Celecoxib, a selective cyclo-oxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic acid in rats.

Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leukocyte infiltration, upregulation of the expression of intercellular adhesion molecule 1 (ICAM-1) and upregulation of P-selectin in the colon. Here, we investigate the effects of the selective cyclo-oxygenase-2 inhibitor, celecoxib, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Rats experienced hemorrhagic diarrhoea and weight loss. At 4 days after administration of DNBS, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology, as well as an increase in myeloperoxidase activity in the mucosa) was associated with upregulation of ICAM-1 and P-selectin, as well as high tissue levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) polymerase showed intense staining in the inflamed colon. Celecoxib (5 mg/kg twice a day orally) significantly reduced the degree of hemorrhagic diarrhoea and the weight loss caused by administration of DNBS. Celecoxib also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde, (iv) the increase in staining (immunohistochemistry) for nitrotyrosine, as well as (v) the upregulation of ICAM-1 and P-selectin caused by DNBS in the colon. Thus, we provide the first evidence that a selective cyclo-oxygenase-2 inhibitor celecoxib reduces the degree of colitis caused by DNBS.

The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat.

BACKGROUND: We have recently demonstrated that 17beta-estradiol (E2) inhibits the increase of inducible nitric oxide synthetase (iNOS) activity in selected model systems such as macrophages, microglia, smooth muscle cells, and proposed that this effect might be associated with an anti-inflammatory activity of this hormone. Here we investigate the effects of endogenous estrogens in rats subjected to carrageenan-induced pleurisy. MATERIALS AND METHODS: Adult female rats were ovariectomized 3 weeks before the experiments to deplete circulating estrogens. Selected inflammatory markers, landmarks of the delayed phase of carrageenan-induced pleurisy, were measured in intact (N-OVX), and ovariectomized (OVX) female rats. In addition, the effect of hormone replacement was evaluated in ovariectomized rats with intraperitoneal injection of 17beta-estradiol (E2; 50 microg/kg) 1 hr before carrageenan treatment (OVX + E2). RESULTS: Ovariectomy enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly increased in estrogens-deprived rats. The iNOS in lung samples was significantly increased by the surgery. The increase of iNOS activity was correlated with a marked enhancement in the production of TNF-alpha and IL-1beta. Immunohistochemical analysis for P-selectin and ICAM-I, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) revealed a positive staining in lungs from carrageenan-treated rats, which was markedly enhanced in ovariectomized rats when compared to cycling rats, particularly in the estrous phase of the cycle. Estrogen replacement counteracted the effect of surgery on all of the above indicators of lung inflammation, suggesting that in the cycling rat this hormone plays a key role in the increased sensitivity to inflammatory injury observed in the OVX rat. CONCLUSION: This study demonstrates that endogenous estrogens production plays an important protective role against carrageenan-induced acute inflammation by decreasing the expression of specific markers of the delayed phase of this well-known model of acute inflammation.

Effect of melatonin on cellular energy depletion mediated by peroxynitrite and poly (ADP-ribose) synthetase activation in an acute model of inflammation.

DNA single-strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) triggers an energy-consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Recently, we proposed that during an acute model (pleurisy), cellular injury is mediated by peroxynitrite formation and consequent PARS activation. Here, we investigated whether in vivo melatonin treatment inhibits cellular injury induced by peroxynitrite production and PARS activation in macrophages collected from rats subjected to carrageenan-induced pleurisy. Macrophages harvested from the pleural cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, carrageenan-induced pleurisy caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, activation of PARS, and reduction of cellular levels of NAD+. In vivo treatment with melatonin [12.5 or 25 or 50 mg/kg, intraperitoneally (i.p.), 30 min before carrageenan] significantly reduced peroxynitrite formation in a dose-dependent manner and prevented the appearance of DNA damage, decrease in mitochondrial respiration, loss of cellular levels of NAD+, and PARS activation. Our study supports the view that the antioxidant and anti-inflammatory effect of melatonin is also correlated with the inhibition of peroxynitrite production and PARS activation. In conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in acute inflammation.

Protective effects of n-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat.

Oxidative stress has been suggested as a potential mechanism in the pathogenesis of lung inflammation. The pharmacological profile of n-acetylcysteine (NAC), a free radical scavenger, was evaluated in an experimental model of lung injury (carrageenan-induced pleurisy). Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many neutrophils (PMNs), an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, tumor necrosis factor alpha, and interleukin 1beta. All parameters of inflammation were attenuated by NAC treatment. Furthermore, carrageenan induced an up-regulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine, and PARS was reduced by NAC. In vivo NAC treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine-123, prevented the appearance of DNA damage, an decrease in mitochondrial respiration, and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. A significant alteration in the morphology of red blood cells was observed 24 h after carrageenan administration. NAC treatment has the ability to significantly diminish the red blood cell alteration. Our results clearly demonstrate that NAC treatment exerts a protective effect and clearly indicate that NAC offers a novel therapeutic approach for the management of lung injury where radicals have been postulated to play a role.

GPI 6150, a poly (ADP-ribose) polymerase inhibitor, exhibits an anti-inflammatory effect in rat models of inflammation.

Poly (ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, has been show to play an important role in the pathogenesis of inflammation. Here, we investigate the effects of GPI 6150 (1,11b-dihydro-[2H]benzopyrano [4,3,2-de]isoquinolin-3-one), a new poly (ADP-ribose) polymerase inhibitor, in animal models of acute and chronic inflammation (carrageenan-induced paw edema, adjuvant-induced arthritis and zymosan-induced multiple organ failure) where oxygen radicals, nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. The results show that the poly (ADP-ribose) polymerase inhibitor GPI 6150 inhibits the inflammatory response (paw swelling, and organ injury). The present results demonstrate that inhibition of poly (ADP-ribose) polymerase by GPI 6150 exerts potent anti-inflammatory effects. Part of these anti-inflammatory effects may be related to a reduction of neutrophil recruitment into the inflammatory site.

Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403.

1. M40403 is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (SODm) that removes superoxide anions (*O2-) without interfering with other reactive species known to be involved in inflammatory responses (e.g. nitric oxide, NO and peroxynitrite, ONOO-). 2. As such, M40403 represents an important pharmacological tool to dissect the roles of *O2- in acute and chronic inflammation. For this purpose, the pharmacological profile of M40403 was evaluated in carrageenan-induced pleurisy. 3. Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by: fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), prostaglandin E2 (PGE2), tumour necrosis factor alpha, (TNFalpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interleukin-10 (IL-10). 4. All parameters of inflammation were attenuated by M40403 except for NOx, PGE2 and IL-10 which remained unaltered. Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. 5. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PARS was reduced by M40403. 6. These results clearly indicate that *O2- plays a critical role in the development of the inflammatory response by altering key components of the inflammatory cascade. Therefore, synthetic enzymes of SOD such as M40403, offers a novel therapeutic approach for the management of various inflammatory diseases where these radicals have been postulated to play a role.

Melatonin reduces dinitrobenzene sulfonic acid-induced colitis.

Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of this study was to examine the effects of the pineal secretory product melatonin in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Rats experienced bloody diarrhea and a significant loss of body weight. Four days after DNBS administration, the colon damage was characterized by areas of mucosal necrosis. Neutrophil infiltration (indicated by myeloperoxidase [MPO] activity in the mucosa) was associated with up-regulation of ICAM-1, expression of P-selectin, and high levels of malondialdehyde (MDA). Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) synthetase (PARS) showed an intense staining in the inflamed colon. Staining of colon tissue sections obtained from DNBS-treated rats with an anti-cycloxygenase-2 (COX-2) antibody showed a diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase (iNOS) was found mainly in the macrophages of the inflamed colons from DNBS-treated rats. Treatment with melatonin (15 mg/kg daily, intraperitoneally) significantly reduced the appearance of diarrhea and the loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture, as well as a significant reduction of colonic MPO activity and MDA levels. Melatonin also reduced the appearance of nitrotyrosine and PARS immunoreactivity in the colon, as well as reducing the up-regulation of ICAM-1 and the expression of P-selectin. The intensity and degree of the stainings for COX-2 and iNOS were markedly reduced in tissue sections obtained from melatonin-treated rats. The results of the this study suggest that the administration of melatonin might be beneficial for the treatment of IBD.

Absence of endogeneous interleukin-10 enhances the evolution of murine type-II collagen-induced arthritis.

Interleukin-10 (IL-10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL-10 in the modulation of the inflammatory response in mice subjected to collagen-induced arthritis. Collagen-induced arthritis (CIA) was induced in mice lacking the gene for IL-10 (IL-10 "knock-out", IL-10KO) and in wild-type control (IL-10WT) mice by an intradermal injection of 100 mul of the emulsion (containing 100 mug of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. IL-10 wild type (WT) mice developed an erosive, hind paw arthritis when immunised with CII in CFA. Macroscopic clinical evidence of CIA first appeared as peri-articular erythema and oedema in the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged IL-10WT. The severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint margins. IL-10KO mice experienced higher rates of clinical signs and more severe knee and paw injury as compared to IL-10WT. The degree of oxidative and nitrosative damage was significantly higher in IL-10KO mice than in wild-type littermates, as indicated by elevated malondialdehyde levels and formation of nitrotyrosine and poly (ADP-ribose) synthetase (PARS). Plasma levels of the proinflammatory cytokines, tumour necrosis factor, interleukin-1beta and interleukin-6 were also greatly enhanced in comparison to wild-type mice. These data demonstrate that endogenous IL-10 exerts an anti-inflammatory role during chronic inflammation and tissue damage associated with collagen-induced arthritis, possibly by regulating neutrophil recruitment, and the subsequent cytokine and oxidant generation.

Amelioration of joint disease in a rat model of collagen-induced arthritis by M40403, a superoxide dismutase mimetic.

OBJECTIVE: To investigate the effects of M40403, a synthetic mimetic of superoxide dismutase (SOD), on collagen-induced arthritis (CIA) in rats. METHODS: CIA was elicited in Lewis rats by intradermal injection of 100 microl of an emulsion of bovine type II collagen (CII) in Freund's incomplete adjuvant at the base of the tail. A second injection was given on day 21. RESULTS: Immunization induced an erosive arthritis of the hind paws. Macroscopic evidence of CIA first appeared as periarticular erythema and edema in the hind paws by days 24-26 after the first injection, with a 100% incidence by days 27. Severity progressed over a 35-day period. Radiography revealed soft tissue swelling and focal resorption of bone, together with osteophyte formation in the tibiotarsal joint. Histopathologic features included erosion of the articular cartilage at the joint margins and subchondral bone resorption associated with bone-derived multinucleated cell-containing granulomatous lesions. Treatment with M40403 (2-10 mg/kg/day) starting at the onset of arthritis (day 25) ameliorated the clinical signs on days 26-35 and improved the histologic findings in the joint and paw. Immunohistochemical analysis for nitrotyrosine (a marker of peroxynitrite formation) and poly(ADP-ribose) polymerase (PARP; a nuclear enzyme activated by DNA single-strand damage) revealed positive staining in the inflamed joints of CII-treated rats, suggestive of the formation of peroxynitrite and DNA damage, both of which were markedly reduced by M40403 treatment. Radiographic evidence of protection from bone resorption, osteophyte formation, and soft tissue swelling was apparent in the tibiotarsal joints of M40403-treated rats. Arthritic rats treated with M40403 gained weight at the same rate and to the same extent as normal, nonarthritic rats. CONCLUSION: This study shows that a low molecular weight mimetic of SOD, M40403, attenuates the degree of chronic inflammation, tissue damage, and bone damage associated with CIA in the rat, and supports the possible use of SOD mimetics as therapeutic agents for the management of chronic diseases such as rheumatoid arthritis.

Calpain inhibitor I reduces the development of acute and chronic inflammation.

There is limited evidence that inhibition of the activity of the protease calpain I reduces inflammation. Here we investigate the effects of calpain inhibitor I in animal models of acute and chronic inflammation (carrageenan-induced pleurisy and collagen-induced arthritis). We report here for the first time that calpain inhibitor I (given at 5, 10, or 20 mg/kg i.p. in the pleurisy model or at 5 mg/kg i.p every 48 hours in the arthritis model) exerts potent anti-inflammatory effects (eg, inhibition of pleural exudate formation, mononuclear cell infiltration, delayed the development of the clinical signs and histological injury) in vivo. Furthermore, calpain inhibitor I reduced (1) the staining for nitrotyrosine and poly (ADP-ribose) polymerase (immunohistochemistry) and (2) the expression of inducible nitric oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated rats and in joints from collagen-treated rats. Thus, prevention of the activation of calpain I reduces the development of acute and chronic inflammation. Inhibition of calpain I activity may represent a novel therapeutic approach for the therapy of inflammation.

Protective effects of melatonin in ischemic brain injury.

Recent studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. NO, peroxynitrite (formed from NO and superoxide anion), and poly (ADP-Ribose) synthetase (PARS) have been implicated as mediators of neuronal damage following focal ischemia. In the present study, we have investigated the effects of melatonin treatment in Mongolian gerbils subjected to cerebral ischemia. Treatment of gerbils with melatonin (10 mg kg(-1), 30 min before reperfusion and 1, 2, and 6 hr after reperfusion) reduced the formation of post-ischemic brain edema, evaluated by water content. Melatonin also attenuated the increase in the brain levels malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischemia. Positive staining for nitrotyrosine was found in the hippocampus of Mongolian gerbils subjected to cerebral ischemia. Hippocampus tissue sections, from Mongolian gerbils subjected to cerebral ischemia, also showed positive staining for PARS. The degrees of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received melatonin. Melatonin treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA-1 showed a reduction of neuronal loss in animals that received melatonin. These results show that melatonin improves brain injury induced by transient cerebral ischemia.

Effects of n-acetylcysteine in a rat model of ischemia and reperfusion injury.

OBJECTIVE: Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock. METHODS AND RESULTS: Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival. CONCLUSION: Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Beneficial effects of n-acetylcysteine on ischaemic brain injury.

1. Nitric oxide (NO), peroxynitrite, formed from NO and superoxide anion, poly (ADP-ribole) synthetase have been implicated as mediators of neuronal damage following focal ischaemia. Here we have investigated the effects of n-acetylcysteine (NAC) treatment in Mongolian gerbils subjected to cerebral ischaemia. 2. Treatment of gerbils with NAC (20 mg kg(-1) 30 min before reperfusion and 1, 2 and 6 h after reperfusion) reduced the formation of post-ischaemic brain oedema, evaluated by water content. 3. NAC also attenuated the increase in the brain levels of malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischaemia. 4. Positive staining for nitrotyrosine was found in the hippocampus in Mongolian gerbils subjected to cerebral ischaemia. Hippocampus tissue sections from Mongolian gerbils subjected to cerebral ischaemia also showed positive staining for poly (ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received NAC. 5. NAC treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischaemia and reperfusion. 6. Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received NAC. 7. These results show that NAC improves brain injury induced by transient cerebral ischaemia.

17beta-estradiol antiinflammatory activity in carrageenan-induced pleurisy.

We have recently demonstrated that 17beta-estradiol (E2) opposes cytokine-dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclear cells migration as shown by cell counting and myeloperoxidase measurement. In addition, E2 pretreatment opposes carrageneen-induced high lipid peroxidation maintaining malondialdehyde activity at control levels. E2 treatment decreases NO production and the activity of iNOS with consequent diminished nitrite synthesis and nitrosine accumulation. Finally, immunohistochemical analysis for poly (ADP-ribose) synthase revealed a positive staining in lungs from carrageenan-treated rats that was blocked by estradiol treatment. We conclude that E2 attenuates the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in the rat.