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. 2017 Feb;21(2):324-335.
doi: 10.1111/jcmm.12967. Epub 2016 Oct 4.

HYDAMTIQ, a Selective PARP-1 Inhibitor, Improves Bleomycin-Induced Lung Fibrosis by Dampening the TGF-β/SMAD Signalling Pathway

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Free PMC article

HYDAMTIQ, a Selective PARP-1 Inhibitor, Improves Bleomycin-Induced Lung Fibrosis by Dampening the TGF-β/SMAD Signalling Pathway

Laura Lucarini et al. J Cell Mol Med. .
Free PMC article

Abstract

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 (PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-β (TGF-β) expression and TGF-β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-β/SMAD signalling pathway with alpha-smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1β, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-β expression and the TGF-β/SMAD transduction pathway.

Keywords: HYDAMTIQ; PAO; ROS; SMAD; αSMA.

Figures

Figure 1
Figure 1
PARP activity. (A) Western blot analysis of PARylated protein content in lung samples from each experimental group. (B) Densitometric analysis was normalized with tubulin (n = 10 animals per group). **P < 0.01 and ***P < 0.001 versus bleomycin + vehicle. 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 2
Figure 2
Lung function and hydroxyproline content. (A) Lung resistance to airflow measured through the evaluation of pressure at airway opening (PAO) (n = 10 animals per group). (B) Percentage increase of hydroxyproline content. Basal content of hydroxyproline in controls (naïve): 15.0 ± 1 μg/ml; bleomycin + vehicle: 26.85 ± 1.05 μg/ml. Data are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. Bleo: bleomycin; 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 3
Figure 3
Lung histology. (A) Histopathological evaluation of fibrosis by Azan‐stained lung analysis. By computer‐aided densitometry analysis, it is possible to obtain a semi‐quantitative measure of this accumulation. n = 10 animals per group. Data are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ. (B) Histopathological evaluation of airway remodelling by haematoxylin and eosin staining. n = 10 animals per group. Data are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ. (C) Goblet cell number in PAS‐stained lung sections (see the arrows) is evaluated in each experimental group. n = 10 animals per group. Data are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 4
Figure 4
Evaluation of TGF‐β signalling pathway. (A) Determination of the pro‐fibrotic marker TGF‐β. Values are expressed as pg of protein/μg of total proteins (n = 10 animals per group). Data are mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 versus bleomycin + vehicle. (B) pSMAD3 expression level, assayed by Western blotting. Densitometric analysis was normalized on β‐actin (n = 10 animals per group). Data are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 5
Figure 5
Evaluation of fibroblast activation. Immunofluorescence staining of lung tissue sections labelled with alpha‐smooth muscle actin (αSMA) (red) and nuclei (blue) counterstained with DAPI (40×). Images in the panels show the inhibition of αSMA expression, a marker of the transformation of fibroblasts into myofibroblasts (n = 10 animals/group). *P < 0.05, **P < 0.001 versus bleomycin + vehicle. Bleo: bleomycin; 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 6
Figure 6
Determination of pro‐inflammatory markers. (A and B) Analysis of IL‐1β and TNF‐α content (respectively) in the supernatant of lung tissue homogenates. The values are expressed as pg or ng/μg of total proteins (n = 10 animals per group). Values are mean ± S.E.M. *P < 0.05 and **P < 0.01 versus bleomycin + vehicle. (C and D) Levels of pro‐inflammatory proteins iNOS and COX‐2 (respectively) are evaluated by Western blot analysis from total cell extracts of lung samples homogenates. The densitometric analysis was normalized to tubulin and each point report data obtained in a single animal (n = 4 animals per group). Bleo: bleomycin; 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.
Figure 7
Figure 7
Evaluation of oxidative stress parameter in lung tissue. Levels of 8‐OH dG, a marker of free radicals‐induced DNA damage (n = 10 animals per group). Values are mean ± S.E.M. *P < 0.05 versus bleomycin + vehicle. Bleo: bleomycin; 1HYD, 3HYD, 10HYD: 1, 3, 10 mg/kg of HYDAMTIQ.

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