Tempol, a membrane-permeable radical scavenger, reduces dinitrobenzene sulfonic acid-induced colitis

Inflammatory bowel disease 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 the present study was to examine the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid. Rats experienced bloody diarrhea and significant loss of body weight. At 4 days after the administration of dinitrobenzene sulfonic acid, the colon injury comprised of large areas of mucosal necrosis. Neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1 and expression of P-selectin and high levels of malondialdehyde (an indicator of lipid peroxidation). Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) synthetase showed an intense staining in the inflamed colon. Treatment of rats with tempol (15 mg/kg daily i.p.) significantly reduced the appearance of diarrhea and the loss in body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture as well as a significant reduction in the degree of both neutrophil infiltration and lipid peroxidation in the inflamed colon. Tempol also reduced the appearance of nitrotyrosine and poly (ADP-ribose) synthetase immunoreactivity in the colon as well as the up-regulation of ICAM-1 and P-selectin. The results of this study suggest that membrane-permeable radical scavengers, such as tempol, exert beneficial effects in experimental colitis and may, hence, be useful in the treatment of inflammatory bowel disease.     

TEMPOL, a membrane-permeable radical scavenger, attenuates peroxynitrite- and superoxide anion-enhanced carrageenan-induced paw edema and hyperalgesia: a key role for superoxide anion

Carrageenan produces both inflammation and pain when injected in rat paws via enhancement of the formation of reactive oxygen species. We have tested the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable superoxide dismutase (SOD) mimetic in carrageenan-induced rat paw edema. Treatment of rats with TEMPOL (15, 30, and 60 mg/kg, 15 min prior to carrageenan) inhibited the paw edema. Furthermore, treatment of rats with the SOD inhibitor diethylthiocarbamate (DETCA, 100 mg/kg, 1 h before carrageenan) enhanced the carrageenan-induced paw edema. Co-administration of peroxynitrite with carrageenan produced a similar fortification of the carrageenan-induced edema. Prior treatment of rats with TEMPOL (30 mg/kg) inhibited the enhancement produced by DETCA treatment (endogenous superoxide anion stress) as well as that produced by the peroxynitrite stress. The effect of TEMPOL as well as the influence of superoxide anion and peroxynitrite stresses was also tested in carrageenan-induced hyperalgesia model. Carrageenan (500 mug/paw) produced significant hyperalgesia presented as shortening of withdrawal latency times using hot plate (52 degrees C) starting 30 min after carrageenan and lasting for 3 h. TEMPOL (60 mg/kg, injected 15 min before carrageenan) ameliorated this hyperalgesia during the first 2 h. Concurrent administration of peroxynitrite promptly intensified the carrageenan hyperalgesia. TEMPOL (60 mg/kg, 15 min before peroxynitrite-carrageenan) inhibited the peroxynitrite enhancement of carrageenan hyperalgesia when tested at 60 min after injection of the cocktail. The present investigation gives the proof for the effectiveness of TEMPOL as anti-inflammation and analgesic agents in carrageenan-induced model of inflammation and hyperalgesia. It further indicated the importance of superoxide anion and peroxynitrite in acute inflammation and inflammatory pain. This raises the chances for considering pharmacologic interventions that interrupt superoxide anion and peroxynitrite stress for putative alternative agents as anti-inflammatory analgesic new medical strategies.     

Esenbeckia leiocarpa Engl. inhibits inflammation in a carrageenan-induced murine model of pleurisy

Objectives The aim of this study was to investigate the anti‐inflammatory effects of the crude hydroalcoholic extract (CHE) isolated from Esenbeckia leiocarpa Engl., and fractions and subfractions derived from it. Methods Dried E. leiocarpa Engl. bark was macerated and extracted with ethanol to obtain the CHE. The n‐hexane, ethyl acetate, aqueous and alkaloid fractions, as well as two alkaloid subfractions (polar and nonpolar) were obtained from the CHE. A preliminary analysis using thin‐layer chromatography was performed. Capillary electrophoresis, physical characteristics and spectral data produced by IR analysis and nuclear magnetic resonance (¹H and ¹³C NMR), and mass spectrometry analysis were used to identify and elucidate the structure of the major compounds. Swiss mice were used in a carrageenan‐induced pleurisy model. Pro‐inflammatory parameters (leukocyte and exudate concentrations, myeloperoxidase and adenosine‐deaminase activity, and nitrate/nitrite, interleukin 1β and tumour necrosis factor α levels) were quantified in exudates at 4 h after carrageenan‐induced pleurisy in mice. Key findings The dihydrocorynantheol alkaloid was isolated as the majority compound in the CHE, ethyl acetate and alkaloid fractions, and in the polar and nonpolar alkaloid subfractions. The CHE, fractions and subfractions inhibited the increases in leukocyte and exudate concentrations, myeloperoxidase and adenosine‐deaminase activity, and nitrite/nitrate, interleukin 1β, and tumour necrosis factor α levels (P < 0.05) in the fluid secreted from the pleural cavity of the carrageenan‐treated mice. Conclusions E. leiocarpa Engl. showed significant in vivo anti‐inflammatory action by inhibiting the inflammation caused by carrageenan. This effect may be, in part, due to the dihydrocorynantheol alkaloid, which was identified as the majority compound isolated from E. leiocarpa bark.     

Jungia sellowii suppresses the carrageenan-induced inflammatory response in the mouse model of pleurisy

This study was conducted to explore the anti-inflammatory effect of Jungia sellowii (Asteraceae) using a murine model of pleurisy induced by carrageenan (Cg). This plant is used in southern Brazil to treat inflammatory diseases. J. sellowii leaves were extracted with ethanol/water to obtain the crude extract (CE), which was fractionated with different solvents, yielding n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH) fractions, and aqueous fraction (Aq). The major compounds succinic acid (SA) and lactic acid (LA) were isolated from Aq fraction, and their structures were determined by ¹H and ¹³C NMR. Pleurisy was induced by Cg (Saleh et al. 1996). The leukocytes, exudation, myeloperoxidase (MPO) and adenosine–deaminase (ADA) activities, metabolites of nitric oxide (NO _x ) levels, protein levels and mRNA expression for interleukin 1 beta (IL-1β), tumour necrosis factor alpha (TNF-α), interleukin 17A (IL17A) and inducible of nitric oxide synthase (iNOs), and p65 protein phosphorylation (NF-κB) were analysed 4 h after pleurisy induction. Animals pre-treated with CE, BuOH, Aq, SA, or LA inhibited leukocytes, exudation, MPO and ADA activities, NO _x , IL-1β, TNF-α, and IL-17A levels, and the mRNA expression for IL-1β, TNF-α, IL-17A, iNOS, and p65 protein phosphorylation (NF-κB) (p < 0.05). Our study demonstrated that J. sellowii can protect against inflammation induced by Cg by decreasing the leukocytes and exudation. Its effects are related to the decrease of either proinflammatory cytokines and/or NO _x . The isolated compounds SA and LA may play an important role in this anti-inflammatory action by inhibiting all the studied parameters. The anti-inflammatory properties of these compounds are due to the downregulation of NF-κB.     

Anti-inflammatory activity of the non-peptidyl low molecular weight radical scavenger IAC in carrageenan-induced oedema in rats

Objective In this research we investigated the anti‐inflammatory activity of a non‐peptidyl low molecular weight radical scavenger (IAC) in an acute and chronic animal model of inflammation. Methods For this purpose the effect of IAC (10, 25, 50 mg/kg) was tested in rats on the associated behavioral responses to subsequent inflammatory and noxious challenges, such as hind paw oedema induced by intra‐plantar injection of carrageenan and granuloma induced by subcutaneous implant of a cotton pellet, using indometacin (2.5 mg/kg) as reference drug. Moreover, the serum level of several cytokines was tested in the animal treated (or not) with IAC (50 mg/kg) both in the absence and presence of carrageenan‐induced inflammation. Key findings IAC showed a significant anti‐inflammatory activity in both in acute and chronic models of inflammation. In addition IAC down regulated significantly the serum levels of interleukin (IL) 2 and IL6 whereas it increased the serum concentration of IL1α and glutathione. Conclusion Although it remains to be elucidated whether or not the antioxidant property of IAC is directly responsible for the modulation of the tested cytokines, these results suggest IAC to be a possible candidate for a novel anti‐inflammatory compound     

Prostacyclin and thromboxanes in carrageenan-induced pleurisy in the rat

The cellular origin and kinetics of TXB2 and 6-keto PGF1 alpha in carrageenan-induced pleurisy has been studied. Maximum levels of these prostanoids occurred 1 hour after induction of pleurisy. Mononuclear cells initially present in the pleural cavity synthesized TXB2 and 6-keto PGF1 alpha from (14C) arachidonic acid. By contrast, PMN cells harvested 6 hours after the induction of inflammation did not produce 6-keto-PGF1 alpha. Selective inhibition of thromboxane synthetase with drugs in vitro and in vivo increased the formation of 6-keto-PGF1 alpha, the stable breakdown product of PGI2. This metabolic effect was parallel to an increase in the volume of exudate and in PMN migration. These results suggest that TXA2 seems to be implicated not only as a chemotactic agent but also as an antagonist of PGI2 vasodilator effects.     

Effect of compound IMMLG5521, a novel coumarin derivative, on carrageenan-induced pleurisy in rats

Accumulative evidences have showed that some coumarin derivatives have significantly anti-inflammatory effects. To investigate the potential anti-inflammatory effect of compound IMMLG5521, a novel coumarin derivative, carrageenan-induced pleurisy model in rats was employed. The results showed that IMMLG5521 (5, 10 and 20 mg/kg) exhibited anti-inflammatory effects, reducing pleural exudate formation, decreasing total number of inflammation cells and polymorphonuclear leukocytes infiltration, attenuating histological injury and reducing TNF-α, IL-1β, MIP-2 and IL-8 release. Further investigation revealed that the compound may exert its anti-inflammatory effect via inhibiting nuclear translocation of NF-кB in inflammatory cells collected from pleural exudates. Taken together, the present results suggested that IMMLG5521 inhibited acute inflammation in carrageenan-induced pleurisy model that could be, in part, related to a reduction of release of inflammatory factors, another part may be related to an inhibition of NF-кB activation.     

荆芥挥发油对胸膜炎模型大鼠的抗炎作用研究

目的:研究荆芥挥发油对急性胸膜炎模型大鼠的抗炎作用及机制,为其祛风散寒之功效与治疗表证的临床应用提供药理学依据.方法:采用胸腔注射角叉菜胶制备大鼠急性胸膜炎模型,荆芥挥发油(0.4、0.2 mL·kg-1)于致炎前后1小时分别灌胃给药1次,造模后5小时处死大鼠,测定胸腔渗出液体积,进行渗出液中白细胞计数与分类、蛋白质含量测定来观察药物的抗炎作用;测定渗出液中前列腺素E2(PGE2)、白细胞介素1(IL-1)与肿瘤坏死因子(TNF-d)含量以及肺组织匀浆中丙二醛(MDA)、髓过氧化物酶(MPO)含量,来观察药物的抗炎作用机制.结果:荆芥挥发油对模型大鼠胸腔炎性渗出液体积有降低趋势,能显著降低渗出液中白细胞计数与蛋白质含量;能显著降低渗出液中炎症介质PGE2、IL-1与TNF-α含量,同时对模型大鼠肺组织中MDA的生成有明显抑制作用,并表现出降低MPO含量的趋势.结论:荆芥挥发油对角叉菜胶所致大鼠急性胸膜炎具有良好的抗炎作用,其作用机制与抑制炎症介质的生成及抗氧化作用有关.     

Disappearance and metabolism of leukotriene B4 during carrageenan-induced pleurisy

Leukotriene B4 (LTB4) has been implicated as a mediator in the inflammatory process by virtue of its potent chemotactic activity. At present, very little is known of the stability of this compound in vivo; therefore, the present study was designed to determine the half-life and metabolic fate of radiolabeled LTB4 during a 2-hr intrapleural incubation in rats with acute carrageenan pleurisy. After injection of 0.2 ml of 1% sodium carrageenan (Viscarin), inflammation was allowed to develop for 4 hr. A small polyethylene cannula was then inserted into the chest, and 0.1 microCi of [14C]LTB4 was injected into the chest. Samples for radioactivity determination were taken at 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 45, 60, 90 and 120 min via the cannula, and at 120 min the entire content of the chest was collected. The half-life for the disappearance of radioactivity from the chest was 45.8 +/- 3.5 min. The 120-min samples were treated with acetone to precipitate protein and extracted with Sep-Paks. The extracts were analyzed by reversed phase high performance liquid chromatography using an ultraviolet detector set at 269 nm and a radioactivity detector. An additional experiment was run using multi-[3H]LTB4, and the only major metabolites detected were omega-hydroxylated compounds. It can be concluded from these results that LTB4 is relatively stable in vivo and could be present for long enough at the inflammatory site to have an influence upon inflammatory cell migration.     

大鼠角叉菜胶胸膜炎渗出白细胞磷脂酶D活性的变化

目的　观察在整体炎症过程中炎症白细胞磷脂酶D(PLD)活性的变化和炎症的关系。方法　采用大鼠角叉菜胶胸膜炎模型 ,以渗出液量和细胞数及渗出液中髓过氧化物酶活性 (中性粒细胞脱颗粒指标 )作为炎症程度。用酶偶联比色法测定白细胞PLD活性。结果　正常大鼠外周血白细胞PLD活性极低 ,为 ( 0 14± 0 0 3) μmol·g-1·min-1。致炎后各时间点胸膜腔渗出白细胞的PLD活性明显升高 ,分别可达 40～ 6 0倍 ,并在 3h达峰值 ,明显早于炎症高峰 ( 12h)。不同剂量 ( 5 0 0 μg和 10 0 0 μg)角叉菜胶可引起致炎 12h明显不同程度的炎症 ,但渗出白细胞PLD活性两者差别不大。低剂量吲哚美辛 ( 2mg·kg-1,ip)和地塞米松 ( 0 1mg·kg-1,ip)均明显抑制致炎 6h大鼠胸膜腔的渗出 ,但渗出白细胞PLD活性与对照组相比差别无显著性。结论　大鼠角叉菜胶性胸膜炎白细胞PLD活性显著升高 ,提示PLD活性升高在该炎症模型中是原发性表现 ,低剂量吲哚美辛和地塞米松的抗炎机制与PLD无关     

Anti-inflammatory effects of fructose-1,6-bisphosphate on carrageenan-induced pleurisy in rat.

In the present study, we evaluated the effect of fructose-1,6-bisphosphate (FBP), a high energy intermediate metabolite of glycolysis, in an acute model of lung injury. Injection of carrageenan into the pleural cavity of rats elicited an acute inflammation response characterized by a fluid accumulation in the pleural cavity which contained a large number of polymorphonuclear neutrophils. FBP (500mg/kg) attenuated the inflammation parameters: exudate volume, total leukocytes and the number of polymorphonuclear leukocytes, but the protein concentration in the exudate was not significantly affected by treatment with FBP. The precise site and mechanism of the anti-inflammatory effect was not addressed, considering the diverse pharmacological actions of FBP. This drug has anti-inflammatory actions suggesting that it may represent a novel strategy for the modulation of inflammatory response.     

Nitric oxide and effect of a radical scavenger N-tert-butyl-alpha-phenylnitrone on stroke in a rat model

To characterize the role of nitric oxide (NO) in stroke, NO was measured using an in vivo microdialysis technique and electron spin resonance spectrometry in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The brain dialysate NO level was higher in SHRSP than in WKY. NO was not detected in M-SHRSP hippocampus microdialysate after stroke except after the administration of N-tert-butyl-alpha-phenylnitrone (PBN). In addition, very little NO was generated in M-SHRSP brain tissue with hemorrhage. These data demonstrate an association between NO and stroke in M-SHRSP. Further, PBN administration results in maintenance of NO levels after stroke in M-SHRSP.     

Decline in the expression of copper/zinc superoxide dismutase in the kidney of rats with endotoxic shock: Effects of the superoxide anion radical scavenger,tempol, on organ injury

1. Endotoxaemia causes an enhanced formation of reactive oxygen species (ROS) which contribute to the multiple organ dysfunction syndrome (MODS) in septic shock. Here we investigate (i) the effects of endotoxin on the expression of two isoforms of superoxide dismutase (SOD), namely Cu/Zn-SOD (cytosol) and Mn-SOD (mitochondria) in the rat kidney, and (ii) the effects of the radical scavenger tempol on the MODS caused by lipopolysaccharide (LPS, E. coli, 6 mg kg⁻¹ i.v.) in the rat.
2. Endotoxaemia resulted in a rapid, but transient, decline in the expression of both mRNA and protein of Cu/Zn-SOD as well as an increase in the expression of the mRNA of Mn-SOD in the kidney. Endotoxaemia for 6 h also caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of nitrite/nitrate.
3. Pretreatment of rats with tempol (100 mg kg⁻¹ i.v. bolus injection, 15 min prior to LPS followed by an infusion of 30 mg kg⁻¹ i.v., n=9) did not affect the circulatory failure, but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LPS. Tempol did not affect the rise in nitrite/nitrate caused by endotoxin.
4. These results imply that an enhanced formation of ROS (including superoxide anions) in conjunction with inadequate defences against such ROS contributes to the injury and dysfunction of the kidney and the liver in endotoxic shock.

     

Beneficial effects of raxofelast (IRFI 016), a new hydrophilic vitamin E-like antioxidant, in carrageenan-induced pleurisy

1. Peroxynitrite is a strong oxidant that results from reaction between NO and superoxide. It has been recently proposed that peroxynitrite plays a pathogenetic role in inflammatory processes. Here we have investigated the therapeutic efficacy of raxofelast, a new hydrophilic vitamin E-like antioxidant agent, in rats subjected to carrageenan-induced pleurisy. 2. In vivo treatment with raxofelast (5, 10, 20 mg kg(-1) intraperitoneally 5 min before carrageenan) prevented in a dose dependent manner carrageenan-induced pleural exudation and polymorphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, as well as histological organ injury were significantly reduced by raxofelast. 3. Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan-treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan-treated rats, which received raxofelast (20 mg kg 1) treatment. 4. Furthermore, in vivo raxofelast (5, 10, 20 mg kg(-1)) treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine 123, prevented the appearance of DNA damage, the 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. 5. In conclusion, our study demonstrates that raxofelast, a new hydrophilic vitamin E-like antioxidant agent, exerts multiple protective effects in carrageenan-induced acute inflammation.     

The protective role of endogenous glutathione in carrageenan-induced pleurisy in the rat.

In the present study we investigated the protective role of endogenous glutathione, a known free radical scavenger, in rats subjected to carrageenan-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g/kg for 24 h, intraperitoneally) enhances the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in BSO pretreated rats. However, the inducible nitric oxide (NO) synthase in lung samples was unaffected by BSO pretreatment. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats, which was massively enhanced by BSO pretreatment. Furthermore, in vivo BSO pretreatment significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced the appearance of DNA damage, the decrease in mitochondrial respiration and partially decreased the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathione (50 mg/kg i.p.) significantly reverts the effects of BSO and exerts anti-inflammatory effects. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.     

Effects of CV-3611, a new free radical scavenger, on ischemic heart failure in conscious beagle dogs

The effects of CV-3611, a new free radical scavenger, on coronary circulation failure and infarct size after ischemia/reperfusion were studied in conscious beagle dogs. The dogs underwent occlusion of the left circumflex coronary artery for 60 min and then were reperfused for 14 days. The dogs were divided into three groups: a control group, a pre-treated group that received CV-3611 or alpha-tocopherol, and a post-treated group that received CV-3611. During occlusion, varying degrees of ventricular arrhythmia were noted; after reperfusion, the arrhythmia tended to become severe. CV-3611 at a daily dose of 10 mg/kg or 30 mg/kg and alpha-tocopherol at a daily dose of 60 mg/kg reduced the incidence of overall post-occlusion arrhythmia. Coronary blood flow in the control group was reduced to 20% of the preocclusion level at 7 days after reperfusion, whereas in the CV-3611 and alpha-tocopherol treated groups, the decreased coronary flow was remarkably suppressed. The infarct size for the CV-3611- and alpha-tocopherol-treated groups, measured at 14 days after reperfusion, was reduced by 70% when compared with the control group. Based on these observations, it is proposed that CV-3611 exerts its beneficial effects on ischemic tissue by protecting against oxygen free radical-mediated damage induced by ischemia/reperfusion.     

Captopril: a free radical scavenger.

The use of captopril in heart failure and hypertension is becoming increasingly accepted. Captopril has a sulphydryl group in its molecular structure. We wondered if this might confer free radical scavenging activity on the drug and have investigated this in an in vitro system. Results show that captopril is a free radical scavenger and we suggest that this action might be relevant in its use in heart failure and other vascular diseases.     

Effects of recombinant human alpha-interferon in a rodent cardiotoxicity model

Recombinant human alpha-interferon infused intravenously into rats at doses of 10(6) (1st infusion) and 10(5) IU (2nd and 3rd infusion) produced marginal evidence of liver damage but no serious toxicity. During the 2nd and 3rd infusions an increased incidence of cardiac arrhythmias of various types was observed. In 2 rats electrocardiographic evidence of myocardial ischemia was noted. No changes in myocardial structure were demonstrated by light and electron microscopy. With the high dose the decrease in body temperature resulting from anesthesia was significantly reduced. Antibodies to interferon were demonstrated in the majority of the animals.     

Chronic treatment with tempol does not significantly ameliorate renal tissue hypoxia or disease progression in a rodent model of polycystic kidney disease

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Ascorbic acid: a scavenger of superoxide radical

The second order rate constant for the reaction between ascorbic acid and superoxide at pH 7.4 using the xanthine-xanthine oxidase system was estimated to be 5.4 x 10(6) M-1 sec-1. The results indicate that the efficacies of superoxide dismutase and ascorbic acid for catalyzing the decay of superoxide radical in animal tissues are similar. The significance of ascorbic acid as a scavenger of superoxide is discussed from the point of view of evolution of ascorbic acid synthesizing capacity in the terrestrial vertebrates.