Effects of lipopolysaccharide, multiwalled carbon nantoubes, and the combination on lung alveolar epithelial cells - PubMed (original) (raw)

. 2017 Feb;32(2):445-455.

doi: 10.1002/tox.22248. Epub 2016 Feb 16.

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Effects of lipopolysaccharide, multiwalled carbon nantoubes, and the combination on lung alveolar epithelial cells

M Pacurari et al. Environ Toxicol. 2017 Feb.

Abstract

Multiwalled carbon nanotubes (MWCNT) have been shown to induce lung fibrosis in animal models, however the underlying molecular factors/mechanisms are still unclear. In this study, we investigated the effects of lipopolysaccharide (LPS), MWCNT, and the combination of LPS and MWCNT on the expression of matrix metalloproteinase-9 and metalloproteinase-12 (MMP-9, MMP-12), collagen 3A1 (Col3A1), and transforming growth factor beta (TGFβ) in alveolar epithelial A549 cells. MMPs are proteinases that degrade extracellular matrix and play a role in lung fibrosis. A549 cells were exposed to LPS (1 ng/mL), MWCNT (20 μg/mL), and the combination and analyzed for paracellular permeability, TGFβ, Col3A1, MMP-9, MMP-12, NF-κB activation, and cell migration by real-time PCR and immunofluorescence. LPS, the combination of LPS and MWCNT, and MWCNT only at the highest tested dose induced blue dextran extravasation. LPS and MWCNT increased the expression of TGFβ and its downstream target gene Col3A, and MMP-9 and MMP-12 mRNA. MWCNT potently induced cell migration toward wound healing, whereas LPS slightly induced cell migration. Both, LPS and MWCNT, induced NF-κB nuclear translocation. Our results indicate that MWCNT activated alveolar epithelial cells to promote fibrogenesis, and that LPS differentially primes molecular factors involved in lung remodeling. These findings suggest a role of alveolar epithelial cells in fibrogenesis and also may aid in the design and development of tests for screening of fibrogenic agents. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 445-455, 2017.

Keywords: ECM; LPS; MMP-12; MMP-9; MWCNT; NF-κB; TGFβ; alveolar epithelial cells; cell migration; collagen.

© 2016 Wiley Periodicals, Inc.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG. 1

FIG. 1

Elemental analysis of MWCNT using ECS 4010 platform. As shown in the figure, ECS analysis shows three peaks for carbon (95%), nitrogen (0.59%), and hydrogen (0.32%).

FIG. 2

FIG. 2

Transmission electron micrograph of MWCNT. The MWCNT sample was prepared as described in Materials and Methods. As shown in the figure, single MWCNT as well as few agglomerates of MWCNT and few impurities are visible.

FIG. 3

FIG. 3

Paracellular permeability of A549 cell monolayer after exposure to LPS (1 or 10 ng/ml), MWCNT (20 or 100 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) for 48 h. Cell layer paracellular permeability was assed using blue dextran extravasation (10 nM). The data represents the mean ± SEM, n=4, relative to control. *, Statistically significant versus control, ANOVA, p < 0.05.

FIG. 4

FIG. 4

Regulation of TGFβ (A) and Col3A1 (B) genes in A549 cells treated LPS (1 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) for 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=4. *, Statistically significant versus control, ANOVA, p <0.05.

FIG. 4

FIG. 4

Regulation of TGFβ (A) and Col3A1 (B) genes in A549 cells treated LPS (1 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) for 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=4. *, Statistically significant versus control, ANOVA, p <0.05.

FIG. 5

FIG. 5

Regulation of MMP-9 (A) and MMP-12 (B) genes in A549 cells treated with LPS (1 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) for 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=4. *, Statistically significant versus control, ANOVA, p <0.05.

FIG. 5

FIG. 5

Regulation of MMP-9 (A) and MMP-12 (B) genes in A549 cells treated with LPS (1 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) for 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=4. *, Statistically significant versus control, ANOVA, p <0.05.

FIG. 6

FIG. 6

Cell migration in wound healing of A549 cell layer after exposure to LPS (1 or 10 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) after 48 h. Cell wound healing was assed using wound scratch assay (A), and cell migration assay (B). One representative micrograph is shown. The data is mean ± SEM, n=4. *, Statistically different vs control, ANOVA, p < 0.05.

FIG. 6

FIG. 6

Cell migration in wound healing of A549 cell layer after exposure to LPS (1 or 10 ng/ml), MWCNT (20 μg/ml), or the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) after 48 h. Cell wound healing was assed using wound scratch assay (A), and cell migration assay (B). One representative micrograph is shown. The data is mean ± SEM, n=4. *, Statistically different vs control, ANOVA, p < 0.05.

FIG. 7

FIG. 7

NF-κB p65 subunit nuclear translocation in A549 cells following treatment with LPS (1 ng/ml), MWCNT (20 μg/ml), and the combination of LPS (1 ng/ml) and MWCNT (20 μg/ml) after 24 h. Nuclear translocation of p65 subunit was detected using immunofluorescence (green) and DAPI (nucleus). The arrow points to immunofluorescence of p65 in the nucleus. One representative micrograph is shown, n=3.

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