Showing papers by "Achille P. Caputi published in 2001"

Antioxidant Therapy: A New Pharmacological Approach in Shock, Inflammation, and Ischemia/Reperfusion Injury

Abstract: A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation, shock, and ischemia/reperfusion injury. The aim of this review is to describe recent developments in the field of oxidative stress research. The first part of the review focuses on the roles of reactive oxygen species (ROS) in shock, inflammation, and ischemia/reperfusion injury. The second part of the review deals with the novel findings using recently identified pharmacological tools (e.g., peroxynitrite decomposition catalysts and selective superoxide dismutase mimetics (SODm) in shock, ischemia/reperfusion, and inflammation. 1) The role of ROS consists of immunohistochemical and biochemical evidence that demonstrates the production of ROS in shock, inflammation, and ischemia/reperfusion injury. ROS can initiate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATPase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation, and ischemia/reperfusion. 2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm, which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, have been shown to prevent in vivo the delayed vascular decompensation and the cellular energetic failure associated with shock, inflammation, and ischemia/reperfusion injury. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical, and hydrogen peroxide) are all potential reactants capable of initiating DNA single-strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase, leading to eventual severe energy depletion of the cells and necrotic-type cell death. Antioxidant treatment inhibits the activation of poly(ADP-ribose) synthetase and prevents the organ injury associated with shock, inflammation, and ischemia/reperfusion.

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

Abstract: 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-). 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. 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 α, (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10). 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. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PARS was reduced by M40403. 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. Keywords: Inflammation, cytokines, adhesion molecules, superoxide anions, superoxide dismutase mimetic Introduction Under normal circumstances, formation of superoxide anion (.O2−; the one-electron reduction product of oxygen) is kept under tight control by endogenous superoxide dismutase (SOD) enzymes. These include: the Mn enzyme in mitochondria (SOD2) and Cu/Zn enzyme present in the cytosol (SOD1) or extracellular surfaces (SOD3). The importance of SOD2 is highlighted by the findings that in contrast to SOD1 (Reaume et al., 1996) and SOD3 (Carlsson et al., 1995), SOD2 knockout is lethal to mice (Li et al., 1995; Lebovitz et al., 1996; Melov et al., 1999). In acute and chronic inflammation, the production of .O2− anion is increased at a rate that overwhelms the capacity of the endogenous SOD enzyme defence system to remove them. The result of such imbalance results in .O2− mediated damage. A proposal that .O2− was intimately involved with the inflammatory response was raised as early as the 1970s through the pioneering work of McCord & Fridovich (1969). Some important pro-inflammatory roles for .O2− include: endothelial cell damage and increased microvascular permeability (Droy-Lefaix et al., 1991; Haglind et al., 1994; Xia et al., 1995), formation of chemotactic factors such as leukotriene B4 (Fantone & Ward, 1982; Deitch et al., 1990), recruitment of neutrophils at sites of inflammation (Boughton-Smith et al., 1993; Salvemini et al., 1999Salvemini et al., 1999), lipid peroxidation and oxidation, DNA single-strand damage (Dix et al., 1996) and formation of peroxynitrite (ONOO−), a potent cytotoxic and proinflammatory molecule (Crow & Beckman, 1995; Salvemini et al., 1998a,1998b; 1999a; Beckman et al., 1990; Ischiropoulos et al., 1992; Beckman & Crow, 1993). Most of the knowledge gathered about the roles of superoxide in disease has been collected by the use of the native SOD enzyme and, more recently, by data generated in transgenic animals that overexpress the human enzyme (Huber et al., 1980; Flohe, 1988; Uematsu et al., 1994; Fridovich, 1995). Protective and beneficial roles of SOD have been demonstrated in a broad range of disease, both preclinically and clinically (Halliwell & Gutteridge, 1985; Maxwell, 1995; McCord, 1974). Orgotein® (bovine CuZnSOD) showed promising results as a human therapeutic in acute and chronic conditions including rheumatoid arthritis and osteoarthritis as well as side effects associated with chemotherapy and radiation therapy (Flohe, 1988; Babior, 1982; Niwa et al., 1985). There are drawbacks or problematic issues associated with the use of the native enzymes as therapeutic agents (e.g., solution instability, immunogenicity of non-human enzymes, bell-shaped dose response curves, high susceptibility to proteolytic digestion) and as pharmacological tools (e.g., they do not penetrate cells or cross the blood brain barrier, limiting the dismutation of superoxide only to the extracellular space or compartments). To overcome the limitations associated with native enzyme therapy we have developed a series of superoxide dismutase mimetics (SODm) that catalytically remove .O2−. M40403 (see chemical structure Figure 1) is a prototypic example of our stable, low molecular weight, manganese-containing, non-peptidic molecules possessing the function and catalytic rate of native SOD enzymes, but with the advantage of being a much smaller molecule (MW 483 vs MW 30,000 for the mimetic and native enzyme, respectively) (Salvemini et al., 1999b). An important property of these SODm is that they catalytically remove superoxide at a high rate without interacting with other reactive species including nitric oxide, peroxynitrite, hydrogen peroxide, oxygen or hydroxyl radicals (Riley et al., 1996; 1997). This property is not shared by other classes of SOD mimetics or scavengers including several metalloporphyrins such as tetrakis-(N-ethyl-2-pyridyl) porphyrin (MnTE-2-PyP) and tetrakis-(benzoic acid) porphyrin (MnTBAP), that interact with other reactive species such as nitric oxide (NO) and ONOO− which clearly play important roles in inflammation (Patel & Day, 1999). Furthermore, SODm are not deactivated by ONOO−, an added advantage over the native MnSOD enzyme that is nitrated and deactivated by ONOO− (Yamakura et al., 1998; Macmillan-Crow & Thompson 1999). We have recently shown that M40403 is anti-inflammatory and protective in ischaemia-reperfusion injury (Salvemini et al., 1999b). Figure 1 Structure of M40403. In the current study we have extended our initial observations to a well characterized model of acute pleurisy in rats and show that M40403 inhibits the inflammatory response following the intrapleural injection of carrageenan in rats. Results obtained in this study support the use of SODm as therapeutic agents in diseases associated with overt production of superoxide.

Melatonin reduces dinitrobenzene sulfonic acid-induced colitis.

Abstract: 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.

Protective effects of a new stable, highly active SOD mimetic, M40401 in splanchnic artery occlusion and reperfusion.

Abstract: 1. 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 M40401, a new S:,S:-dimethyl substituted biscyclohexylpyridine Mn-based superoxide dismutase mimetic (SODm, k(cat)=1.2x10(+9) M(-1) s(-1) at pH=7.4), in rats subjected to SAO shock. 2. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 microg kg(-1), 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. M40401 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. 3. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO-shocked rats which had received M40401. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue sections from SAO-shocked rats. M40401 treatment significantly improved survival. 4. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose ( approximately microg kg(-1)) required to afford protection in this SAO model of ischemia reperfusion injury. 5. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite-related pathways with subsequent reduction of neutrophil-mediated cellular injury.

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

Abstract: 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.

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

Abstract: 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 μl 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.

Serotonin, norepinephrine and dopamine involvement in the antidepressant action of hypericum perforatum.

Abstract: Hypericum perforatum is considered an effective alternative to the synthetic antidepressants in the treatment of mild-to-moderate depression. Recently, we showed that the effects on neurotransmitter contents in different brain regions of laboratory animals are more evident after administration of hypericum extracts containing a higher concentration of flavonoids, thus suggesting that these compounds are important in the antidepressant action of hypericum perforatum. We studied the effects of Ph-50, a hypericum extract standardized to flavonoids (50%) and containing 0.3% hypericin and 4.5% hyperforin on brain serotonin content, norepinephrine and dopamine by a high-performance liquid chromatography method in discrete brain areas (cortex, diencephalon and brainstem) in male Sprague-Dawley rats. Moreover, we evaluated the effects of Ph-50 alone or in association with sulpiride (a dopamine receptor antagonist), metergoline (a serotonin receptor antagonist) and 6-hydroxydopamine (6-OH-DA, destroying norepinephrine-containing neurons) using a forced-swimming test in the rat. Hypericum extract (Ph-50; 250-500 mg/kg) with acute oral administration enhanced serotonin, norepinephrine and dopamine content in the brain and reduced the immobility time of rats in the forced-swimming test. Sulpiride, metergoline and 6-OH-DA significantly increased the period of immobility in the forced-swimming test for the rats receiving hypericum extract (Ph-50). The results indicate that the neurotransmitters studied could be involved in the anti-immobility effects of hypericum, and suggest that its antidepressant action is probably mediated by serotonergic, noradrenergic and dopaminergic system activation.

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

Abstract: We have recently demonstrated that 17β-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 antiinflammatory activity of this hormone Here we investigate the effects of endogenous estrogens in rats subjected to carrageenan-induced pleurisy 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 17β-estradiol (E2; 50 µg/kg) 1 hr before carrageenan treatment (OVX + E2) 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-α and IL-1β 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 carrageenantreated 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 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

Inducible nitric oxide synthase-deficient mice exhibit resistance to the acute pancreatitis induced by cerulein.

Abstract: Oxidative stress plays an important role in the early stage of acute pancreatitis as well as the associated multiple organ injury. Here we compare the degree of pancreatitis caused by cerulein in mice lacking the inducible (or type 2) nitric oxide synthase (iNOS) and in the corresponding wild-type mice. Intraperitoneal injection of cerulein resulted in wild-type mice in a severe, acute pancreatitis, which was characterized by edema, neutrophil infiltration, tissue hemorrhage and cell necrosis as well as increases in the serum levels of amylase and/or lipase. The infiltration of the pancreatic tissue of these animals with neutrophils (measured as increase in myeloperoxidase activity) was associated with up-regulation/expression of the adhesion molecules ICAM-1 and P-selectin as well as signs of enhanced lipid peroxidation (e.g., increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for nitrotyrosine and poly (ADP-ribose) synthetase (PARS) in the pancreas of cerulein-treated iNOS wild-type mice. In contrast, the degree of pancreatic inflammation and tissue injury (histological score), upregulation/expression of P-selectin and ICAM-1, the staining for nitrotyrosine and PARS, and lipid peroxidation was markedly reduced in pancreatic tissue sections obtained from cerulein-treated iNOS-deficient mice. These findings support the view that iNOS plays an important, pro-inflammatory role in the acute pancreatitis caused by cerulein in mice.

Beneficial effects of tempol, a membrane-permeable radical scavenger, on the multiple organ failure induced by zymosan in the rat.

Abstract: Background and Methods We investigated the effects of tempol, a membrane-permeable radical scavenger, on the multiple organ failure (MOF) caused by zymosan in the rat. Zymosan (500 mg/kg, suspended in saline solution, ip) enhances formation of reactive oxygen species, which contribute to the pathophysiology of MOF. After zymosan or saline administration, animals were monitored for 12 days. Results Treatment of rats with tempol (10, 30, or 100 mg/kg ip, 1 and 6 hrs after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan in a dose-dependent fashion. Tempol also attenuated the lung, liver, and intestinal injury (histology) as well as the increase in the concentrations of myeloperoxidase and malondialdehyde caused by zymosan in the lung, liver, and intestine. Immunohistochemical analysis for nitrotyrosine and for poly(adenosine 5′-diphosphate-ribose)synthetase demonstrated a positive staining in lung, liver, and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine and for poly(adenosine 5′-diphosphate-ribose) synthetase was markedly reduced in tissue sections obtained from zymosan-treated rats that had received tempol (100 mg/kg ip). Furthermore, treatment of rats with tempol significantly reduced the following: a) the formation of peroxynitrite, b) the DNA damage, c) the impairment in mitochondrial respiration, and d) the decrease in the cellular concentration of oxidized nicotinamide adenine dinucleotide observed in macrophages harvested from the peritoneal cavity of rats treated with zymosan. Conclusion This study provides the first evidence that tempol, a small molecule that permeates biological membranes and scavenges reactive oxygen species, attenuates the degree of MOF associated with zymosan-induced peritonitis in the rat.

Inducible nitric oxide synthase knockout mice exhibit resistance to the multiple organ failure induced by zymosan

Abstract: In the present study, by comparing the responses in wild-type mice (+/+) and mice lacking (-/-) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the development of non-septic shock. A severe inflammatory response characterized by peritoneal exudation, high peritoneal levels of nitrate/nitrite, and leukocyte infiltration into peritoneal exudate was induced by zymosan administration in iNOS +/+ mice. This inflammatory process coincided with the damage of lung, liver, and small intestine, as assessed by histological examination. Lung, small intestine, and liver myeloperoxidase (MPO) activity, indicative of neutrophil infiltration and lipid peroxidation, were significantly increased in zymosan-treated iNOS +/+ mice. Peritoneal administration of zymosan in the iNOS +/+ mice induced also a significant increase in the plasma levels of nitrite/nitrate and in the levels of peroxynitrite at 18 h after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine and to poly ADP-ribose synthetase (PARS) in the lung, liver, and intestine of zymosan-treated iNOS +/+ mice. The intensity and degree of nitrotyrosine and PARS were markedly reduced in tissue section from zymosan-iNOS -/- mice. Zymosan-treated iNOS -/- mice showed a significantly decreased mortality and inhibition of the development of peritonitis. In addition, iNOS -/- mice showed a significant protection on the development of organ failure since tissue injury and MPO were reduced in lung, small intestine, and liver. Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage, and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of iNOS -/- mice subjected to zymosan-induced non-septic shock. In vivo treatment with aminoguanidine (300 mg/kg 1 and 6 h after zymosan administration) significantly prevents the inflammatory process. Taken together, our results clearly demonstrate that iNOS plays an important role in zymosan-induced non-septic shock.

Calpain inhibitor I reduces colon injury caused by dinitrobenzene sulphonic acid in the rat

Abstract: BACKGROUND AND AIMS—Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leucocyte infiltration, upregulation of expression of intercellular adhesion molecule 1 (ICAM-1), and upregulation of P-selectin in the colon. The aim of the present study was to examine the effects of calpain inhibitor I in rats subjected to experimental colitis. METHODS—Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). RESULTS—Rats experienced haemorrhagic diarrhoea and weight loss. Four days after administration of DNAB, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology as well as by 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 (PARP) showed intense staining in the inflamed colon. Staining of sections of colon obtained from DNBS treated rats with an anti-cyclooxygenase 2 antibody showed diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase was found mainly in macrophages located within the inflamed colon of DNBS treated rats. Calpain inhibitor I (5 mg/kg daily intraperitoneally) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. Calpain inhibitor I also caused a substantial reduction in (i) degree of colon injury, (ii) rise in myeloperoxidase activity (mucosa), (iii) increase in tissue levels of malondialdehyde, (iv) increase in staining (immunohistochemistry) for nitrotyrosine and PARP, as well as (v) upregulation of ICAM-1 and P-selectin caused by DNBS in the colon. CONCLUSION—Calpain inhibitor I reduces the degree of colitis caused by DNBS. We propose that calpain inhibitor I may be useful in the treatment of inflammatory bowel disease. Keywords: calpain; calpain inhibitor I; cyclooxygenase; nitric oxide; inflammatory bowel disease; rat

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

Abstract: 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.

Interleukin-6 involvement in antidepressant action of Hypericum perforatum.

Abstract: Hypericum, a plant widely used as antidepressant has been shown to interact with the immune system. We studied the effects of the administration of the Hypericum perforatum extract Ph-50, a Hypericum extract, standardized to flavonoids (50%) and containing 0.3% of hypericin and 4.5% of hyperforin in a forced swimming test and tryptophan, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) diencephalic content using a high performance liquid chromatography method in male interleukin-6 (IL-6) knock-out (IL-6 -/- ) and wild type (IL-6 +/+ ) mice. Hypericum extract (Ph-50; 500 mg/kg) oral acute administration reduced the immobility time of wild type, but not of knockout mice. Tryptophan content was not modified by Hypericum in all the animal groups. Serotonin and 5-HIAA diencephalic content was increased by Hypericum in both wild type and knockout mice. However, the increase observed in the wild type was greater than in knockout mice. These data indicate that IL-6 could be necessary to the antidepressant action of Hypericum, and that this cytokine (probably) mediates the effects of Hypericum through activation of the serotonin system.

Beneficial effects of gpi 6150, an inhibitor of poly(adp-ribose) polymerase in a rat model of splanchnic artery occlusion and reperfusion.: 174

Abstract: The aim of the present study was to investigate the effects of GPI 6150, a new poly(ADP-ribose) polymerase (PARP) inhibitor, in the pathogenesis of splanchnic artery occlusion (SAO) shock. SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by reperfusion. At 60 min after reperfusion, SAO-shocked rats developed a significant fall in mean arterial blood pressure, significant increase of tissue myeloperoxidase activity (111 ± 4.3 U/100 mg wet tissue vs. 28 ± 3.2 U/100 mg wet tissue of sham-operated rats), and marked histological injury to the distal ileum and a significant mortality (0% survival at 2 h after reperfusion). Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to PARP, P-selectin, and intercellular adhesion molecule (ICAM-1) in the necrotic ileum. GPI 6150 treatment significantly improved mean arterial blood pressure, prevented the infiltration of neutrophils (72 ± 3.6 U/100 mg wet tissue) into the reperfused intestine, improved the histological status of the reperfused tissues, markedly reduced the intensity of P-selectin and ICAM-1 in tissue section from SAO-shocked rats, and improved survival. In conclusion, our study demonstrates that GPI 6150 exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock.