Effect of miR-124a on collagen-induced arthritis in mice and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms.@*METHODS@#Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group.@*RESULTS@#Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001).@*CONCLUSIONS@#Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.

Rhoifolin regulates oxidative stress and proinflammatory cytokine levels in Freund's adjuvant-induced rheumatoid arthritis via inhibition of NF-κB

Rheumatoid arthritis (RA) is an autoimmune disease of knee joints involving pain and inflammation. Rhoifolin is a plant flavonoid known to have antioxidant and anti-inflammatory properties. This study was taken to identify the effect of rhoifolin on complete Freund's adjuvant (CFA)-induced arthritis in the rat model. Treatment with rhoifolin (10 and 20 mg/kg) showed a significant improvement in the overall health parameters such as paw edema and weight loss. This improvement in morphological parameters corroborated the findings with gross morphological changes observed in the histopathological analysis. Rhoifolin treatment also caused a significant decrease in oxidative stress, evident from changes in intracellular levels of glutathione, glutathione peroxidase, malondialdehyde, and superoxide dismutase in the articular cartilage tissue. Moreover, proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin(IL)-1β, and IL-6 showed a significant downregulation of gene expression and intracellular protein concentration levels. The NF-κB pathway showed a significant attenuation as evident in the significant reduction in the levels of NF-κB p65 and p-IκB-α. These results indicated that rhoifolin can be a natural therapeutic alternative to the extant regimens, which include non-steroidal anti-inflammatory drugs and immunosuppressants. Additionally, the antioxidant and anti-inflammatory action of rhoifolin was probably mediated by the NF-κB pathway. However, the exact target molecules of this pathway need to be determined in further studies.

Repercussions of adjuvant-induced arthritis on body composition, soleus muscle, and heart muscle of rats

This study investigated the repercussions of adjuvant-induced arthritis (AIA) on body composition and the structural organization of the soleus and cardiac muscles, including their vascularization, at different times of disease manifestation. Male rats were submitted to AIA induction by intradermal administration of 100 μL of Mycobacterium tuberculosis (50 mg/mL), in the right hind paw. Animals submitted to AIA were studied 4 (AIA4), 15 (AIA15), and 40 (AIA40) days after AIA induction as well as a control group of animals not submitted to AIA. Unlike the control animals, AIA animals did not gain body mass throughout the evolution of the disease. AIA reduced food consumption, but only on the 40th day after induction. In the soleus muscle, AIA reduced the wet mass in a time-dependent manner but increased the capillary density by the 15th day and the fiber density by both 15 and 40 days after induction. The diameter of the soleus fiber decreased from the 4th day after AIA induction as well as the capillary/fiber ratio, which was most evident on the 40th day. Moreover, AIA induced slight histopathological changes in the cardiac muscle that were more evident on the 15th day after induction. In conclusion, AIA-induced changes in body composition as well as in the soleus muscle fibers and vasculature have early onset but are more evident by the 15th day after induction. Moreover, the heart may be a target organ of AIA, although less sensitive than skeletal muscles.

Programmed cell death 5 transgenic mice attenuates adjuvant induced arthritis by 2 modifying the T lymphocytes balance

BACKGROUND: Programmed cell death 5 (PDCD5) is an apoptosis-related gene cloned from TF-1 cells whose primary biological functions are to promote apoptosis and immune regulation. The effects and mechanisms exerted by key mediators of arthritic inflammation remain unclear in PDCD5 transgenic (PDCD5 tg) mice. RESULTS: In the current study, PDCD5 tg mice inhibited the progression of adjuvant-induced arthritis, specifically decreasing clinical signs and histological damage, compared with arthritis control mice. Additionally, the ratio of CD4+IFN-γ+ cells (Th1) and CD4+IL-17A+ cells (Th17), as well as the mRNA expression of the pro-inflammatory mediators IFN-γ, IL-6, IL-17A and TNF-α, were decreased in PDCD5 tg mice, while CD4+CD25+Foxp3+ regulatory T (Treg) cells and the anti-inflammatory mediators IL-4 and IL-10 were increased. Furthermore, PDCD5 tg mice demonstrated reduced serum levels of IFN-γ, IL-6, IL-17A and TNF-α and increased levels of IL-4. CONCLUSIONS: Based on our data, PDCD5 exerts anti-inflammatory effects by modifying the T lymphocytes balance, inhibiting the production of pro-inflammatory mediators and promoting the secretion of anti-inflammatory cytokines, validating PDCD5 protein as a possible treatment for RA.

Abnormal lipid metabolism in collagen-induced arthritis rat model: In vitro, high resolution NMR spectroscopy based analysis.

Collagen-induced arthritis (CIA) was induced in female Wistar rats by intradermal injection of porcine immunization grade native collagen type II (Chondrex). Development and progression of CIA was monitored by studying histopathological, radiographical and biochemical features of arthritic manifestations in the knee joints, hind limb and blood plasma. In addition, oxidative stress status of arthritic animals was determined by measuring lipid peroxidation and the antioxidant enzymes: catalase, superoxide dismutase and glutathione peroxidase. High resolution proton NMR spectroscopy was employed for the analysis of lipid components in the lipid extracts of the joint tissue and plasma of collagen-induced arthritic and control rats. Triglyceride levels showed significant decreases in plasma (1.7 times) but were unchanged in the joint tissue of CIA rats as compared to control. One-dimensional proton NMR spectra showed a 6.2 times reduction in the quantity of choline-containing phospholipids in the plasma of CIA as compared to control rats. There was a 1.6 times elevation of choline-containing phospholipids in the joint tissue of CIA rats as compared to controls. Induction of arthritis showed a 4.0 times reduction in the level of total cholesterol in the plasma and 1.6 times elevation in the joint tissue of CIA rats as compared to controls. The ratio of saturated fatty acids to unsaturated fatty acids was 1.5 times significantly higher in joint tissue and 2.1 times significantly higher in plasma of CIA rats as compared to controls. The results demonstrated significantly altered lipid patterns in the joint tissue and plasma of collagen-induced arthritic rats as detected by one- and two-dimensional NMR spectroscopy compared with controls.

Molecular MR Imaging for Visualizing ICAM-1 Expression in the Inflamed Synovium of Collagen-Induced Arthritic Mice

OBJECTIVE: To determine the utility of intercellular adhesion molecule (ICAM)-1 antibody-conjugated gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA-anti-ICAM-1) as a targeted contrast agent for the molecular magnetic resonance imaging (MRI) in collagen-induced arthritis (CIA). MATERIALS AND METHODS: Three groups of mice were used: non-arthritic normal, CIA mice in both the early inflammatory and chronic destructive phases. The MR images of knee joints were obtained before and after injection of Gd-DTPA-anti-ICAM-1, Gd-DTPA, and Gd-DTPA-Immunoglobulin G (Ig G) and were analyzed quantitatively. The patterns of enhancement on the MR images were compared with the histological and immunohistochemical ICAM-1 staining. RESULTS: The images obtained after injection of Gd-DTPA-anti-ICAM-1 displayed gradually increasing signal enhancement from the moment following injection (mean +/- standard deviation [SD]: 424.3 +/- 35.2, n = 3) to 24 hours (532 +/- 11.3), rather than on pre-enhanced images (293 +/- 37.6) in the early inflammatory phase of CIA mice. However, signal enhancement by Gd-DTPA and Gd-DTPA-IgG disappeared after 80 minutes and 24 hours, respectively. In addition, no significant enhancement was seen in the chronic destructive phase of CIA mice, even though they also showed inflammatory changes on T2-weighted MR images. ICAM-1 expression was demonstrated in the endothelium and proliferating synovium of the early inflammatory phase of CIA mice, but not in the chronic destructive phase. CONCLUSION: Molecular MRI with Gd-DTPA-anti-ICAM-1 displays specific images targeted to ICAM-1 that is expressed in the inflamed synovium of CIA. This novel tool may be useful for the early diagnosis and differentiation of the various stages of rheumatoid arthritis.

Parallel changes in fibronectin and alpha5beta1 integrin in articular cartilage in type II collagen-induced arthritis.

Interactions of cells with extracellular matrix (ECM) are mediated through specific cell surface receptors, belonging to the integrin family of transmembrane proteins. Integrins have been shown to be involved in chondrocyte-matrix interactions in the cartilage. In this study, the status of a matrix glycoprotein fibronectin (FN) and its receptor alpha5beta1 integrin in the articular cartilage in collagen type II-induced experimental arthritis in rats, as well as in synovial fluid from osteoarthritic patients was investigated. Experimental arthritis was induced by intradermal injection of type-II collagen (300 microg/100 g body wt) and Freund's complete adjuvant. Saline-treated animals served as control. Clinical severity was indicated by increase in paw volume. Significant increase in the activities of lysosomal enzymes beta-glucuronidase and beta-hexosaminidase was observed in synovial effusate, serum and cartilage of arthritic animals, when compared to untreated control, indicating dysfunction of cartilage. Changes in FN and alpha5beta1 integrin were studied by ELISA. A progressive increase was observed in the FN level in synovial effusate and cartilage of arthritic animals, when compared to untreated controls. FN levels were also significantly high in synovial fluid of osteoarthritic patients. A significant increase in the levels of alpha5beta1 integrin was found in cartilage of arthritic rats. Parallel changes in FN and alpha5beta1 integrin indicated that alterations in FN and alpha5beta1 integrin in chondrocytes constituted one of the molecular mechanisms during progression of arthritis.

Reversible plasma testosterone levels reduction after gentamicin administration and freund's adjuvant arthritis in rats.

The present study was undertaken in order to investigate the influence of gentamicin on plasma testosterone levels of healthy and with Freund's adjuvant arthritis rats. Gentamicin (40 mg/day for 4 days) induced significant decrease of testosterone levels in comparison with the control group (P<0.025). Intraperitoneal calcium administration (30 mg/ kg bw) prevented gentamicin effect and maintained testosterone levels to that of the control. Decreased testosterone levels were also observed in gentamicin received Freund's adjuvant arthritic rats, in the acute stage of the inflammatory disease (P<0.025), and in the acute stage of Freund's adjuvant arthritis (P<0.001). It is concluded that the administration of gentamicin decreases plasma testosterone levels without any effect on body and seminal vesicles weight. Calcium loading counteracts gentamicin reducing effect on plasma testosterone levels. Freund's adjuvant arthritis influences the function of body and seminal vesicles as it was shown by the reduction of testosterone levels, body and seminal vesicles weight during the acute phase of the inflammatory disease. In any case the effect was reversible.

RANK, RANKL and osteoprotegerin in arthritic bone loss

Rheumatoid arthritis is characterized by the presence of inflammatory synovitis and destruction of joint cartilage and bone. Tissue proteinases released by synovia, chondrocytes and pannus can cause cartilage destruction and cytokine-activated osteoclasts have been implicated in bone erosions. Rheumatoid arthritis synovial tissues produce a variety of cytokines and growth factors that induce monocyte differentiation to osteoclasts and their proliferation, activation and longer survival in tissues. More recently, a major role in bone erosion has been attributed to the receptor activator of nuclear factor kappa B ligand (RANKL) released by activated lymphocytes and osteoblasts. In fact, osteoclasts are markedly activated after RANKL binding to the cognate RANK expressed on the surface of these cells. RANKL expression can be upregulated by bone-resorbing factors such as glucocorticoids, vitamin D3, interleukin 1 (IL-1), IL-6, IL-11, IL-17, tumor necrosis factor-alpha, prostaglandin E subscrito 2, or parathyroid hormone-related peptide. Supporting this idea, inhibition of RANKL by osteoprotegerin, a natural soluble RANKL receptor, prevents bone loss in experimental models. Tumor growth factor-ß released from bone during active bone resorption has been suggested as one feedback mechanism for upregulating osteoprotegerin and estrogen can increase its production on osteoblasts. Modulation of these systems provides the opportunity to inhibit bone loss and deformity in chronic arthritis.

Effect of superoxide dismutase from bovine erithrocytes on different activity parameters in Ajuvant-Induced arthritis

Background. The purpose of this work was to evaluate the effect of superoxide dismutase (SOD) on primary swelling, lipoperoxidation, body thymus, and spleen weight in the adjuvants-induced arthritis (AIA) model in rats. Methods. Orally and intraperitoneally administered SOD (100 U/kg) from bovine erythrocytes, as well as naproxen (40 mg/kg) and dexamethasone (25 mg/kg), were evaluated againts placebo. Results. Primary edema was not decreased by SOD; in contrast, naproxen and dexamethasone showed good anti-inflammatory activity. Lipoperoxidation increased 1.8, 2.5, and 2.8 times with intraperitoneal SOD, naproxen, and dexamethasone administration, respectively, while oral SOD decreased lipoperoxidation levels to approximately one-half of that found in the control group. Body weight increased with SOD but decreased with dexamethasone. Naproxen did not change the animal weight. Thymus weight remained unchanged with SOD and naproxen, while it decreased with dezamethasone. Splee weight remained the same wih SOD, but increased with naproxen and decreased with dezamethasone. No side of gastrointestinal hemorrhage, and 50 percent of the rats in the dexamethasone group, of pulmonary infection. Conclusions. In conclusion, SOD showed no anti-inflammatory activity but decreased lipoperoxidation when administered orally. No deleterious effects in primary and secondary immunologic organs were observed with this agent

Molecular characteristics of soluble arthritic collagens.

The molecular characteristics of acid-soluble collagen prepared from animals rendered adjuvant arthritis and matched controls have been thoroughly investigated. The arthritic process was induced in rats by the injection of paraffin oil containing the suspension of heat-killed Mycobacterium tuberculosis at the dorsum of the tail root. In comparison to the normal, the arthritic collagens consistently demonstrated increased solubility of reconstituted fibrils and marked decrease in intrinsic viscosity. Digestion of arthritic collagens with various proteolytic enzymes showed an increased susceptibility over normal collagens. Electrophoresis on SDS-polyacrylamide gels revealed a marked decrease in the concentration of beta-components of arthritic collagens and appreciable increase in the ratio of alpha to beta subunit component. The data clearly indicate that the arthritic collagens are very poor in polymerization due to the impairment of intra and inter-molecular interactions. These changes could explain in part, the altered response of the connective tissues to inflammation and arthritis.

Influencia del tratamiento con Mycobacterium leprae sobre la respuesta inmune en ratas: su evaluacion en un modelo de artritis experimental / The influence of treatment with Mycobacterium leprae on immune response in rats: its evaluation in a model of experimental arthritis

Ratas endocriadas de línea "m" machos adultos, fueron tratadas con Mycobacterium leprae (Ml) muerto por calor (4 x 10**7 bacilos/ml, extraído de lepromas humanos) de acuerdo con el siguiente esquema: una inyección intraperitoneal -ip- o subcutánea -sc- de 2 ml de Ml dos días anters de la inducción artrítica; una inyección ip o sc de 0.5 ml de Ml 2 veces por semana, durante 15 días previos a la inducción. La artritis por adyuvante (AA) fue provocada 48 horas después, inoculando 0.1 ml de Adyuvante Completo de Freund (ACF) en la almohadilla plantar de la pata trasera derecha. Se constató una significativa reducción del proceso artrítico, a los 15 días de la inducción, en las ratas tratados con 4 inyecciones ip de 0.5 ml de Ml. Un efecto depresivo similar también se obtuvo en ratas singeneicas que recibieron 1 x 10**8 células esplénicas de animales tratados con el mismo protocolo. En otra serie de experimentos, 48 horas antes de la inducción de la AA, 2 grupos de ratas recibieron 1 x 10**8 totales, o 1 x 10**7 células esplénicas no adherentes de animales tratados con Ml. Se pudo constatar que la población de células no adherentes también fue capaz de producir una notable reducción del proceso artrítico. A su vez, esta población no modificó la AA en el receptor cuando la transferencia se efectuó 48 horas después de la inducción. Sobre la base de nuestros resultados, concluimos que la exposición reiterada al Ml induciría células supresoras con actividad regulatoria sobre la AA que operaría...