IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-alpha, IL-1beta, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.     

Arthritis Induces Early Bone High Turnover,Structural Degradation and Mechanical Weakness

BackgroundWe have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone.MethodsTwenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for compar-ison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers.ResultsAIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phos-phorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular sepa-ration (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, re-spectively) in the arthritic group.ConclusionsWe have shown in an AIA rat model that arthritis induc-es early bone high turnover, structural degradation, mineral loss and mechanical weak-ness.     

Intra-articular injections of high-molecular-weight hyaluronic acid have biphasic effects on joint inflammation and destruction in rat antigen-induced arthritis

To assess the potential use of hyaluronic acid (HA) as adjuvant therapy in rheumatoid arthritis, the anti-inflammatory and chondroprotective effects of HA were analysed in experimental rat antigen-induced arthritis (AIA). Lewis rats with AIA were subjected to short-term (days 1 and 8, n = 10) or long-term (days 1, 8, 15 and 22, n = 10) intra-articular treatment with microbially manufactured, high-molecular-weight HA (molecular weight, 1.7 x 10(6) Da; 0.5 mg/dose). In both tests, 10 buffer-treated AIA rats served as arthritic controls and six healthy animals served as normal controls. Arthritis was monitored by weekly assessment of joint swelling and histological evaluation in the short-term test (day 8) and in the long-term test (day 29). Safranin O staining was employed to detect proteoglycan loss from the epiphyseal growth plate and the articular cartilage of the arthritic knee joint. Serum levels of IL-6, tumour necrosis factor alpha and glycosaminoglycans were measured by ELISA/kit systems (days 8 and 29). HA treatment did not significantly influence AIA in the short-term test (days 1 and 8) but did suppress early chronic AIA (day 15, P < 0.05); however, HA treatment tended to aggravate chronic AIA in the long-term test (day 29). HA completely prevented proteoglycan loss from the epiphyseal growth plate and articular cartilage on day 8, but induced proteoglycan loss from the epiphyseal growth plate on day 29. Similarly, HA inhibited the histological signs of acute inflammation and cartilage damage in the short-term test, but augmented acute and chronic inflammation as well as cartilage damage in the long-term test. Serum levels of IL-6, tumour necrosis factor alpha, and glycosaminoglycans were not influenced by HA. Local therapeutic effects of HA in AIA are clearly biphasic, with inhibition of inflammation and cartilage damage in the early chronic phase but with promotion of joint swelling, inflammation and cartilage damage in the late chronic phase.     

Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis

Introduction

Recent studies revealed that co-morbidity and mortality due to cardiovascular disease are increased in patients with rheumatoid arthritis (RA) but little is known about factors involved in these manifestations. This study aimed at characterizing the impact of arthritis on oxidative stress status and tissue fibrosis in the heart of rats with adjuvant-induced arthritis (AIA).

Methods

AIA was induced with complete Freund's adjuvant in female Lewis rats. Animals were treated by oral administration of vehicle or angiotensin-converting enzyme inhibitor ramipril (10 mg/kg/day) for 28 days, beginning 1 day after arthritis induction. Isolated adult cardiomyocytes were exposed to 10 μM 4-hydroxynonenal (HNE) for 24 hours in the presence or absence of 10 μM ramipril.

Results

Compared to controls, AIA rats showed significant 55 and 30% increase of 4-HNE/protein adducts in serum and left ventricular (LV) tissues, respectively. Cardiac mitochondrial NADP+-isocitrate dehydrogenase (mNADP-ICDH) activity decreased by 25% in AIA rats without any changes in its protein and mRNA expression. The loss of mNADP-ICDH activity was correlated with enhanced accumulation of HNE/mNADP-ICDH adducts as well as with decrease of glutathione and NADPH. Angiotensin II type 1 receptor (AT₁R) expression and tissue fibrosis were induced in LV tissues from AIA rats. In isolated cardiomyocytes, HNE significantly decreased mNADP-ICDH activity and enhanced type I collagen and connective tissue growth factor expression. The oral administration of ramipril significantly reduced HNE and AT₁R levels and restored mNADP-ICDH activity and redox status in LV tissues of AIA rats. The protective effects of this drug were also evident from the decrease in arthritis scoring and inflammatory markers.

Conclusion

Collectively, our findings disclosed that AIA induced oxidative stress and fibrosis in the heart. The fact that ramipril attenuates inflammation, oxidative stress and tissue fibrosis may provide a novel strategy to prevent heart diseases in RA.     

Anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, MAS, in experimental models of arthritis

Activation of the renin-angiotensin (Ang) system induces inflammation via interaction between Ang II and type 1 receptor on leukocytes. The relevance of the new arm of the renin-Ang system, namely Ang-converting enzyme-2/Ang-(1-7)/Mas receptor, for inflammatory responses is not known and was investigated in this study. For this purpose, two experimental models were used: Ag-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Male C57BL/6 wild-type or Mas(-/-) mice were subjected to AIA and treated with Ang-(1-7), the Mas agonist AVE 0991, or vehicle. AdIA was performed in female rats that were given AVE 0991 or vehicle. In wild-type mice, Mas protein is expressed in arthritic joints. Administration of AVE 0991 or Ang-(1-7) decreased AIA-induced neutrophil accumulation, hypernociception, and production of TNF-α, IL-1β, and CXCL1. Histopathological analysis showed significant reduction of inflammation. Mechanistically, AVE 0991 reduced leukocyte rolling and adhesion, even when given after Ag challenge. Mas(-/-) mice subjected to AIA developed slightly more pronounced inflammation, as observed by greater neutrophil accumulation and cytokine release. Administration of AVE 0991 was without effect in Mas(-/-) mice subjected to AIA. In rats, administration of AVE 0991 decreased edema, neutrophil accumulation, histopathological score, and production of IL-1β and CXCL1 induced by AdIA. Therefore, activation of Mas receptors decreases neutrophil influx and cytokine production and causes significant amelioration of arthritis in experimental models of arthritis in rats and mice. This approach might represent a novel therapeutic opportunity for arthritis.     

Suppressive effects of QFGJS, a preparation from an anti-arthritic herbal formula, on rat experimental adjuvant-induced arthritis

To analyze the anti-arthritic effects of QFGJS (a pharmaceutical preparation from herbs) on rheumatoid arthritis, adjuvant-induced arthritis (AIA) was established in male SD rats, and two administration protocols, i.e., oral treatment with different doses of QFGJS on the day of arthritis induction or on the day when visible clinical signs of arthritis occurred, were initiated and continued until day 30. Treatments with QFGJS using both administration protocols significantly suppressed the incidence and severity of arthritis in a dose-dependent manner, showing dramatic reduction of paw swelling and ESR throughout the disease progression of AIA. Radiological and histopathological examinations showed markedly decreased tissue and bone destruction of ankle joints in the QFGJS-treated rats. The serum levels of TNF-alpha, IL-1beta, and IL-6 were significantly decreased in the QFGJS-treated rats. QFGJS demonstrates pronounced anti-arthritic effects on AIA, indicating that this herbal preparation would be a potent candidate as a novel botanical drug for further investigation.     

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFα or anti-IL-1 therapies

Introduction

Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFα, IL-1β, and receptor activator of NF-κB ligand (RANKL). Anti-IL-1 or anti-TNFα therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.

Methods

Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFα inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E₂ (PGE₂), acute-phase protein alpha-1-acid glycoprotein (α₁AGP), multiple cytokines) were measured in serum (day 14 post onset).

Results

Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFα reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFα and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFα and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum α₁AGP, IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE₂, TNFα and IL-17. Anti-TNFα reduced systemic α₁AGP, CCL2 and PGE₂ in AIA rats, while anti-IL-1 decreased systemic α₁AGP, IL-8 and PGE₂. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.

Conclusions

Anti-TNFα or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.     

Suppression of up-regulated LXRα by silybin ameliorates experimental rheumatoid arthritis and abnormal lipid metabolism

BackgroundAs dysregulation of immunometabolism plays a key role in the immunological diseases, dyslipidemia frequently observed in rheumatoid arthritis (RA) patients (60%) is associated with the disease activity and has been considered as the potential target of anti-inflammatory strategy. However, targeting of metabolic events to develop novel anti-inflammatory therapeutics are far from clear as well as the mechanism of dyslipidemia in RA.PurposeTo explore the therapeutic potential and mechanisms of silybin again RA through the regulation of lipid metabolism.MethodsAdjuvant-induced arthritis (AIA) rat model was used to examine the effects of silybin on modulating dysregulated lipid metabolism and arthritis. Metabolomics, docking technology, and biochemical methods such as western blots, qRT-PCR, immunofluorescence staining were performed to understanding the underlying mechanisms. Moreover, knock-down of LXRα and LXRα agonist were used on LO2 cell lines to understand the action of silybin.ResultsWe are the first to demonstrate that silybin can ameliorate dyslipidemia and arthritis in AIA rats. Overexpression of LXRα and several key lipogenic enzymes regulated by LXRα, including lipoprotein lipase (LPL), cholesterol 7α and 27α hydroxylase (CYP7A, CYP27A), adipocyte fatty acid-binding protein (aP2/FABP4) and fatty acid translocase (CD36/FAT), were observed in AIA rats, which mostly accounted for dyslipidemia during arthritis development. Metabolomics, docking technology, and biochemical results indicated that anti-arthritis effects of silybin related to suppressing the up-regulated LXRα and abnormal lipid metabolism. Notably, activation of LXRα could potentiate cell inflammatory process induced by LPS through the regulation of NF-κB pathway, however, suppression of LXRα agonism by siRNA or silybin reduced the nuclear translocation of NF-κB as well as the induction of downstream cytokines, indicating LXRα agonism is the important factor for the arthritis development and could be a potential target.ConclusionThe up-regulation of LXRα can activate lipogenesis enzymes to worsen the inflammatory process in AIA rats as well as the development of dyslipidemia, therefore, rectifying lipid disorder via suppression of LXRα agonism pertains the capacity of drug target, which enables to discover and develop new drugs to treat rheumatoid arthritis with dyslipidaemia.     

Delayed resolution of acute inflammation during zymosan-induced arthritis in leptin-deficient mice

The severity of antigen-induced arthritis (AIA) is decreased in leptin-deficient ob/ob mice. However, joint inflammation in AIA depends on the immune response, which is impaired in ob/ob mice. In the present study we investigated the effects of leptin deficiency on zymosan-induced arthritis (ZIA), which is independent of adaptive immunity. Arthritis was induced by injection of zymosan into the knee joint. Joint swelling was similar after 6 and 24 hours in ob/ob and control mice. However, it remained elevated in ob/ob animals on day 3 whereas values normalized in controls. Histology revealed similar articular lesions in all animals on day 3, but on days 14 and 21 arthritis tended to be more severe in ob/ob mice. The acute phase response, reflected by circulating levels of IL-6 and serum amyloid A, was also more pronounced in ob/ob mice, although corticosterone was significantly elevated in these animals. Similar results were obtained in leptin receptor-deficient db/db mice. Thus, in contrast to AIA, ZIA is not impaired in leptin-deficient animals. On the contrary, resolution of acute inflammation appears to be delayed in the absence of leptin or leptin signalling, suggesting that chronic leptin deficiency interferes with adequate control of the inflammatory response in ZIA.     

Therapeutic effects of lactosyl derivative Gu-4 in a collagen-induced arthritis rat model

Rheumatoid arthritis (RA) is an inflammatory disorder that is characterized by persistent recurrence of joint inflammation leading to cartilage and bone destruction. The present anti-arthritis therapies failed to achieve satisfactory remission in all patients; therefore, it is still necessary to develop novel approaches to fulfill the demand in clinic. Here, we reported the therapeutic effects of lactosyl derivative Gu-4, a synthetic compound that was previously identified as a selective inhibitor against leukocyte integrin CD11b, in a bovine type II collagen induced arthritis (CIA) rat model. First, prophylactic administration of Gu-4 (1.2728?mg/kg) to rats by intraperitoneal injection every 2?days from the first day of collagen immunization significantly decreased the incidence of CIA, diminished the mean paw volume increase, and reduced the number of swollen paws. Second, administration of Gu-4 (1.2728?mg/kg) to rats at early-onset stage of CIA prevented the progression of the pathological process of RA, accelerated the remission of paw edema, and declined the arthritis score; after 5?weeks treatment, X-ray and histological examinations were carried out, the ankle joint of hind limb of Gu-4 treated CIA rats exhibited slighter bone erosion and much less inflammatory cell infiltration compared to those of saline treated animals; furthermore, Gu-4 remarkably attenuated the production of rheumatoid factor (RF) in the serum of CIA rats as determined by ELISA. Moreover, we performed in vitro lymphocyte proliferation assay and found that Gu-4 significantly inhibited the proliferation of splenic lymphocytes isolated from CIA rats in a dose-dependent manner. Our results suggest that Gu-4 can effectively ameliorate CIA and might be an alternative option for the treatment of RA.     

Anti‐arthritis activity of cationic materials

Cationic materials exhibit remarkable anti‐inflammatory activity in experimental arthritis models. Our aim was to confirm this character of cationic materials and investigate its possible mechanism. Adjuvant‐induced arthritis (AIA) models were used to test cationic materials for their anti‐inflammatory activity. Cationic dextran (C‐dextran) with different cationic degrees was used to investigate the influence of the cationic elements of materials on their anti‐inflammatory ability. Peritoneal macrophages and spleen cells were used to test the expression of cytokines stimulated by cationic materials. Interferon (IFN)‐γ receptor‐deficient mice and macrophage‐depleted rats were used to examine the possible mechanisms of the anti‐inflammatory activity of cationic materials. In AIA models, different cationic materials shared similar anti‐inflammatory characters. The anti‐inflammatory activity of C‐dextran increased with as the cationic degree increased. Cationic materials stimulated interleukin (IL)‐12 expression in peritoneal macrophages, and strong stimulation of IFN‐γ secretion was subsequently observed in spleen cells. In vivo experiments revealed that circulating IL‐12 and IFN‐γ were enhanced by the cationic materials. Using IFN‐γ receptor knockout mice and macrophage‐depleted rats, we found that IFN‐γ and macrophages played key roles in the anti‐inflammatory activity of the materials towards cells. We also found that neutrophil infiltration at inflammatory sites was reduced when AIA animals were treated with C‐dextran. We propose that cationic signals act through an unknown receptor on macrophages to induce IL‐12 secretion, and that IL‐12 promotes the expression of IFN‐γ by natural killer cells (or T cells). The resulting elevated systemic levels of IFN‐γ inhibit arthritis development by preventing neutrophil recruitment to inflammatory sites.     

The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.     

Differential roles of VLA-4(CD49d/CD29) and LFA-1(CD11a/CD18) integrins and E- and P-selectin during developing and established active or adoptively transferred adjuvant arthritis in the rat

The role of the integrins VLA-4 and LFA-1 and of the selectin adhesion molecules in autoimmune arthritis was investigated. Adjuvant arthritis was induced in Lewis rats by active immunization (s.c.) with Mycobacterium butyricum or by adoptive transfer of immune T cells. With active adjuvant arthritis, Lewis rats develop maximal polyarticular joint inflammation and migration of radiolabelled (111In and 51Cr) blood neutrophils and monocytes to the joints 14 days post Mycobacterium butyricum immunization. Using blocking monoclonal antibodies we osbserved that at this stage monocyte recruitment was dependent (85%) on P-selectin plus VLA-4 (alpha4B1) and neutrophil recruitment depended (> 80%) on P-selectin plus LFA-1 (CD11a/CD18). E-selectin played a minimal role in inflammatory cell recruitment to the already inflamed joint. In contrast, during the development of active adjuvant arthritis, blockade of P-selectin beginning at day 5 post-immunization had no effect on subsequent arthritis. However, E-selectin blockade at this stage reduced arthritic scores by 70% (P < 0.01) and combined E-selectin plus VLA-4 blockade prevented development of arthritis. Either treatment nearly abolished neutrophil and monocyte recruitment to joints at day 14 and prevented cartilage damage. VLA-4 blockade alone was less effective. Adoptive T-cell transfer of adjuvant arthritis to naive rats employed spleen/lymph node lymphocytes from Mycobacterium butyricum immunized rats stimulated with Concanavalin A in vitro (48 h). E-selectin +/- P-selectin blockade had no effect on the development of adoptive arthritis. However, VLA-4 integrin blockade inhibited adoptive arthritis severity by 55% (P < 0.01). LFA-1 blockade had no effect. In adoptive adjuvant arthritis, inhibition of arthritis clinically and by histology was essentially complete (> 90%) when E- and P-selectin blockade was combined with VLA-4 blockade. Thus, in the development of actively induced arthritis E-selectin plays an important role, likely mediating early antigen reactive T-cell recruitment to joints. In contrast, VLA-4 and multiple selectin mechanisms are involved in arthritis induction by ex vivo restimulated arthritogenic T cells. Furthermore, in actively induced adjuvant arthritis, P- and E-selectin and VLA-4 are differently important in the initiation of arthritis, and at the time of fully developed joint inflammation.     

Differential effects of locally and systemically administered soluble glycoprotein 130 on pain and inflammation in experimental arthritis

Introduction  

Interleukin-6 (IL-6) is a key player in systemic arthritis, involved in inflammation and joint destruction. IL-6 signalling has also been revealed in nerve cells. Recently, IL-6 and in particular IL-6 together with its soluble IL-6 receptor (sIL-6R) were shown to induce a long-lasting robust sensitization of joint nociceptors for mechanical stimuli which was difficult to reverse, suggesting that IL-6 signalling plays a significant role in the generation and maintenance of arthritic pain. Here we tested in a preclinical model of arthritis, antigen-induced arthritis (AIA) in the rat, whether systemic or local neutralization of IL-6/sIL-6R complexes with soluble glycoprotein 130 (sgp130) alters arthritic pain and how sgp130 influences the inflammatory process in AIA.     

Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine restores endothelial function in rat adjuvant-induced arthritis

Introduction

Endothelial dysfunction (ED) participates to atherogenesis associated to rheumatoid arthritis. We recently reported increased arginase activity/expression in vessels from adjuvant-induced arthritis (AIA) rats. In the present study, we investigated the effects of a curative treatment with the arginase inhibitor N_w-hydroxy-nor-L-arginine (nor-NOHA) on vascular dysfunction in AIA rats.

Methods

AIA rats were treated with nor-NOHA (40 mg/kg/d, ip) for 21 days after the onset of arthritis. A group of untreated AIA rats and a group of healthy rats served as controls. ED was assessed by the vasodilatory effect of acetylcholine (Ach) on aortic rings. The role of superoxide anions, prostanoids, endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide synthase (NOS) pathway was studied. Plasma levels of IL-6 and vascular endothelial growth factor (VEGF) were determined by ELISA kits. Arthritis severity was estimated by a clinical, radiological and histological analysis.

Results

Nor-NOHA treatment fully restored the aortic response to Ach to that of healthy controls. The results showed that this beneficial effect is mediated by an increase in NOS activity and EDHF and reduced superoxide anion production as well as a decrease in the activity of cyclooxygenase (COX)-2, thromboxane and prostacyclins synthases. In addition, nor-NOHA decreased IL-6 and VEGF plasma levels in AIA rats. By contrast, the treatment did not modify arthritis severity in AIA rats.

Conclusions

The treatment with an arginase inhibitor has a potent effect on ED in AIA independently of the severity of the disease. Our results suggest that this new pharmacological approach has the potential as a novel add-on therapy in the treatment of RA.     

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats

Background

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA).

Methods

Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively.

Results

Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects.

Conclusions

Our results validate celastrol as a promising compound for the treatment of arthritis.     

Variation of substance P-like immunoreactivity in plasma and cerebrospinal fluid in the course of arthritis induced by Freund adjuvant in rats, a model for the study of chronic pain

Parallel time courses of clinical and behavioural parameters and levels of plasma substance P-like immunoreactivity (SPLI-PI) were studied in arthritic rats (adjuvant induced arthritis, AIA, a chronic pain model). Acute (14 and 21 post-inoculation days,PI) and post-acute (42 days PI) phases of the syndrome were investigated. These data were compared with those obtained in a control situation (inoculation day). In a second experimental series, levels of substance P-like immunoreactivity in cerebrospinal fluid (SPLI-CSF) were determined at the same stages of AIA. In arthritic rats SPLI-PI was strongly enhanced (X4) as early as 14 days PI and remained increased (X4) at all stages studied, whereas SPLI-LCR was significantly increased (X2) only 21 days PI and returned to control levels at 42 days PI. These data suggest that SP could be distributed in two different pools, a peripheral one of inflammatory origin, and a central one which could be more specific to the chronic pain situation.     

Wutou decoction ameliorates experimental rheumatoid arthritis via regulating NF-kB and Nrf2: Integrating efficacy-oriented compatibility of traditional Chinese medicine

BackgroundThousands of years of clinical application of Wutou decoction (WTD) support its reliable efficacy and safety in treating rheumatoid arthritis (RA). However, the underlying molecular mechanism remains unclear, and the synergistic involvement of assistant herbs in WTD in enhancing the sovereign herb in treating RA is unknown.PurposeThis study aimed to investigate the efficacy-oriented compatibility of five herbs in WTD and the underlying mechanisms.MethodsThe anti-arthritic effects of WTD and the compatibilities of the five herbs in WTD were studied in vivo with adjuvant-induced arthritis (AIA) rat model and in vitro with LPS-induced RAW264.7 macrophage. Network pharmacology analysis was conducted to identify the dominant pathways involved in the anti-arthritis mechanisms of WTD and how the five herbs work synergistically. The results were further verified by in vivo and in vitro experiments.ResultsOur data revealed that the five herbs in WTD exert synergistic anti-arthritic effects on RA. Moreover, Radix Aconite (AC) is the principal anti-inflammatory component in WTD according to the extent of therapeutic effects exerted on the AIA rats. In vivo and in vitro experiments demonstrated that WTD inhibited NF-κB phosphorylation and simultaneously increased the expression of Nrf2, which were the major pathways identified by the network pharmacology analysis. The major assistant component, Herba Ephedrae (EP), evidently inhibited NF-κB mediated inflammatory response. The other assistant component, Radix Astragali (AS), considerably enhanced the expression of Nrf2 when used alone or in combination with AC. These combinations improved the anti-arthritis effects on the AIA rats better than that of AC alone. Nevertheless, WTD always achieved the best effects than any combinations both in vivo and in vitro.ConclusionThe ministerial herbs EP and AS intensify the anti-arthritic effects of AC by regulating the NF-κB-mediated inflammatory pathway and the Nrf2-mediated anti-oxidation pathway which are the major pathways of WTD for alleviating the symptoms of RA.     

Palmitoylethanolamide and luteolin ameliorate development of arthritis caused by injection of collagen type II in mice

IntroductionN-palmitoylethanolamine (PEA) is an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs). Recently, several studies demonstrated that PEA is an important analgesic, antiinflammatory, and neuroprotective mediator. The aim of this study was to investigate the effect of co-ultramicronized PEA + luteolin formulation on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA).MethodsCIA was induced by an intradermally injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen (CII)) and complete Freund adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice subjected to CIA were administered PEA (10 mg/kg 10% ethanol, intraperitoneally (i.p.)) or co-ultramicronized PEA + luteolin (1 mg/kg, i.p.) every 24 hours, starting from day 25 to 35.ResultsMice developed erosive hind-paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hindpaws. The incidence of CIA was 100% by day 28 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period with a resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with PEA or PEA + luteolin ameliorated the clinical signs at days 26 to 35 and improved histologic status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in PEA + luteolin-treated mice, as indicated by nitrotyrosine and malondialdehyde (MDA) levels. Plasma levels of the proinflammatory cytokines and chemokines were significantly reduced by PEA + luteolin treatment.ConclusionsWe demonstrated that PEA co-ultramicronized with luteolin exerts an antiinflammatory effect during chronic inflammation and ameliorates CIA.