Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury

Abstract

Background and purpose: The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in 'preventive' and 'therapeutic' protocols and compared with glucocorticoids.

Experimental approach: Roflumilast (1 and 5 mg.kg(-1).d(-1), p.o.) or dexamethasone (2.5 mg.kg(-1).d(-1), p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U.kg(-1)). In Wistar rats, roflumilast (1 mg.kg(-1).d(-1), p.o.) was compared with methylprednisolone (10 mg.kg(-1).d(-1), p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillation of bleomycin (7.5 U.kg(-1)). Analyses were performed at the end of the treatment periods.

Key results: Preventive. Roflumilast reduced bleomycin-induced lung hydroxyproline, lung fibrosis and right ventricular hypertrophy; muscularization of intraacinar pulmonary vessels was also attenuated. The PDE4 inhibitor diminished bleomycin-induced transcripts for tumour necrosis factor (TNFalpha), transforming growth factor (TGFbeta), connective tissue growth factor, alphaI(I)collagen, endothelin-1 and the mucin, Muc5ac, in lung, and reduced bronchoalveolar lavage fluid levels of TNFalpha, interleukin-13, TGFbeta, Muc5ac, lipid hydroperoxides and inflammatory cell counts. Therapeutic. In mice, roflumilast but not dexamethasone reduced bleomycin-induced lung alphaI(I)collagen transcripts, fibrosis and right ventricular hypertrophy. Similar results were found in the rat.

Conclusions and implications: Roflumilast prevented the development of bleomycin-induced lung injury, and alleviated the lung fibrotic and vascular remodeling response to bleomycin in a therapeutic protocol, the latter being resistant to glucocorticoids.

Figure 1

Effects of roflumilast on bleomycin-induced fibrotic response in mouse lung. Mice received a single dose of bleomycin (3.75 U·kg⁻¹) intratracheally at day 1 and roflumilast (0.5, 1 or 5 mg·kg⁻¹·d⁻¹ p.o., once daily) or vehicle was administered from day 1 to 14 (preventive protocol) until analysis at day 14. Histology (A), lung hydroxyproline content (µg per lung) (B) and fibrosis score (C) were assessed as described in Methods. In A, upper panels (a–d) show H&E staining (original magnification ×40) and lower panels (e–h) Masson's trichrome (original magnification ×10; collagen is stained in blue) for controls (a,e), bleomycin (b,f), bleomycin + roflumilast 1 mg·kg⁻¹·d⁻¹ (c,g) and bleomycin + roflumilast 5 mg·kg⁻¹·d⁻¹ (d,h). #P < 0.05 versus control, *P < 0.05 versus bleomycin. Results are given as mean ± SEM from n = 6 (B, C).

Figure 2

Analysis of bleomycin-induced pulmonary vascular remodeling and right ventricular hypertrophy. Mice received a single dose of bleomycin (BLM; 3.75 U·kg⁻¹) intratracheally at day 1 and roflumilast (ROF) was administered at 0.5, 1 or 5 mg·kg⁻¹·d⁻¹ p.o., once daily from day 1 to 14 until analysis in a preventive protocol. Right ventricular hypertrophy (expressed as RV/LV + S ratio) in A, histology of intra-acinar pulmonary arteries in B and, in C, the percentage of fully muscularized (F), partially muscularized (P) and non-muscularized (N) distal pulmonary vessels were determined, as described in Methods. #P < 0.05 versus control, *P < 0.05 versus bleomycin. Results are shown as mean ± SEM from nine to 10 (controls) or five to nine (bleomycin) mice (A) or three mice (C). LV + S, left ventricle + septum; RV, right ventricle.

Figure 3

Effects of roflumilast on Muc5ac mRNA (lung), protein (BAL fluid) and mucin-forming cells. Mice received a single dose of bleomycin (3.75 U·kg⁻¹) intratracheally at day 1 and roflumilast was given at 1 or 5 mg·kg⁻¹·d⁻¹ p.o. from day 1 to 14 (preventive protocol). Muc5ac mRNA in lung extracts (A) and protein in BAL fluid (B) was measured at day 14. Muc5ac mRNA or protein were quantified by real-time RT-PCR or ELISA and data were given as relative expression (i.e. x-fold change over control). Results are shown as mean ± SEM from four to five (mRNA) and six (protein) mice. #P < 0.05 versus control, *P < 0.05 versus bleomycin. Representative photomicrographs of airway epithelium stained with Alcian blue to detect mucus-forming cells were taken at day 14. Mucus producing cells are stained in blue, magnification was ×40. (a) control, (b) bleomycin, (c) bleomycin and roflumilast 5 mg·kg⁻¹·d⁻¹ (C). BAL, bronchoalveolar lavage; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 4

Comparison of roflumilast and dexamethasone on lung αI(I)collagen mRNA and right ventricular hypertrophy associated with bleomycin in a therapeutic protocol in mice. Mice received a single dose of intratracheal bleomycin (3.75 U·kg⁻¹) at day 1, and roflumilast (ROF: 1 or 5 mg·kg⁻¹·d⁻¹ p.o.) or dexamethasone (DEX; 2.5 mg·kg⁻¹·d⁻¹ p.o.) either from day 1 to 14 with analyses at day 14 (preventive protocol) or from day 7 to 21 with analyses at day 21 (therapeutic protocol). αI(I)collagen was quantitated in lung extracts by real-time RT-PCR and data are given as relative expression levels (i.e. x-fold increase over control) (A). Lung histology shows H&E staining (original magnification ×40) in the upper panels (a–c) and Masson's trichrome (original magnification ×40; collagen is stained in blue) in the lower panels (d–f) for bleomycin (a,d), bleomycin + roflumilast 1 mg·kg⁻¹·d⁻¹ (b,e) and bleomycin + roflumilast 5 mg·kg⁻¹·d⁻¹ (c,f) with roflumilast from day 7 to 21 and analyses at day 21 (B). The RV/LV + S ratio was calculated as a measure of right ventricular hypertrophy (C). Results are given as the means ± SEM from three to five [αI(I)collagen] or nine (RV/LV + S) animals. #P < 0.05 versus control, *P < 0.05 versus bleomycin. LV + S, left ventricle + septum; RT-PCR, reverse transcription-polymerase chain reaction; RV, right ventricle.

Figure 5

Comparison of roflumilast (ROF) and methylprednisolone (MP) on lung αI(I)collagen mRNA and right ventricular hypertrophy associated with bleomycin in a therapeutic protocol in rats. Wistar rats were exposed to a single intratracheal dose of bleomycin (7.5 U·kg⁻¹) at day 1 and roflumilast (1 mg·kg⁻¹·d⁻¹ p.o.) or methylprednisolone (10 mg·kg⁻¹·d⁻¹ p.o.) was administered either from day 1 to 21 (preventive protocol) or from day 10 to 21 (therapeutic protocol). Lung extracts for determination of αI(I)collagen, TGFβ1 and CTGF mRNA by real-time RT-PCR were prepared at day 21. Results are shown as relative expression levels (x-fold increase over control) and given as mean ± SEM from six to eight animals per treatment group. #P < 0.05 versus control, *P < 0.05 versus bleomycin. CTGF, connective tissue growth factor; TGFβ1, transforming growth factor-β1; RT-PCR, reverse transcription-polymerase chain reaction.