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Anti-fibrotic effect of meloxicam in a murine lung fibrosis model
European Journal of Pharmacology 564 (2007) 181 – 189 www.elsevier.com/locate/ejphar
Anti-fibrotic effect of meloxicam in a murine lung fibrosis model Hossam M.M. Arafa a,⁎, Mohamed H. Abdel-Wahab a , Mohamed F. El-Shafeey a , Osama A. Badary b , Farid M.A. Hamada a a b
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Cairo, Egypt Received 28 January 2007; received in revised form 16 February 2007; accepted 26 February 2007 Available online 20 March 2007
Abstract A murine lung fibrosis model has been induced by challenging male Swiss albino mice with a fibrotic dose of bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 weeks. The model has been characterized and confirmed biochemically, histologically and morphometrically. Keeping in mind that inflammation is the forerunner of lung fibrosis, we have investigated the possible anti-fibrotic effect of meloxicam; a selective COX-2 inhibitor, in this lung fibrosis paradigm. When administered ahead of bleomycin challenge, meloxicam significantly reduced the lung content of hydroxyproline; the backbone of collagen matrix. This was further confirmed by the lower collagen deposition as revealed by histochemical examination of lung sections. Meloxicam had also anti-oxidant effect as shown by increase in lung reduced glutathione (GSH) level and decreases in lung malonedialdehyde (MDA) content and myeloperoxidase (MPO) activity. Besides, meloxicam has shown an apparent angiostatic activity. Histologically, meloxicam lessened lung inflammation and fibrotic changes induced by bleomycin. Taken together, one could conclude that meloxicam has shown anti-fibrotic effect in the bleomycin lung fibrosis model. Apart from its well-known anti-inflammatory potential, this anti-fibrotic action of meloxicam resides most probably, at least partly, in its anti-oxidant and angiostatic effects. © 2007 Elsevier B.V. All rights reserved. Keywords: Lung fibrosis; Bleomycin; Collagen; Meloxicam; Anti-oxidant; Angiogenesis
1. Introduction Bleomycin is an antibiotic anti-cancer drug that has shown efficacy in an array of tumors both in man and animals (Chen and Stubbe, 2005). The therapeutic utility of the drug has, however, been precluded by severe toxicities especially of the lung and skin (Azambuja et al., 2005). Lung fibrosis has been considered the dose-limiting toxicity of the anti-neoplastic drug. Bleomycin-induced pulmonary fibrosis in rodents has been used as a surrogate model for human lung fibrosis (Giri et al., 2002). The pathogenesis of bleomycin-induced pneumopathy is not yet fully explored. Inflammation and immune processes are, however, considered among the major mechanisms that injure lung tissue and induce fibrosis (Hagiwara et al., 2000). There is also considerable evidence that oxidative stress due to oxygengenerated free radicals plays a major role in inflammatory and
⁎ Corresponding author. Tel.: +20 202 4577202; fax: +20 202 2014052. E-mail address: [email protected] (H.M.M. Arafa). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.065
immune-mediated bleomycin-induced lung damage (Oury et al., 2001). Meloxicam is a selective cyclooxygenase (COX-2) inhibitor that is used effectively for the management of many rheumatic disorders including rheumatoid arthritis and osteoarthritis. The drug has shown improved gastric and renal tolerability and higher therapeutic index both experimentally and clinically compared to conventional non-steroidal anti-inflammatory drugs (NSAIDs) (Gates et al., 2005). The forerunner for bleomycin-induced pulmonary fibrosis has been considered the inflammatory responses induced by the anti-neoplastic agent. Therefore, meloxicam has been utilized in the current study initially for its anti-inflammatory effects. Besides, the notion that the COX-2 inhibitor has shown some antiradical effects (Agha et al., 1999; Gupta et al., 2002) prompted us to investigate its modulatory effects on bleomycin-induced lung fibrosis possibly by regulating the oxidant/anti-oxidant imbalance. Taking all that into consideration, we have addressed in the present work whether or not meloxicam could ameliorate
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bleomycin-induced lung injury using a mouse model of lung fibrosis. In this sense, we have induced pulmonary fibrosis in male Swiss albino mice by challenging the animals with a fibrotic dose of bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 consecutive weeks. The possible protective effects of meloxicam have been studied and the possible mechanism(s) whereby meloxicam could attenuate such bleomycin-induced pneumopathy were also elucidated. The design of the current work involved biochemical, morphological and morphometric analyses. The biochemical parameters encompassed the lung contents of hydroxyproline as a marker of collagen deposition, reduced glutathione (GSH), and malonedialdehyde (MDA) as a measure of lipid peroxidation, and the activities of lung myeloperoxidase (MPO) and superoxide dismutase (SOD). Histopathological examination as well as morphometric analysis of lung tissue sections for collagen accumulation and deposition was achieved. The finding that angiogenesis is a central hallmark of pulmonary fibrosis (Burdick et al., 2005) warranted our attention to assess if meloxicam has a possible angiostatic effect as a plausible mechanism for any possible anti-fibrotic action. 2. Materials and methods 2.1. Drugs Bleomycin hydrochloride was supplied as bleocin ampoules (15 mg/ml) from Nippon Kayaku Co., Ltd (Tokyo, Japan). Meloxicam was kindly supplied as a yellow pure powder from (ADWIA, Cairo, Egypt). 2.2. Chemicals 4-hydroxy-L-proline was purchased from Sigma-Aldrich Chemical Co., Ltd (Gillingham, Dorset, England). Chloramine-t (N-chloro-p-toluene sulfonamide sodium), dimethoxybenzidine, Evan's blue, and all other chemicals were of the finest analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Animals Swiss albino mice weighing 25–30 g were housed in the animal facility of the Pharmacy College, Al-Azhar University. The animals were kept at temperature of 25 ± 1 °C and a relative humidity of 55% with a regular 12 h light/12 h dark cycle. The animals were fed standard chow (El-Nasr Co., Abou-Zaabal, Cairo, Egypt) and water was ad libitum. All animal experiments were conducted according to the regulations of the Committee on Bioethics for Animal Experiments of Al-Azhar University. 2.4. Ehrlich ascites carcinoma (EAC) cells It is a murine mammary cell line that was a kind gift from the Cancer Biology Department, National Cancer Institute, Cairo University. A cell population of 2.5 × 106 was used to induce angiogenesis.
2.5. Design of work 2.5.1. Lung toxicity study This study was designed to address the possible protective effects of meloxicam in bleomycin-induced lung fibrosis model in male Swiss albino mice. In this context, sixty male Swiss albino mice were classified into 4 groups; 15 mice each. One group received saline s.c. (0.1 ml/20 g body weight) twice weekly for 6 consecutive weeks, and served as control group. Another group was injected i.p. with meloxicam in a dose of 10 mg/kg body weight (Engelhardt et al., 1995) twice weekly for 6 consecutive weeks. A third group was challenged with bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 consecutive weeks (Filderman et al., 1988). A fourth group was injected with meloxicam (10 mg/kg body weight, i.p.) 1 h before bleomycin (10 mg/kg body weight, s.c.) that was given twice weekly for 6 consecutive weeks. Five days after the last bleomycin injection, animals were euthanized by cervical dislocation, lungs were dissected out and processed as in methodology for biochemical, histological and histochemical analyses. Each biochemical parameter was presented as a mean of 10 animals. Two animals were used for lung histopathology, while the remaining three animals were used for the morphometric analysis of collagen. A numerical mean of optical density of 10 fields per group was computed. 2.5.1.1. Lung hydroxyproline content. Total lung collagen was determined as hydroxyproline according to the method earlier described by Woessner (1961). In brief, lung homogenate was acid digested and the digest was mixed with a mixture of chloramine-t and Ehrlich's solution. The color developed was measured at 550 nm using a Shimadzu Spectrophotometer UV 1201 (Japan). 2.5.1.2. Lung myeloperoxidase (MPO) activity. MPO activity was assessed according to Manktelow and Meyer (1986). Lung MPO was extracted with hexadecyl trimethylammonium bromide. Then, dimethoxybenzidine (DMB) was oxidized by MPO in presence of hydrogen peroxide, and the optical density was measured at 460 nm. 2.5.1.3. Reduced glutathione (GSH) level. Reduced glutathione (GSH) was determined according to the method described earlier by Ellman (1959). The procedure is based on the reduction of Ellman's reagent by SH groups to form 2-nitro-5mercaptobenzoic acid, which has an intense yellow color that is measured spectrophotometrically at 412 nm using Shimadzu Spectrophotometer UV 1201 (Japan). 2.5.1.4. Lung superoxide dismutase (SOD) activity. The enzymatic activity of lung SOD was assessed according to Marklund (1985). In brief, SOD activity was determined by computing the difference between auto-oxidation of pyrogallol alone and in presence of SOD. One unit of SOD activity is defined as the amount of the enzyme causing 50% inhibition of auto-oxidation of pyrogallol.
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(1947). Tissues were then embedded in paraffin blocks. 5 micron-thick sections were obtained from the blocks and stained by routine hematoxylin and eosin. They were then examined microscopically.
Fig. 1. Effects of bleomycin (BLM) and/or meloxicam on lung hydroxyproline content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
2.5.1.5. Lung lipid peroxidation. Lipid peroxidation was assessed as MDA content of the lung according to the method of Uchiyama and Mihara (1978). In short, the colorimetric determination of MDA is based on the reaction of one molecule of the reactive aldehyde with two molecules of thiobarbituric acid at low pH (2–3), and at a temperature of 95 °C for 45 min. The resultant pink color was extracted by n-butanol, and the absorbance was determined at 535 nm and 520 nm spectrophotometrically. The difference in optical density between both wavelengths was used as a measure of lung MDA content. 2.5.1.6. Histochemical localization of collagen. Morphometric analysis was carried out using Masson's trichrome technique by means of CAS-200 image analyzer (Masson, 1929). Simply, deparaffinized lung sections fixed in formol-saline were subjected to triple stain including acid fuschin, phosphomolybdic acid and methyl blue. Collagen deposits were visualized as a green fluorescence that was computed as numerical values.
2.5.2. Angiostatic activity The angiostatic activity of meloxicam was assessed according to Lee et al. (1990). A total of 20 Swiss albino mice were allocated to 2 groups; 10 mice each. Each animal was inoculated i.d. at 4 to 6 sites bilaterally on the lower ventral side. Each site was injected with 0.1 ml saline containing 2.5 × 106 EAC cells. Animals were assigned to 2 groups, and treatments were initiated 2 h after tumor inoculation that was designated day 0. Animals received the treatment regimens as such: 1) One group received a single dose of PBS (0.2 ml/20 g body weight, s.c.). A second group was injected meloxicam (10 mg/kg body weight, i.p.). Twenty four hours after the administration of the last dose, each mouse received 0.25 ml (1% w/v) Evan's blue through the tail vein. Animals were then anesthetized by thiopental (1 mg/mouse) and orbital blood samples were collected using heparinized microcapillaries and layered on a solution of 0.5% sodium sulfate/acetone (2/3 v/v) at a concentration of 2 μl blood/ml. Percentage angiogenesis was calculated as follows: %Ang: ¼ ½ð A−BÞ=ðC−BÞ 100 Where, A, B, C represent the optical density, measured at 620 nm, of the treated tumor, background skin, and control tumor, respectively.
2.5.1.7. Histopathological examination. Right lungs were kept in 10% formaline solution for 24 h using Hartz Technique
2.5.3. Statistical analysis Data were analyzed using a software program (GraphPad InStat, version 2.0, 1993, Philadelphia, USA). Data were presented as mean ± SE. Multiple comparisons were achieved by one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. Independent Student's t-test was used to address the statistical significance in the
Fig. 2. Effects of bleomycin (BLM) and/or meloxicam on lung myeloperoxidase activity of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
Fig. 3. Effects of bleomycin (BLM) and/or meloxicam on lung SOD activity of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
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Fig. 4. Effects of bleomycin (BLM) and/or meloxicam on lung content of GSH in male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
angiogenesis assay. P b 0.05 was chosen as the criterion for significance. 3. Results 3.1. Estimation of lung hydroxyproline content Meloxicam had no effect on the lung content of hydroxyproline compared to control animals (Fig. 1). However, bleomycin resulted in a marked increase in lung hydroxyproline content amounted to 521% compared to control value (Fig. 1). Prior administration of meloxicam ahead of bleomycin challenge significantly decreased the lung hydroxyproline content by about 52% compared to animals that received bleomycin alone. However, this value was still significantly higher than normal value by about 198% (Fig. 1). 3.2. Lung myeloperoxidase (MPO activity Meloxicam did not affect the lung activity of myeloperoxidase compared to control mice (Fig. 2). MPO
Fig. 5. Effects of bleomycin (BLM) and/or meloxicam on lung MDA content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
Fig. 6. Effects of bleomycin (BLM) and/or meloxicam on lung collagen content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
activity has been greatly increased by about 228% following administration of bleomycin compared to control animals (Fig. 2). Pre-treatment with meloxicam before bleomycin administration reduced the lung MPO activity by about 35% compared to bleomycin-treated animals. However, such combination modality exhibited still a higher lung myeloperoxidase activity amounted to about 114% compared to control value (Fig. 2). 3.3. Lung superoxide dismutase activity (SOD) Meloxicam had no effect on the lung activity of SOD compared to control value (Fig. 3). Bleomycin, however, caused an apparent elevation in the lung activity of SOD amounted to about 163% compared to control animals (Fig. 3). Prior administration of meloxicam ahead of bleomycin injection significantly lowered the lung SOD activity by about 44% compared to animals that received the cytotoxic drug alone. The combination regimen, however, showed a higher SOD activity amounted to 48.5% compared to the control one (Fig. 3).
Fig. 7. Effect of meloxicam on percentage angiogenesis in male Swiss albino mice. Data present mean ± SE (n = 10). ⁎Significantly different from control at p b 0.05 using two-tailed independent Student's t-test.
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3.4. Reduced glutathione (GSH) level Meloxicam showed no effect on the lung GSH content comzpared to control mice (Fig. 4). Bleomycin significantly reduced the lung content of GSH by about 43% compared to control animals (Fig. 4). Prior administration of meloxicam before bleomycin resulted in a notable increase in lung GSH content amounted to about 26% compared to mice treated with bleomycin alone. The lung GSH, however, was still lower than the control value by about 28% (Fig. 4).
Fig. 9. A. Normal lung tissues showing normal open alveoli and interalveolar space with normal terminal bronchi (H & E × 125). B. Meloxicam-treated lung sections having the same normal parenchymal architecture (H & E × 125). C. Bleomycin-treated lung sections showing severe congestion and edema of interalveolar space, inflammation and thickened and collapsed alveoli with marked fibrosis (H & E × 125). D. Meloxicam prior to bleomycin showing mild inflammation, moderate edema and minimal fibrotic changes (H & E × 125).
3.5. Lung lipid peroxidation
Fig. 8. A. Normal lung tissue showing normal open pattern of alveoli and interalveolar space and normal bronchi with minimal collagen deposition (MT ×125). B. Meloxicam-treated lung sections having the same tissue architecture and collagen profile as in control animals (MT × 125). C. Bleomycin-treated lung sections showing extensive peribronchial and perialveolar collagen deposition that obliterates the alveolar spaces (MT × 125). D. Meloxicam ahead of bleomycin showing mild peribronchial and perialveolar collagen deposition (MT ×125).
Meloxicam resulted in virtually the same level of lung MDA as in control mice (Fig. 5). Bleomycin caused a substantial increase in lung content of MDA amounted to about 451% compared to control level (Fig. 5). Combined administration of meloxicam and bleomycin reduced significantly the lung MDA level by about 77% compared to animals that received bleomycin alone (Fig. 5).
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3.6. Histochemical localization of collagen in lung tissue Normal lung tissues showed normal open patterns of alveoli and interalveolar spaces and normal terminal bronchi with minimal collagen deposition (Fig. 8A). Meloxicam-treated mice had virtually the same tissue architecture and collagen profile as in control animals (Figs. 6 and 8B). Bleomycin-induced extensive peribronchial and perialveolar collagen deposition (green fluorescence) that obliterated the alveolar spaces (Fig. 8C). The collagen deposition was so abundant and amounted to more than 16-fold that of control animals (Fig. 6). Prior administration of meloxicam ahead of bleomycin challenge caused mild peribronchial and perialveolar collagen deposition (Fig. 8D). The combination modality markedly reduced the collagen deposition by about 50% compared to bleomycin-treated animals. However, it resulted in more than a 7-fold increase in collagen deposition compared to normal collagen value (Fig. 6) 3.7. Angiostatic activity Meloxicam inhibited significantly tumor-induced angiogenesis by about 25% compared to untreated control animals (Fig. 7). 3.8. Hisopathological examination of lung tissue Normal lung tissues showed normal open alveoli and interalveolar spaces with normal terminal bronchi (Fig. 9A). Meloxicam had no injurious effects on the lung tissue with virtually the same normal parenchymal architecture (Fig. 9B). Repeated exposure to bleomycin-induced severe lung injury manifested as congestion and edema of interalveolar spaces, interalveolar inflammation and thickened alveolar wall, collapsed alveoli with bronchial erosion and patchy cheesy fibrotic areas (Fig. 9C). Pre-treatment with meloxicam before bleomycin showed mild inflammation of peribronchial and perialveolar tissues with moderate edema of interalveolar spaces and minimal fibrotic changes (Fig. 9D). 4. Discussion Bleomycin is an antibiotic anti-cancer drug that has proven efficacy in a variety of human malignancies including germ-cell tumors, lymphomas, Kaposi's sarcoma, cervical cancer, and squamous cell carcinomas of the head and neck (Sleijfer, 2001). The clinical usefulness of bleomycin has been, however, hampered by detrimental side effects. Pulmonary fibrosis has been considered the dose-limiting toxicity of the drug. Pulmonary fibrosis, idiopathic or otherwise, is commonly progressive and essentially an untreatable disease, with an increasingly fatal outcome (Coker and Laurent, 1998). The bleomycin-rodent paradigm of lung fibrosis is an established and widely used surrogate model of human lung fibrosis (Keane et al., 2001).
There has been a wealth of studies employing bleomycin in different animal models including mice, rats, hamsters and dogs. The use of these animal models has been helpful in partly establishing pathways of lung damage leading to fibrosis, and comparison studies of patients with lung pneumopathy have validated many of these animal studies (Cooper, 2000). In the current study we have used a mouse model of lung fibrosis by challenging the animals subcutaneously with a fibrogenic dose of bleomycin (10 mg/kg body weight) twice weekly for 6 consecutive weeks (Filderman et al., 1988). The present work has been conducted in an attempt to address whether or not meloxicam; a selective COX-2 enzyme inhibitor, would protect against bleomycin-induced lung fibrosis in male Swiss albino mice, thus conferring a possible anti-fibrotic potential in this animal model. In this sense, lung content of hydroxyproline, an index of collagen deposition (Keane et al., 1999), lung activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), and lung contents of reduced glutathione (GSH) and MDA; as a measure of lipid peroxidation, have been undertaken in the current study as biochemical markers. Histochemical localization of collagen in lung tissue has been further done to confirm the assessment of collagen deposition. Assessment of the angiostatic activity of meloxicam was further determined to establish a link between its possible angiostatic effect and anti-fibrotic potential (if any). Lung histopathology was also done to confirm the model and to unravel the possible anti-fibrogenic activity of meloxicam. Bleomycin-induced lung fibrosis model that was clearly characterized biochemically, morphometrically and histopa thologically. Bleomycin produced more than 5-fold increase in lung content of hydroxyproline compared to control values. Indeed, 4-hydroxyproline is the building block of collagen, and, thus, measurement of the amino acid following acid digestion of collagen would be a good representative of collagen content. This finding is in accord with many previous reports that demonstrated apparent elevation in lung hydroxyproline content as an index of collagen accumulation and deposition (Giri et al., 2002; Lossos et al., 2002; Tanino et al., 2002). This was further affirmed by morphometric analysis of lung sections for collagen deposition. Bleomycin, in the present work, induced collagen accumulation and deposition in peribronchial and perialveolar tissues that obliterated alveolar spaces as tiny fibrils. The lung collagen content in bleomycin-treated animals was abundant reaching about 16-fold that of control value. Actually, the deposition of excess or abnormal collagen is characteristic of lung fibrosis as reported by many previous studies (Daba et al., 2002; Pardo et al., 2003; Serrano-Mollar et al., 2003). Bleomycin enhanced myeloperoxidase activity; a specific heme peroxidase belonging to the mammalian family of peroxidases that exerts oxidizing activities leading to essential protein inactivation and cytotoxicity. This finding is coping with many previous similar data that demonstrated the importance of the enzyme as a biochemical marker of bleomycininduced lung fibrosis (Azuma et al., 2000; Azoulay et al., 2003; Serrano-Moller et al., 2003).
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Bleomycin challenge significantly increased the lung activity of SOD. This finding is contradictory to the popular belief that the SOD family confers anti-oxidant potential. Consistent with that was the finding that the lecithinized SOD type has been used to suppress the progression of pulmonary fibrosis following bleomycin challenge in mice (Tamagawa et al., 2000). However, our finding is in line with other reports that demonstrated an increase in lung activity of SOD after bleomycin administration (Arslan et al., 2002; Bowler et al., 2002). Such debate could be explained in virtue of the postulate adopted by Giri and Hollinger (1996) that the lung SOD is rather an oxidant marker of lung injury especially in the early events. Bleomycin notably reduced the lung content of GSH. This is in harmony with other reports that demonstrated GSH reduction or depletion following bleomycin challenge (Arslan et al., 2002; Pardo et al., 2003; Serrano-Moller et al., 2003). Such nadir in GSH level could be indirectly attributed to the increased activity of MPO system. Consistent with that is the fact that GSH is an important biological target of HOCl of the MPO system. In addition to giving the disulfide (GSSG), which can be recycled by the activity of GSH reductase, HOCl attack of GSH also gives products identified as internal sulfonamide and thiosulphonate, which are likely to be irreversible, depleting the cell of GSH and compromising the anti-oxidant defense (Winterbourn and Brennan 1997). Bleomycin increased lipid peroxidation in lung tissue as evidenced by about 4.5-fold increase in MDA level above normal value. Similar findings have been previously reported (Hagiwara et al., 2000; Wang et al., 2000; Giri et al., 2003). The lung fibrosis paradigm has been further confirmed morphologically. Histological analysis of lung tissue sections following repeated exposure to a fibrogenic dose of bleomycin in the present work revealed severe lung injury manifested as congestion and edema of interalveolar spaces, interalveolar inflammation and thickened alveolar wall, collapsed alveoli with bronchial erosion and patchy cheesy fibrotic areas. This is in harmony with previous studies that demonstrated the injurious effects of bleomycin on lung tissues (Genovese et al., 2005; Gharaee-Kermani et al., 2005; Li et al., 2006). The pathogenesis of bleomycin-induced pulmonary fibrosis has been extensively studied in different animal species, however the underlying mechanisms for such pneumopathy are not clearly understood. Though the pathophysiology of such a lung injury is thought to be multifactorial, potential mechanisms include the infiltration of inflammatory cells to the lungs and the generation of free radicals and proinflammatory mediators (Sleijfer, 2001; Nagase et al., 2002). Reactive oxygen species and its sequela; oxidative stress, play an important role in the development of fibrotic responses in the lung especially those induced by bleomycin challenge. Daba et al. (2002) have used two anti-oxidants for the abatement of bleomycin-induced lung fibrosis in rats. Indeed, traditional approaches used to attenuate bleomycininduced lung fibrosis in animal models have focused on ameliorating the inflammatory response (Tokuda et al., 2000), abrogating epithelial injury (Kuwano et al., 1999), and targeting
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the transforming growth factor β pathways and the associated fibrotic responses (Munger et al., 1999). Since inflammation is the forerunner of fibrosis (Iyer et al., 2000), controlling the degree of inflammation could minimize the release of various inflammatory mediators known to be involved in collagen synthesis and accumulation, and thus attenuating the development of bleomycin-induced lung fibrosis. Hence, the current study has been designed in an attempt to evaluate if meloxicam; a selective COX-2 inhibitor has the ability to initially ameliorate the inflammatory events and, thus contributing to its possible anti-fibrotic effect, if any, in the bleomycin-mouse model of lung fibrosis used in the present work. When administered ahead of bleomycin, meloxicam ameliorated all the biochemical parameters that have been altered by the anti-cancer drug. It is noteworthy to say that we have administered meloxicam 1 h ahead of each bleomycin injection based on its pharmacokinetic profile (Degner et al., 1998). In this context, meloxicam significantly reduced the lung hydroxyproline level. This was confirmed by the finding in the current study that meloxicam halved lung collagen content. This finding is coping with the report of El-Chaar et al. (2005) who demonstrated that meloxicam reduced collagen biosynthesis in renal fibrosis. This effect was ascribed to the COX-2 inhibitory effect of meloxicam, since it is known that reactive oxygen species can stimulate the synthesis of prostaglandins and COX2 (Lu and Wahl, 2005). Meloxicam notably inhibited the lung activity of myeloperoxidase when given ahead of bleomycin challenge. This is in harmony with other previous data that reported significant reduction of myeloperoxidase activity in other tissue inflammation models (Saleh et al., 1999; Khan et al., 2002). Meloxicam pre-treatment increased the lung content of GSH, but notably reduced the lung lipid peroxide content. These results suggest an anti-radical effect of meloxicam. This finding is coping with previous reports that demonstrated anti-oxidant effects of meloxicam (Agha et al., 1999; Burak-Cimen et al., 2003; Van Antwerpen and Neve, 2004). The concept that free radical generation is crucial for lung fibrosis (Manoury et al., 2005) and the fact that inflammation is the forerunner of fibrosis may highlight the importance of the anti-radical effect of meloxicam observed in the current study as a possible mechanism for its anti-fibrotic effect in the bleomycin-mouse pulmonary fibrosis model. Meloxicam significantly reduced bleomycin-induced increase in lung SOD, and this effect could further add to its anti-radical effects observed in the current study if the rise in lung SOD was considered primarily as oxidant marker as discussed before. Meloxicam administered ahead of bleomycin challenge reduced the peribronchial and perialveolar tissue inflammation and resulted in moderate edema of interalveolar spaces and minimal fibrotic changes. This is in line with the finding of Wang et al. (1999) who reported that meloxicam alleviated the histopathologic damage induced by lipoploysaccharide in rat lung injury. Such protective effect, though not complete, reveals an anti-fibrotic effect of meloxicam that is further supported biochemically and morphometrically.
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Of major interest was the finding in the present work that meloxicam had an apparent angiostatic effect using mouse tumor angiogenesis model. This is coping with other previous reports that demonstrated angiostatic effects of COX-2 inhibitors (Wu et al., 2005; Klenke et al., 2006). Such anti-angiogenic effect observed in the current study for meloxicam could possibly explain, at least partly, why the the COX-2 inhibitor attenuated bleomycin-induced pneumopathy in mice. Indeed, a wealth of studies have addressed the link between pulmonary inflammation and fibrosis, angiogenesis and COX-2 expression (Brewer et al., 2003; Keane, 2004; Bhatt et al., 2006). In conclusion, meloxicam has shown anti-fibrotic effect when it was administered ahead of bleomycin challenge. The COX-2 inhibitor abrogated all the injurious lung effects of the anti-neoplastic drug, whether biochemical, histologic or histochemical, in this murine model of pulmonary fibrosis. Apart from its well-known anti-inflammatory effect, the anti-fibrotic effect of meloxicam could be possibly ascribed to its antioxidant and/or anti-angiogenic effects that modulated the inflammatory responses and fibrotic changes induced by bleomycin. Irrespective of the pathway, or the molecular mechanism involved, the fact that meloxicam therapy could so apparently inhibit bleomycin-induced fibrosis raises the exciting possibility of using COX-2 inhibition approach as an adjuvant for the abatement of this bleomycin-induced pneumopathy. References Agha, A.M., El-Khatib, A.S., Al-Zuhair, H., 1999. Modulation of oxidant status by meloxicam in experimentally induced arthritis. Pharmacol. Res. 40, 385–392. Arslan, N., Miller, T.R., Dehashti, F., Battafarano, R.J., Siegel, B.A., 2002. Evaluation of response to neoadjuvant therapy by quantitative 2-deoxy-2 (18F), fluoro-d-glucose with position emission tomography in patients with esophageal cancer. Mol. Imaging Biol. 4, 301–310. Azambuja, E., Fleck, J.F., Batista, R.G., Menna, S.S., Barreto, 2005. Bleomycin lung toxicity: who are the patients with increased risk? Pulm. Pharmacol. Ther. 18, 363–366. Azoulay, S., Levane, M., Brochard, L., Schlemmer, B., Harf, A., Delclanx, C., 2003. Effect of granulocyte colony-stimulating factor on bleomycin-induced acute lung injury and pulmonary fibrosis. Crit. Care Med. 31, 1442–1448. Azuma, A., Takahashi, S., Nose, M., Araki, K., Araki, M., Takahashi, T., Hirose, M., Kawashima, H., Miyasaka, M., Kudoh, S., 2000. Role of E-selectin in bleomycin induced lung fibrosis in mice. Thora 55, 147–152. Bhatt, N., Baran, C.P., Allen, J., Magro, C., Marsh, C.B., 2006. Promising pharmacologic innovations in treating pulmonary fibrosis. Curr. Opin. Pharmacol. 6, 284–292. Bowler, R.P., Nicks, M., Warnick, K., Crapo, J.D., 2002. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am. J. Physiol., Lung Cell. Mol. Physiol. 282, L719–L726. Brewer, G.J., Ullenbruch, M.R., Dick, R., Olivarez, L., Phan, S.H., 2003. Tetrathiomolybdate therapy protects against bleomycin-induced pulmonary fibrosis in mice. J. Lab. Clin. Med. 141, 210–216. Burak-Cimen, M.Y., Cimen, O.B., Eskandari, G., Sahin, G., Erdogan, C., Atik, U., 2003. In vivo effects of meloxicam, celecoxib, and ibuprofen on free radical metabolism in human erythrocytes. Drug Chem. Toxicol. 26, 169–176. Burdick, M.D., Murray, L.A., Keane, M.P., Xue, Y.Y., Zisman, D.A., Belperio, J.A., Strieter, R.M., 2005. CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling. Am. J. Respir. Crit. Care Med. 171, 261–268. Chen, J., Stubbe, J., 2005. Bleomycins: towards better therapeutics. Nat. Rev., Cancer 5, 102–112.
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Anti-fibrotic effect of meloxicam in a murine lung fibrosis model Hossam M.M. Arafa a,⁎, Mohamed H. Abdel-Wahab a , Mohamed F. El-Shafeey a , Osama A. Badary b , Farid M.A. Hamada a a b
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Cairo, Egypt Received 28 January 2007; received in revised form 16 February 2007; accepted 26 February 2007 Available online 20 March 2007
Abstract A murine lung fibrosis model has been induced by challenging male Swiss albino mice with a fibrotic dose of bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 weeks. The model has been characterized and confirmed biochemically, histologically and morphometrically. Keeping in mind that inflammation is the forerunner of lung fibrosis, we have investigated the possible anti-fibrotic effect of meloxicam; a selective COX-2 inhibitor, in this lung fibrosis paradigm. When administered ahead of bleomycin challenge, meloxicam significantly reduced the lung content of hydroxyproline; the backbone of collagen matrix. This was further confirmed by the lower collagen deposition as revealed by histochemical examination of lung sections. Meloxicam had also anti-oxidant effect as shown by increase in lung reduced glutathione (GSH) level and decreases in lung malonedialdehyde (MDA) content and myeloperoxidase (MPO) activity. Besides, meloxicam has shown an apparent angiostatic activity. Histologically, meloxicam lessened lung inflammation and fibrotic changes induced by bleomycin. Taken together, one could conclude that meloxicam has shown anti-fibrotic effect in the bleomycin lung fibrosis model. Apart from its well-known anti-inflammatory potential, this anti-fibrotic action of meloxicam resides most probably, at least partly, in its anti-oxidant and angiostatic effects. © 2007 Elsevier B.V. All rights reserved. Keywords: Lung fibrosis; Bleomycin; Collagen; Meloxicam; Anti-oxidant; Angiogenesis
1. Introduction Bleomycin is an antibiotic anti-cancer drug that has shown efficacy in an array of tumors both in man and animals (Chen and Stubbe, 2005). The therapeutic utility of the drug has, however, been precluded by severe toxicities especially of the lung and skin (Azambuja et al., 2005). Lung fibrosis has been considered the dose-limiting toxicity of the anti-neoplastic drug. Bleomycin-induced pulmonary fibrosis in rodents has been used as a surrogate model for human lung fibrosis (Giri et al., 2002). The pathogenesis of bleomycin-induced pneumopathy is not yet fully explored. Inflammation and immune processes are, however, considered among the major mechanisms that injure lung tissue and induce fibrosis (Hagiwara et al., 2000). There is also considerable evidence that oxidative stress due to oxygengenerated free radicals plays a major role in inflammatory and
⁎ Corresponding author. Tel.: +20 202 4577202; fax: +20 202 2014052. E-mail address: [email protected] (H.M.M. Arafa). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.065
immune-mediated bleomycin-induced lung damage (Oury et al., 2001). Meloxicam is a selective cyclooxygenase (COX-2) inhibitor that is used effectively for the management of many rheumatic disorders including rheumatoid arthritis and osteoarthritis. The drug has shown improved gastric and renal tolerability and higher therapeutic index both experimentally and clinically compared to conventional non-steroidal anti-inflammatory drugs (NSAIDs) (Gates et al., 2005). The forerunner for bleomycin-induced pulmonary fibrosis has been considered the inflammatory responses induced by the anti-neoplastic agent. Therefore, meloxicam has been utilized in the current study initially for its anti-inflammatory effects. Besides, the notion that the COX-2 inhibitor has shown some antiradical effects (Agha et al., 1999; Gupta et al., 2002) prompted us to investigate its modulatory effects on bleomycin-induced lung fibrosis possibly by regulating the oxidant/anti-oxidant imbalance. Taking all that into consideration, we have addressed in the present work whether or not meloxicam could ameliorate
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bleomycin-induced lung injury using a mouse model of lung fibrosis. In this sense, we have induced pulmonary fibrosis in male Swiss albino mice by challenging the animals with a fibrotic dose of bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 consecutive weeks. The possible protective effects of meloxicam have been studied and the possible mechanism(s) whereby meloxicam could attenuate such bleomycin-induced pneumopathy were also elucidated. The design of the current work involved biochemical, morphological and morphometric analyses. The biochemical parameters encompassed the lung contents of hydroxyproline as a marker of collagen deposition, reduced glutathione (GSH), and malonedialdehyde (MDA) as a measure of lipid peroxidation, and the activities of lung myeloperoxidase (MPO) and superoxide dismutase (SOD). Histopathological examination as well as morphometric analysis of lung tissue sections for collagen accumulation and deposition was achieved. The finding that angiogenesis is a central hallmark of pulmonary fibrosis (Burdick et al., 2005) warranted our attention to assess if meloxicam has a possible angiostatic effect as a plausible mechanism for any possible anti-fibrotic action. 2. Materials and methods 2.1. Drugs Bleomycin hydrochloride was supplied as bleocin ampoules (15 mg/ml) from Nippon Kayaku Co., Ltd (Tokyo, Japan). Meloxicam was kindly supplied as a yellow pure powder from (ADWIA, Cairo, Egypt). 2.2. Chemicals 4-hydroxy-L-proline was purchased from Sigma-Aldrich Chemical Co., Ltd (Gillingham, Dorset, England). Chloramine-t (N-chloro-p-toluene sulfonamide sodium), dimethoxybenzidine, Evan's blue, and all other chemicals were of the finest analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Animals Swiss albino mice weighing 25–30 g were housed in the animal facility of the Pharmacy College, Al-Azhar University. The animals were kept at temperature of 25 ± 1 °C and a relative humidity of 55% with a regular 12 h light/12 h dark cycle. The animals were fed standard chow (El-Nasr Co., Abou-Zaabal, Cairo, Egypt) and water was ad libitum. All animal experiments were conducted according to the regulations of the Committee on Bioethics for Animal Experiments of Al-Azhar University. 2.4. Ehrlich ascites carcinoma (EAC) cells It is a murine mammary cell line that was a kind gift from the Cancer Biology Department, National Cancer Institute, Cairo University. A cell population of 2.5 × 106 was used to induce angiogenesis.
2.5. Design of work 2.5.1. Lung toxicity study This study was designed to address the possible protective effects of meloxicam in bleomycin-induced lung fibrosis model in male Swiss albino mice. In this context, sixty male Swiss albino mice were classified into 4 groups; 15 mice each. One group received saline s.c. (0.1 ml/20 g body weight) twice weekly for 6 consecutive weeks, and served as control group. Another group was injected i.p. with meloxicam in a dose of 10 mg/kg body weight (Engelhardt et al., 1995) twice weekly for 6 consecutive weeks. A third group was challenged with bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 consecutive weeks (Filderman et al., 1988). A fourth group was injected with meloxicam (10 mg/kg body weight, i.p.) 1 h before bleomycin (10 mg/kg body weight, s.c.) that was given twice weekly for 6 consecutive weeks. Five days after the last bleomycin injection, animals were euthanized by cervical dislocation, lungs were dissected out and processed as in methodology for biochemical, histological and histochemical analyses. Each biochemical parameter was presented as a mean of 10 animals. Two animals were used for lung histopathology, while the remaining three animals were used for the morphometric analysis of collagen. A numerical mean of optical density of 10 fields per group was computed. 2.5.1.1. Lung hydroxyproline content. Total lung collagen was determined as hydroxyproline according to the method earlier described by Woessner (1961). In brief, lung homogenate was acid digested and the digest was mixed with a mixture of chloramine-t and Ehrlich's solution. The color developed was measured at 550 nm using a Shimadzu Spectrophotometer UV 1201 (Japan). 2.5.1.2. Lung myeloperoxidase (MPO) activity. MPO activity was assessed according to Manktelow and Meyer (1986). Lung MPO was extracted with hexadecyl trimethylammonium bromide. Then, dimethoxybenzidine (DMB) was oxidized by MPO in presence of hydrogen peroxide, and the optical density was measured at 460 nm. 2.5.1.3. Reduced glutathione (GSH) level. Reduced glutathione (GSH) was determined according to the method described earlier by Ellman (1959). The procedure is based on the reduction of Ellman's reagent by SH groups to form 2-nitro-5mercaptobenzoic acid, which has an intense yellow color that is measured spectrophotometrically at 412 nm using Shimadzu Spectrophotometer UV 1201 (Japan). 2.5.1.4. Lung superoxide dismutase (SOD) activity. The enzymatic activity of lung SOD was assessed according to Marklund (1985). In brief, SOD activity was determined by computing the difference between auto-oxidation of pyrogallol alone and in presence of SOD. One unit of SOD activity is defined as the amount of the enzyme causing 50% inhibition of auto-oxidation of pyrogallol.
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(1947). Tissues were then embedded in paraffin blocks. 5 micron-thick sections were obtained from the blocks and stained by routine hematoxylin and eosin. They were then examined microscopically.
Fig. 1. Effects of bleomycin (BLM) and/or meloxicam on lung hydroxyproline content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
2.5.1.5. Lung lipid peroxidation. Lipid peroxidation was assessed as MDA content of the lung according to the method of Uchiyama and Mihara (1978). In short, the colorimetric determination of MDA is based on the reaction of one molecule of the reactive aldehyde with two molecules of thiobarbituric acid at low pH (2–3), and at a temperature of 95 °C for 45 min. The resultant pink color was extracted by n-butanol, and the absorbance was determined at 535 nm and 520 nm spectrophotometrically. The difference in optical density between both wavelengths was used as a measure of lung MDA content. 2.5.1.6. Histochemical localization of collagen. Morphometric analysis was carried out using Masson's trichrome technique by means of CAS-200 image analyzer (Masson, 1929). Simply, deparaffinized lung sections fixed in formol-saline were subjected to triple stain including acid fuschin, phosphomolybdic acid and methyl blue. Collagen deposits were visualized as a green fluorescence that was computed as numerical values.
2.5.2. Angiostatic activity The angiostatic activity of meloxicam was assessed according to Lee et al. (1990). A total of 20 Swiss albino mice were allocated to 2 groups; 10 mice each. Each animal was inoculated i.d. at 4 to 6 sites bilaterally on the lower ventral side. Each site was injected with 0.1 ml saline containing 2.5 × 106 EAC cells. Animals were assigned to 2 groups, and treatments were initiated 2 h after tumor inoculation that was designated day 0. Animals received the treatment regimens as such: 1) One group received a single dose of PBS (0.2 ml/20 g body weight, s.c.). A second group was injected meloxicam (10 mg/kg body weight, i.p.). Twenty four hours after the administration of the last dose, each mouse received 0.25 ml (1% w/v) Evan's blue through the tail vein. Animals were then anesthetized by thiopental (1 mg/mouse) and orbital blood samples were collected using heparinized microcapillaries and layered on a solution of 0.5% sodium sulfate/acetone (2/3 v/v) at a concentration of 2 μl blood/ml. Percentage angiogenesis was calculated as follows: %Ang: ¼ ½ð A−BÞ=ðC−BÞ 100 Where, A, B, C represent the optical density, measured at 620 nm, of the treated tumor, background skin, and control tumor, respectively.
2.5.1.7. Histopathological examination. Right lungs were kept in 10% formaline solution for 24 h using Hartz Technique
2.5.3. Statistical analysis Data were analyzed using a software program (GraphPad InStat, version 2.0, 1993, Philadelphia, USA). Data were presented as mean ± SE. Multiple comparisons were achieved by one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. Independent Student's t-test was used to address the statistical significance in the
Fig. 2. Effects of bleomycin (BLM) and/or meloxicam on lung myeloperoxidase activity of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
Fig. 3. Effects of bleomycin (BLM) and/or meloxicam on lung SOD activity of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
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Fig. 4. Effects of bleomycin (BLM) and/or meloxicam on lung content of GSH in male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
angiogenesis assay. P b 0.05 was chosen as the criterion for significance. 3. Results 3.1. Estimation of lung hydroxyproline content Meloxicam had no effect on the lung content of hydroxyproline compared to control animals (Fig. 1). However, bleomycin resulted in a marked increase in lung hydroxyproline content amounted to 521% compared to control value (Fig. 1). Prior administration of meloxicam ahead of bleomycin challenge significantly decreased the lung hydroxyproline content by about 52% compared to animals that received bleomycin alone. However, this value was still significantly higher than normal value by about 198% (Fig. 1). 3.2. Lung myeloperoxidase (MPO activity Meloxicam did not affect the lung activity of myeloperoxidase compared to control mice (Fig. 2). MPO
Fig. 5. Effects of bleomycin (BLM) and/or meloxicam on lung MDA content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
Fig. 6. Effects of bleomycin (BLM) and/or meloxicam on lung collagen content of male Swiss albino mice. Data present mean ± SE (n = 10). Multiple comparisons were done using one way ANOVA followed by Bonferroni test for selected pairs as post-ANOVA test. ⁎Significantly different from control at p b 0.05. ⁎⁎Significantly different from bleomycin at p b 0.05.
activity has been greatly increased by about 228% following administration of bleomycin compared to control animals (Fig. 2). Pre-treatment with meloxicam before bleomycin administration reduced the lung MPO activity by about 35% compared to bleomycin-treated animals. However, such combination modality exhibited still a higher lung myeloperoxidase activity amounted to about 114% compared to control value (Fig. 2). 3.3. Lung superoxide dismutase activity (SOD) Meloxicam had no effect on the lung activity of SOD compared to control value (Fig. 3). Bleomycin, however, caused an apparent elevation in the lung activity of SOD amounted to about 163% compared to control animals (Fig. 3). Prior administration of meloxicam ahead of bleomycin injection significantly lowered the lung SOD activity by about 44% compared to animals that received the cytotoxic drug alone. The combination regimen, however, showed a higher SOD activity amounted to 48.5% compared to the control one (Fig. 3).
Fig. 7. Effect of meloxicam on percentage angiogenesis in male Swiss albino mice. Data present mean ± SE (n = 10). ⁎Significantly different from control at p b 0.05 using two-tailed independent Student's t-test.
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3.4. Reduced glutathione (GSH) level Meloxicam showed no effect on the lung GSH content comzpared to control mice (Fig. 4). Bleomycin significantly reduced the lung content of GSH by about 43% compared to control animals (Fig. 4). Prior administration of meloxicam before bleomycin resulted in a notable increase in lung GSH content amounted to about 26% compared to mice treated with bleomycin alone. The lung GSH, however, was still lower than the control value by about 28% (Fig. 4).
Fig. 9. A. Normal lung tissues showing normal open alveoli and interalveolar space with normal terminal bronchi (H & E × 125). B. Meloxicam-treated lung sections having the same normal parenchymal architecture (H & E × 125). C. Bleomycin-treated lung sections showing severe congestion and edema of interalveolar space, inflammation and thickened and collapsed alveoli with marked fibrosis (H & E × 125). D. Meloxicam prior to bleomycin showing mild inflammation, moderate edema and minimal fibrotic changes (H & E × 125).
3.5. Lung lipid peroxidation
Fig. 8. A. Normal lung tissue showing normal open pattern of alveoli and interalveolar space and normal bronchi with minimal collagen deposition (MT ×125). B. Meloxicam-treated lung sections having the same tissue architecture and collagen profile as in control animals (MT × 125). C. Bleomycin-treated lung sections showing extensive peribronchial and perialveolar collagen deposition that obliterates the alveolar spaces (MT × 125). D. Meloxicam ahead of bleomycin showing mild peribronchial and perialveolar collagen deposition (MT ×125).
Meloxicam resulted in virtually the same level of lung MDA as in control mice (Fig. 5). Bleomycin caused a substantial increase in lung content of MDA amounted to about 451% compared to control level (Fig. 5). Combined administration of meloxicam and bleomycin reduced significantly the lung MDA level by about 77% compared to animals that received bleomycin alone (Fig. 5).
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3.6. Histochemical localization of collagen in lung tissue Normal lung tissues showed normal open patterns of alveoli and interalveolar spaces and normal terminal bronchi with minimal collagen deposition (Fig. 8A). Meloxicam-treated mice had virtually the same tissue architecture and collagen profile as in control animals (Figs. 6 and 8B). Bleomycin-induced extensive peribronchial and perialveolar collagen deposition (green fluorescence) that obliterated the alveolar spaces (Fig. 8C). The collagen deposition was so abundant and amounted to more than 16-fold that of control animals (Fig. 6). Prior administration of meloxicam ahead of bleomycin challenge caused mild peribronchial and perialveolar collagen deposition (Fig. 8D). The combination modality markedly reduced the collagen deposition by about 50% compared to bleomycin-treated animals. However, it resulted in more than a 7-fold increase in collagen deposition compared to normal collagen value (Fig. 6) 3.7. Angiostatic activity Meloxicam inhibited significantly tumor-induced angiogenesis by about 25% compared to untreated control animals (Fig. 7). 3.8. Hisopathological examination of lung tissue Normal lung tissues showed normal open alveoli and interalveolar spaces with normal terminal bronchi (Fig. 9A). Meloxicam had no injurious effects on the lung tissue with virtually the same normal parenchymal architecture (Fig. 9B). Repeated exposure to bleomycin-induced severe lung injury manifested as congestion and edema of interalveolar spaces, interalveolar inflammation and thickened alveolar wall, collapsed alveoli with bronchial erosion and patchy cheesy fibrotic areas (Fig. 9C). Pre-treatment with meloxicam before bleomycin showed mild inflammation of peribronchial and perialveolar tissues with moderate edema of interalveolar spaces and minimal fibrotic changes (Fig. 9D). 4. Discussion Bleomycin is an antibiotic anti-cancer drug that has proven efficacy in a variety of human malignancies including germ-cell tumors, lymphomas, Kaposi's sarcoma, cervical cancer, and squamous cell carcinomas of the head and neck (Sleijfer, 2001). The clinical usefulness of bleomycin has been, however, hampered by detrimental side effects. Pulmonary fibrosis has been considered the dose-limiting toxicity of the drug. Pulmonary fibrosis, idiopathic or otherwise, is commonly progressive and essentially an untreatable disease, with an increasingly fatal outcome (Coker and Laurent, 1998). The bleomycin-rodent paradigm of lung fibrosis is an established and widely used surrogate model of human lung fibrosis (Keane et al., 2001).
There has been a wealth of studies employing bleomycin in different animal models including mice, rats, hamsters and dogs. The use of these animal models has been helpful in partly establishing pathways of lung damage leading to fibrosis, and comparison studies of patients with lung pneumopathy have validated many of these animal studies (Cooper, 2000). In the current study we have used a mouse model of lung fibrosis by challenging the animals subcutaneously with a fibrogenic dose of bleomycin (10 mg/kg body weight) twice weekly for 6 consecutive weeks (Filderman et al., 1988). The present work has been conducted in an attempt to address whether or not meloxicam; a selective COX-2 enzyme inhibitor, would protect against bleomycin-induced lung fibrosis in male Swiss albino mice, thus conferring a possible anti-fibrotic potential in this animal model. In this sense, lung content of hydroxyproline, an index of collagen deposition (Keane et al., 1999), lung activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), and lung contents of reduced glutathione (GSH) and MDA; as a measure of lipid peroxidation, have been undertaken in the current study as biochemical markers. Histochemical localization of collagen in lung tissue has been further done to confirm the assessment of collagen deposition. Assessment of the angiostatic activity of meloxicam was further determined to establish a link between its possible angiostatic effect and anti-fibrotic potential (if any). Lung histopathology was also done to confirm the model and to unravel the possible anti-fibrogenic activity of meloxicam. Bleomycin-induced lung fibrosis model that was clearly characterized biochemically, morphometrically and histopa thologically. Bleomycin produced more than 5-fold increase in lung content of hydroxyproline compared to control values. Indeed, 4-hydroxyproline is the building block of collagen, and, thus, measurement of the amino acid following acid digestion of collagen would be a good representative of collagen content. This finding is in accord with many previous reports that demonstrated apparent elevation in lung hydroxyproline content as an index of collagen accumulation and deposition (Giri et al., 2002; Lossos et al., 2002; Tanino et al., 2002). This was further affirmed by morphometric analysis of lung sections for collagen deposition. Bleomycin, in the present work, induced collagen accumulation and deposition in peribronchial and perialveolar tissues that obliterated alveolar spaces as tiny fibrils. The lung collagen content in bleomycin-treated animals was abundant reaching about 16-fold that of control value. Actually, the deposition of excess or abnormal collagen is characteristic of lung fibrosis as reported by many previous studies (Daba et al., 2002; Pardo et al., 2003; Serrano-Mollar et al., 2003). Bleomycin enhanced myeloperoxidase activity; a specific heme peroxidase belonging to the mammalian family of peroxidases that exerts oxidizing activities leading to essential protein inactivation and cytotoxicity. This finding is coping with many previous similar data that demonstrated the importance of the enzyme as a biochemical marker of bleomycininduced lung fibrosis (Azuma et al., 2000; Azoulay et al., 2003; Serrano-Moller et al., 2003).
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Bleomycin challenge significantly increased the lung activity of SOD. This finding is contradictory to the popular belief that the SOD family confers anti-oxidant potential. Consistent with that was the finding that the lecithinized SOD type has been used to suppress the progression of pulmonary fibrosis following bleomycin challenge in mice (Tamagawa et al., 2000). However, our finding is in line with other reports that demonstrated an increase in lung activity of SOD after bleomycin administration (Arslan et al., 2002; Bowler et al., 2002). Such debate could be explained in virtue of the postulate adopted by Giri and Hollinger (1996) that the lung SOD is rather an oxidant marker of lung injury especially in the early events. Bleomycin notably reduced the lung content of GSH. This is in harmony with other reports that demonstrated GSH reduction or depletion following bleomycin challenge (Arslan et al., 2002; Pardo et al., 2003; Serrano-Moller et al., 2003). Such nadir in GSH level could be indirectly attributed to the increased activity of MPO system. Consistent with that is the fact that GSH is an important biological target of HOCl of the MPO system. In addition to giving the disulfide (GSSG), which can be recycled by the activity of GSH reductase, HOCl attack of GSH also gives products identified as internal sulfonamide and thiosulphonate, which are likely to be irreversible, depleting the cell of GSH and compromising the anti-oxidant defense (Winterbourn and Brennan 1997). Bleomycin increased lipid peroxidation in lung tissue as evidenced by about 4.5-fold increase in MDA level above normal value. Similar findings have been previously reported (Hagiwara et al., 2000; Wang et al., 2000; Giri et al., 2003). The lung fibrosis paradigm has been further confirmed morphologically. Histological analysis of lung tissue sections following repeated exposure to a fibrogenic dose of bleomycin in the present work revealed severe lung injury manifested as congestion and edema of interalveolar spaces, interalveolar inflammation and thickened alveolar wall, collapsed alveoli with bronchial erosion and patchy cheesy fibrotic areas. This is in harmony with previous studies that demonstrated the injurious effects of bleomycin on lung tissues (Genovese et al., 2005; Gharaee-Kermani et al., 2005; Li et al., 2006). The pathogenesis of bleomycin-induced pulmonary fibrosis has been extensively studied in different animal species, however the underlying mechanisms for such pneumopathy are not clearly understood. Though the pathophysiology of such a lung injury is thought to be multifactorial, potential mechanisms include the infiltration of inflammatory cells to the lungs and the generation of free radicals and proinflammatory mediators (Sleijfer, 2001; Nagase et al., 2002). Reactive oxygen species and its sequela; oxidative stress, play an important role in the development of fibrotic responses in the lung especially those induced by bleomycin challenge. Daba et al. (2002) have used two anti-oxidants for the abatement of bleomycin-induced lung fibrosis in rats. Indeed, traditional approaches used to attenuate bleomycininduced lung fibrosis in animal models have focused on ameliorating the inflammatory response (Tokuda et al., 2000), abrogating epithelial injury (Kuwano et al., 1999), and targeting
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the transforming growth factor β pathways and the associated fibrotic responses (Munger et al., 1999). Since inflammation is the forerunner of fibrosis (Iyer et al., 2000), controlling the degree of inflammation could minimize the release of various inflammatory mediators known to be involved in collagen synthesis and accumulation, and thus attenuating the development of bleomycin-induced lung fibrosis. Hence, the current study has been designed in an attempt to evaluate if meloxicam; a selective COX-2 inhibitor has the ability to initially ameliorate the inflammatory events and, thus contributing to its possible anti-fibrotic effect, if any, in the bleomycin-mouse model of lung fibrosis used in the present work. When administered ahead of bleomycin, meloxicam ameliorated all the biochemical parameters that have been altered by the anti-cancer drug. It is noteworthy to say that we have administered meloxicam 1 h ahead of each bleomycin injection based on its pharmacokinetic profile (Degner et al., 1998). In this context, meloxicam significantly reduced the lung hydroxyproline level. This was confirmed by the finding in the current study that meloxicam halved lung collagen content. This finding is coping with the report of El-Chaar et al. (2005) who demonstrated that meloxicam reduced collagen biosynthesis in renal fibrosis. This effect was ascribed to the COX-2 inhibitory effect of meloxicam, since it is known that reactive oxygen species can stimulate the synthesis of prostaglandins and COX2 (Lu and Wahl, 2005). Meloxicam notably inhibited the lung activity of myeloperoxidase when given ahead of bleomycin challenge. This is in harmony with other previous data that reported significant reduction of myeloperoxidase activity in other tissue inflammation models (Saleh et al., 1999; Khan et al., 2002). Meloxicam pre-treatment increased the lung content of GSH, but notably reduced the lung lipid peroxide content. These results suggest an anti-radical effect of meloxicam. This finding is coping with previous reports that demonstrated anti-oxidant effects of meloxicam (Agha et al., 1999; Burak-Cimen et al., 2003; Van Antwerpen and Neve, 2004). The concept that free radical generation is crucial for lung fibrosis (Manoury et al., 2005) and the fact that inflammation is the forerunner of fibrosis may highlight the importance of the anti-radical effect of meloxicam observed in the current study as a possible mechanism for its anti-fibrotic effect in the bleomycin-mouse pulmonary fibrosis model. Meloxicam significantly reduced bleomycin-induced increase in lung SOD, and this effect could further add to its anti-radical effects observed in the current study if the rise in lung SOD was considered primarily as oxidant marker as discussed before. Meloxicam administered ahead of bleomycin challenge reduced the peribronchial and perialveolar tissue inflammation and resulted in moderate edema of interalveolar spaces and minimal fibrotic changes. This is in line with the finding of Wang et al. (1999) who reported that meloxicam alleviated the histopathologic damage induced by lipoploysaccharide in rat lung injury. Such protective effect, though not complete, reveals an anti-fibrotic effect of meloxicam that is further supported biochemically and morphometrically.
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Of major interest was the finding in the present work that meloxicam had an apparent angiostatic effect using mouse tumor angiogenesis model. This is coping with other previous reports that demonstrated angiostatic effects of COX-2 inhibitors (Wu et al., 2005; Klenke et al., 2006). Such anti-angiogenic effect observed in the current study for meloxicam could possibly explain, at least partly, why the the COX-2 inhibitor attenuated bleomycin-induced pneumopathy in mice. Indeed, a wealth of studies have addressed the link between pulmonary inflammation and fibrosis, angiogenesis and COX-2 expression (Brewer et al., 2003; Keane, 2004; Bhatt et al., 2006). In conclusion, meloxicam has shown anti-fibrotic effect when it was administered ahead of bleomycin challenge. The COX-2 inhibitor abrogated all the injurious lung effects of the anti-neoplastic drug, whether biochemical, histologic or histochemical, in this murine model of pulmonary fibrosis. Apart from its well-known anti-inflammatory effect, the anti-fibrotic effect of meloxicam could be possibly ascribed to its antioxidant and/or anti-angiogenic effects that modulated the inflammatory responses and fibrotic changes induced by bleomycin. Irrespective of the pathway, or the molecular mechanism involved, the fact that meloxicam therapy could so apparently inhibit bleomycin-induced fibrosis raises the exciting possibility of using COX-2 inhibition approach as an adjuvant for the abatement of this bleomycin-induced pneumopathy. References Agha, A.M., El-Khatib, A.S., Al-Zuhair, H., 1999. Modulation of oxidant status by meloxicam in experimentally induced arthritis. Pharmacol. Res. 40, 385–392. Arslan, N., Miller, T.R., Dehashti, F., Battafarano, R.J., Siegel, B.A., 2002. Evaluation of response to neoadjuvant therapy by quantitative 2-deoxy-2 (18F), fluoro-d-glucose with position emission tomography in patients with esophageal cancer. Mol. Imaging Biol. 4, 301–310. Azambuja, E., Fleck, J.F., Batista, R.G., Menna, S.S., Barreto, 2005. Bleomycin lung toxicity: who are the patients with increased risk? Pulm. Pharmacol. Ther. 18, 363–366. Azoulay, S., Levane, M., Brochard, L., Schlemmer, B., Harf, A., Delclanx, C., 2003. Effect of granulocyte colony-stimulating factor on bleomycin-induced acute lung injury and pulmonary fibrosis. Crit. Care Med. 31, 1442–1448. Azuma, A., Takahashi, S., Nose, M., Araki, K., Araki, M., Takahashi, T., Hirose, M., Kawashima, H., Miyasaka, M., Kudoh, S., 2000. Role of E-selectin in bleomycin induced lung fibrosis in mice. Thora 55, 147–152. Bhatt, N., Baran, C.P., Allen, J., Magro, C., Marsh, C.B., 2006. Promising pharmacologic innovations in treating pulmonary fibrosis. Curr. Opin. Pharmacol. 6, 284–292. Bowler, R.P., Nicks, M., Warnick, K., Crapo, J.D., 2002. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am. J. Physiol., Lung Cell. Mol. Physiol. 282, L719–L726. Brewer, G.J., Ullenbruch, M.R., Dick, R., Olivarez, L., Phan, S.H., 2003. Tetrathiomolybdate therapy protects against bleomycin-induced pulmonary fibrosis in mice. J. Lab. Clin. Med. 141, 210–216. Burak-Cimen, M.Y., Cimen, O.B., Eskandari, G., Sahin, G., Erdogan, C., Atik, U., 2003. In vivo effects of meloxicam, celecoxib, and ibuprofen on free radical metabolism in human erythrocytes. Drug Chem. Toxicol. 26, 169–176. Burdick, M.D., Murray, L.A., Keane, M.P., Xue, Y.Y., Zisman, D.A., Belperio, J.A., Strieter, R.M., 2005. CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling. Am. J. Respir. Crit. Care Med. 171, 261–268. Chen, J., Stubbe, J., 2005. Bleomycins: towards better therapeutics. Nat. Rev., Cancer 5, 102–112.
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