Pulmonary fibrosis is a common response to various insults to the lung and it is the end-point of a numerous and heterogeneous group of disorders known as interstitial lung diseases (ILD) that are characterized by chronic inflammation and progressive fibrosis of the pulmonary interstitium: alveolar walls (including epithelial cells and capillaries), septae, and the perivascular, perilymphatic, and peribronchiolar connective tissues . While the pathogenesis is incompletely understood, a growing body of evidence suggests two different pathogenic routes for developing pulmonary fibrosis. The inflammatory pathway, where a shift to the so-called T-helper 2 type cytokine networks is critical, and the epithelial pathway represented by idiopathic pulmonary fibrosis, by far the most aggressive ILD. Both routes may trigger a number of cytokines/growth factors inducing fibroblast migration/proliferation and phenotype change to myofibroblasts, with a consequent accumulation of extracellular matrix . In addition, various evidences have point out an important role for IL-6 and IL-11 in the pulmonary fibrosis [34, 35].
The common pathologic features in ILD, include the fibrosis of the interstitium, involve collagen, elastic and smooth muscle elements, architectural remodeling and chronic inflammation of the interstitium (ie, variable increases in lymphocytes, neutrophils, plasma cells, macrophages, eosinophils, and mast cells), hyperplasia of type II cells and hyperplasia of endothelial cells . Intratracheal instillation of the antitumour agent BLM is the most commonly used animal model for pulmonary fibrosis . The bleomycin-oxygen complex is thought to bind to DNA and lead to the efficient cleavage of the phosphodiester-deoxyribose backbone and the generation of ROS  In the presence of oxygen and a reducing agent in fact, the ferrous ion-BLM complex becomes activated and functions mechanically as a ferrous oxidase, transferring electrons from ferrous ion to molecular oxygen to produce ROS that cause scission of DNA [38, 39]. Therefore, earlier reports [40, 41] point out that the pathogenesis of BLM-induced fibrosis, at least in part, is mediated through the generation of reactive oxygen species (ROS) which cause the peroxidation of membrane lipids and DNA damage. If, that perspective is true, then antioxidant therapy may prevent the lung fibrosis caused by BLM and may prevent other diseases related with interstitial pulmonary fibrosis. Because BLM administration results in increased lipid peroxidation (LPO) and alters activities of antioxidant enzymes in bronchoalveolar lavage fluids (BALFs) and lung tissue [42, 43], in previous studies [44, 45] some natural or synthetic antioxidants have been used to protect against BLM oxidative lung toxicity both in vivo and also in vitro. In addition to ROS, an overproduction of nitric oxide (NO) due to the expression of the inducible isoform of NO synthase (iNOS) also plays important role in various models of inflammation [5, 9, 46]. Pharmacological inhibitors of NOS, and also ablation of the gene for iNOS has been shown to reduce the development of the inflammatory response [47–49]. In addition, various studies have point out that iNOS play an important role in the pulmomary fibrosis induced by bleomycin [50–52]. Therefore, some study have also demonstrated that non-selective and partial selective iNOS inhibitor like aminoguanidine exert beneficial effect against lung injury induced by bleomycin [53–57]. However, frequently a misunderstanding is the definition of an inhibitor as selective for, e.g. iNOS versus eNOS, and then ignoring its non-selectivity for nNOS or completely distinct enzyme targets. An interesting example is aminoguanidine that is a partially selective for iNOS versus eNOS , while the selectivity over nNOS is minimal. Moreover it has a wide range of other effects, inhibiting advanced glycosylation end-product formation, diamine oxidase and polyamine metabolism [59, 60], catalase  and having anti-oxidant effects [62, 63]. For this reason aminoguanidine should not be described as a selective inhibitor.
In contrast, GW274150 is a novel, potent and selective inhibitor of iNOS activity and previous studies have demonstrated its protective effect in organ injury in hemorrhagic shock, in renal ischemia and reperfusion and in a model of collagen-induced arthritis [24–26]. This study provides the first evidence of the protective role of GW274150 in an experimental model of lung fibrosis. Here we demonstrate that the lack of iNOS gene as well as the pharmacological inhibition of iNOS (GW274150 treatment) reduces: i) the development of bleomycin-induced lung injury, (ii) the infiltration of the lung with inflammatory cells, (iii) the degree of nitrosative stress in the lung and (iv) the mortality rate. All of these findings support the view that NO plays an important role in the degree of inflammation and lung fibrosis caused by bleomycin in the mice. We report in the present study a reduction of the tissue damage in the lung of bleomycin-treated iNOSKO mice as well as bleomycin-treated iNOSWT mice which received the treatment with GW274150.
Two of the most recognized fibrosis markers, lung collagen deposition and TGF-β1 expression, were significantly reduced in these animals. Evidence from animal models and human studies suggests that TGF-β1 plays a central role in a variety of fibroproliferative disorders, including pulmonary fibrosis. TGF-β1 plays a critical role in the pathogenesis of lung fibrosis through stimulation of collagen and fibronectin production in fibroblasts , as well as through inhibition of biosynthesis of proteases that degrade the extracellular matrix . TGF-β1 promotes wound healing  and its presence has been shown to be increased in bleomycin -induced lung fibrosis [67, 68] and particularly in lung macrophages . In addition it has been shown that during the course of pulmonary fibrosis the increase of TGF-β1 mRNA precede the increase of type I and type III procollagen mRNAs The secretion of biologically active TGF-β1 by alveolar macrophages is transiently elevated in bleomycin -induced pulmonary inflammation, whereas latent (L)-TGF-β1 secretion remains elevated for a prolonged length of time and it is probable that the extent of inflammation and fibrosis in this model depends on the quantity of active TGF-β1 available .
In BAL of iNOSWT animals, that underwent bleomycin instillation we observed, a strong increase of inflammatory cells such as macrophages and neutrophils. This could justify, at least in part, the significantly higher TGF-β1 expression and the increased lung collagen content in these mice. Bleomycin-treated iNOSKO mice as well as bleomycin-treated iNOSWT mice which received the treatment with GW274150 showed both significantly reduced TGF-β1 expression as well as lung collagen deposition, together with a significantly reduced inflammatory cells presence in the BAL. Furthermore we report in the present study in the lung tissue of bleomycin-treated iNOSKO mice as well as bleomycin-treated iNOSWT mice which received the treatment with GW274150 a significant reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPO. Neutrophils recruited into the tissue can contribute to tissue destruction by the production of reactive oxygen metabolites, granule enzymes, and cytokines that further amplify the inflammatory response by their effects on macrophages and lymphocytes . Furthermore, we found that the tissue damage induced by bleomycin in vehicle-treated mice was associated with an intense immunostaining of nitrotyrosine formation also suggesting that a structural alteration of the lung had occurred, most probably due to the formation of highly reactive nitrogen-derivatives. Recent evidence indicates, that bleomycin is a well-known cause of intracellular oxidative stress, several findings in this study suggest that extracellular oxidative stress may also play a role in the pathogenesis of bleomycin-induced lung injury [72, 73]. Therefore, in this study we clearly demonstrate that the genetic and pharmacological inhibition of iNOS prevent the formation of peroxynitrite. Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for the detection of the endogenous formation "footprint" of peroxynitrite . There is, however, recent evidence that certain other reactions can also induce tyrosine nitration; e.g., the reaction of nitrite with hypoclorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine . Increased nitrotyrosine staining is considered, therefore, as an indication of "increased nitrative stress" rather than a specific marker of the generation of peroxynitrite. From the present data, we cannot determine the mechanism of tyrosine nitration: inhibition of NOS by NOS inhibitor would inhibit both NO formation (and thus, reduce the generation of peroxynitrite) as well as it would suppress nitrite formation (and thereby attenuate the peroxidase dependent mechanisms of tyrosine nitration). Nevertheless, we can certainly conclude from the current data that the absence of iNOS significantly reduced tyrosine nitration in vivo.