Effect of anthocyanins contained in a blackberry extract on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat.

Anthocyanins are a group of naturally occurring phenolic compounds related to the colouring of plants, flowers and fruits. These pigments are important as quality indicators, chemotaxonomic markers and for their antioxidant activities. Here we have investigated the therapeutic efficacy of anthocyanins contained in a blackberry extract on (i) circulatory failure, (ii), multiple organ dysfunction and (iii) activity of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclooxygenase (COX-2) in anaesthetised rats with endotoxic shock. In a model of endotoxic shock induced by lipopolysaccharide (LPS, E. coli, 10 mg/kg, i.v.) in the rat, pretreatment with anthocyanins present in the blackberry extract (5 mg/kg, i. v. 30 min before LPS) prevented the hypotension induced by LPS. Endotoxaemia also caused rises in the serum levels of (i) glutamyl oxaloacetic transaminase (GOT), glutamyl pyruvic transaminase (GPT), alkaline phosphates and bilirubin (hepatic dysfunction) (ii) creatinine (renal dysfunction), (iii) amylase and lipase (pancreatic injury), (iii) NOx and 6-keto-PGF1 alpha. Anthocyanins attenuated the hepatic and pancreatic injury, the renal dysfunction and decreased NOx and 6-keto-PGF1 alpha levels. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX activity in rat lung, which was attenuated in rats pretreated with anthocyanins. Moreover, anthocyanins (0.02 - 0.32 mg/mL) inhibited in vitro iNOS and COX activity from lung of LPS-treated rats. Polymorphonuclear (PMN) infiltration (myeloperoxidase activity), lipid peroxidation (malondialdehyde levels), as well as tissue injury (histological examination) induced by LPS in rat lung and ileum was reduced by anthocyanins (5 mg/kg, i. v. 30 min before LPS). Furthermore, endotoxaemia induced the formation of nitrotyrosine and poly(ADP-ribose) synthetase (PARS) activation as determined by immunohistochemical analysis of lung and ileum tissues. The degree of staining was lowered by anthocyanin treatment. These results indicate that the anthocyanins contained in the blackberry extract exert multiple protective effects in endotoxic shock.

Role of 5-lipoxygenase in the multiple organ failure induced by zymosan.

OBJECTIVE: This study investigated the role of 5-lipoxygenase in the pathogenesis of multiple organ failure (MOF) induced by zymosan. DESIGN: Male mice with a targeted disruption of the 5-lipoxygenase gene (5-LOKO) and littermate wild-type (WT) controls (5-LOWT) were used to evaluate the role of 5-lipoxygenase (5-LO) in the pathogenesis of MOF. SETTING: University research laboratory. INTERVENTIONS AND MEASUREMENTS: MOF was induced by peritoneal injection of zymosan (500 mg/kg i.p. as a suspension in saline) in 5-LOWT and in 5-LOKO mice. MOF was assessed 18 h after administration of zymosan and monitored for 12 days (for loss of body weight and mortality). RESULTS: A severe inflammatory process induced by zymosan administration in WT mice coincided with the damage of lung and small intestine, as assessed by histological examination. Myeloperoxidase activity indicative of neutrophil infiltration and lipid peroxidation were significantly increased in zymosan-treated WT mice. Zymosan in the WT mice also induced a significant increase in the plasma level of nitrite/nitrate. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to ICAM-1 and P-selectin in the lung and intestine of zymosan-treated WT mice. In contrast, the degree of (a) peritoneal inflammation and tissue injury, (b) upregulation/expression of P-selectin and ICAM-1, and (c) neutrophil infiltration were markedly reduced in intestine and lung tissue obtained from zymosan-treated 5-LO deficient mice. Zymosan-treated 5-LOKO showed also a significantly decreased mortality. CONCLUSIONS: These findings clearly demonstrate that 5-LO exerts a role in zymosan-induced nonseptic shock.

Calpain I inhibitor ameliorates the indices of disease severity in a murine model of cerulein-induced acute pancreatitis.

OBJECTIVE: Nuclear factor-kappaB (NF-kappaB) is a transcription factor which plays a pivotal role in the induction of genes involved in the response to injury and inflammation. Calpain I inhibitor is a potent antioxidant which is an effective inhibitor of NF-kappaB. This study examined whether the postulate that calpain I inhibitor attenuates experimental acute pancreatitis. DESIGN AND SETTING: In a murine model we measured NF-kappaB activation, expression of intercellular adhesion molecule (ICAM) 1, nitrotyrosine, inducible nitric oxide synthase (iNOS), nuclear enzyme poly(ADP-ribose) synthetase (PARS), myeloperoxidase, malondialdehyde, amylase and lipase and determined histological evidence of lung and pancreas injury in four groups: control (saline only), cerulein, calpain I inhibitor plus cerulein and calpain I inhibitor plus saline. MEASUREMENTS AND RESULTS: Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterised by oedema, neutrophil infiltration, tissue haemorrhage and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS and PARS in the pancreas and lung of cerulein-treated mice. In contrast, pre-treatment with calpain I inhibitor markedly reduced: the degree of pancreas and lung injury; upregulation/expression of ICAM-1; staining for iNOS, nitrotyrosine and PARS; and lipid peroxidation. Additionally, calpain I inhibitor treatment significantly prevented the activation of NF-kappaB as suggested by the inhibition of IkappaB-alpha; degradation in the pancreas tissues after cerulein administration. CONCLUSIONS: Taken together, our results clearly demonstrate that prevention of the activation of NF-kappaB by calpain I inhibitor ameliorates experimental murine acute pancreatitis.

Protective effects of M40401, a selective superoxide dismutase mimetic, on zymosan-induced nonseptic shock.

OBJECTIVE: Zymosan enhances formation of reactive oxygen species, which contributes to the pathophysiology of organ failure during nonseptic shock. Here we have investigated the effects of M40401, a new superoxide dismutase mimetic, on the organ failure associated with nonseptic shock caused by zymosan in rats. DESIGN: Experimental study. SETTING: Laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: We investigated the effects of M40401 on the organ failure associated with nonseptic shock caused by zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) in rats. MEASUREMENTS AND MAIN RESULTS: Organ failure and systemic inflammation in rats were assessed 18 hrs after administration of zymosan and/or M40401 and were monitored for 12 days (for loss of body weight and mortality). Treatment of rats with M40401 (10 mg/kg intraperitoneally, 1 and 6 hrs after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. M40401 administration also attenuated the lung and intestinal injury (histology) as well as the increase in myeloperoxidase activity and malondialdehyde concentrations caused by zymosan in lung and intestine. Immunohistochemical analysis for nitrotyrosine and for poly(adenosine 5'-diphosphate-ribose) revealed positive staining in lung and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine and poly(adenosine 5'-diphosphate-ribose) was markedly reduced in tissue sections obtained from zymosan-treated rats administered with M40401. CONCLUSION: This study provides the first evidence that M40401 attenuates the degree of zymosan-induced nonseptic shock in the rat.

Effect of methylguanidine in carrageenan-induced acute inflammation in the rats.

In vitro and in vivo studies have demonstrated that methylguanidine, an inhibitor of nitric oxide synthase (NOS), is also able to reduce tumour necrosis factor-alpha (TNF-alpha) release. In the present study, we evaluated the anti-inflammatory potential of methylguanidine treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy) where oxyradical, nitric oxide (NO) and prostaglandins play a crucial role in the inflammatory processes. Our data show that methylguanidine, given intraperitoneally at the dose of 30 mg/kg, inhibits the inflammatory response reducing significantly (P<0.05) paw swelling, pleural exudates formation, mononuclear cell infiltration and histological injury. Furthermore, our data suggests that there is a significant (P<0.05) reduction in the activity and expression both of the inducible NOS (iNOS) and of cyclooxygenase-2 in lung tissue of pleurisy model. Methylguanidine is also able to reduce the appearance of nitrotyrosine and of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) synthase immunoreactivity in the inflamed lung tissues. Treatment with aminoguanidine, the reference drug, significantly reduced all the evaluated pro-inflammatory parameters in carrageenan-treated rats. Taken together, the present results demonstrate that methylguanidine exerts potent anti-inflammatory effects that could be, in part, related to an inhibition of the expression/activity of the iNOS and cyclooxygenase-2 and, another part, may be related to a reduction of TNF-alpha release.

Reduction in the evolution of murine type II collagen-induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma.

OBJECTIVE: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. The thiazolidinedione rosiglitazone is a PPARgamma ligand that modulates the transcription of target genes. The aim of this study was to investigate the effects of rosiglitazone on the inflammatory response in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1J mice by an intradermal injection of 100 microl of an emulsion of bovine type II collagen (CII; 100 microg) in Freund's complete adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Rosiglitazone (10 mg/kg/day) or vehicle (10% DMSO) was administered beginning on day 25 (arthritis onset) until day 35. Clinical, radiographic, histopathologic, and laboratory assessments were performed. RESULTS: Mice immunized with CII in CFA developed erosive arthritis of the hind paws. Macroscopic evidence of CIA first appeared as periarticular erythema and edema of the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged mice, and the severity progressed over the 35-day study period. Radiographic evaluation revealed focal resorption of bone. Histopathologic features of CIA included erosion of cartilage at the joint margins. Rosiglitazone treatment ameliorated the clinical signs on days 26-35 and improved the histologic findings in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in rosiglitazone-treated mice, as indicated by elevation of malondialdehyde levels, formation of nitrotyrosine, and activation of poly(ADP-ribose) polymerase. Plasma levels of the proinflammatory cytokines tumor necrosis factor, interleukin-1beta, and interleukin-6 were also significantly reduced by rosiglitazone treatment. CONCLUSION: These data demonstrate that rosiglitazone exerts an antiinflammatory effect on chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

Pyrrolidine dithiocarbamate reduces the severity of cerulein-induced murine acute pancreatitis.

The nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in the induction of genes involved in the response to injury and inflammation. Dithiocarbamates are antioxidants that are potent inhibitors of NF-kappaB. This study tested the hypothesis that pyrrolidine dithiocarbamate (PDTC) attenuates experimental acute pancreatitis. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by edema, neutrophil infiltration, tissue hemorrhage and necrosis, and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine and intracellular adhesion molecule-1 in the pancreas and lung of cerulein-treated mice. In contrast, the degree of 1) pancreas and lung injury, 2) upregulation/expression of intracellular adhesion molecule-1, 3) staining for nitrotyrosine, and 4) lipid peroxidation was markedly reduced by pretreatment with PDTC. This study demonstrates that prevention of the activation of NF-kappaB by PDTC ameliorates the tissue injury associated with experimental murine acute pancreatitis and provides an important insight into the molecular biology of acute pancreatitis.

Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation.

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E2 (PGE2). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg(-1) 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.

5-lipoxygenase knockout mice exhibit a resistance to acute pancreatitis induced by cerulein.

Here we compare the degree of pancreatitis caused by cerulein in mice lacking 5-lipoxygenase (5-LO) and in the corresponding wild-type mice. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for intracellular adhesion molecule-1 (ICAM-1), P-selectin and E-selectin in the pancreas and lung of cerulein-treated mice. In contrast, the degree of (1) pancreatic inflammation and tissue injury (histological score), (2) up-regulation/expression of P-selectin, E-selectin and ICAM-1, and (3) neutrophil infiltration was markedly reduced in pancreatic and lung tissue obtained from cerulein-treated 5-LO-deficient mice. These findings support the view that 5-LO plays an important, pro-inflammatory role in the acute pancreatitis caused by cerulein in mice.

5-lipoxygenase knockout mice exhibit a resistance to splanchnic artery occlusion shock.

In the present study, we used 5-lipoxygenase (5-LO) knockout (KO) mice to evaluate the possible role of 5-LO on the pathogenesis of splanchnic artery occlusion (SAO) shock. SAO shock was induced in mice by clamping both the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 120 min after reperfusion, animals were sacrificed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum as well as in the lung of the SAO-shocked 5-LO wild-type (WT) mice after reperfusion. The absence of 5-LO did not reduce the lipid peroxidation in the intestine or the lung. SAO-shocked WT mice developed a significant increase of tissue (ileum and lung) myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (50% survival at 5 h after reperfusion). Reperfused ileum and lung tissue sections from SAO-shocked WT mice showed positive staining for P-selectin, ICAM-1, and E-selectin that was mainly localized in the vascular endothelial cells. The intensity and degree of P-selectin, E-selectin, and ICAM-1 were markedly reduced in tissue section from SAO-shocked 5-LOKO mice. SAO-shocked 5-LOKO mice showed also a significant reduction of the neutrophils infiltration into the reperfused intestine as well as in the lung as evidenced by reduced myeloperoxidase activity, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that 5-LO plays an important role in ischemia and reperfusion injury and put forward the hypothesis that inhibition of 5-LO may represent a novel and possible strategy in the treatment of ischemia and reperfusion injury. Part of this effect may be due to inhibition of the expression of adhesion molecules and subsequent reduction of neutrophil-mediated cellular injury.

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure.

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants. The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM). In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.

5-Lipoxygenase knockout mice exhibit a resistance to pleurisy and lung injury caused by carrageenan.

In the present study, by comparing the responses in wild-type (WT) mice and mice lacking [knockout (KO)] the 5-lipoxygenase (5-LO), we investigated the role played by 5-LO in the development of acute inflammation. When compared with carragenan-treated 5-LOWT mice, 5-LOKO mice, which had received carrageenan, exhibited a reduced degree of pleural exudation, polymorphonuclear cell migration. Lung myeloperoxidase activity, an index of neutrophil infiltration, was significantly reduced in 5-LOKO mice in comparison with 5-LOWT. Lung-tissue sections from carrageenan-treated 5-LOWT mice showed positive staining for intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and E-selectin, which were mainly localized around vessels. The intensity and degree of ICAM-1, VCAM-1, P-selectin, and E-selectin were markedly reduced in tissue section from carrageenan-5-LOKO mice, which also improved the histological status of the inflamed lungs. Taken together, our results clearly demonstrate that 5-LO modulates neutrophil infiltration in the acute lung inflammation.

The cyclopentenone prostaglandin 15-deoxy-delta(12,14)- PGJ2 attenuates the development of colon injury caused by dinitrobenzene sulphonic acid in the rat.

1. Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and increased expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) in the colon. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15-deoxy-delta(12,14)-PGJ(2) (15d- PGJ(2)) functions as an early anti-inflammatory signal. 2. The aim of the present paper is to investigate the effects of 15d-PGJ(2) in rats subjected to experimental colitis. 3. Colitis was induced in rats by intra-colonic instillation of dinitrobenzene sulphonic acid (DNBS). 15d-PGJ(2) was administered daily as intraperitoneal injection (20 or 40 microg kg(-1)). On day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. 4. 15d-PGJ(2) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. 15d-PGJ(2) also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde (MDA) and (iv) of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). 5. Furthermore, 15d-PGJ(2) reduced the increase in immunohistochemical staining for (i) inducible nitric oxide synthase (iNOS), (ii) nitrotyrosine and (iii) poly (ADP-ribose) polymerase (PARP), as well as (iv) the increased expression of ICAM-1 caused by DNBS in the colon. 6. Electrophoresis mobility shift assay (EMSA) of inflamed colon revealed that 15d- PGJ(2) also caused a substantial reduction of the activation of nuclear factor-kappaB (NF-kappaB). Furthermore, 15d-PGJ(2) stimulates the activation of heat shock protein 72 (hsp72) in the inflamed colon, as assessed by Western blot analysis. 7. In conclusion, 15d-PGJ(2) reduces the development of experimental colitis.

Regulation of prostaglandin generation in carrageenan-induced pleurisy by inducible nitric oxide synthase in knockout mice.

In the present study, by comparing the responses in wild-type mice (iNOSWT) and mice lacking (iNOSKO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the correlation between endogenous nitric oxide (NO) and prostaglandin (PG) generation in carrageenan-induced pleurisy. The inflammatory response in iNOSKO mice was significantly reduced in respect to iNOSWT animals, as demonstrated by the exudate volume (-63%) and numbers of infiltrating cells (-62%). The levels of NOx in the pleural exudate from carrageenan-treated mice were significantly (p < 0.01) decreased in iNOSKO mice (16 +/- 7.6 nmoles/mice) compared to iNOSWT animals (133 +/- 9 nmoles/mice). Similarly, the amounts of PGE2 in the pleural exudates of carrageenan-treated animals were significantly (p < 0.01) lower in iNOSKO compared to iNOSWT mice (120 +/- 20 pg/mice vs. 308 +/- 51 pg/mice). Also the amounts of 6-keto-PGF(1 alpha) produced by lungs from carrageenan-treated iNOSKO mice (1.01 +/- 0.10 ng/tissue mg) were significantly (p < 0.01) reduced compared to iNOSWT carrageenan-treated mice (2.1 +/- 0.09 ng/tissue mg). In conclusion our results confirm, by the use of iNOSKO mice that in carrageenan-induced pleurisy NO positively modulates PG biosynthesis.

TEMPONE reduces renal dysfunction and injury mediated by oxidative stress of the rat kidney.

Here we investigate the effects of the stable, water-soluble nitroxyl radical, TEMPONE, on renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the rat kidney in vivo. TEMPONE significantly improved both glomerular and tubular function (serum urea, creatinine, creatinine clearance, and fractional excretion of Na(+)) in a dose-dependent manner and significantly attenuated the reperfusion-injury associated with I/R (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase, assessment of renal histology). TEMPONE also markedly reduced the immunohistochemical evidence of the formation of nitrotyrosine and poly(ADP-ribose), indicating reduction of nitrosative and oxidative stress, respectively. The latter was reflected in vitro, where TEMPONE significantly reduced cellular injury of primary cultures of rat renal proximal tubular (PT) cells caused by hydrogen peroxide in a dose-dependent manner. Importantly, in contrast to its in vivo metabolite TEMPOL (which also provided protective effects against renal I/R and oxidative stress of PT cells), TEMPONE reduced renal dysfunction and injury without causing a significant reduction in blood pressure upon administration. These results suggest, for the first time, that TEMPONE can reduce the renal dysfunction and injury caused by I/R and the injury caused to PT cells by oxidative stress without producing the adverse cardiovascular effects observed when using other nitroxyl radicals.

Effects of calpain inhibitor I on multiple organ failure induced by zymosan in the rat.

OBJECTIVE: Zymosan enhances the formation of reactive oxygen species, which contributes to the pathophysiology of multiple organ failure. We investigated the effects of calpain inhibitor I (5, 10, or 20 mg/kg) on the multiple organ failure caused by zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) in rats. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats.INTERVENTIONS Multiple organ failure in rats was assessed 18 hrs after administration of zymosan and/or calpain inhibitor I and was monitored for 12 days (for loss of body weight and mortality rate). MEASUREMENT AND MAIN RESULTS: Treatment of rats with calpain inhibitor I (5, 10, or 20 mg/kg intraperitoneally, 1 and 6 hrs after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan in a dose-dependent fashion. Calpain inhibitor I also attenuated the lung, liver, and intestinal injury (histology) as well as the increase in myeloperoxidase activity and malondialdehyde concentrations caused by zymosan in the lung, liver, and intestine. Immunohistochemical analysis for nitrotyrosine and for poly(adenosine-disphosphate-ribose) revealed positive staining in lung, liver, and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine and poly(adenosine-disphosphate-ribose) was reduced markedly in tissue sections obtained from zymosan-treated rats administered calpain inhibitor I (20 mg/kg intraperitoneally). Furthermore, treatment of rats with calpain inhibitor I significantly reduced the expression of inducible nitric oxide synthase and cyclooxygenase-2 in lung, liver, and intestine. CONCLUSION: This study provides the first evidence that calpain inhibitor I attenuates the degree of zymosan-induced multiple organ failure in the rat.

Beneficial effects of GW274150, a novel, potent and selective inhibitor of iNOS activity, in a rodent model of collagen-induced arthritis.

The aim of this study was to investigate the role of inducible nitric oxide synthase (iNOS) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis. Collagen-induced arthritis was induced in wild-type mice (iNOS-WT) treated with GW274150, a novel, potent and selective inhibitor of iNOS activity, and in mice lacking the gene for iNOS (iNOS 'knock-out', iNOS-KO), by an intradermal injection of 100 microl of emulsion containing 100 microg of bovine type II collagen and complete Freund's adjuvant at the base of the tail. After 21 days, a second injection of type II collagen in complete Freund's adjuvant was administered. iNOS-WT mice developed erosive hind paw arthritis when immunised with type II collagen in complete Freund's adjuvant. Over a 35-day period, macroscopic clinical evidence of collagen-induced arthritis first appeared as periarticular erythema and oedema in the hind paws. By day 28, the incidence of collagen-induced arthritis was 100% in type II collagen-challenged iNOS-WT mice and the severity of collagen-induced arthritis progressed with radiographic evaluation revealing resorption of bone. Histopathology of collagen-induced arthritis mice demonstrated erosion of the cartilage at the joint margins. iNOS-WT mice treated with GW274150 (5 mg/kg, i.p. daily) starting at the onset of arthritis (day 23), and iNOS-KO mice showed a delay of the development of the clinical signs at days 24-35 and an improvement of the histological status in the knee and paw. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) polymerase revealed positive staining in inflamed joints from type II collagen-treated iNOS-WT mice. The degree of staining for nitrotyrosine and poly(ADP-ribose) polymerase were markedly reduced in tissue sections obtained from type II collagen-treated iNOS-WT mice, who had received GW274150 and from iNOS-KO mice. Furthermore, radiographic signs of protection against bone resorption were present in the joints of iNOS-WT mice treated with GW274150 as well as in the joint from iNOS-KO mice. This study provides the first evidence that GW274150, a novel, potent and selective inhibitor of iNOS activity, attenuates the degree of chronic inflammation and tissue damage associated with collagen-induced arthritis in mice. Furthermore, these results suggest that the induction of iNOS and NO production are essential for the up-regulation of the inflammatory response during experimental collagen-induced arthritis.

Absence of endogenous interleukin-10 enhances the evolution of acute lung injury.

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 on the modulation of the inflammatory response in mice subjected to carrageenan-induced lung injury. When compared to carrageenan-treated IL-10 wild-type (WT) mice, carrageenan-treated IL-10 knock-out mice (IL-10KO) mice experienced a higher rate of pleural exudation, and polymorphonuclear cell migration. Exudate levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Lung myeloperoxidase (MPO) activity was significantly reduced in IL-10WT mice when compared to IL-10KO mice-treated with carrageenan. 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). Staining of lung tissue sections obtained from carrageenan-treated IL-10WT with an anti-COX-2 antibody showed a positive staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase (iNOS) was found mainly in the macrophages of the inflamed lungs from carrageenan-treated IL-10WT mice. The intensity and degree of the staining for COX-2 and iNOS were markedly enhanced in tissue sections obtained from carrageenan-treated IL-10KO mice. Most notably, the degree of lung injury caused by carrageenan was also enhanced in IL-10KO mice. Taken together, our results clearly demonstrate that endogenous IL-10 exerts an anti-inflammatory role during acute inflammation and tissue damage associated with carrageenan-induced pleurisy, possibly by regulating neutrophil recruitment, and the subsequent cytokine and oxidant generation.