Experimental Adjuvant Arthritis Research Articles

Traditional Chinese Medicine Rhodiola Sachalinensis Borissova from Baekdu Mountain (RsBBM) for Rheumatoid Arthritis: Therapeutic Effect and Underlying Molecular Mechanisms.

The lack of effective rheumatoid arthritis (RA) therapies is a persistent challenge worldwide, prompting researchers to urgently evaluate traditional Chinese medicines (TCMs) as potential clinical RA treatments. The present investigation was conducted to evaluate the therapeutic effects and potential molecular mechanisms of the active components isolated from TCM Rhodiola sachalinensis Borissova from Baekdu Mountain (RsBBM) using an experimental adjuvant arthritis model induced by injection of rats with Freund’s complete adjuvant. After induction of the adjuvant arthritis rat model, the extract-treated and untreated groups of arthritic rats were evaluated for RsBBM therapeutic effects based on comparisons of ankle circumferences and ELISA-determined blood serum inflammatory factor levels (TNF-α, IL-1β, and PGE2). In addition, the joint health of rats was evaluated via microscopic examination of hematoxylin-eosin-stained synovial tissues. Furthermore, to explore whether NF-κB and RANK/RANKL/OPG signaling pathways participated in observed therapeutic effects from a molecular mechanistic viewpoint, mRNA and protein levels related to the expression of nuclear factor kappa-B (NF-κB), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-Β ligand (RANKL) were analyzed via quantitative RT-PCR and Western blot analysis, respectively. Treatment of arthritic rats with the extract of RsBBM was shown to reduce ankle swelling, reduce blood serum levels of inflammatory factors, and alleviate arthritis-associated synovial inflammation and joint damage. Moreover, an RsBBM 50% ethanol extract treatment inhibited bone destruction by up-regulating OPG-related mRNA and protein expression and down-regulating RANKL-related mRNA and protein expression, while also reducing inflammation by the down-regulating of the NF-κB pathway activity. The results clearly demonstrated that the extract of RsBBM alleviated adjuvant arthritis-associated joint damage by altering activities of inflammation-associated NF-κB and the RANK/RANKL/OPG signaling pathways. Due to its beneficial effects for alleviating adjuvant arthritis, this RsBBM 50% ethanol extract should be further evaluated as a promising new therapeutic TCM treatment for RA.

Evaluation of the anti-inflammatory activity of melatonin in a model of experimental arthritis

The epiphyseal hormone melatonin (1 and 5 mg/kg) significantly limited locomotor and psychoemotional disorders, indicators of inflammation and immunological reactivity in rats with experimental adjuvant arthritis.

Sorafenib suppresses proliferation rate of fibroblast-like synoviocytes through the arrest of cell cycle in experimental adjuvant arthritis.

Rheumatoid arthritis, a recurrent incendiary autoimmune joint syndrome, features by prominent synovial hyperplasia. Fibroblast-like synoviocytes are the executive components in the pathogenesis of rheumatoid arthritis. It is generally accepted that excessive proliferation and reduced apoptosis of fibroblast-like synoviocytes lead to synovial hyperplasia. Our previously studies found that sorafenib could inhibit adjuvant arthritis in rats and induced adjuvant arthritis fibroblast-like synoviocytes apoptosis. Presently, we aim to investigate the inhibitory effect with mechanisms of action of sorafenib on adjuvant arthritis fibroblast-like synoviocytes proliferation. Cell counting kit-8 and flow cytometry detection were conducted to monitor FLSs proliferation and cell cycle. Western blotting and qPCR assays were performed to detect P21, P53, CDK4, CyclinD1 and proliferating cell nuclear antigen content levels. Sorafenib significantly inhibited adjuvant arthritis fibroblast-like synoviocytes proliferation with an IC50 value of 4 µmol/L by a concentration-dependent pattern, which accompanies by G1 cell cycle arrest. Also, sorafenib significantly decreased the levels of P21, CyclinD1, CDK4 and proliferating cell nuclear antigen, as well as up-regulated P53 expression in adjuvant arthritis fibroblast-like synoviocytes. Sorafenib could inhibit adjuvant arthritis fibroblast-like synoviocytes proliferation via arresting G1/S cell cycle progression, which was partially through CDK4/CyclinD1-mediated pathway, as well as up-regulating P53 and down-regulating proliferating cell nuclear antigen expressions. These results suggest that sorafenib may provide a new paradigm for rheumatoid arthritis treatment.

Investigation of anti-inflammatory effects of bee venom in experimentally induced adjuvant arthritis.

ObjectivesRheumatoid arthritis is a multisystemic inflammatory disease characterized by destruction of the joints. An effective treatment method of the disease has not been developed yet. The aim of the present study is to evaluate the effects of bee (Apis mellifera anatoliaca) venom (BV) on serum inflammatory parameters, serum antioxidant load and clinical parameters of experimentally induced adjuvant arthritis in rats.Material and methodsA total of 35 Wistar albino male rats were used. The animals were divided into 5 groups. First group animals served as negative controls. The second, third, fourth and fifth groups were used for experimental arthritis induction. Following clinical development of arthritis, the first group was subcutaneously administered 0.2 ml of physiological saline, and the second, third and fourth groups were treated subcutaneously with 2 µg/kg, 4 µg/kg and 20 µg/kg once a week three times. Physiological saline injected fifth group animals were used as a sham-treatment group. Clinical observations and evaluation of arthritis were made at the 15th day, and at the end of the experiment. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, paraoxonase, serum aryl esterase, high-sensitivity C reactive protein, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) were determined in cardiac blood samples taken at the end of the 29th day.ResultsFrom the data, total oxidant level (TOL) and oxidative stress index (OSI) were calculated. Significant improvements were observed in the clinical signs of arthritis and inflammatory markers such as in IL-1β, TNF-α, IL-6 and TOL and OSI in the 20.0 µg/kg BV-administered group. Bee venom administration did not cause any significant increase in ALT and AST values or signs of liver toxicity.ConclusionsBee venom treatment was effective in alleviation of symptoms of the experimental rat adjuvant arthritis by means of clinical observation and serum inflammatory markers.

Antiinflammatory effect of herbal preparations on adjuvant arthritis in rats

The present study aimed to investigate the antiinflammatory effects of 2 herbal extracts (CDS and CDSM) in rats with experimental adjuvant arthritis (AA). Joint swelling, development of polyarthritis, blood indices, pro/antioxidant activity, and proinflammatory cytokine IL-17 in blood serum, as well as the liver and paw histopathology and histochemistry, were assessed. Oral injections of extracts showed an antiinflammatory effect analogous to that of diclofenac (DF), improved blood indices and suppressed level of IL-17, significantly diminished joint swelling, and histological changes in the joints. Increase in CAT activity and AOA and reduction in lipid peroxidation was noted. The preparations did not show toxic effects on the liver. The obtained results suggest that the prophylactic use of CDS and CDSM have a significant effect on slowing AA progression, similar to the effect of DF. Both preparations could be potential preventive or therapeutic candidates for the treatment of autoimmune processes in combination with other drugs.

Role of Sinomenine on Complete Freund's Adjuvant-Induced Arthritis in Rats.

The investigation was undertaken to evaluate the effect of sinomenine (Sin) on experimental adjuvant arthritis rats stimulated by Freund's complete adjuvant and explore the corresponding potential molecular mechanism. The content of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6 were detected. Besides, canonical nuclear factor kappa B (NF-κB) pathway was also assessed to evaluate the antiarthritic potential of sinomenine. Pathological sections of rat paws showed sinomenine and diclofenac sodium significantly alleviated articular cartilage lesion, cellular infiltration, epithelial cell degeneration, synovial tissue vasodilation and congestion. The phosphorylations of inhibitor of kappaB alpha and NF-κB subunit p65 were downregulated with the treatment of sinomenine in dose dependent manners, as well as proinflammatory cytokines. Therefore, it was assumed that sinomenine might be a new therapeutic candidate to treat arthritis. © 2016 IUBMB Life, 68(6):429-435, 2016.

Effect of Uncaria tomentosa extract on purinergic enzyme activities in lymphocytes of rats submitted to experimental adjuvant arthritis model.

BackgroundConsidering that adjuvant arthritis is an experimental model of arthritis widely used for preclinical testing of numerous anti-arthritic agents, which were taken by a large number of patients worldwide, it is of great interest to investigate the therapeutic action of compounds with anti-inflammatory properties, such as Uncaria tomentosa extract. Moreover, there are no studies demonstrating the effect of U. tomentosa on the metabolism of adenine nucleotides published so far. Thus, the purpose of the present study is to investigate the effects of U. tomentosa extract on E-NTPDase and E-ADA activities in lymphocytes of Complete Freund’s Adjuvant (CFA) arthritis induced rats.MethodsTo evaluate the effect of U. tomentosa extract on the activity of E-NTPDase and ADA in lymphocytes, the rats were submitted to an experimental adjuvant arthritis model. Peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Data were analyzed by a one- or two-way ANOVA. Post hoc analyses were carried out by the Student-Newman-Keuls (SNK) Multiple Comparison Test.ResultsE-NTPDase activity was increased in arthritic untreated. Arthritic rats which received U. tomentosa extract, presented similar results to the control group. However, results obtained for adenosine hydrolysis by E-ADA were not altered in arthritic rats. U. tomentosa extract did not alter E-NTPDase and E-ADA activity in healthy animals.ConclusionsThe present investigation supports the hypothesis that the increased E-NTPDase activity verified in arthritic rats might be an attempt to maintain basal levels of ATP and ADP in the extracellular medium, since the arthritis induction causes tissue damage and, consequently, large amounts of ATP are released into this milieu. Also, it highlights the possibility to use U. tomentosa extract as an adjuvant to treat arthritis.

FULLERENE C60 INHIBITED FREE RADICAL AND DESTRUCTIVE PROCESSES IN CONNECTIVE TISSUE DURING ADJUVANT ARTHRITIS IN RATS

The effects of fullerene C60 (FC60) on the level of free radical and destruction processes were studied in rats with experimental adjuvant arthritis (AA). It was shown the protective effect of FC60 during AA. The effect was accompanied by an increase of the antioxidant enzymes activity, superoxide dismutase in the liver (15.96 ± 0.38 μmol/kg x s) and in the kidneys (5.36 ± 0.27 μmol/kg x s) and catalase in the kidneys (9.56 ± 0.78 μmol/kg x s) and in the heart (2.26 ± 0.41 μmol/kg x s) in comparison to control group (43.83± 5.69%; 54.55 ± 6.18%; 11.68 ± 0.52 μmol/kg x s; 3.43 ± 0.47 μmol/kg x s; 4.77 ± 0.5 μmol/kg x s; 0.98 ± 0.12 μmol/kg x s accordingly). It was shown a protective effect of FC60 during AA directed on the depression of the destructive processes in connective tissue that was expressed through the reduction of the total collagenolitic activity level in cartilage (10.05 ± 0.06 μmol/g/min) and bone (11.21 ± 0.04 μmol/g/min) tissues, free hydroxyproline contents (1.54 ± 0.04 μg/ml) and alkaline phasphatase activity (1.24 ± 0,14 μmol/l x sec) in comparison to control group (11.91 ± 0.49 μmol/g/min; 13.19 ± 0.15 μmol/g/min; 2.25 ± 0.07 μg/ ml; 2.19 ± 0.24 μmol/l x sec accordingly). Taken together, these results accentuate the perspective of future investigations of action FC60 during rheumatoid arthritis as a feasible therapeutic agent.

Anti-inflammatory and anti-oxidative effects of herbal preparation EM 1201 in adjuvant arthritic rats

Background and objectiveThe purpose of the present study was to examine the anti-arthritic and antioxidant effects of herbal and active organic ingredient complex (EM 1201) in rats with experimental adjuvant arthritis (AA). Materials and methodsAA was induced in 30 male Wistar rats by intradermal injection of complete Freund's adjuvant into the left hind paw. The course of disease in 30 rats in response to the treatment with EM 1201 and diclofenac, the parameters including body weight, joint swelling, blood indices pro-/antioxidant status of blood serum, and histology of joints and the liver, were investigated. ResultsPreparation EM 1201 showed anti-inflammatory effect analogous to diclofenac, improved blood indices, significantly decreased joint swelling and histological changes in them. Joint swelling was suppressed by 29%–42.8% and 9.3%–34.4% in response to administration of EM 1201 and diclofenac during the entire experiment. Both preparations significantly suppressed pannus formation, general inflammatory reaction and edema in soft periarticular tissues and synovium, diminished MDA level and elevated AOA in the blood serum. Significantly lower absolute and relative weight of the liver and lower dystrophic processes in it, and general inflammatory infiltration of hepatic stroma proved the positive effect of treatment with EM 1201. ConclusionsThe present study suggests that EM 1201 has protective activity against arthritis and demonstrated its potential beneficiary effect analogical to diclofenac. Anti-inflammatory and anti-oxidative effect of EM 1201 in rats with AA support the need of further investigations by using it as supplementary agent alone or together with other anti-arthritic drugs in the treatment of rheumatoid arthritis.

Content of IL-10 and CD4+CD210+ cells in mice with adjuvant arthritis before and after treatment with cryopreserved placental cells.

We analyzed the serum level of IL-10, blood content of CD4+СD210+ cells, and expression of IL-10 receptors in adjuvant arthritis before and after treatment with native and cryopreserved placental cells. In animals with experimental adjuvant arthritis, a negative correlation was found between the index of arthritis and IL-10 concentration (r=-0.7; p=0.03); in addition, a considerable decrease in the content of CD4+СD210+ cells and changes in the expression of IL-10 receptors on cells of the lymph nodes and spleen were demonstrated. After administration of cryopreserved DMSO-protected placental cells (in contrast to administration of cryopreserved propanedioxysacharol-protected cell suspension), the content of CD4+СD210+ cells in the spleen of animals with experimental adjuvant arthritis returned to normal.

Swertiamarin attenuates inflammation mediators via modulating NF-κB/I κB and JAK2/STAT3 transcription factors in adjuvant induced arthritis

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that leads to pannus formation followed by severe joint destruction, characterized by synovial hyperplasia, inflammation and angiogenesis. Swertiamarin is a secoiridoid glycoside that is used as an anti-inflammatory compound, mainly found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in Indian system of traditional medicine. In the present study, the effect of swertiamarin was evlauated in experimental adjuvant arthritis animal model by the estimation of biochemical (paw thickness, lysosomal enzymes, and urinary degradative products) parameters, proinflammatory cytokines and enzymes along with histopathological and radiographic observations. The proteins of phosphorylated NF-κB/IκB and JAK2/STAT3 transcription factors were also quantified from experimental animals as well as LPS induced RAW 264.7 macrophage cells. In in silico analysis, swertiamarin was docked with proinflammatory enzymes to confirm its potential. The administration of swertiamarin (2, 5, 10mg/kg bw) significantly (P⩽0.05) inhibited the levels of paw thickness, lysosomal enzymes and increased the body weight of experimental animals in a dose dependent manner. In molecular analysis, the treatment decreased the release of proinflammatory cytokines (IL1, TNF, IL-6) and proangiogenic enzymes (MMPs, iNOS, PGE2, PPARγ and COX-2); and also significantly (P⩽0.05) increased the levels of antiinflammatory proteins (IL-10, IL-4) when compared to the disease groups. The swertiamarin treatment significantly (P⩽0.05) inhibited the release of NF-κB p65, p-IκBα, p-JAK2 and p-STAT3 signaling proteins levels on both experimental animals and LPS induced cells. Histopathological and radiological analysis evidenced the curative effect of swertiamarin on bone destruction. The docking studies of swertiamarin on proinflammatory enzymes supported the results from the in vivo experiments. Thus the swertiamarin inhibited the development of arthritis by modulating NF-κB/IκB and JAK2/STAT3 signaling. These findings suggested that swertiamarin acted as an anti-rheumatic agent.

Efecto del alendronato sobre el perfil de citoquinas y metaloproteinasas 2 y 9 en un modelo múrido de artritis experimental

IntroductionArthritis is one of the most common arthropathies, characterized by cartilage damage associated with subchondral bone resorption. Early diagnosis is crucial for the prevention of subchondral bone lesions. ObjectivesTo use an experimental adjuvant arthritis rat model, to measure joint biomarkers, interleukins (IL-4, IL-6 and TNF-α), and metalloproteases (MMP-2 and MMP-9) to follow the progression of the disease and to analyze the possible changes in the different treatment groups, with preventive or curative alendronate, Materials and methodsPreventive alendronate was applied on day 0, and a curative regimen 2 months post-adjuvant. A clinical scale score was used for characterizing clinical symptoms, and, at sacrifice, joint biomarkers and histopathological and radiographic studies were determined. ResultsThe most notable result was that the group that received preventive alendronate reached a normal clinical score faster than control adjuvant group. All alendronate groups showed significantly lower MMPs levels. ConclusionsAlendronate apparently decreases proteases activity linked to the pathophysiology of joint disease, this could be extremely beneficial for the clinical outcome of arthritis.

Протизапальна дія фулерену С60 при ад’ювантному артриті у щурів

The anti-inflammatory properties of non-modified fullerene C60 (FC60) by adjuvant arthritis in Wistar rats have been studied. It was shown that the intraperitoneal introduction of FC60 (50 ng) reveals an anti-inflammatory and chondroprotective actions in the phase of systemic manifestation of adjuvant arthritis. The effect was carried out by limitation of inflammation of damaged limb, normalization of body weight, the decrease in body temperature. Introduction of FC60 promote the reduction of leukocyte level, the erythrocyte sedimentation rate, concentrations of sialic acids and the ceruloplasmin levels, processes degeneration of cartilaginous tissues of the joint of rats. It has been concluded that the therapeutic effectiveness of non-modified FC60 in experimental adjuvant arthritis is comparable with the action of water-soluble forms of fullerenes. The results substantiate the future investigations of non-modified FC60 for design of therapeutic agents for treatment of rheumatoid arthritis in clinics.

Cerebral Biochemical Pathways in Experimental Autoimmune Encephalomyelitis and Adjuvant Arthritis: A Comparative Metabolomic Study

Many diseases, including brain disorders, are associated with perturbations of tissue metabolism. However, an often overlooked issue is the impact that inflammations outside the brain may have on brain metabolism. Our main goal was to study similarities and differences between brain metabolite profiles of animals suffering from experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rat models. Our principal objective was the determination of molecular protagonists involved in the metabolism underlying these diseases. EAE was induced by intraplantar injection of complete Freund’s adjuvant (CFA) and spinal-cord homogenate (SC-H), whereas AA was induced by CFA only. Naive rats served as controls (n = 9 for each group). Two weeks after inoculation, animals were sacrificed, and brains were removed and processed for metabolomic analysis by NMR spectroscopy or for immunohistochemistry. Interestingly, both inflammatory diseases caused similar, though not identical, changes in metabolites involved in regulation of brain cell size and membrane production: among the osmolytes, taurine and the neuronal marker, N-acetylaspartate, were decreased, and the astrocyte marker, myo-inositol, slightly increased in both inoculated groups compared with controls. Also ethanolamine-containing phospholipids, sources of inflammatory agents, and several glycolytic metabolites were increased in both inoculated groups. By contrast, the amino acids, aspartate and isoleucine, were less concentrated in CFA/SC-H and control vs. CFA rats. Our results suggest that inflammatory brain metabolite profiles may indicate the existence of either cerebral (EAE) or extra-cerebral (AA) inflammation. These inflammatory processes may act through distinct pathways that converge toward similar brain metabolic profiles. Our findings open new avenues for future studies aimed at demonstrating whether brain metabolic effects provoked by AA are pain/stress-mediated and/or due to the presence of systemic proinflammatory molecules. Regardless of the nature of these mechanisms, our findings may be of interest for future clinical studies, e.g. by in-vivo magnetic resonance spectroscopy.

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

RAT MODEL FOR POLY-AUTOIMMUNITY

Around 30% of the male PVG rats have very low testosterone levels. Upon immunisation with myelin basic protein in complete Freund’s adjuvant they develop experimental autoimmune encephalomyelitis and adjuvant arthritis. The androgen insensitive syndrome in these rats could be used in studies on poly-autoimmunity as well as in studies on variations in the sexual differentiation of brain and behavior.

Effect of methotrexate on inflammatory cells redistribution in experimental adjuvant arthritis

The aim of this study was to evaluate the morphological changes in the spleen, the thymus and the knee joints of rats with experimental adjuvant arthritis induced by Mycobacterium butyricum in the incomplete Freund's adjuvant and the effect of treatment with methotrexate (MTX). Particular attention was aimed on the redistribution of granulocytes in the tissues during the inflammatory process. Clinical parameters, e.g., joint edema, body weight and of gamma glutamyl transferase (GGT) activity as an inflammatory marker, have also been determined. Induction of adjuvant arthritis caused a significant decrease in granulocyte number in the spleen and vice versa a significant increase in the knee joints, but without significant changes in the thymus. Treatment with methotrexate reversed this phenomenon by increasing the granulocyte number in the spleen and decreasing it in knee joints. MTX decreased the joint edema as well as the activity of GGT in the spleen, modified the size of the white pulp of the spleen and increased the cortex/medulla ratio in the thymus. The observed changes support the anti-inflammatory and immunomodulatory properties of MTX supporting its use as the first-line medication in patients with rheumatoid arthritis.

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate

IntroductionFolate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin.MethodsUsing a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans.ResultsEC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems.ConclusionsEC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

Quercetin Ameliorates Experimental Autoimmune Myocarditis in Rats

Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and post-myocarditis dilated cardiomyopathy. The pathogenesis of EAM has not been elucidated, but there is accumulating evidence that cytokines secreted from monocytes/macrophages and T cells play a crucial role in the induction and progression of disease. Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, which scavenge oxygen radicals and have anti-inflammatory activities. Having in mind in vivo beneficial effects of flavonoid quercetin in different animal models of immunoinflammatory diseases such as experimental autoimmune encephalomyelitis and adjuvant arthritis, on the one side, and its in vitro suppressive effect on production of tumor necrosis factor-alpha (TNF-α) on the other side, we investigated the effects of quercetin on EAM in rats. Myocarditis was induced in Dark Agouti (DA) rats by injection of porcine cardiac myosin and quercetin at doses of 10 or 20 mg/kg was orally administered from days 0 to 21 after induction of disease. The severity of myocarditis was evaluated by determination of heart weight/body weight ratio (Hw/Bw) and histopathological examination of hearts. The levels of cytokines (TNF-α, IL-12, IL-17 and IL-10) in serum and lymph node cells (LNC) culture supernatants were measured by ELISA. The rats treated with 20 mg/kg of quercetin had significantly decreased incidence of EAM, Hw/Bw, macroscopic and microscopic scores of hearts. Further, in EAM rats treated with quercetin levels of TNF-α and IL-17 were significantly lower, while the level of IL-10 was significantly higher both in serum and culture supernatants of LNC stimulated with concanavalin A compared with vehicle-treated animals. The present study suggests that quercetin ameliorates EAM, at least in part, by interfering production of proinflammatory (TNF-α and IL-17) and/or anti-inflammatory (IL-10) cytokines.

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

IntroductionThe objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.MethodsEfficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.ResultsLiposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.ConclusionsThis new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.