirfenidone intervention inhibited the epithelial-mesenchymal transition and pulmonary fibrosis in rat silicosis model, which effects may be related to the TGF-β1/smad pathway.

1. Introduction Silicosis is an interstitial pulmonary fibrosis disease caused by inhalation of crystalline silica, a component of certain types of rocks or sand (Abdelaziz et al., 2016; Yan et al., 2016). So occupations with a high risk of silicosis are usually special occupations, such as mining, clay manufacturing and stone carving (Braz et al., 2016; Yang et al., 2016b). Occupational personnel were exposed to silica dust directly or indirectly in their work environments (Liang et al., 2016). Once they are diagnosed with this disease, the fibrotic lesions of lung tissue are

almost irreversible (Fernández et al., 2015). Silica particles, ingested by the human body, can be swallowed by alveolar macrophages (Yang et al., 2016b). In turn, macrophages are activated and released some inflammatory mediators, oxygen free radicals, and fibrogenic factors, including transforming growth factor-1 (TGF-β1) (Li et al., 2013). These factors can stimulate the proliferation of lung fibroblasts, the production of collagen and eventually lead to the formation of fibrosis. The irreversible fibrosis may increase the risk of pulmonary heart disease, seriously affecting the patients’ health and quality of life (Yu et al., 2016). Therefore it is necessary to explore new effective medicines that

Abbreviations: TGF-β1, transforming growth factor-β1; EMT, epithelial-mesenchymal transition; PFD, pirfenidone; Col-I, collagen I ⁎ Corresponding author. E-mail addresses: [email protected] (H. Shao), [email protected] (Z. Zhang). https://doi.org/10.1016/j.toxlet.2018.10.019 Received 18 May 2018; Received in revised form 4 September 2018; Accepted 17 October 2018 Available online 28 October 2018 0378-4274/ © 2018 Elsevier B.V. All rights reserved.

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can alleviate silicosis fibrosis (Yan et al., 2016). Pirfenidone (5-methyl-N-phenyl-2-(1H)-pyridone, PFD) is a broadspectrum antifibrotic medicine (Sun et al., 2018; Taguchi et al., 2015). In 2014, it was approved for the treatment of the idiopathic pulmonary fibrosis (IPF) by U.S. Food and Drug Administration. In addition, various clinical and animal experimental studies have shown that PFD can also be used to treat fibrotic diseases of various organs (Li et al., 2016b). According to reports, pirfenidone reduced CCl4-induced deposition of collagen in mouse liver tissues and had significant antifibrotic effect in early liver fibrosis (Seniutkin et al., 2017). Sun et al. found that PFD inhibited the proliferation and differentiation of intestinal fibroblasts and reduced the increase of collagen I (Col-I) and α-smooth muscle actin (α-SMA) induced by TGF-β1 (Sun et al., 2018). Pirfenidone also ameliorated the fibrosis of experimental Crohn's disease by activating myofibroblast TRPA1 (Kurahara et al., 2017). In addition, Yuan et al. found that PFD regulated the Nrf2/Bach1 balance, which is associated with oxidative stress, and reduced bleomycin-induced the infiltration of inflammatory cells and lung fibrosis in mice lung tissues (Liu et al., 2017a). The results of PFD against various organs’ fibrosis provide a possibility for studying the intervention of PFD in silicosis fibrosis. Epithelial-mesenchymal transition (EMT) played an important role in the development of pulmonary fibrosis (Song et al., 2013). In the process of transition, growth factor-activated alveolar epithelial cells lost the epithelial phenotype and acquired the mesenchymal phenotype, which in turn promoted the formation of extracellular matrix and pulmonary fibrosis (Câmara and Jarai, 2010). EMT could be induced by many signaling molecules, including epidermal growth factor and transforming growth factor β1 (Wang et al., 2015). Among them, TGFβ1, a fibrogenic factor, was considered to be a crucial factor in promoting EMT and the development of pulmonary fibrosis (Akhurst and Hata, 2012; Yang et al., 2013). Inomata et al. found that administration of pirfenidone inhibited bleomycin-induced accumulation of fibrocytes and the increase of total TGF-β1 level in the lung tissues of mice (Inomata et al., 2014; Tanaka et al., 2012). In addition, pirfenidone can inhibit the increase of TGF-β1 levels and EMT process in carboplatininduced non-small cell lung cancer cell lines (Fujiwara et al., 2017). However, there are relatively few studies on the effect of pirfenidone on the EMT process in silicosis fibrosis. In this study, we established the rats silicosis model, using a tracheal perfusion method, and treated the rats with pirfenidone to elucidate the effect of PFD on EMT and antifibrotic property in silicosis.

2.3. Preparation of silica particles and induction of experimental silicosis Silica (SiO2) particles were grinded for 2 h and were prepared a 50 mg/mL standard suspension of silica dust in distilled water. Prior to tracheal instillation, silica suspension were autoclaved at 120 °C for 30 min. To perform instillation, rats were lightly anesthetized with 10% chloral hydrate and was intratracheally instilled with 1 mL this suspension by using a syringe with a plastic tube. Normal control rats received equal volume of sterilized saline. 2.4. Groups and treatments Following induction of experimental silicosis, all rats were randomly allocated to four different groups: the control group, the model group and the pirfenidone group (50, 100 mg/kg/day). Each group included 16 rats. Gastric gavage was given once daily starting from the second day after silica injection. Rats in the control group and model group were treated with 1% CMC daily, rats in the pirfenidone group were treated with 50 mg/kg or 100 mg/kg pirfenidone daily. At 14 and 28 days after medicine intervention, 8 rats in each group were deeply anesthetized using intraperitoneal injection of 10% chloral hydrate method. Afterwards, the lungs were separated and rinsed clean in ice cold saline. Left lung tissues of rats were fixed with 4% paraformaldehyde solution and right lung tissues were stored at −80 °C. 2.5. Histopathological observation Left lung tissues having been fixed with 4% formaldehyde solution were dehydrated and embedded in paraffin. The sample blocks were sliced into 4 μM and stained with hematoxylin and eosin (H&E) to observe the inflammatory infiltration and integrity of the alveolar structure under anoptical microscope (Nikon Corporation, Japan). Masson trichrome staining was used to verify silicosis induction by collagen deposition. The degree of alveolar inflammation and pulmonary fibrosis were evaluated using the Szapiel's method (Szapiel et al., 1979). 2.6. Enzyme-linked immunosorbent assay (ELISA) The right lung lobes from each rat were homogenized with a tissue homogenizer. After centrifugation of all homogenized samples at 3000 rpm at 4 °C for 10 min, the supernatant was taken and stored at −80 °C. Supernatant samples were thawed and analyzed for the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β (Thermo Fisher Technology Co., Ltd. USA) and IL-6 (Multisciences Co., Ltd. Hangzhou, China) in lung tissues using rat ELISA assay kits. All test's procedures were performed according to the manufacturers’ instructions. And the level of each cytokine in the supernatant of the lung homogenate was standardized with the protein concentration determined by Coomassie blue staining kit (Beyotime Biotechnology Co., Ltd. Shanghai, China). Results were expressed as ng or pg cytokine/mg of protein.

2. Materials and methods 2.1. Medicines and reagents Silica particles were purchased from Sigma Aldrich. Carboxymethylcellulose (CMC) was obtained from Sinopharm Chemical Reagent Limited Company (Shanghai, China). Pirfenidone was purchased from Beijing Continent Pharmaceutical Limited Company (Beijing, China) and was dissolved in 1% CMC for intragastric administration.

2.7. Immunohistochemical analysis Firstly, the tissue sections were deparaffinized, and retrieved antigen using the citrate buffer or digestive enzymes. The sections for antigen retrieval were incubated with H2O2 for 25 min to block endogenous peroxidase activity. Then, the sections were blocked with 3% bovine serum albumin (BSA) for 30 min. Lung tissue sections were incubated with primary antibodies against TGF-β1 (Abcam, USA), smad2/3 (Boster Biological Technology Co., Ltd. China), vimentin (Cell Signaling Technology, USA) or Col-I (Wuhan Goodbio Technology Limited Company, Wuhan, China) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP) labeled secondary antibody in 37 °C for 50 min. The sections were visualized by DAB and counterstained with hematoxylin for 3 min. Finally, images were

2.2. Animals Forty male Sprague Dawley rats, weighting 200–230 g, were purchased from Beijing Vital River Laboratory Animal Technology Limited Company (Beijing, China). Rats were housed in air-conditioned room and acclimated for 1 week at 23 ± 3 °C and humidity (40–70%) with a regular 12 h light/12 h dark cycle before the experiments. And rats were permitted free access to food and water. All animal experimental procedures were approved by the Animal Experimentation Ethics Committee of the Shandong academy of medical sciences. All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 60

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acquired by microscopy for analysis.

Table 1 Alveolar inflammation and pulmonary fibrosis score of lung tissues in different groups.

2.8. Western blot analysis

Group

Upper lobe of right lung was homogenized and then was centrifuged at 12 000 rpm for 10 min to collect the supernatant. The supernatant was collected and protein content determined with a protein assay kit. The protein concentration was determined using a BCA protein assay kit (Wuhan Goodbio Technology Limited Company, Wuhan, China). Following, equal amounts of proteins from the right lung in each groups were separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes (Millipore, MA, USA). Membranes were blocked with 5% fat-free milk and incubated overnight at 4 °C with the primary antibody, namely TGF-β1 (Abcam, USA), smad2/3, E-cadherin (E-cad), vimentin (Cell Signaling Technology, USA) and Col-I (Servicebio, Wuhan, China), followed by alkaline phosphatase-conjugated secondary antibodies (Servicebio, Wuhan, China) for 30 min at 37 °C. Subsequently, the blots were visualized by developing reagents. The optical density of the target bands was analyzed with Alpha software.

Control Model 50 mg/kg PFD 100 mg/ kg PFD a b

Day 14

Day 28

Alveolar inflammation

Pulmonary fibrosis

Alveolar inflammation

Pulmonary fibrosis

0 2.60 ± 0.55a 1.40 ± 0.55b

0 2.20 ± 0.84a 1.20 ± 0.45b

0 2.40 ± 0.89a 1.20 ± 0.84b

0 2.40 ± 0.55a 1.60 ± 0.89b

1.00 ± 0.71b

1.00 ± 0.00b

0.80 ± 0.45b

1.20 ± 0.45b

P < 0.05 vs. control group. P < 0.05 vs. model group.

model group. These results indicated that pirfenidone intervention effectively attenuated silica-induced alveolar inflammation in rats (Table 1). 3.2. Effect of PFD on the level of TNF-α, IL-1β and IL-6 in lung tissues

2.9. Statistical analysis To further investigate inflammatory changes in the lungs, we measured the level of three representative pro-inflammatory cytokines TNFα, IL-1β and IL-6 in lung tissues by ELISA assay kits. As shown in Fig. 2, ELISA showed that TNF-α, IL-1β and IL-6 level in lung tissues was obviously increased on the 14th and 28th days after crystalline silica instillation. However, TNF-α level in lung tissues was decreased by PFD intervention, particularly at day 14. And the administration of 50 and 100 mg/kg PFD also caused significant suppression of tissue level of IL1β and IL-6 compared with those in the model group. This suggested that PFD inhibited the up-regulated effects of inflammation-related factors in rat lung tissues induced by silica, thereby attenuating the alveolar inflammation.

In this study, the SPSS 22.0 software was used to evaluate statistical significance. Differences between groups were evaluated by one-way ANOVA followed by LSD-test. All data are expressed as mean ± SEM, and P < 0.05 was considered statistically significant. 3. Results 3.1. Lung morphometry and inflammation HE staining (Fig. 1) revealed that the lungs showed ideal lung architecture and thin alveolar septum without inflammation in the control group. Lung specimen of the model group revealed severe inflammatory reaction with significant increase of alveolar septal thickness and the infiltration of neutrophils, mononuclear cells and mainly macrophages around the bronchial and alveolar interstitial. In contrast, pirfenidone intervention for 14 days and 28 days significantly relieved the alveolar inflammation and improved the alveolar structure. And based on the Szapiel's method, the statistical classification of alveolar inflammation showed that the alveolar inflammation score of the model group was significantly higher than that of the control group. However, after 14 and 28 days of pirfenidone intervention, the alveolar inflammation score of the pirfenidone group was lower than that of the

3.3. Lung fibrosis As shown in Fig. 3, Masson staining revealed that marked lung fibrosis was observed following exposure of the lungs to silica. The collagen deposition (blue areas) was significantly higher in model group compared with the control group. However, pirfenidone intervention reduced cellular nodules and the inflammation, as well as the accumulation of collagenous fibers. And based on the Szapiel's method, the statistical classification of pulmonary fibrosis showed that the pulmonary fibrosis score of the model group was significantly higher than

Fig. 1. HE staining in rat lungs after intervention for 14 days and 28 days (×200). 61

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Fig. 2. ELISA of TNF-α (A), IL-1β (B) and IL-6 (C) in rat lungs after pirfenidone intervention for 14 days and 28 days. Results showed pirfenidone (50, 100 mg/kg/ day) inhibited the increase of TNF-α, IL-1β and IL-6 in rat lung tissues induced by silica. *P < 0.05 vs. control group, #P < 0.05 vs. model group.

that of the control group. The pulmonary fibrosis score was lower in the pirfenidone group compared to the model group. The results indicated that pirfenidone intervention for 14 and 28 days effectively reduced the degree of silica-induced pulmonary fibrosis in rats (Table 1). Besides, the lung fibrosis can be evidenced by increased collagen I deposition in the lung tissues, so we detect the effect of pirfenidone on expression of collagen type I by western blot. Results showed, compared with the control animals, SiO2 stimulation increased the protein expression of collagen I, but this increase was attenuated by pirfenidone administration (Fig. 4).

in fibrotic lung disease. As shown in C of Fig. 5, silica inhalation could increase the protein expression of TGF-β1 and its downstream smad2/3 in lungs, while PFD intervention for 14 days and 28 days significantly decreased TGF-β1 and smad2/3 protein level. Results showed that PFD undermined the effect of silica on increasing the level of TGF-β1 and smad2/3 and PFD decreased the level of Smad2/3 by reducing the protein expression of TGF-β1. Besides, immunohistochemistry confirmed similar results (Fig. 5A and B).

3.4. Effect of PFD on the expression of TGF-β1 and smad2/3

Epithelial-mesenchymal transition (EMT) is the important approach to the formation of silicosis fibrosis. EMT can stimulate fibroblast proliferation and the production of collagen protein and promote ulteriorly the progress of pulmonary fibrosis. So we detected the expression of the epithelial cells marker E-cadherin and the acquisition of mesenchymal cells marker vimentin in the rat silicosis model with or

3.5. Effect of PFD on the expression of E-cad and vimentin

To further investigate the antifibrotic effect of PFD on the silicosis rats, the western blot was used to evaluate the expression of fibrosis related cytokines in lung tissues. TGF-β1 as the most important profibrotic cytokine, the signals relative to TGF-β1 are commonly activated

Fig. 3. Masson staining in rat lungs after intervention for 14 days and 28 days (×200). 62

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Fig. 4. Intervention of pirfenidone inhibited silica-induced Col-I expression in rat lungs. Western blot showed silica increased the protein expression of Col-I in the lungs compared with the control group, pirfenidone (50, 100 mg/kg/day) intervention for 14 days and 28 days inhibited Col-I expression. *P < 0.05 vs. control group, #P < 0.05 vs. model group.

tissues and further impairment of lung function (Leung et al., 2012; Greenberg et al., 2007). Therefore, to elucidate the anti-inflammatory and anti-fibrotic efficacy of PFD, we first determine if the silicosis model was successfully constructed by analyzing the lung histopathological changes of the control and model groups from H&E and Masson stains. Compared with the control group, there were a lot of inflammatory cells infiltration and damage of alveolar structure in the lung tissues of the model group from H&E staining. At the same time, Masson staining revealed that cell nodules and collagen fibers were evident in the model group. All of these indicators confirmed that the rat silicosis model successfully replicated. Excessive deposition of collagen synthesized and secreted by fibroblasts can lead to the formation of pulmonary fibrosis (Sun et al., 2010). In this study, the protein expression of type I collagen was significantly increased in the model group and it further confirmed the formation of silicosis fibrosis. However, these changes were significantly inhibited by the intervention of PFD for 14 days and 28 days. The above data were consistent with the reported literature, which showed that pirfenidone reduced overexpressed collagen I in radiation-induced intestinal fibrosis in rats (Sun et al., 2018). In addition, PFD inhibited the level of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in lung tissues. These findings suggested the potential anti-inflammatory and antifibrotic function of PFD in the rat model of silicosis. To further investigate the antifibrotic effect of PFD, we studied the epithelial-mesenchymal transition (EMT) process of rat lung tissues. According to reports, EMT is assumed to be a potential source of fibroblasts and an important link in fibrotic diseases such as heart and kidney fibrosis (Yang et al., 2016a). EMT is also one of the important mechanisms of pulmonary fibrosis (Wang et al., 2015). The loss of the

without pirfenidone intervention. Western blot results indicated that the protein expression of E-cad was decreased and vimentin were increased in the model group. Pirfenidone intervention for 14 days and 28 days attenuated the downregulation of E-cadherin and reversed the upregulation of vimentin induced by silica (Fig. 6B). Immunohistochemical results were similar to the western blot (Fig. 6A), pirfenidone significantly decreased the expression of vimentin in the lung tissues. These suggested that PFD intervention groups could inhibit the process of EMT and the progression of fibrosis. 4. Discussion Silicosis is a health problem all around the world, especially in developing countries (Yan et al., 2016). It is an occupational respiratory disorder characterized by lung inflammation and progressive fibrosis, which has significant and harmful effects on the patients’ health and quality of life (Abdelaziz et al., 2016; Li et al., 2016a). But it is difficult to find an effective treatment at the present stage. Pirfenidone has been approved of treating the IPF. In addition, PFD has been shown to have significant anti-inflammatory and anti-fibrotic properties in fibrotic models such as heart, kidney and liver (Oku et al., 2008; Schaefer et al., 2011). We found that PFD had the same effect on SiO2-induced pulmonary fibrosis in rats. In this study, intratracheal administration of SiO2 was used to establish the rat model of silicosis fibrosis in vivo and showed that PFD intervention alleviated SiO2-induced inflammation and fibrosis in the lungs. Silicosis is mainly caused by long-term inhalation of silica dust in occupational environment, which induces the activation and infiltration of inflammatory cells, the formation of silicosis nodules in lung 63

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Fig. 5. Immunohistochemical of TGF-β1 (A) and smad2/3 (B) in rat lungs after pirfenidone intervention for 14 days and 28 days (×400), and Western blot (C) was analyzed the protein expression of TGF-β1 and smad2/3. Results showed silica exposure upregulated the protein expression of TGF-β1 and smad2/3 in the lungs compared with the control group, pirfenidone (50, 100 mg/kg/day) downregulated TGF-β1 and smad2/3 expression. *P < 0.05 vs. control group, #P < 0.05 vs. model group.

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Fig. 6. Immunohistochemical of vimentin (A) in rat lungs after pirfenidone intervention for 14 days and 28 days (×400), and Western blot (B) was analyzed the protein expression of E-cad and vimentin. Results showed silica exposure upregulated the expression of vimentin and downregulated the expression of E-cad in the lungs compared with the control group, pirfenidone (50, 100 mg/kg/day) inhibited the increase of Vimentin and the decrease of E-cad. *P < 0.05 vs. control group, # P < 0.05 vs. model group.

effects of PFD in the rat model of silicosis and then we used immunohistochemistry and Western blot methods to analyze the localization and protein expression of E-cad and vimentin in rat lung tissues. The results showed that the protein expression of vimentin was significantly down-regulated after PFD intervention for 14 days and 28 days, and accordingly, E-cadherin expression was up-regulated. This study demonstrated that PFD can inhibit SiO2-induced EMT in the rat model of silicosis fibrosis. The results were similar to those of Zhang et al., which showed pirfenidone attenuated epithelial-mesenchymal transition and pulmonary fibrosis in a rat model of bleomycin-induced pulmonary fibrosis (Zhang et al., 2018).

epithelial cell phenotype and acquisition of mesenchymal cell characteristics are the main feature of EMT, which contributes to the development of silicosis (Deng et al., 2016). In this study, exposure to silica reduced the expression of the epithelial marker E-cadherin and increased the expression of the mesenchymal marker vimentin in the lung tissues of rats. The result indicated that EMT was occurred in vivo in the rat model of pulmonary fibrosis induced by silica. Studies have demonstrated that inhibition of abnormal EMT is a favorable strategy for lung fibrosis (Tang et al., 2015). However, whether PFD inhibits silica-induced EMT in this experiment was still required to further study. We hypothesized that EMT was involved in the antifibrotic 65

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TGF-β1, the profibrotic cytokine, can induce EMT in alveolar epithelial cells and endothelial cells through various signaling pathways, which thereby stimulates lung fibroblast proliferation and collagen synthesis (Yan et al., 2016; Wang et al., 2015; Sun et al., 2010). Among them, Smad2/3 is the first downstream signaling molecule of TGF-β1 signaling, and increasing evidences indicate that Smad2/3 is widely activated in fibrotic diseases and animal experiments, regulating a variety of genes including α-SMA and Col-I (Liu et al., 2017b). Kyungsun et al. found that pirfenidone inhibited TGF-β1 signaling by preventing nuclear accumulation of the active Smad2/3 complex in the human retinal pigment epithelial cell line ARPE-19 (Kyungsun et al., 2012). To elucidate the potential mechanism of PFD to inhibit silicainduced EMT, we further analyzed the protein expression of TGF-β1 and Smad2/3 in rat lung tissues. It was found that the protein levels of TGF-β1 and Smad2/3 in the model group were significantly higher than those in the control group at weeks 2 and 4 after stimulation with silica, indicating that the TGF-β1/Smad2/3 pathway may be involved in the silicosis model. However, the administration of pirfenidone inhibited the protein expression of TGF-β1 and Smad2/3 in lung tissues of rats. This study suggested that the inhibitory effect of PFD on SiO2-induced EMT in rat lung tissues may be related to the TGF-β1/Smad2/3 pathway.

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5. Conclusion In summary, the present study suggests that pirfenidone can inhibit silica-induced EMT and the formation of pulmonary fibrosis in vivo, which may be related to the regulation of the TGF-β1/Smad2/3 signaling pathway. These findings suggested possible mechanisms by which PFD intervention on silica-induced pulmonary fibrosis in rats and provided a basis for PFD to be useful medicine in treating silicosis fibrosis in the future. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by grants from the Shandong Academy of Medical Science Medical Health Science and Technology Innovation Project and 2018 Shandong Public Welfare Technology Public Relations Project (2018GSF118102). I would like to express my gratitude for the guidance of Professor Zhenling Zhang, my supervisor and Professor Hua Shao, and the technical assistance of teachers: Zhifeng Yang, Qiang Jia, Cunxiang Bo. References Abdelaziz, R.R., Elkashef, W.F., Said, E., 2016. Tadalafil reduces airway hyperactivity and protects against lung and respiratory airways dysfunction in a rat model of silicosis. Int. Immunopharmacol. 40, 530–541. Akhurst, R.J., Hata, A., 2012. Targeting the TGFβ signalling pathway in disease. Nat. Rev. Drug. Discov. 11 (10), 790–811. Braz, N.F., Carneiro, A.P., Avelar, N.C., Miranda, A.S., Lacerda, A.C., Teixeira, M.M., Teixeira, A.L., Mendonça, V.A., 2016. Influence of cytokines and soluble receptors in the quality of life and functional capacity of workers exposed to silica. J. Occup. Environ. Med. 58 (3), 272–276. Câmara, J., Jarai, G., 2010. Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-α. Fibrogenesis Tissue Repair 3 (1), 2. Deng, H., Xu, H., Zhang, X., Sun, Y., Wang, R., Brann, D., Yang, F., 2016. Protective effect of Ac-SDKP on alveolar epithelial cells through inhibition of EMT via TGF-β1/ROCK1 pathway in silicosis in rat. Toxicol. Appl. Pharmacol. 294, 1–10. Fernández, Álvarez, R., Martínez, González, C., Quero, Martínez, A., Blanco, Pérez, J.J., Carazo, Fernández, L., Prieto, Fernández, A., 2015. Guidelines for the diagnosis and monitoring of silicosis. Arch. Bronconeumol. 51 (2), 86–93. Fujiwara, A., Shintani, Y., Funaki, S., Kawamura, T., Kimura, T., Minami, M., Okumura, M., 2017. Pirfenidone plays a biphasic role in inhibition of epithelial-mesenchymal transition in non-small cell lung cancer. Lung Cancer 106, 8–16. Greenberg, M.I., Waksman, J., Curtis, J., 2007. Silicosis: a review. Dis. Month 53 (8),

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