SB203580 inhibits epithelial–mesenchymal transition and pulmonary fibrosis in a rat silicosis model.

Author: Wang Yan Li Xiaoli An Guoliang Zhu Zhonghui Liang Di Lian Ximeng Niu Piye Chen Li Tian Lin

PII: S0378-4274(16)32954-X
DOI: http://dx.doi.org/doi:10.1016/j.toxlet.2016.07.591
Reference: TOXLET 9552

To appear in: Toxicology Letters
Received date: 21-4-2016
Revised date: 21-7-2016
Accepted date: 25-7-2016
Please cite this article as: Yan, Wang, Xiaoli, Li, Guoliang, An, Zhonghui, Zhu, Di, Liang, Ximeng, Lian, Piye, Niu, Li, Chen, Lin, Tian, SB203580 inhibits epithelial–mesenchymal transition and pulmonary fibrosis in a rat silicosis model.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.07.591
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SB203580 inhibits epithelial–mesenchymal transition and pulmonary fibrosis in a rat silicosis model.

Wang Yan1, 2* Li Xiaoli1, 2* An Guoliang1, 2 Zhu Zhonghui1, 2 Liang Di1, 2

Lian Ximeng1, 2 Niu Piye1, 2 Chen Li1, 2 Tian Lin1, 2

1Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China; 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University,Beijing 100069, China.

*These authors contributed equally to this work. Electronic address: [email protected].
Corresponding author: Tian Lin. Address: Department of Occupational and Environmental Health, School of Public Health, Capital Medical University, No. 10, Xi toutiao outside You anmen Street, Beijing, 100069, People′s Republic of China. Fax: +86(10)-83911506

Graphical abstract

p38 MAPK was pivotal for EMT in silicosis fibrosis in rats. At 7 days after silica instillation, p38 MAPK/ZEB-1 (ZEB-2, Twist) was involved in EMT and pulmonary fibrosis, while SB203580 could inhibit the activity of p38 MAPK/ZEB-1 (ZEB-2, Twist) pathway by restraining the phosphorylation of MK-2.

Highlights

⦁ We found silica induced EMT and the activity of p38 MAPK in vivo.

⦁ SB203580 could inhibit EMT and pulmonary fibrosis in silicosis model.
⦁ p38 MAPK/ZEB-1 (ZEB-2, Twist) was involved at 7 days after silica

instillation.

Abstract To investigate the role of p38 MAPK in silicosis, we explored the effects of SB203580 as a specific inhibitor of p38 MAPK in the silicosis model in rats. Rats were exposed to 50mg/ml silica intratracheally. From the first day after instillation, rats were injected with SB203580 1mg/kg/d. Rats were sacrificed at 7 and 15 days after exposure of silica. The results demonstrated SB203580 could prevent the activation of p38. TGF-β1 in bronchoalveolar lavage fluid, the expression of vimentin and α-SMA in the lung tissue was down-regulated and
E-cadherin was up-regulated after intervention with SB203580 at 7 days and 15 days. The percentage of the cells staining with SP-C and vimentin doubly was lower in SB203580 treated group than in silica group at 7 days and 15days. SB203580 also inhibited the increase of ZEB-1, ZEB-2 and Twist at 7 days. Histopathologic examination showed the decrease in the number of nodules and the blue areas of collagen fibers in the lung after SB203580 treatment. The content of hydroxyproline and the expression of collagen Ⅰ and Ⅲ decreased in SB203580 treated group
than in silica group. These results suggested that p38 MAPK/ZEB-1

(ZEB-2, Twist) pathway was involved at 7 days after silica instillation and p38 MAPK was pivotal for EMT in silicosis fibrosis in rats.

Abbreviations p38 MAPK, p38 mitogen-activated protein kinase; EMT, epithelial-mesenchymal transition; TGF-β, transforming growth factor beta; MK-2, MAPKAPK-2;

Keywords: p38 MAPK; SB203580; EMT; pulmonary fibrosis; silica

⦁ Introduction

Silicosis, induced by the inhalation and deposition of silica particles, is a kind of interstitial pulmonary fibrosis disease(Leung et al., 2012). It usually occurs in people working in the environments where crystalline silica is fractured or smashed by machines(Ziskind et al., 1976). Although efforts to prevent silicosis have been made for many years, silicosis is still a problem around the world, especially in developing countries(Greenberg et al., 2007). So it is necessary to explore the pathogenesis of silicosis and find effective approaches to alleviate silicosis fibrosis.
A number of studies have indicated that the damage of alveolar epithelial cells followed by a diffuse inflammatory response and epithelial–mesenchymal transition (EMT) are involved in the pathogenesis of pulmonary fibrosis(Tanjore et al., 2009). Many previous investigations have found EMT occurred in pathological tissue fibrosis and that myofibroblasts may be directly derived from alveolar epithelial

cells(Vaughan and Chapman, 2013). However, the mechanisms inducing EMT and subsequent progression of lung fibrosis have not been fully clarified. Accumulating evidence about pulmonary fibrosis has proved that persistent activation of MAPK pathway is related with recruitment of fibroblast cells and deposition of extracellular matrix. MAPK pathway can be activated in response to different extracellular stimuli, such as cytokines, growth factors, oxidants stress and hormones. p38 MAPK is one of the MAPK members and was found to be involved in EMT induced by TGF-β in alveolar epithelial cells(Chen et al., 2013). Some scholars found that the inhibition of p38 MAPK could inhibit
radiation-induced fibroblast differentiation(Park et al., 2010). p38 MAPK was also found to be involved in bleomycin-induced EMT and fibrosis(Guo et al., 2015). In our previous study, we have detected p38 MAPK inhibitor could inhibit EMT induced by silica in A549 cells(Wang et al., 2015).
The aim of this study was to investigate the role of p38 MAPK on early pulmonary EMT and subsequent fibrosis in the process of silicosis. To explore the role of p38 MAPK pathway in the development of silicosis fibrosis, we used SB203580, a specific p38 MAPK pathway inhibitor, to block the activation of p38 MAPK. Our results showed that the inhibition of p38 MAPK pathway with SB203580 alleviated early pulmonary EMT response and fibrosis in the lungs. We demonstrated that p38 MAPK

pathway was involved in silica-induced EMT and fibrosis.

⦁ Material

⦁ Silica

Crystalline silica was provided by the Center of Occupational Health and Poisoning Control, Chinese Center for Diseases Control and Prevention (Beijing, China). Approximately 95% of particle diameter was below 5 µm and the silica content was >99%.The samples were weighed and suspended in saline (50 mg/ml) and autoclaved to sterilize. Before use the silica dust was supplied with final concentration of 5000 IU/ml penicillin.
⦁ Rats

Adult male Wistar rats, weighing 200–240 g, were provided by the Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All procedures involving animals were approved by the Laboratory Animal Care and Use Committee at Capital Medical University. All animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Rats were housed under a 12 h light/dark cycle with free water access and commercial diet.
⦁ Experimental design

A total of 80 adult rats were classified into control group (n=16), 1%DMSO group (n=16), SB203580 group (n=16), silica group (n=16)

and silica+SB203580 group (n=16). The rats were anesthetized with ether and administered with silica suspension 1ml intra-tracheally except for control, 1%DMSO and SB203580 group which received 1ml of saline instead. We selected 1 mg/kg/d as the dose by reviewing relevant literatures of SB203580 injected in rats(Engel et al., 2006; Sigala et al., 2012; Sigala et al., 2011; Yin et al., 2008). From the first day after instillation, rats in SB203580 and silica+SB203580 group were injected with 1mg/kg/d SB203580 (Selleck Chemicals, US) intraperitoneally every day. Rats in 1%DMSO and silica group were given 1ml 1%DMSO intraperitoneally, and rats in the control group were given 1 ml saline instead. They were sacrificed and the lungs were removed for further examination on day 7 or day 15 after silica instillation.
⦁ Measurement of TGF-β1 in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay (ELISA)
On day 7 and 15 after silica instillation, the left lungs were lavaged 3 times with 2 ml ice-cold PBS each time, and the recovery ratio was above 80%. BALF from each rat was centrifuged at 1500 g for 10 min at 4°C, and the supernatant was stored at −80°C for the cytokine measurements. TGF-β1 levels in BALF (pg/ml) were measured with a rat TGF-β1 ELISA kit (Westang Bio-Tech Company, Shanghai, China) according to the instructions of manufacturer. In brief, samples which have been activated were applied onto each well of 96-well plates, and the plates

were incubated at 37 °C for 40min. Calibration curves were constructed with eight points by serially diluting a solution of recombinant rat
TGF-β1 (20ng/ml). The plates were washed six times with washing solution (PBS, pH 7.4 containing 0.5 % of Tween). Purified anti-rat TGF-β1 IgG was applied to each well and the plates were incubated for
20min at 37°C. The plates were washed six times with the washing buffer. One hundred μl of HRP-linked streptavidin was added to each well and the plates were placed at 37 °C for 10min. After further washing, the amount of TGF-β1 was determined by addition of 3, 3’, 5,
5’-tetramethylbenzidine and was read at 450 nm after stopping the reaction by adding 2 mol/L H2SO4. All procedures were performed in accordance with the manufacturer’s instructions.
⦁ HE and Masson staining

The middle of the right lung was fixed in 10% formalin for 48h, embedded in paraffin and cut into 5 µm thick slices. The slices were stained with hematoxylin and eosin (H&E) and Masson trichrome staining for evaluation of histopathological changes in the lung. A light microscope (Olympus D72, Japan) was used to examine slides with 200×magnification.
⦁ Preparation of whole lung single cell suspension

The single lung cells from the inferior lobe of right lung were prepared as previously described(Liang et al., 2014). After cutting into 1

× 1 × 1 cm3 pieces, the lung tissues were incubated with 5ml digest solution containing 0.4% collagenase type I (Sigma-Aldrich, US) with water bath shaking table at 37℃, digesting 50 min. The digest solution was then filtered through a 200μm cell trainer. The filtrate was
centrifuged with 1500 rpm for 10 min. After abandoning the supernatant, the cells were re-suspended with red blood cell lysis buffer (Solarbio, Beijing, China) for 3min. The single lung cells were re-suspended using
0.35 ml fixing liquid containing 4% paraformaldehyde (BD Bioscience, US), stored at 4℃ until flow cytometry analysis.
⦁ Flow cytometry

To quantify the occurrence of EMT, antigens including SP-C and vimentin were examined by flow cytometry with the FACSCalibur flow cytometer (BD Biosciences). To stain intracellular antigens, the cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences). Antibody against SP-C (1:100; Bioss Biotechnology, Beijing, China) and vimentin (1μg/1×106
cell, Santa Cruz, USA) were used to stain the cells. SP-C and vimentin

positive cells were acquired, and the co-expressed markers on the resulting populations were examined in a two-color analysis with a FACS Calibur flow cytometer (BD BioScience) and FlowJo software (Tree Star). All experiments included appropriate isotype-matched controls. A minimum of 50,000 events were analyzed for each sample.

⦁ Hydroxyproline assay

Hydroxyproline assay was experimented to analyze the levels of collagen content in lung with a commercial kit (Nanjing Jiancheng Bio engineering Institute, Nanjing, China). The test was conducted according to the manufacturer’s instructions. Lung tissues (50mg) from each rat were weighed and hydrolyzed in 1 ml NaOH at 95°C for 20min. The data was expressed as micrograms of hydroxyproline per gram of lung weight.
⦁ Western blot analysis

Equal amounts of protein (40µg) were separated by 10%

SDS-polyacrylamide gels electrophoresis and transferred to a PVDF membrane(Millipore, Billerica, MA, USA). Nonspecific binding to membranes was blocked with 5% BSA in TBST for 1hat room temperature. The membranes were then incubated at 4°C overnight with the following primary antibodies: E-cadherin, vimentin, phosphorylation-MAPKAPK-2 (P-MK-2), MK-2 and GAPDH were purchased from Cell Signaling Technology Inc (Beverly, MA, USA),
α-SMA was purchased from Abcam Technology Inc and ZEB-1/2, Twist were from Santa Cruz Biotechnology (Santa Cruz, CA). After washes with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit (Cell Signaling Technology, Beverly, MA, USA) for 1h at room temperature and then washed three times. The membranes were incubated with the substrate and exposed to X-ray film. All Western blots

were repeated at least 3 times. Western Blot images were acquired and analyzed by Molecular Imager BIO-RAD GelDocTM XR+ with Image lab TM Software (BIO-RAD, California, USA).
⦁ Quantitative real-time RT-PCR

Total RNA was extracted from lung tissues using SV Total Isolation System (Promega, Madison, WI, USA). The RNA was reverse transcribed to cDNA using the TransScript First-Strand cDNA Synthesis SuperMix (Transgen biotech, Beijing, China). The primers used for SYBR Green quantitative real-time PCR were as follows: E-cadherin sense sequence,
5-ATGAGGTCGGTGCCCGTATT-3 and antisense sequence,

5-CTCGTTGGTCTTGGGGTCTGT-3; vimentin sense sequence, 5-CTGCTGGAAGGGGAGGAGAG-3 and antisense sequence, 5-GGTCATCGTGGTGCTGAGAAG-3; a-SMA sense sequence, 5-CACGGCATCATCACCAACTG-3 and antisense sequence,
5-CCACGCGAAGCTCGTTATAGA-3; Collagen Ⅰsense sequence,

5-CAATGGCACGGCTGTGTGCG-3 and antisense sequence,

5-CACTCGCCCTCCCGTCTTTGG-3; Collagen Ⅲ sense sequence, 5-TGAATGGTGGTTTTCAGTTCAG-3 and antisense sequence,
5-GATCCCATCAGCTTCAGAGACT-3; β-actin sense sequence,

5-AGAGGTCTTTACGGATGTCAACGT-3 and antisense sequence,

5-GTCAGGTCATCACTATCGGCAAT-3. Gene expression was detected with the Tip Green qPCR Kit (Transgen biotech, Beijing, China), and was

determined by normalizing to glyceraldehyde-3-phosphate dehydrogenase using the 2−ΔΔCT method. The levels of Gene transcript were determined using the CFX96Tm real-time quantitative PCR detection system (BioRadInc, Hercules, CA). Data were shown normalized to expression of β-actin.
⦁ Statistical analysis

All experiments were performed at least 3 times. The data were expressed as means±SD. Data were analyzed by LSD-test between any two groups. The difference of means among groups was assessed by
one-way ANOVA analysis of variance. P<0.05 was considered to indicate statistical significance.
⦁ Results

⦁ Inhibition of p38 reduced silica-induced EMT.

The binding of SB203580 does not inter-fere the phosphorylation of p38 but blocks the activity of the downstream kinases. We evaluated the activation of p38 MAPK by examining the protein expression of P-MK-2, a downstream mediator of p38 pathway. The expression of P-MK-2 was up-regulated in the silica group, while this increase was reduced in SB203580 treated group. This verified that the inhibitor could prevent the activation of p38 (Fig. 1).
TGF-β1 is a crucial cytokine in the regulation of extracellular matrix and EMT, as it occupies central fibrogenic activities. In order to evaluate

the anti-EMT effects of SB203580, we measured levels of TGF-β1 in the BALF. Silica exposure upregulated the levels of TGF-β1 in the BALF on day 7 and day 15, and infusion of SB203580 significantly reduced the secretion of the pro-fibrotic cytokine TGF-β1(Fig. 2A).
In order to examine the involvement of p38 in the EMT induced by silica, the mRNA and protein expression of epithelial and mesenchymal markers in the lung was tested. At day 7 and 15, the mRNA level of vimentin (Fig. 2B) and α-SMA (Fig. 2C) in the silica group was significantly higher than that in the1% DMSO group (Fig. 2). However, the mRNA expression of vimentin and α-SMA was significantly reduced in SB203580-treated rats than when exposed to silica alone (Fig.2). While E-cadherin expression (Fig. 2D) was significantly higher in SB203580 treated groups compared with silica groups on day 7 and day 15 post instillation. Likewise, the protein expression of α-SMA (Fig. 3) was upregulated in silica group compared to the 1% DMSO group. But in SB203580 treated group, α-SMA was down-regulated compared with silica group (Fig. 3). The protein expression of E-cadherin in
SB203580-treated groups increased compared with that in silica groups at all time points (Fig. 3).
To further assess the induction of EMT during the development of silicosis fibrosis, the analysis of flow cytometry was conducted. Cells from the lung single cell suspensions were stained with SP-C and

vimentin doubly and were evaluated whether mesenchymal cells could derive from alveolar epithelial cells at 7 days and 15 days. In this study, 41.7% and 37.1% of cells staining with SP-C were positive for vimentin in the silica group at 7 and 15 days, respectively (Fig. 4). But in SB203580 treated group, the percentage was much lower (Fig. 4).
To observe the influence of DMSO in the lung, we designed the saline control group and found the mRNA of vimentin (Fig. 2B) increased and E-cadherin (Fig. 2D) decreased in the saline control group compared with 1% DMSO group at 7 days. This implied the protective role of DMSO on the injury of lung at early time.
The results suggest that alveolar epithelial cells contributed to the formation of the fibroblast foci during silicosis fibrosis and p38 MAPK pathway was involved in the EMT.
⦁ Inhibition of p38 reduced transcriptional factors expression in silicosis rats.
To further evaluate the roles of transcriptional factors in vivo, the protein expression of ZEB-1, ZEB-2, Twist and Snail was examined. The results showed that silica induced a marked upregulation of ZEB-1,
ZEB-2 and Twist on days 7 after instillation, but SB203580 treatment markedly reduced the upregulation on days 7(Fig. 3). By contrast, silica couldn’t induce the increase of them on days 15 (Fig. 3). While Snail was not only decreased on days 7 but also on days 15(Fig. 3), further

indicating that Snail may not be involved in the observed time points.

It shows that p38/ZEB-1 (ZEB-2, Twist) was involved in the early pathophysiological events in the development of silicosis.
⦁ Blocking p38 MAPK signaling pathway inhibited silica-induced fibrosis.
HE staining (Fig. 5) showed that there were mainly normal findings in the lungs of rats in the saline, 1%DMSO and SB203580 control group. While histologic analysis of the silicosis rats showed thickening alveolar septal, infiltration of inflammatory cells and the cellular nodules were gradually increased after silica instillation. Similarly, Masson staining (Fig. 5) revealed that the collagen deposition (blue areas) was significantly higher in silica group compared with 1% DMSO group.
However, after treatment of SB203580, the lung showed a significant decrease in extracellular matrix deposition, as assessed by less cellular nodules, slighter inflammation and decrease in blue stained areas. To assess the effect of SB203580 on silica-induced pulmonary fibrosis in rats, the hydroxyproline levels were measured as well. The hydroxyproline content of the lung was obviously increased after silica instillation across all study days compared with 1% DMSO group (Fig. 6A), however, it
was reduced in the SB203580 treated rats on day 7 and 15 (Fig. 6A), compared with the silica group. Accordingly, this reduction in extracellular matrix deposition was associated with a decrease in collagen

Ⅰand Ⅲ mRNA expression (Fig. 6B and 6C). The mRNA of collagenⅠ and Ⅲ increased in the control group compared with 1% DMSO group
at 7 days, which also showed the protective role of DMSO (Fig. 6B and 6C).
It confirmed that collagen deposition was increased in silica group but was reduced by the inhibition of p38 MAPK pathway.
⦁ Discussion

Increasing evidence demonstrates that p38is involved in the pathogenesis of the inflammatory and fibrosis diseases. A recent study showed that p38 was involved in EMT and pulmonary fibrosis induced by bleomycin(Tan et al., 2016). Some studies have found p38 occupied a critical role in the development of cardiac fibrosis(Yin et al., 2008).
According to an earlier report, inhibition of p38 attenuated renal fibrosis in a murine renal artery stenosis model(Wang et al., 2013). p38 MAPK inhibition has also been shown to improve dyspnea and lung function parameters in patients with COPD(MacNee et al., 2013). The present study laid emphasis on the role of p38 MAPK signaling pathway on early EMT and subsequent fibrosis in the lung during the pathogenesis of silicosis fibrosis.
In the previous study, we confirmed that silica exposure induces the activation of p38 MAPK in vivo and in vitro(Wang et al., 2015). While in this study, we found that silica exposure induces phosphorylation of

MK-2, a substrate of p38 MAPK, in rats instilled with silica. MK-2 is considered to be regulated by p38 MAPK exclusively. It would bind with p38 MAPK upon activation of p38, leading to the downstream molecular phosphorylated which then regulates various cellular functions(Qian et al., 2015). The specific inhibitor, SB203580, inhibited phosphorylation of
MK-2. Moreover, in this study, SB203580 exerted anti-EMT and

anti-fibrotic effects in the rat model of silicosis fibrosis. These results strongly showed that p38 MAPK played a pivotal role in EMT and pulmonary fibrosis induced by silica, and the advantageous effect of SB203580 in the early time may be relevant to suppression of ZEB-1, ZEB-2 and Twist expression. These observations indicate that p38 MAPK is a crucial target of regulation of silicosis fibrosis.
p38 MAPK was demonstrated to be activated by various extracellular stimuli, and the present study revealed that silica also activated p38 MAPK pathway. After silica exposure, MK-2 was phosphorylated on day 7. TGF-β1, the pro-fibrotic cytokines, plays important roles in EMT and pulmonary fibrosis. The expression of
TGF-β1 was tested to further confirm whether EMT could be induced by silica or inhibited by SB203580. As shown in Fig. 2A, SB203580 administration obviously suppressed the elevated TGF-β1 induced by silica during the observing phase (day 7 and 15) of silica instillation.
Current evidence indicates that abnormally alveolar epithelial cells

(AECs) might directly contribute to the expansion of the population of fibroblasts through EMT in pulmonary fibrosis(Marmai et al., 2011). To accomplish this, we assessed whether EMT of AECs played avital role during silica-induced pulmonary fibrosis using the flow cytometry analysis of the lung cell suspensions. The biomarkers of EMT were measured at 7 days and 15 days. The results showed that much more
SP-C positive cells were transitioned to mesenchymal cells in silica group than that in 1% DMSO group. Therefore, AECs-derived mesenchymal cells are prevalent in the rats instilled with silica. While the rate decreased markedly after treatment with SB203580. Furthermore, we used real-time PCR and Western blot to analyze the expression of the epithelial and mesenchymal markers in the lung. It showed that vimentin and a-SMA expression was significantly upregulated 7 d and 15 d after silica exposure. Correspondingly, the expression of E-cadherin was downregulated. However, SB203580 obviously attenuated silica-induced EMT, as evidenced by its induction of E-cadherin and repression of vimentin and a-SMA.
The presence of EMT is related to the stimulation of transcription factors which are considered as inducers of EMT such as Snail, ZEB-1, ZEB-2 or Twist(Thiery et al., 2009), and they have been found to directly or indirectly repress E-cadherin and activate the transcriptomes of mesenchyme (Kalluri and Weinberg, 2009). In this study, ZEB-1, ZEB-2

and Twist were activated after exposure with silica and were decreased obviously with inhibition of p38 at 7 days, which indicated p38/ZEB-1 (ZEB-2, Twist) was involved in the early development of silicosis fibrosis.
However, ZEB-1, ZEB-2 and Twist were not upregulated at 15 days and it needs further investigation what happened in the later time. Some scholars found high levels of Snail have been detected in fibrotic kidneys from patients(Boutet et al., 2006), and our previous study has also found the upregulation of Snail was vital in silica-induced EMT in A549 cells(Wang et al., 2015). However, on day 7 and day 15 after instillation of silica, Snail levels did not increase compared to the 1% DMSO group. Further study of snail in the earlier time after silica instillation is needed.
We also found p38 inhibition reduced the fibrosis in the histologic analysis. In the lungs harvested from rats of the silica+ SB203580 group on day 7 and 15, the decrease in the mRNA expression of collagenⅠ and
Ⅲ were detected compared with the silica group. The assays of HYP

contents also indicated an inhibitory effect of SB203580 on silica-induced pulmonary fibrosis. The results revealed the important role of p38 MAPK on silica-induced pulmonary fibrosis. It is natural that p38 MAPK is one of the signal transducers to contribute to the formation of silica-induced pulmonary fibrosis. The results also shows DMSO treatment could reduce the levels of vimentin, collagen Ⅰ and Ⅲ and increase E-cadherin
mRNA expression induced by saline at day 7, which coincided with the

previous study(Boybeyi et al., 2014).

In conclusion, the present study demonstrates that p38 MAPK inhibitor SB203580 ameliorates the formation of silica-induced EMT and pulmonary fibrosis. The present findings suggested that p38
MAPK/ZEB-1 (ZEB-2, Twist) was involved in the early time of silicosis fibrosis in rats. This study provides evidence that regulation of p38 MAPK is a novel appealing therapeutic target for silicosis fibrosis.

Conflict of Interest statement

The authors declare that there are no conflicts of interest

Acknowledgement

This work was supported by grants of National Natural Science Foundation of China (No. 81273047) and Key Projects of Science and Technology Program by Beijing Municipal Education Commission (No. KZ201610025020).

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Fig.1 Treatment of SB203580 inhibited silica-induced p38 activity. Western blot showed silica exposure upregulated the levels of P-MK-2 on day 7 and day 15 after instillation, and infusion of SB203580 reduced the expression of P-MK-2, especially on day 7. GAPDH or MK-2 was used as a loading control. *P<0.05 vs. 1% DMSO group, # P<0.05 vs. silica group.
Fig.2 SB203580 inhibited silica-induced EMT.
Silica significantly increased TGF-β1 in the BALF (A) and the mRNA expression of vimentin (B) and α-SMA (C) in the lung, accordingly, silica

decreased the mRNA expression of E-cadherin (D) in the lung. However, SB203580 could inhibit the increase of TGF-β1 (A), vimentin (B),
α-SMA (C) and the decrease of E-cadherin (D). *P<0.05 vs. 1% DMSO group, # P<0.05 vs. silica group.
Fig.3 Inhibition of p38 attenuated silica-induced EMT and transcriptional factors expression in silicosis rats.
Western blot analysis showed the protein expression of α-SMA was upregulated and E-cadherin was downregulated in the silica group compared with 1% DMSO group, while α-SMA decreased and
E-cadherin increased in the silica+SB203580 group compared with the silica group. SB203580 inhibited the upregulation of ZEB-1, ZEB-2 and Twist after silica exposure at 7 days, and silica did not increase the expression of snail.
Fig.4 SB203580 inhibited EMT of AECs.

Wistar rats were exposed to silica or saline instillation intra-tracheally and the whole lung cell suspensions were obtained at 7 and 15 days
post-exposure. (A) Quantitative results of the percentage of SP-C positive cells stained for vimentin. *P<0.05 vs. 1% DMSO group, # P<0.05 vs. silica group. (B) and (C) are representative flow cytometry plots with gating strategies on day 7 and day 15 post-exposure, respectively; gating strategies was applied equally for all samples.

Fig.5 Histopathologic changes in rat lungs at 7 days and 15 days after instillation (×200). (A) HE and Masson staining at 7 days after instillation;
(B) HE and Masson staining at 15 days after instillation.

Fig.6 SB203580 reduced silica-induced lung fibrosis in rats.

(A) The hydroxyproline content of rat lungs; (B) The mRNA expression of collagenⅠof rat lungs; (C) The mRNA expression of collagen Ⅲ of rat lungs. *P<0.05 vs. 1% DMSO group, # P<0.05 vs. silica group.

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