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.

Efeitos de um minuto e dez minutos de deambulação em ratos com artrite induzida por adjuvante completo de Freund sobre os sintomas de dor e edema / Effects of one minute and ten minutes of walking activity in rats with arthritis induced by complete Freund's adjuvant on pain and edema symptoms

Este estudo avaliou o efeito de dois protocolos de exercício na nocicepção, edema e migração celular em ratos com artrite induzida por CFA. Ratos Wistar fêmeas (200 - 250 g, n = 50) foram induzidos à monoartrite por adjuvante completo de Freund (CFA, Mycobacterium butyricum; 0,5 mg/mL; 50 μL) na articulação do joelho direito (TF; n = 24) ou tornozelo direito (TT; n = 26). A incapacitação articular foi mensurada pelo tempo de elevação da pata (TEP; s) em 1 minuto de avaliação. O edema do joelho ou tornozelo foi avaliado pela medida do diâmetro articular (AD, cm) e pelo edema de pata (EP, mL), respectivamente. Ambos foram avaliados durante 10 dias consecutivos. Dois protocolos de exercício foram realizados: (a) exercício constante (TF, n = 6; TT, n = 6), realizando 1 minuto diário de exercício no cilindro (3 r.p.m.); (b) exercício variável (TF, n = 6; TT, n = 7), exercício com aumento de 1 minuto por dia, totalizando 10 minutos no último dia. Os grupos-controle (TF, n = 12; TT, n = 13) não realizaram exercício. Após 10 dias, os animais foram eutanasiados para contagem total (células/mm3) e diferencial (mononucleares e polimorfos nucleares; células/mm3) de leucócitos do tecido inflamado. O exercício variável inibiu a incapacitação e o edema em ambas as articulações. Entretanto, reduziu a migração total de leucócitos apenas na articulação TF. O exercício constante inibiu o edema nas duas articulações e reduziu a migração total de leucócitos da articulação TT. Porém, não reduziu a incapacitação. O exercício variável pareceu ser mais efetivo em reduzir os parâmetros inflamatórios em comparação com o exercício constante.

This study evaluated the effects of two protocols of exercise on nociception, edema and cell migration in rats with CFA-induced arthritis. Female Wistar rats (200 - 250 g, n = 50) was monoarthritis-induced by complete Freund's adjuvant (CFA; Mycobacterium butyricum, 0.5 mg/mL; 50 μL) into the right knee joint (TF; n = 24) or right ankle joint (TT; n = 26). Incapacitation was measured by the paw elevation time (TEP; s) in 1-min periods of observation. The edema of the knee or ankle joints was evaluated by the variation of the articular diameter (DA, cm) and by the paw volume variation (EP, mL), respectively. Both were measured during 10 consecutive days. Two protocols of exercise were performed: (a) in the constant exercise group (TF, n = 6; TT, n = 6) performing 1 minute of daily exercise on the cylinder; (b) variable exercise group (TF, n = 6; TT, n = 7), the exercise increased by 1 minute per day. The control groups (TF, n = 12; TT, n = 13) didn´t perform the exercise. After 10 days, the animals were euthanized for total (CT; cells/mm3) and differential leukocyte counts (mononuclear - MON, and polymorphonuclear - PMN, cells/mm3) of the articular inflammatory exudate. The variable exercise protocol inhibited incapacitation and edema for both joints. However, cell migration decreased only in the TF.The constant exercise reduced edema in both joints, and cell migration was decreased in the TT. However, the incapacitation was not reduced. Variable exercise seemed to be more effective in reducing the inflammatory parameters than constant exercise.

Padronização de modelo experimental adequado a estudos do efeito do exercício na artrite / Standardization of an experimental model suitable for studies on the effect of exercise on arthritis

OBJETIVO: Padronizar um modelo experimental de monoartrite crônica induzida por adjuvante completo de Freund apropriado à análise do efeito da deambulação na nocicepção e no edema articular. MÉTODOS: Foram avaliados os seguintes fatores para a indução da monoartrite: via e local de administração, número e intervalo das inoculações, espécie de micobactéria e gênero dos animais. Para tanto, ratos Wistar machos e fêmeas (200 a 250g) receberam duas injeções de adjuvante completo de Freund contendo Mycobacterium tuberculosis (1,0mg/mL; 50µL) ou Mycobacterium butiricum (0,5mg/mL; 50µL) intra-articular nas articulações tibiotársica ou tibiofemural ou, ainda, uma injeção de adjuvante completo de Freund (Mycobacterium butiricum ou tuberculosis) intradérmica na base da cauda e outra intra-articular (tibiotársica ou tibiofemural). Os animais foram submetidos à avaliação da incapacitação e edema articulares. A incapacitação articular foi avaliada pelo tempo de elevação da pata (em segundos) durante a marcha de 1 minuto. O edema da articulação tibiofemural foi avaliado pela variação do diâmetro articular (cm). O edema da articulação tibiotársica foi medido pelo volume da pata (mL). RESULTADOS: A administração de adjuvante completo de Freund, contendo Mycobacterium butiricum, aumentou o tempo de elevação da pata e o edema, em ambas as articulações. CONCLUSÃO: Esses dados possibilitaram a padronização de um modelo animal de monoartrite crônica, adequado à análise dos efeitos do exercício no tratamento da artrite reumatoide.

OBJECTIVE: To standardize an experimental model of chronic monoarthritis induced by complete Freund's adjuvant appropriate for the analysis of the effect of walking on nociception and on joint edema. METHODS: The following factors were evaluated as to monoarthritis induction: route and site of administration, number and interval of inoculations, Mycobacterium species, and animal gender. Wistar male and female rats (200 to 250g) received two injections of complete Freund's adjuvant containing Mycobacterium tuberculosis (1.0mg/mL; 50µL) or Mycobacterium butyricum (0.5mg/mL; 50µL) intra-articularly in the tibiotarsal or tibiofemoral joints, or an injection of complete Freund's adjuvant (Mycobacterium butyricum or tuberculosis) intradermally at the base of the tail and another intra-articularly (tibiotarsal or tibiofemoral). The animals were submitted to evaluations of articular disability and edema. Articular disability was assessed by paw elevation time (in seconds) during the one-minute walk test. Edema of the tibiofemoral joint was assessed by variation of joint diameter (cm). Tibiotarsal joint edema was measured by the volume of the paw (mL). RESULTS: Administration of complete Freund's adjuvant containing Mycobacterium butyricum increased paw elevation time and edema in both joints. CONCLUSIONS: These data allow standardization of an animal model of chronic monoarthritis adequate for analysis of the effects of exercise on treatment of rheumatoid arthritis.

Effect of lipoarabinomannan from Mycobacterium avium subsp avium in Freund’s incomplete adjuvant on the immune response of cattle

The aim of the present study was to determine whether lipoarabinomannan (LAM), in combination with Freund’s incomplete adjuvant (FIA), was able to improve cell-mediated and antibody-mediated immune responses against ovalbumin (OVA) in cattle. Twenty-three calves were assigned to four treatment groups, which were subcutaneously immunized with either OVA plus FIA, OVA plus FIA and LAM from Mycobacterium avium subsp avium, FIA plus LAM, or FIA alone. Lymphoproliferation, IFN-γ production and cell subpopulations on peripheral blood mononuclear cells before and 15 days after treatment were evaluated. Delayed hypersensitivity was evaluated on day 57. Specific humoral immune response was measured by ELISA. Inoculation with LAM induced higher levels of lymphoproliferation and IFN-γ production in response to ConA and OVA (P < 0.05). Specific antibody titers were similar in both OVA-immunized groups. Interestingly, our results showed that the use of LAM in vaccine preparations improved specific cell immune response evaluated by lymphoproliferation and IFN-γ production by at least 50 and 25 percent, respectively, in cattle without interfering with tuberculosis and paratuberculosis diagnosis.

Evaluation of disease modifying activity of Coriandrum sativum in experimental models.

Background & objectives: Coriandrum sativum (CS), has been widely used in traditional systems of medicine for treatment of rheumatoid arthritis. However, the mechanism of action for its antiarthritic effects is not clearly known. Therefore, the present study was carried out to evaluate the antiarthritic activity of CS in rats in two experimental models. Methods: The antiarthritic activity of CS seed hydroalcoholic extract (CSHE) was evaluated in adult Wistar rats by using two experimental models, viz. formaldehyde and Complete Freund's adjuvant (CFA) induced arthritis. The expression of pro-inflammatory cytokines (predominantly contributed by macrophages) was also evaluated. TNF-α level was estimated in serum by ELISA method. TNF-R1, IL-1 β and IL-6 expression in the synovium was analysed by immunohistochemistry. Results: CSHE produced a dose dependent inhibition of joint swelling as compared to control animals in both, formaldehyde and CFA induced arthritis. Although there was a dose dependent increase in serum TNF-α levels in the CSHE treated groups as compared to control, the synovial expression of macrophage derived pro-inflammatory cytokines/cytokine receptor was found to be lower in the CSHE treated groups as compared to control. Interpretation & conclusions: Our results demonstrate that the antiarthritic activity of CSHE may be attributed to the modulation of pro-inflammatory cytokines in the synovium. In further studies CSHE could be explored to be developed as a disease modifying agent in the treatment of RA.

Effects of soluble antigen-induced immune cell activation on steroidogenesis in murine lymphoid organ.

The effect of soluble antigenic (bovine serum albumin, BSA) stimulation to induce steroidogenesis in murine lymphoid organs with concomitant changes in proinflammatory or inflammatory cytokine levels and its implication in the alteration of T-cell response was studied in the mice. Male Swiss albino mice (6-8 weeks old) with average body weight (20 +/- 4 g) were randomly assigned to 3 groups and injected with BSA in presence and absence of Freund's complete or incomplete adjuvant, whereas the control group received only saline. After 3 weeks, animals were sacrificed, and serums as well as lymphoid organs were collected. From the lymphoid tissue homogenate, the activities of steroidogenic enzymes and corticosterone and cytokine levels of the serum were estimated. Steroidogenic enzyme activities in murine lymphoid organs, as well as the pro-inflammatory and inflammatory cytokines levels in serum increased after Freund's complete adjuvant-emulsified BSA administration, as compared to control. The serum corticosterone and serum cytokine profile were also elevated. Results suggested that soluble protein antigen (BSA) administration stimulated steroidogenesis in murine lymphoid tissues and rise in the pro-inflammatory or inflammatory cytokine levels might indicate monocyte recruitment as well as TH1 activation.

Procedimientos para la obtención de ratones Balb/c hiperinmunes contra el antígeno B del sistema ABO de grupos sanguíneos humanos / Production of Balb-c mice hyperimmunized against B antigen from ABO human blood groups

Los antígenos del sistema ABO de grupos sanguíneos son oligosacáridos presentes en la membrana celular que forman parte de las glicoproteínas y glicolípidos. La introducción de la tecnología de hibridomas murinos para la obtención de anticuerpos monoclonales (AcM) con especificidad para los antígenos ABO ha permitido iniciar en Cuba la producción de reactivos hemoclasificadores basados en AcM. El perfeccionamiento de los reactivos requiere la búsqueda de nuevos clones de hibridomas, para lo que se necesita la sensibilización de ratones Balb/c con los antígenos. Se presentan los resultados de la evaluación de dos vías de inoculación (intraperitoneal e intraesplénica) y tres formas de presentación del antígeno (hematíes completos del grupo B, sustancia específica B y sustancia B tratada con alúmina) en ratones Balb/c a los que se les determinó el título de anticuerpos hemaglutinantes por la técnica de microplacas frente a un panel de hematíes de los grupos A, B y O antes y durante el período de inmunización. La vía intraperitoneal con hematíes completos del grupo B resultó adecuada para alcanzar altos títulos de anticuerpos aglutinantes contra hematíes B, aunque también reaccionaron con los hematíes de otros grupos.

Effects of immunization with sperm specific lactate dehydrogenase with & without muramyl dipeptide as adjuvant.

The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

A study of the efficacy of liposomes in comparison to new and established adjuvants in potentiating the antibody response against hepatitis B virus surface antigen.

The dehydration-rehydration vesicle (DRV) method was used to encapsulate hepatitis B surface antigen (HBsAg) in phosphatidylcholine (PC) and distearoyl phosphatidylcholine (DSPC) liposomes giving entrapment values of 31.7% and 33.1% respectively. A comparison of antibody levels, as determined by ELISA, in the primary and secondary immune responses in mice immunized twice with 1 microgram HBsAg free, or in formulations of PC DRV, DSPC DRV, Syntex Adjuvant Formulation (SAF), alum and Freund's Complete Adjuvant (FCA) showed that by far, FCA was the best adjuvant in both the primary and secondary IgG1, IgG2a and IgG2b responses. In the secondary response, apart from FCA, DSPC DRV and SAF were equally efficacious and better adjuvants than alum and PC DRV for the IgG2a and IgG2b subclasses. SAF was a better adjuvant for HBsAg than alum, DSPC DRV and PC DRV (in descending order of efficacy) in the secondary IgG1 response.