Effects of tempol, a membrane-permeable radical scavenger, in a rodent model of carrageenan-induced pleurisy

Carrageenan causes enhanced formation of reactive oxygen species, which contribute to the pathophysiology of inflammation. We have investigated the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to carrageenan-induced pleurisy. Treatment of rats with tempol (10, 30, or 100 mg/kg 15 min prior to carrageenan) attenuated the pleural exudation and the migration of polymorphonuclear cells caused by carrageenan dose dependently. Tempol also attenuated the lung injury (histology) as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by carrageenan in the lung. However, tempol did not inhibit the activity of inducible nitric oxide synthase in the lungs. Immunohistochemical analysis for nitrotyrosine revealed positive staining in lungs from carrageenan-treated rats. Lung tissue sections from carrageenan-treated rats also showed positive staining for poly-(ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and PARS was markedly reduced in tissue sections obtained from carrageenan-treated rats, which had received tempol (100 mg/kg). Furthermore, treatment of rats with tempol significantly reduced (i) the formation of peroxynitrite, (ii) the DNA damage, (iii) the impairment in mitochondrial respiration, and (iv) the fall in the cellular level of NAD(+) observed in macrophages harvested from the pleural cavity of rats treated with carrageenan. Tempol also attenuated the cell injury caused by hydrogen peroxide (1 mM) in cultured human endothelial cells. This study provides the first evidence that tempol, a small molecule which permeates biological membranes and scavenges ROS, attenuates the degree of inflammation and tissue damage associated with carageenan-induced pleurisy in the rat. The mechanisms of the anti-inflammatory effect of tempol are discussed.     

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.     

Protective effects of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic, in paw oedema induced by carrageenan in the rat.

In the present study we investigated the therapeutic efficacy of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic which possesses peroxynitrite scavenging effects, in rats subjected to carrageenan-induced paw oedema. Local administration of MnTBAP (5, 25, and 50 microg/paw) significantly and dose dependently reduced carrageenan-induced paw oedema at all time points. MnTBAP also caused a significant dose-dependent reduction in paw myeloperoxidase activity and lipid peroxidation, as well as preventing histological injury. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in paw from carrageenan-treated rats. No positive nitrotyrosine staining was found in the paws of the carrageenan-treated rats that received MnTBAP. Our study demonstrates that MnTBAP exerts protective effects in carrageenan-induced paw oedema. Part of these anti-inflammatory effects may be related to: 1) reduction of superoxide formation due to the superoxide dismutase-like activity of the compound; and 2) scavenging of peroxynitrite.     

Effect of FK506 (tacrolimus) in animal models of inflammation

Inflammation is the response of living tissue to damage. Cytokines play an important role in inflammatory processes. FK506 (tacrolimus), an immunosuppressant, is known to reduce the activation of microglia in vitro and affect the expression of various cytokines like interleukin-1, interleukin-6 and tumor necrosis factor. With this background the present study was designed to explore the effect of FK506 in animal models of acute inflammation and experimental pleurisy. Acute inflammation in rats was induced by intraplantar injection of carrageenan (1%, w/v). Experimental pleurisy was induced in rats by intrapleural injection of carrageenan (2%, w/v). Pretreatment with FK506 (0.5–3 mg/kg p.o.) significantly and dose-dependently reduced carrageenan-induced increase in paw volume, as well as carrageenan-induced inflammatory nociception. FK506 (1 and 3 mg/kg p.o.) inhibits exudate formation and migration of polymorhonuclear leukocytes and monocytes in carrageenan-induced experimental pleurisy. The myeloperoxidase enzyme level was significantly increased in carrageenan-treated animals, which was significantly reversed by FK506 treatment. The results of the present study suggest the potential anti-inflammatory properties of FK506 against carrageenan-induced acute inflammation and experimental pleurisy.     

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.     

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.     

The depression of hepatic drug-metabolizing enzyme activity in rats with carrageenan-induced inflammation. II. The effect on the status of plasma antipyrine concentration in rats]

The variation of hepatic drug-metabolism was investigated in male Wistar rats bearing hind paw edema induced by carrageenan (1%, 0.1 ml, s.c.). The content of cytochrome P-450 and the biotransformation of aminopyrine in the hepatic 9000 x g supernatant (S-9) decreased after the subcutaneous injection of carrageenan to rat hind paw. In carrageenan-treated rats the plasma concentration of antipyrine after the administration of antipyrine (20 mg/kg, i.v.) was higher than that of control rats. In comparison with the control groups, the pharmacokinetic parameters of antipyrine in carrageenan-treated rats was altered. AUC0-200 and t 1/2 significantly increased and CLtot and ke significantly decreased. On the other hand, no change of the volume of distribution (Vd) was observed. The results indicate that the hepatic drug metabolism under the carrageenan-induced inflammation is inhibited both in vitro and in vivo.     

Effect of invertebrate serine proteinase inhibitors on carrageenan-induced pleural exudation and bradykinin release

The carrageenan model of pleurisy is described as temporal plasma exudation (1-5 h) with extensive neutrophil infiltration and release of proteinases into the pleural cavity. The aim of this work was to study the effects of serine proteinase inhibitors on the inflammatory process induced by administration of carrageenan to the rat pleural cavity and on release of kinins in pleural exudate. Pleurisy was induced by injecting carrageenan and serine proteinase inhibitors simultaneously into the pleural cavity. The proteinase inhibitors used were: aprotinin, a plasma kallikrein inhibitor; recombinant leech derived tryptase inhibitor-2PL (LDTI-2PL), a plasmin inhibitor; Boophilus microplus trypsin inhibitors (BmTIs); trypsin; plasma kallikrein; plasmin and neutrophil elastase inhibitors; and a synthetic neutrophil elastase inhibitor (EIsynt). Administration of carrageenan with LDTI-2PL and BmTIs induced a marked increase in exudation (143% and 201%) and leukocyte migration (288% and 408%), respectively, when compared to the control group. Pleural exudate from LDTI-2PL and BmTIs plus carrageenan-treated rats showed a significant increase in plasma kallikrein-like activity, measured by chromogenic substrate hydrolysis. The specific inhibition of enzymatic activity with aprotinin confirmed that 50% of S2302 hydrolysis was produced by plasma kallikrein-like enzymes. Kinin release was increased by 97% and 103% in exudates from LDTI-2PL and BmTIs plus carrageenan-treated rats, respectively. Considering that the plasmin inhibitors LDTI-2PL and BmTIs increased exudation, leukocyte migration and bradykinin release, our results suggest an anti-inflammatory role for plasmin in the pleurisy model.     

Inhibition of inducible nitric oxide synthase in vitro and in vivo by a water-soluble extract of Wendita calysina leaves

Wendita calysina is a Paraguayan herbaceous plant commonly known as burrito. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside. From W. calysina leaves, a standardized, water-soluble extract (WSE) rich in phenylpropanoid glycosides has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. In this study, we investigated the effect of the W. calysina WSE both in vitro in murine macrophage cell line J774.A1 stimulated with lipopolysaccharide (LPS) and, in vivo in an animal model of acute inflammation, carrageenan-induced pleurisy. Here we report that W. calysina WSE (0.05, 0.1, and 0.5 mg/ml) inhibited inducible nitric oxide synthase (iNOS) expression and activity in LPS-stimulated J774.A1. In vivo experiments showed that injection of carrageenan (2%) into the pleural cavity of rats elicited an acute inflammatory response characterized by iNOS expression, intercellular adhesion molecule-1 (ICAM-1) up-regulation, nitrotyrosine and poly (ADP-ribose) synthase (PARS) formation, and lung tissue damage—all parameters significantly reduced by W. calysina WSE (500 mg/kg per os). These results report, for the first time, that a treatment with W. calysina WSE exerts anti-inflammatory effects both in vitro and in vivo.     

水杨酸甲酯糖苷抗大鼠急性胸膜炎的作用研究

目的水杨酸甲酯糖苷(DL0309)是来源于民族药滇白珠的新型非甾体抗炎药,本实验的主要目的是评价其对角叉菜胶致大鼠胸膜炎模型的抗炎作用及其可能的机制。方法将48只♂SD大鼠按体重随机分为正常对照组、模型对照组、阳性对照组(地塞米松)、DL0309低、中、高剂量组。通过注射角叉菜胶,建立大鼠急性胸膜炎模型。造模后5 h处死大鼠,通过测定胸腔渗出液的体积,对渗出液中的白细胞计数,并测定渗出液中蛋白质的含量来观察药物对该模型的抗炎作用;通过测定渗出液中一氧化氮(NO)、肿瘤坏死因子(TNF-α)、白介素1β(IL-1β)和前列腺素E2(PGE2)以及血浆中超氧化物歧化酶(SOD)和丙二醛(MDA)的含量,来考察DL0309对该模型的抗炎作用机制。结果结果显示地塞米松和DL0309均可明显降低胸膜炎大鼠胸腔炎性渗出液的体积以及渗出液中的白细胞数量和蛋白质含量,同时对于胸腔炎性渗出液中的NO、TNF-α、IL-1β和PGE2的含量均有不同程度的抑制,能不同程度地降低血浆中MDA含量,升高SOD活力。结论 DL0309具有抗角叉菜胶诱导的大鼠急性胸膜炎作用。     

Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model

An intrapleural injection of carrageenan in rats induced LTB₄ and LTC₄/D₄/E₄ biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100nmol) 16–18h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30mg/kg) inhibited the enhanced LTB₄ and LTC₄/D₄/E₄ (1 to 10mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r ²=0.989) between pleural fluid concentration of LTB₄ and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB₄ and LTC₄/D₄/E₄ in an ongoing inflammatory response. Received: 9 February 1995 / Accepted: 20 December 1996     

Copper and zinc status in rats with acute inflammation: focus on the inflamed area

Status of copper and zinc in plasma, blood cells, liver and hind paws (sectioned at the tibio-tarsal joint) were evaluated in rats with carrageenan-induced paw-oedema; moreover, concentrations of copper and zinc in the supernatant and cell fractions obtained from exudates pooled from rats with carrageenan-induced pleurisy were also determined. The evaluation of copper and zinc status in the blood and in the liver of rats with carrageenan-induced paw oedema, showed that only minor variations differentiated this experimental pathology from the previously studied carrageenan-induced pleurisy in rat. In inflammatory exudates withdrawn from pleural cavity, copper concentrations were found to be higher than the basal values measured in the whole paw, whereas zinc concentrations were found to be dramatically lower. Thus, the induction of the carrageenan paw-oedema determined an increase in copper and a decrease in zinc concentrations in the inflamed paw; however, in the inflamed paw, the total amounts of both copper and zinc were found to be significantly increased.     

Inhibition of glycogen synthase kinase-3beta attenuates the development of carrageenan-induced lung injury in mice

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) is a ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and has recently been implicated in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition in a model of acute inflammation. Here, we have investigated the effects of TDZD-8, a potent and selective GSK-3beta inhibitor, in a mouse model of carrageenan-induced pleurisy. EXPERIMENTAL APPROACH: Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), prostaglandin E2 (PGE2), tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, iNOS, COX-2 as well as nitrotyrosine as determined by immunohistochemical analysis of lung tissues. KEY RESULTS: Administration of TDZD-8 (1, 3 or 10 mg kg(-1), i.p.), 30 min prior to injection of carrageenan, caused a dose-dependent reduction in all the parameters of inflammation measured. CONCLUSIONS AND IMPLICATIONS: Thus, based on these findings we propose that inhibitors of the activity of GSK-3beta, such as TDZD-8, may be useful in the treatment of various inflammatory diseases.     

Differential effects of anti-inflammatory drugs on fluid accumulation and cellular infiltration in reverse passive arthus pleurisy and carrageenan pleurisy in rats

At 4 h following induction of pleural inflammation in rats using either an immune stimulus (reverse passive Arthus reaction, RPAR) or a chemical stimulus (carrageenan), the cellular infiltration and fluid accumulation responses were quantitated. The bell-shaped antigen (BSA) dose-response curve describing the fluid response was increased upward as the anti-BSA dose was increased from 0.25 to 1 mg, whereas the dose-response curve for cellular infiltration was both shifted upward and to the right. Both nonsteroidal anti-inflammatory drugs and a mixed lipoxygenase-cyclooxygenase inhibitor (BW 755C) preferentially inhibited fluid accumulation in RPAR pleurisy elicited with 5 mg BSA and 1 mg anti-BSA and in carrageenan pleurisy. In contrast, these drugs inhibited cellular infiltration preferentially in RPAR pleurisy elicited with 1 mg BSA and 1 mg anti-BSA. These results demonstrate that the fluid and cellular responses in rat pleural inflammation can be differentially regulated by anti-inflammatory drugs depending upon the doses of antigen and antibody employed in RPAR pleurisy and the identity of the inflammatory stimulus.     

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.     

Anti-inflammatory activity of Sapindus trifoliatus Linn

Anti-inflammatory activity of the ethanolic extract of the seeds of Sapindus trifoliatus Linn. was studied in wister rats using the carrageenan induced left hind paw edema, carrageenan induced pleurisy and cotton pellet induced granuloma model. The ethanolic extract (150 mg/kg, p.o.) produced the inhibition of carrageenan induced rat paw edema. It also showed an inhibitory effect on leukocyte migration and a reduction on the pleural exudates as well as reduction on the granuloma weight in the cotton pellet granuloma method. The results indicated that the ethanolic extract produced significant (P < 0.001) anti-inflammatory activity when compared with the standard and untreated control.