Suppression of experimental zymosan‐induced arthritis by intraperitoneal administration of adenosine

The anti‐inflammatory effect of methotrexate (MTX) is mediated by the increasing extracellular concentration of adenosine. The response of the immune system to activation of cell membrane subclass receptors for adenosine initiates intracellular signaling pathways, which causes immunomodulatory responses. To test the direct immunomodulatory effects of systemic administration of adenosine on animal model of inflammation, zymosan induced arthritis (ZIA) in mice was employed. Sterile zymosan (30 µg) was injected directly into the knee cleft of 60 mice, divided into 3 equal groups. The experimental groups received, every second day, 0, 0.25, and 0.5 mg/kg adenosine intraperitoneally. Inflammatory intensity was evaluated clinically by knee swelling measurement, histology, the total white blood cell (WBC) count, and serum tumor necrosis factor‐alpha (TNF‐α) levels served as markers of systemic inflammatory reaction. As observed, only the mice that received the higher dose of 0.5 mg/kg adenosine developed a significantly milder arthritis. A 23.9% reduction (P < 0.004) of the mean knee diameter swelling was noted. The histological inflammatory parameters and WBC count in the 0.5 mg/kg adenosine‐treated mice were also decreased (6,555 ± 510/mm³ vs. 9,250 ± 530 WBC/mm³ in the untreated mice, P > 0.006; 9,188 ± 588 WBC/mm³ in the 0.25 mg/kg adenosine‐treated group). Serum TNF‐α levels were significantly reduced (78.1 pg/ml in the 0.5 mg/kg adenosine‐treated group vs. 124.3 pg/ml, in the control group, P < 0.004). The data indicate a remarkable clinical beneficial effect, as well as a local and systemic anti‐inflammatory response that was noted on administration of 0.5 mg/kg adenosine every alternate day to mice with inflammatory arthritis. Drug Dev. Res. 57:182–186, 2002. © 2003 Wiley‐Liss, Inc.     

Ramipril and resveratrol co‐treatment attenuates RhoA/ROCK pathway‐regulated early‐stage diabetic nephropathy‐associated glomerulosclerosis in streptozotocin‐induced diabetic rats

Clinical studies have shown that hyperglycemia can induce early‐stage diabetic nephropathy (DN). Furthermore, oxidative stress, tubular epithelial‐mesenchymal transition and extracellular matrix accumulation promote the progression of DN to chronic kidney disease and tubulointerstitial fibrosis. It is necessary to initiate treatment at the early stages of DN or even during the early stages of diabetes. In this work, rats with streptozotocin (STZ)‐induced diabetes mellitus (DM) presented early DN symptoms within 45 days, and collagen accumulation in the glomerulus of the rats was primarily mediated through the RhoA/ROCK pathway instead of the TGF‐β signaling pathway. Resveratrol (15 mg/kg/day) and ramipril (10 mg/kg/day) co‐treatment of STZ‐induced DN rats showed that glomerulosclerosis in early‐stage DN was reversible (P < .05 compared with that in STZ‐induced DM rats). The results of this study support early intervention in diabetes or DN as a more efficient therapeutic strategy.     

Anti‐inflammatory and antinociceptive effects of tilifodiolide,isolated from Salvia tiliifolia Vahl (Lamiaceae)

Salvia tiliifolia Vahl (Lamiaceae) is used for the empirical treatment of pain and inflammation. The diterpenoid tilifodiolide (TFD) was isolated from Salvia tiliifolia. The in vitro anti‐inflammatory effects of TFD (0.1–200 µM) were assessed using murine macrophages stimulated with LPS and estimating the levels of pro‐inflammatory mediators for 48 h. The in vivo anti‐inflammatory activity of TFD was assessed using the carrageenan‐induced paw edema test for 6 h. The antinociceptive effects of TFD were evaluated using the formalin test and the acetic acid induced‐writhing test. The effects of TFD on locomotor activity were assessed using the open field test and the rotarod test. TFD inhibited the production of TNF‐α (IC₅₀ = 5.66 µM) and IL‐6 (IC₅₀ = 1.21 µM) in macrophages. TFD (200 mg/kg) showed anti‐inflammatory effects with similar activity compared to 10 mg/kg indomethacin. The administration of TFD induced antinociception in the phase 1 (ED₅₀ = 48.2 mg/kg) and the phase 2 (ED₅₀ = 28.9 mg/kg) of the formalin test. In the acetic acid assay, TFD showed antinociceptive effects (ED₅₀ = 32.3 mg/kg) with similar potency compared to naproxen (ED₅₀ = 36.2 mg/kg). In the presence of different inhibitors in the acetic acid assay, only the co‐administration of TFD and naloxone reverted the antinociceptive activity shown by TFD alone. TFD did not affect locomotor activity in mice. TFD exerts in vitro and in vivo anti‐inflammatory activity and in vivo antinociceptive effects.     

Anti‐inflammatory properties of resveratrol in the retinas of type 2 diabetic rats

Resveratrol (trans‐3,5,4′‐trihydroxystilbene) is a nutritional supplement with anti‐inflammatory properties. The present study investigated the long‐term anti‐inflammatory property of resveratrol in the retinas of type 2 diabetic rats. Male Wistar rats were divided into four groups: normal control, diabetic control, resveratrol‐treated normal rats and resveratrol‐treated diabetic rats. Type 2 diabetes was induced by a single dose injection of streptozotocin (50 mg/kg; i.p.) 15 min after the administration of nicotinamide (110 mg/kg; i.p.) in 12‐h fasted rats (the streptozotocin–nicotinamide type 2 diabetic model). Oral resveratrol administration (5 mg/kg per day for 4 months) significantly improved glucose tolerance, and alleviated hyperglycemia and weight loss in diabetic rats. Furthermore, resveratrol administration significantly decreased the elevated levels of nuclear factor‐κB activity, and mRNA expression, tumour necrosis factor alpha level and apoptotic cells in the retinas of the diabetic rats. Furthermore, resveratrol did not significantly affect plasma insulin levels. Long‐term resveratrol administration has beneficial anti‐inflammatory properties in a rat model of diabetes. However, whether resveratrol exerts its effects directly or through reducing blood glucose levels requires further investigation.     

Caffeic acid phenethyl ester (CAPE) prevents methotrexate-induced hepatorenal oxidative injury in rats

Objectives This study aimed to investigate the antioxidant and anti‐inflammatory effects of caffeic acid phenethyl ester (CAPE) on the methotrexate (MTX)‐induced hepatorenal oxidative damage in rats. Methods Following a single dose of methotrexate (20 mg/kg), either vehicle (MTX group) or CAPE (10 µmol/kg, MTX + CAPE group) was administered for five days. In other rats, vehicle (control group) or CAPE was injected for five days, following a single dose of saline injection. After decapitation of the rats, trunk blood was obtained, and the liver and kidney tissues were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) and sodium potassium‐adenosine triphosphatase (Na⁺/K⁺‐ATPase) activity. TNF‐α and IL‐1β levels were measured in the blood. Key findings Methotrexate administration increased the tissue MDA levels, MPO activity and decreased GSH levels and Na⁺/K⁺‐ATPase activity, while these alterations were reversed in the CAPE‐treated MTX group. Elevated TNF‐α and IL‐1β levels were also reduced with CAPE treatment. Conclusions The results of this study revealed that CAPE, through its anti‐inflammatory and antioxidant actions, alleviates methotrexate‐induced oxidative damage, which suggests that CAPE may be of therapeutic benefit when used with methotrexate.     

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

Abstract: We investigated the effects of (1,4)‐α‐D‐glucan (α‐DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro‐ and anti‐inflammatory cytokines (interleukin [IL]‐1β, IL‐6, tumour necrosis factor‐α [TNF‐α], γ‐interferon [IFN‐γ] and IL‐10) in the lung and spleen of endotoxin‐stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non‐lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg α‐DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL‐1β concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar‐septal thickening and 11% decrease in the alveolar‐interstitial space ratio). In the lung, α‐DG treatment reduced concentrations of IL‐1β by 30% (p > 0.05), IL‐6 by 43% (p < 0.01), IFN‐γ by 46% (p < 0.01) and the anti‐inflammatory cytokine, IL‐10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, α‐DG treatment decreased the ratio of IL‐1β to IL‐10 by 55% (p < 0.05), demonstrating an anti‐inflammatory trend. These data suggest that α‐DG differentially modulates cytokine response in the lung and spleen and modifies the pro‐ and anti‐inflammatory balance during an early period of endotoxaemia in juvenile rats.     

Dehydroxymethylepoxyquinomicin,a novel nuclear factor‐κB inhibitor,prevents inflammatory injury induced by interferon‐γ and histamine in NCTC 2544 keratinocytes

1. The novel nuclear factor (NF)‐κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)‐α‐induced activation of NF‐κB by suppressing nuclear translocation of NF‐κB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)‐γ‐ and histamine‐activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN‐γ and 10^?4 mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 μg/mL) or hydrocortisone (10^?5 mol/L), which was used as a reference anti‐inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)‐1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)‐1, RANTES and interleukin (IL)‐8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non‐stimulated NCTC 2544 keratinocytes, the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 μg/mL DHMEQ potently inhibited ICAM‐1 production (by 50%), as well as the release of MCP‐1 (to 25% of control), RANTES (to 5% of control) and IL‐8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN‐γ‐ and histamine‐induced activation of the keratinocyte cell line NCTC 2544. The anti‐inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub‐toxic concentrations of anti‐inflammatory drugs to producer a synergistic effect.     

Antinociceptive and anti‐inflammatory properties of 7‐hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the antinociceptive, anti‐inflammatory and antipyretic effects of 7‐hydroxycoumarin (7‐HC) in animal models. Methods The effects of oral 7‐HC were tested against acetic acid‐induced writhing, formalin test, tail flick test, complete Freund's adjuvant (CFA)‐induced hypernociception, carrageenan‐induced paw oedema, lipopolysaccharide‐induced fever and the rota rod test. Key findings 7‐HC (3–60 mg/kg) produced a dose‐related antinociception against acetic acid‐induced writhing in mice and in the formalin test. In contrast, treatment with 7‐HC did not prevent thermal nociception in the tail flick test. A single treatment with 7‐HC, 60 mg/kg, produced a long‐lasting antinociceptive effect against CFA‐induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7‐HC produced a continuous antinociceptive effect against CFA‐induced hypernociception. 7‐HC (30–120 mg/kg) produced anti‐inflammatory and antipyretic effects against carrageenan‐induced inflammation and lipopolysaccharide‐induced fever, respectively. Moreover, 7‐HC was found to be safe with respect to ulcer induction. In the rota rod test, 7‐HC‐treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti‐inflammatory effects of 7‐HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.     

Antinociceptive and Anti‐Inflammatory Effects of Ketamine and the Relationship to Its Antidepressant Action and GSK3 Inhibition

Ketamine (KET), a NMDA antagonist, exerts an antidepressant effect at subanaesthetic doses and possesses analgesic and anti‐inflammatory activities. We evaluated the involvement of KET antinociceptive and anti‐inflammatory effects with its antidepressant action. Male Swiss mice were subjected to formalin, carrageenan‐induced paw oedema and forced swimming tests, for assessing antinociceptive, anti‐inflammatory and antidepressant effects. The treatment groups were as follows: control, KET (2, 5 and 10 mg/kg), lithium (LI: 5 mg/kg) and KET2 + LI5 combination. Immunohistochemistry analyses (TNF‐α, iNOS, COX‐2 and GSK3) in oedematous paws were performed. KET5 and KET10 reduced licking times in neurogenic (22 and 38%) and inflammatory (67 and 78%) phases of the formalin test, respectively, as related to controls. While LI5 inhibited the second phase by 24%, the licking time was inhibited by 26 and 59% in the KET2 + LI5 group (first and second phases). Furthermore, oedema volumes were reduced by 37 and 45% in the KET5 and KET10 groups, respectively. Oedema reductions were 29% in the LI5 group and 48% in the KET2 + LI5 group. In the forced swimming test, there were 23, 38 and 53% decreases in the immobility time in KET2, KET5 and KET10 groups, respectively. While LI5 caused no significant effect, decreases of 52% were observed with KET2 + LI5. KET also decreased TNF‐α, iNOS, COX‐2 and GSK3 immunostainings in oedematous paws, effects intensified with KET2 + LI5. We showed that KET presents antinociceptive and anti‐inflammatory effects associated with its antidepressant response. Furthermore, our results indicate the close involvement of GSK3 inhibition and blockade of inflammatory responses, in the antidepressant drug effect.     

Adipose‐derived mesenchymal stem cells therapy for acute kidney injury induced by ischemia‐reperfusion in a rat model

Acute kidney injury (AKI) represents a group of complicated syndromes with a high mortality rate. The administration of adipose‐derived mesenchymal stem cells (ADMSCs) has been tested as a possible treatment method for AKI. The long‐term evaluation of AKI induced by ischemia/reperfusion (IR) and the probable renal protection of ADMSCs are limited. In this study we have established a rat AKI model induced by IR and investigated the possible protective effects of ADMSCs. Adult Sprague‐Dawley (SD) rats were divided into three groups (n = 6/each group). The MOCK group was as the normal control. Rats in the IR‐AKI and IR‐AKI+ADMSCs groups were subjected to IR injury by clamping both renal pedicles for 40 minutes. Rats in the MOCK and IR‐AKI groups were injected with PBS via the tail vein as negative treatment controls. Rats in the IR‐AKI+ADMSCs group received ADMSCs therapy (2 × 10⁶ cells were injected into the rats via the tail vein). We found that ADMSC transplantation restored the pathologic morphology induced by IR‐AKI to normal compared with the MOCK group, suggesting the reparative function of ADMSCs in kidney tissues. Compared with IR‐induced AKI alone, ADMSC treatment significantly decreased the number of apoptotic cells, the level of total urinary protein and serum creatinine, the expression of pro‐inflammatory cytokines (IL‐6, TNF‐α, IL‐1β, IFN‐γ, TNF‐α, IFN‐γ, and TGF‐β), and the inflammation‐associated proteins (HGF and SDF1), but increased the expression of the anti‐inflammatory cytokine, IL‐10, and the anti‐apoptotic regulator, Bcl‐2. Our data have indicated that ADMSC transplantation may protect against IR‐induced AKI by anti‐apoptotic and anti‐inflammatory effects.     

Minocycline inhibits neurogenic inflammation by blocking the effects of tumor necrosis factor‐α

It has been well established that neurogenic inflammation is one of the major pathological processes underlying inflammatory pain, but there are few effective anti‐inflammatory drugs to alleviate such pain. The present study shows that minocycline, a widely used glial activation inhibitor, is effective in reducing neurogenic inflammation. Patch‐clamp recordings showed that small sized dorsal root ganglion (DRG) neurons were dramatically excited following intradermal capsaicin injection in the rat hind paw, evidenced by decreased rheobase and membrane threshold. Pretreatment with minocycline (30 mg/kg for 1 day, intraperitoneal injection) blocked the increased neuronal excitability. Western blot and immunostaining of DRG revealed the activation of satellite glial cells (SGCs) following capsaicin injection. The up‐regulation of glial fibrillary acidic protein (GFAP) was significantly inhibited by minocycline pre‐administration. Measurement of tumor necrosis factor α (TNF‐α) and its receptor, TNF‐α receptor 1 (TNFR1), showed that minocycline mainly blocked the up‐regulation of TNF‐α in SGCs and TNFR1s in neurons following capsaicin injection. The pivotal role of TNF‐α in neurogenic inflammation was further supported by the findings that incubation DRG with TNF‐α mimicked the increased excitability of DRG neurons induced by capsaicin injection, and that TNF‐α application enhanced cutaneous vasodilation in the hind paws induced by antidromic electrical stimulation of dorsal roots. Based on these results, we propose that minocycline is a potential therapeutic drug that can reduce neuronal excitability and neurogenic inflammation by working on SGCs to inhibit the expression of TNF‐α.     

Evaluation of the Effect of Hydroalcoholic Extract of Zingiber officinale Rhizomes in Rat Collagen‐induced Arthritis

Abstract: Patients with chronic, painful diseases often seek alternative therapy. The purpose of this study was to investigate the potential of hydroalcoholic extract of Zingiber officinale rhizomes (Z. officinale extract) to ameliorate inflammatory process in rat collagen‐induced arthritis. Our results show that Z. officinale extract in doses higher than 50 mg/kg/day intraperitoneally starting from the dose of booster immunization and for 26 days can ameliorate the clinical scores, disease incidence, joint temperature and swelling, and cartilage destruction, together with reduction of serum levels of interleukin (IL)‐1β, IL‐2, IL‐6, tumour necrosis factor‐α, and anti‐CII antibodies. Moreover, Z. officinale extract at the dose of 200 mg/kg/day was superior to 2 mg/kg/day of indomethacin at most of the measured parameters. These observations might make Z. officinale extract a good alternative to non‐steroidal anti‐inflammatory drugs for patients with rheumatoid arthritis.     

Beneficial effect of aspirin against interferon‐α‐2b—induced depressive behavior in Sprague Dawley rats

Accumulating data advocates that inflammatory mediators may contribute to depression in experimental models as well as in humans. Nonetheless, whether anti‐inflammatory treatments can prevent depression still remains controversial. To substantiate our hypothesis, we used an interferon‐α‐2b model of depression using Sprague Dawley rats. Interferon‐α‐2b is a cytokine which activates immune response and also produces depression. The animals were treated for 21 days with aspirin (10 mg/kg, per oral (p.o.)) dexamethasone (1 mg/kg p.o.) and amitriptyline (10 mg/kg p.o.). Amitriptyline was used as reference standard, and given concurrently with aspirin and dexamethasone to examine any synergy. Interferon‐α‐2b (6000 IU/kg, intraperitoneal (i.p.)) was administered in all the above groups daily, except normal control. Tests performed included sucrose preference test, behavioural tests like forced swim test, elevated plus maze, light dark box and locomotor activity along with biochemical estimations like serum cortisol and brain neurotransmitters. The rats in the group treated with Interferon‐α‐2b produced depressive behaviour in rats. We found that animals treated with aspirin decreased immobility time in forced swim test, increased sucrose preference, decreased serum cortisol and increased brain serotonin levels signifying antidepressant action. In contrast, there was no effect in groups treated with dexamethasone. Our results suggest that aspirin can serve as a potential antidepressant both individually and as adjuvant agent in the treatment of depression. Inhibition of the cyclo‐oxygenase‐2 levels and prostaglandins concentration or any other potential physiological and biochemical mechanisms may be involved in antidepressant effect.     

Physiological Effects of a Novel Immune Stimulator Drug, (1,4)‐α‐d‐Glucan,in Rats

Abstract: The (1,4)‐α‐d ‐glucan (α‐d ‐glucan), derived from medicinal plant, Tinospora cordifolia, activates human lymphocytes with downstream synthesis of the pro‐ and anti‐inflammatory cytokines, in vitro. We investigated physiological and immunological effects of a low and a high dose of α‐d ‐glucan (0.5 and 10 mg/kg), in vivo, testing the hypothesis that intravenous administration of α‐d ‐glucan does not affect haemodynamic, respiratory, haematological, and immune responses in normal rats. Male rats (300–400 g) were anaesthetized, tracheostomized, and catheterized in one femoral artery and vein. The mean arterial blood pressure and heart rate were continuously recorded. The baselines for gas exchange, differential blood cell count, and plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ were determined. Rats were then randomly assigned to controls (n = 7), a low dose (0.5 mg/kg; n = 10), and a high dose (10 mg/kg; n = 7) of α‐d ‐glucan for a six 6 hr study period. Gas exchange, differential cell count, plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ, and mean arterial blood pressure values remained within physiological range. Intravenous administration of 10 mg/kg α‐d ‐glucan created tachycardia, associated with hyperventilation, and significant reductions in the blood haemoglobin and haematocrit concentrations. We suggest that these in vivo effects of α‐d ‐glucan should be considered for future clinical and/or experimental trials.     

Differential Inhibitory Effects of the Polyphenol Ellagic Acid on Inflammatory Mediators NF‐κB,iNOS, COX‐2, TNF‐α, and IL‐6 in 1,2‐Dimethylhydrazine‐Induced Rat Colon Carcinogenesis

Abstract: Dietary polyphenols have been found to possess preventive and therapeutic potential against several types of cancers. We investigated the effect of ellagic acid on colon cancer induced by 1,2‐dimethylhydrazine in rats. Male Wistar albino rats were divided into four groups. Group 1 served as control, group 2 rats received ellagic acid 60 mg/kg bodyweight/every day p.o. throughout the experiment. Rats from groups 3 and 4 were given subcutaneous (s.c.) injections of 1,2‐dimethylhydrazine (20 mg/kg body weight) once a week for the first 15 weeks; rats in group 4 received ellagic acid as in group 2 after the last injection of 1,2‐dimethylhydrazine and continued till the end of the experimental period of 30 weeks. 1,2‐dimethylhydrazine‐induced rats exhibited alterations in cancer tumour markers [5′‐nucleotidase (5′‐ND), gamma glutamyl transpeptidase (γ‐GT), carcinoembryonic antigen (CEA), alphafetoprotein (AFP) and cathepsin‐D (CD)]; pathophysiological markers [alkaline phosphatase (ALP) and lactate dehydrogenase (LDH)] and oral administration of ellagic acid restored the levels of these marker enzymes. Nuclear factor‐kappa B (NF‐κB) actively involved in the regulation of both pro‐inflammatory proteins [inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2)] and pro‐inflammatory cytokines [tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] and in our study 1,2‐dimethylhydrazine‐induced group exhibited elevated expressions of all these inflammatory proteins. Ellagic acid administration reduced the expressions of NF‐κB, COX‐2, iNOS, TNF‐α and IL‐6 as confirmed by immunohistochemical, immunoblot and immunofluorescence analysis during 1,2‐dimethylhydrazine‐induced colon carcinogenesis. In conclusion, ellagic acid demonstrates anti‐inflammatory property by iNOS, COX‐2, TNF‐α and IL‐6 down‐regulation due to inhibition of NF‐κB and exerts its chemopreventive effect on colon carcinogenesis.