Effects of Different Concentrations of Oil Mist Particulate Matter on Pulmonary Fibrosis In Vivo and In Vitro

Abstract

Oil-mist particulate matter (OMPM) refers to oily particles with a small aerodynamic equivalent diameter in ambient air. Since the pathogenesis of pulmonary fibrosis (PF) has not been fully elucidated, this study aims to explore the potential molecular mechanisms of the adverse effects of exposure to OMPM at different concentrations in vivo and in vitro on PF. In this study, rats and cell lines were treated with different concentrations of OMPM in vivo and in vitro. Sirius Red staining analysis shows that OMPM exposure could cause pulmonary lesions and fibrosis symptoms. The expression of TGF-β1, α-SMA, and collagen I was increased in the lung tissue of rats. The activities of MMP2 and TIMP1 were unbalanced, and increased N-Cadherin and decreased E-Cadherin upon OMPM exposure in a dose-dependent manner. In addition, OMPM exposure could activate the TGF-β1/Smad3 and TGF-β1/MAPK p38 signaling pathways, and the differentiation of human lung fibroblast HFL-1 cells. Therefore, OMPM exposure could induce PF by targeting the lung epithelium and fibroblasts, and activating the TGF-β1/Smad3 and TGF-β1/MAPK p38 signaling pathways.

Keywords: OMPM; PF; TGF-β1/MAPK p38; TGF-β1/Smad3.

Figure 1

OMPM-induced PF in Wistar rat lung tissues with Sirius Red staining; 100×, bar: 100 µm; 200×, Bar: 50 µm.

Figure 2

PF biomarkers were determined via ELISA assay after OMPM exposure at different concentrations in rat serum and lung tissues. (A) COL-1 concentration; (B) α-SMA concentration; (C) TGF-β1 concentration; (D) MMP2 concentration; (E) TIMP1 concentration; (F) N-cadherin concentration; (G) E-cadherin concentration. Data represent mean ± SD. * p < 0.05 vs. the control group.

Figure 3

OMPM exposure induced PF via the TGF-β1/Smad3 and TGF-β1/p38 MAPK signalingpathwaysin rat lung tissues. (A) The phosphorylation of Smad3 and p38 after OMPM exposure detected with Western blotting; (B) ratio of p-Smad3/Smad3; (C) ratio of p-p38/p38. Data represent mean ± SD. * p < 0.05 vs. the control group.

Figure 4

OMPMexposure induced fibrotic phenotypes in the BEAS-2B cell line. (A) COL-1 concentration; (B) α-SMA concentration; (C) TGF-β1 concentration; (D) MMP2 concentration; (E) TIMP1 concentration; (F) N-cadherin concentration; (G) E-cadherin concentration. (H) Phosphorylation of Smad3 and p38 after OMPM exposure detected with Western blotting. (I) α-SMA expression after OMPM exposure was detected using immunofluorescence. (J) Collagen I expression after OMPM exposure was detected using immunofluorescence; 100×, bar: 100 µm. Data represent mean ± SD. * p < 0.05 vs. the control group.

Figure 5

OMPM exposure activated the differentiation of the HFL-1 cell line. (A) COL-1 concentration; (B) α-SMA concentration; (C) TGF-β1 concentration; (D) MMP2 concentration; (E) TIMP1 concentration; (F) N-Cadherin concentration; (G) E-cadherin concentration. (H) Phosphorylation of Smad3 and p38 after OMPM exposure detected with Western blotting. (I) α-SMA expression after OMPM exposure was detected using immunofluorescence. (J) Collagen I expression after OMPM exposure was detected using immunofluorescence; 100×, bar: 100 µm. Data represent mean ± SD. * p < 0.05 vs. the control group.

Figure 6

Different ways with which OMPM-exposed BEAS-2B cell lines could induce the differentiation of the HFL-1 cell line. (A) COL-1 concentration; (B) α-SMA concentration; (C) MMP2 concentration; (D) TIMP1 concentration; (E) N-cadherin concentration; (F) E-cadherin concentration. (G) α-SMA expression after the treatment of the medium of non-OMPM-M and OMPM-M cell supernatants of non-OMPM-E and OMPM-E groups of BEAS-2B cell lines was detected using immunofluorescence. (H) Collagen I expression after the treatment of the medium of non-OMPM-M and OMPM-M cell supernatants of non-OMPM-E and OMPM-E groups of BEAS-2B cell lines was detected using immunofluorescence; 100×, bar: 100 µm. (I) Phosphorylation of Smad3 and p38 after the treatment of the medium of non-OMPM-M and OMPM-M cell supernatants of non-OMPM-E and OMPM-E groups of BEAS-2B cell lines was detected with Western blotting. Non-OMPM-M, medium for BEAS-2B without OMPM treatment before it was used for culture; OMPM-M, medium for BEAS-2B with OMPM treatment before it was used for culture; non-OMPM-E, BEAS-2B that was not exposed to OMPM; OMPM-M, BEARS-2B that was exposed to OMPM. Data represent mean ± SD. * p < 0.05 vs. the non-OMPM-M, OMPM-M, and non-OMPM-E groups.