Phosphodiesterase-5 inhibition by sildenafil citrate in a rat model of bleomycin-induced lung fibrosis

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Abstract

Sildenafil, a selective and potent inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE)5, has a relaxant effect on the smooth muscle cells of the arterioles supplying the human corpus cavernosum acting via nitric oxide (NO)-dependent mechanism. This study aimed to investigate the possible protective effect of sildenafil citrate on the extent of tissue integrity, oxidant–antioxidant status and neutrophil infiltration to the inflamed organ in a rat model of bleomycin-induced lung fibrosis. Lung fibrosis was induced by intratracheal administration of 0.1 ml of bleomycin hydrochloride (5 mg/kg in 0.9% NaCl) under anesthesia to Sprague–Dawley rats (200–250 g; n = 7–8 per group). Control rats received an equal volume of saline intratracheally. In the treatment groups, the rats were treated with either sildenafil citrate (10 mg/kg per day; subcutaneously) or saline for 14 days. Another group of rats were administered subcutaneously with N(G)-nitro-l-arginine methyl ester (l-NAME; 20 mg/kg in 0.9% NaCl) 5 min after sildenafil injections. After decapitation, the lungs were excised and taken for microscopic evaluation or stored for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, and myeloperoxidase (MPO) activity, and for the assessment of apoptosis. Trunk blood was collected for the assessment of serum tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels. In the group with lung fibrosis, the lung tissue was characterized by microscopic lesions, increased lipid peroxidation with a concomitant reduction in GSH content, increased MPO activity and apoptosis. Serum TNF-α and IL-1β levels were higher in the lung fibrosis group compared to control values. Sildenafil reversed tissue MDA levels, MPO activity and serum pro-inflammatory cytokine levels, and preserved GSH content although its effect on the extent of tissue lesion and apoptosis was not statistically significant. Treatment with l-NAME reversed the effect of sildenafil on GSH content. In conclusion, sildenafil citrate administration to rats with bleomycin-induced lung fibrosis seems to be beneficial via prevention of lipid peroxidation, cytokine production and/or release and neutrophil accumulation.

Introduction

Sildenafil is a selective and potent inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE)5, which catalyzes the hydrolysis of cGMP and has a relaxant effect on the smooth muscle cells of the arterioles supplying the human corpus cavernosum [1], [2]. The data of a recent study have shown that sildenafil, acting via nitric oxide (NO)-dependent mechanism, prevented indomethacin-induced gastropathy, possibly through a reduction of leukocyte adhesion and maintenance of gastric blood flow [3].

Bleomycin is an anti-cancer agent with limited therapeutic use in the clinics due to its toxicities [4]. Bleomycin-induced pulmonary fibrosis is a widely used animal model of idiopathic pulmonary fibrosis [5]. Intratracheal administration of bleomycin into the lungs of the rats has shown to cause alveolar damage, fibroblast proliferation, inflammatory cell accumulation and collagen deposition all of which contribute to the development of lung fibrosis. The reactive oxygen metabolites were found to have a significant role in the development of lung injury in this model. Indeed, several observations demonstrated that antioxidant regimen including N-acetylcysteine and erdosteine attenuated the extent of tissue injury [5], [6], [7], [8], [9]. Moreover, excess nitric oxide production was also shown to be involved in the bleomycin-induced toxicity in rats [5].

In the light of the above-mentioned findings, this study aimed to investigate the possible protective effect of sildenafil citrate on the extent of tissue integrity, oxidant–antioxidant status and neutrophil infiltration to the inflamed organ in a rat model of bleomycin-induced lung fibrosis.

Section snippets

Animals

Sprague–Dawley rats of either sex (200–250 g; n = 7–8 per group) were kept in a room at a constant temperature (22 ± 1 °C) with 12 h light/dark cycles and fed standard pellet chow and water ad libitum. The study protocol was approved by Marmara University, School of Medicine, Animal Care and Use Committee.

Induction of lung fibrosis

After an overnight fasting, lung fibrosis was induced by intratracheal administration of 0.1 ml of bleomycin hydrochloride (5 mg/kg in 0.9% NaCl) under anesthesia with 60 mg/kg ketamine hydrochloride

Microscopic evaluation of the lung samples

The microscopic lesion score (4.7 ± 0.2) of the group with bleomycin-induced lung fibrosis was reduced by treatment with sildenafil citrate (4.2 ± 0.2), however, this effect did not reach a statistically significant level. Interestingly, in l-NAME-treated sildenafil group, the microscopic lesion score showed a significant decrease when compared to the group that was not treated with l-NAME (3.6 ± 0.2; p < 0.05) (Fig. 1).

The histopathological analysis of the lung tissues from either untreated or

Discussion

The results of the present study demonstrate that daily sildenafil citrate treatment of rats markedly improves bleomycin-induced lung fibrosis as confirmed by biochemical assays.

Recently, experiments have shown the involvement of oxidative mechanisms in the pathogenesis of bleomycin-induced toxicity. It has generally been hypothesized that activated inflammatory cells which accumulate in the lungs release reactive oxygen species that result in lung injury, and proliferation of fibroblasts in

Acknowledgments

Sildenafil citrate was a generous gift of Fako Pharmaceutical Company, Istanbul, Turkey for research use.

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