Dissertations / Theses on the topic 'Adjuvant arthritis'

In this thesis behavioural and electrophysiological techniques in both the rat and the mouse were used to determine the effect of a number of mediators and/or pharmacological receptors reported to be involved in chronic inflammation and nociception. The specific aims of the thesis centred on testing the following hypotheses: a) The neuropeptide somatostatin inhibits sensory nerve function in both normal and arthritic knee joints, b) the endocannabinoid anandamide activates peripheral nociceptors via its reported action at the vanilloid receptor (VR₁), c) chronic unilateral inflammation of the knee joint can be induced in mice using Freund’s Complete Adjuvant (FCA), and d), the purinoceptor P2X₇ plays a role in the induction of inflammation and hyperalgesia associated with experimental arthritis in mice. An additional aim of the thesis was in vivo neural recording from nociceptors innervating the mouse knee joint with a view to examining transgenic mice in future studies. In summary, in relation to the hypothesis being tested, the results showed that: a) the reported anti-nociceptive effect of somatostatin is not mediated by action on peripheral nociceptors or the inhibition of tested algogens, b) anandamide is able to directly activate sensory afferents via VR₁ receptors, c) chronic, unilateral arthritis can be induced in mice by repeated intra-articular injections of FCA, and d P2X₇ purinoceptors do not play a role in the induction of inflammation and hyperalgesia associated with the FCA model of unilateral arthritis. Innovation in this thesis included a novel model of murine unilateral arthritis and the development of a new technique for direct measurement of evoked discharge from peripheral nociceptors innervating the mouse knee joint. These advances in knowledge provide information relevant to understanding inflammatory joint disease and for future drug development.

The hypothalamic-pituitary-adrenal (HPA) axis, a system activated by stress, is traditionally considered to affect the susceptibility to chronic pain via effects on peripheral processes. This study investigates whether the HPA axis contributes to the development of chronic pain in an animal model via direct effects on central pain mechanisms.
First, correlations between pain processes and the susceptibility to chronic pain in an animal model that is correlated with HPA-axis function were examined. The results show that, in the Fischer rat, the amount of pain suppression observed during the formalin interphase depression is negatively correlated with susceptibility to polyarthritis. Since the formalin interphase depression mechanisms are within the central nervous system, the results suggest a role for central pain mechanisms in the development of polyarthritis.
Hypophysectomy inhibits the development of adjuvant-induced arthritis. To test whether hypophysectomy inhibits adjuvant-induced polyarthritis via central pain mechanisms, the analgesic effect of hypophysectomy was examined in the formalin test. The results show that hypophysectomy specifically prolongs the formalin interphase depression, further supporting that the underlying central pain suppression mechanisms are associated with resistance to adjuvant-induced polyarthritis.
Corticotropin-releasing factor (CRF) was then investigated as a possible underlying mechanism of the effects of hypophysectomy. Peripheral injection of CRF into inflamed tissue affects pain mechanisms unrelated to the susceptibility to adjuvant-induced polyarthritis. However, central and intravenous administration of CRF preferentially affect the formalin interphase depression mechanisms. The observed dose-response relationships indicate that these effects are due to direct actions of CRF within the central nervous system.
In conclusion, the results strongly suggest that the HPA axis modulates the susceptibility to adjuvant-induced polyarthritis via direct effects on supraspinal pain suppression mechanisms. Thus, the HPA axis may contribute to the development of chronic pain syndromes associated with HPA-axis abnormalities, such as rheumatoid arthritis and fibromyalgia, via effects on pain mechanisms within the central nervous system.

La cryothérapie est utilisée de manière large et empirique à visée adjuvante dans les rhumatismes inflammatoires, avec un niveau de preuve faible. Dans une revue systématique de la littérature, en poolant les données de 6 études non contrôlées, nous avons pu démontrer que la cryothérapie (locale ou corps entier) appliquée deux fois par jour pendant 7 à 15 jours réduisait significativement l'EVA douleur et le score d'activité DAS25 dans la polyarthrite rhumatoïde. La cryothérapie locale (glace ou gaz froide) montrait par ailleurs des effets taille intra-classes supérieurs à ceux obtenus en utilisant la cryothérapie corps entier. L'objectif de ce travail était de mesurer les effets de la cryothérapie locale sur al douleur, l'inflammation synoviale et systémique chez les patients arthritiques et dans le modèle murin d'arthrite à l'adjuvant. Dans les études randomisées CDRI et ALGGAR, nous avons évalué les effets de deux applications locales de froid (glace versus gaz froid) sur la douleur, l'activité Doppler et les taux protéiques de cytokines intra-articulaires controlatéraux non souffrant d'arthrites de genou non septiques. Les genoux arthritiques controlatéraux non traités étaient utilisés comme contrôles. Nous avons par ailleurs étudié in vitro les effets de l'hypothermie modérée (30°C pendant 2heures) sur l'expression protéique des cytokines dans un modèle de culture de rotules de rats arthritiques. Nous avons enfin étudié in vitro dans l'arthrite à l'adjuvant les effets de l'application sub-chronique de glace ou de gaz froid (2 fois par jour pendant 14 jours versus contrôles arthritiques non traités) sur le score d'arthrite, le diamètre de cheville, la transcription des gènes codant pour les cytokines pro-inflammatoires dans les pattes arrières (Q-RT-PCR) et l'expression protéique des cytokines dans le plasma (Multiplex et ELISA) après 14 jours de traitement. Dans l'étude CDRI, la cryothérapie locale (glace et gaz froid) réduisait significativement l'EVA douleur ainsi que le score Doppler dans les genoux traités, ces effets persistant le lendemain des deux applications. Dans une analyse intermédiaire des résultats de l'étude ALGGAR, en combinant les deux groupes de traitement (glace et gaz de froid), nous avons observé une baisse des taux d'IL-6, d'IL-1β et de VEGF dans le liquide articulaire arès deux applications. dans les cultures d'explants de rotules de rats arthritiques, l'hypothermie ponctuelle réduisait significativement les taux d'IL-6, IL-17A et IL-1β dans les pattes arrières après 14 jours de traitement. Les deux modalités réduisaient significativement les niveaux plasmatiques d'IL-17A et la glace réduisait en outre les taux d'IL-6 et de VEGF. Nous n'avons observé aucun effet de la cryothérapie locale sur le voie du TNF-α chez l'homme ni chez l'animal. Nos résultats démontrent pour la première fois un effet thérapeutique et anti-inflammatoire de la cryothérapie locale dans l'arthrite. Les effets biologiques était IL-6/IL-147 dépendants et TNF-α indépendants. Des études complémentaires permettront de mieux caractériser les mécanismes moléculaires sous-jacents et de déterminer su la cryothérapie locale pourrait être une alternative aux AINS et corticoïdes dans les rhumatismes inflammatoires
Cryotheapy i widely and empirically used in an adjuvant setting in inflammatory rheumatic diseases, with a low level of evidence. We performed a systematic review of the literature and, by pooling data from 6 non-controlled studies, we could show that local cryotherapy (local or whole-body cryotherapy) applaied twice a day for 7-15 days significantly reduced the pain VAS and the DAS28 activity score in rheumatoid arthritis. Furthermore, local cryotherapy (ice packs or cold gas) showed significantly greater intra)class effect-sizes compared to whole-body cryotherapy. The aim of this work was to measure the effects of local cryotherapy on pain, synovial and systemic inflammation in arthrici patients and in the murine model of adjuvant-induced arthritis. First, in the CDRI and ALGGAR randomized studies, we evaluated the effects of 2 local cold applications (ice versus cold gas) on pain, power Doppler activity and intra-joint cytokine protein levels in 46 patients suffering from non-septic knee arthritides. Contralateral arthritic knee were used as control. Secondly, we studied the in vitro effects of mild hypothermia (30°C for 2 hours) on cytokine protein expression in a model of cultured arthritic rat patellae. Thidly, we studied the in vitro effects of sub-chronically applied ice or cold gas (twice a day for 14 days versus non-treated arthritic controls) on the arthritis score, the ankle diameter, pro-inflammatory cytokine gene transcription levelsin hind paws (Q-RT-PCR) and cytokine plasma protein levelx (Multiplex and ELISA) after 14 days of treatment. In the CDRI study, local cryotherapy (ice and cold gas) significantly reduced the pain VAS and the power Doppler score in treated kness, and these effects remained significant the day afetr 2 cold applicaitions. In an intermediate analysis of the ALGGAR study results, by pooling the 2 treatment groups, we could show significant decreases in IL-6 protein, IL-1β and VEGF synovial fluid protein levels after 2 cold applicatios. In arthritic rat patella explangt culture experiments, punctual hypothermia significantly reduced IL-6 protein levels. In vivon ice was more efficient on the clinical parameters and better tolerated compared to cold gas. Both techniques significantly reduced IL-6, IL-17A ans IL-1β gene transcription levels in hind paws after 14 days of treatment. Both techniques redcued IL-17A plasma protein levles, while ice also reduced IL-6 and VEGF plasma protein levels. Conversely, we observed no effect of local cryotherapy on the TNF-α pathway, neither in patients nor in our animal model. Here we demonstrate for the first time therapeutic and anti-inflammatory effet-cts of local cryothepary in arthritis. The biological effects were IL-6/IL-17-driven and TNF-α independent. Further studies will help elucidate the underlying molecular mlechanisms involved and detemrine whether local cryotherapy might be a safer alternative to NSAIDs ans corticosteroids in inflammatory rheumatic diseases

Prostaglandins (PGs) are lipid mediators derived from a cascade initiated by catalyzing arachidonic acid to PGH2 via the cyclooxygenases (COXs). Subsequently, downstream prostanoid synthases convert PGH2 into the biologically active PGs, which are potent mediators of pain and inflammation. Membrane prostaglandin E2 synthase-1 (mPGES-1) is a terminal prostaglandin synthase that isomerizes PGH2 into PGE2. Studies have shown that COX-2 and mPGES-1 are induced following pro-inflammatory stimuli in cell lines and tissues. The focus of this thesis was to better understand the role of mPGES-1 in inflammation, using rat adjuvant-induced arthritis as a model. Our study showed that mPGES-1 followed a similar induction profile as COX-2 by RNA and protein analysis, implying that mPGES-1 is the major terminal PGES enzyme downstream of COX-2 that plays a role in the inflamed rat paws. Immunofluorescence studies also revealed the induction of mPGES-1 along with COX-2, 3 days post-adjuvant treatment. These findings suggest that mPGES-1 may constitute a target for the treatment joint inflammation.

Les patients atteints de polyarthrite rhumatoide (PR) ont une diminution de l'espérance de vie de 10 à 15 ans. Cette augmentation de la mortalité semble liée à un processus athéromateux accéléré. La dysfonction endothéliale (DE) joue un rôle clé dans ce processus. L'arginase est une enzyme qui régule le niveau de NO par compétition avec la NO Synthase (NOS) pour leur substrat commun, la L-arginine.Nous avons montré que la vasodilatation Acétylcholine (ACh) dépendante était altérée dans le modèle d'Arthrite Induite à Adjuvant (AIA) chez le rat Lewis, témoignant d'une DE. L'incubation des anneaux aortiques avec la nor-NOHA un inhibiteur sélectif d'arginase a amélioré la réponse vasculaire à l'ACh chez les rats AIA. L'activité et l'expression vasculaire de l'arginase se sont révélées augmentées chez les rats AIA et corrélées positivement à la sévérité de l'arthrite.Nous avons caractérisé les mécanismes impliqués dans la DE du rat AIA. Nos résultats ont montré que la DE mettait enjeu une diminution de l'activité de la NO synthase, un déficit en EDHF, une suractivité de la COX-2, ainsi qu'une production excessive des anions superoxydes. Le traitement curatif des rats AIA par la nor-NOHA pendant 3 semaines, a permis de restaurer la fonction endothéliale. L'inhibition de l'arginase n'a pas modifié l'atteinte articulaire des rats AIA.Nos travaux ont permis de mieux comprendre la physiopathologie de la DE associée à la PR et ont déterminé pour la première fois le rôle délétère de l'arginase dans cette anomalie. Ils ouvrent des perspectives quant à l'utilisation des inhibiteurs d'arginase comme futurs traitements pharmacologiques de l'atteinte vasculaire du patient PR
Patients with RA are characterized by a decrease in life expectancy of 10 to 15 years. This increase in mortality seems to be related to an accelerated atheroma process. Endothelial dysfunction (ED) has a key role in these processes. The arginase is an enzyme which regulates the level of NO by competing with the NO synthase (NOS) to their common substrate, L-arginine.We showed that acetylcholine (ACh) dependent vasodilation was altered in the model of Adjuvant Induced Arthritis (AIA) in Lewis rats, indicating a endothelial dysfunction. The incubation of aortic rings with nor-NOHA has improved the vascular response to ACh in AIA rats. The activity and expression of vascular arginase are increased in AIA rats and positively correlated with the severity of arthritis.We characterized the mechanisms involved in DE in AIA rats. Our results showed that ED involved a decrease of activity of NO synthase, a decrease of EDHF, overactiviry of COX-2, thromboxane synthase and prostacyclin synthase, as well as excessive superoxide anions. The cure of AIA rats by a selective inhibitor of arginase, nor-NOHA for 3 weeks, has restored endothelial function. In contrast, inhibition of arginase activity did not change the weight, the diameter of ankles, radiological or histological articular damage in AIA rats.Our work has led to a better understanding of pathophysiology of ED associated with rheumatoid arthritis and determined for the first time the deleterious role of arginase in this vascular anomaly. These results open prospects for the use of arginase inhibitors as future pharmacological treatment of vascular patient PR

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Physical activity is thought to be beneficial to arthritis, delaying disability and/or improving joint function. The aim of the present study was to evaluate the effects of exercise on nociception, edema and cell migration in two groups of rats with CFA-induced arthritis, as well as verify whether the possible endogenous liberation of corticoids while exercising can have an influence on the nociception and edema. Arthritis was induced in female Wistar rats (200 - 250 g, n=50) by subcutaneously injecting complete Freund's adjuvant (CFA; Mycobacterium butyricum, 0.5 mg/mL; 50 μL) into the base of the tail and, after 21 days, into the right knee joint (TF) or right ankle joint (TT). Incapacitation was measured daily by the paw elevation time (TEP; s) in 1-min periods of observation on a revolving cylinder (3 r.p.m.). The variation of inflammatory edema of the knee and ankle joints was evaluated by the variation of the articular diameter (DA, cm) and by the paw volume variation (EA, mL), respectively. Both, incapacitation and edema were measured during 10 consecutive days. Two protocols of exercise were evaluated: (a) in the 1-minute exercise group, the animals performed 1 minute of daily exercise on the cylinder, this exercise being the incapacitation test itself; (b) for the progressive exercise group the duration of daily exercise increased by 1 minute per day (the additional exercise was always done just after the daily incapacitation test); (c) in the control groups (TF and TT), incapacitation was evaluated only on the 1st, 5th and 10th days after intra-articular CFA injection. After completion of the trials the animals were euthanized and the articular inflammatory exudates was sampled for total (CT; cells/mm3) and differential leukocyte counts (mononuclear - MON, and polymorphonuclear - PMN, cells/mm3). The progressive exercise protocol inhibited incapacitation and edema for both the knee and ankle joints. However, cell migration was inhibited only in the knee joint. The daily 1-min exercise reduced edema in both knee and ankle joints, while cell migration was inhibited only in the ankle joint. The effect on incapacitation was not significant in this protocol. Aminoglutetimide (50 mg/kg; oral) 1 hour before exercise did not modify the antinociceptive and antiedematogenic effect of exercise in this model. By comparison, progressive exercise seemed to be more effective in reducing the inflammatory parameters than 1-min exercise. Although other exercise protocols need to be evaluated, this study suggests that regular physical activity can consistently contribute to improving joint function, as well as present an anti-inflammatory effect.
O exercício físico apresenta potenciais benefícios na artrite, retardando a incapacidade funcional e melhorando a função das articulações. Sendo assim, o objetivo deste estudo foi avaliar a influência da atividade física sobre a nocicepção, edema e migração celular em ratas com artrite induzida por adjuvante completo de Freund (CFA), como também verificar se a possível liberação endógena de corticóides durante o exercício podem influenciar na nocicepção e no edema. Ratos Wistar fêmeas (200 250 g; n=50) receberam injeção intradérmica de CFA (Mycobacterium butiricum; 0,5 mg/mL; 50 μL) na base da cauda e, após 21 dias, os animais receberam injeção de CFA na articulação tíbio-femural (TF) ou tíbio-társica (TT). A incapacitação articular foi avaliada pelo tempo de elevação da pata (TEP, s) durante marcha forçada de 1 minuto sobre um cilindro em rotação (3 r.p.m.). A variação do edema inflamatório da articulação TF e TT foi avaliada pelo aumento do diâmetro articular (DA, cm) e pelo aumento do volume de pata (EA, mL), respectivamente. Ambos, incapacitação e edema foram avaliados durante 10 dias consecutivos. Dois protocolos de exercício foram avaliados: (a) exercício diário de 1 min, onde os animais realizaram 1 minuto de exercício no cilindro, sendo este o próprio teste de incapacitação; (b) exercício progressivo, onde os animais realizaram o exercício com aumento de 1 minuto por dia (realizado sempre após o teste de incapacitação); (c) Grupo controle (TF e TT), sem exercício, cuja incapacitação foi mensurada apenas no 1°, 5° e 10° dias após a injeção intraarticular de CFA. Finalizado os 10 dias de avaliação, os animais foram eutanasiados para a realização da contagem total (CT; células/mm3) e diferencial (mononucleares - MON e polimorfonucleares - PMN; células/mm3) de leucócitos do tecido inflamado. O exercício progressivo inibiu a incapacitação e o edema em ambas as articulações. Entretanto, houve redução da migração total de leucócitos apenas na articulação TF. O exercício de 1 min inibiu o edema para as duas articulações e reduziu a migração total de leucócitos da articulação TT. Porém, o efeito do exercício de 1 minuto na incapacitação não foi significativo. A administração de aminoglutetimida (50 mg/kg; oral) 1 hora antes do exercício mostrou não ter efeito sobre a redução da nocicepção e edema ocasionados pelo exercício. O exercício progressivo parece ser mais efetivo em reduzir os parâmetros inflamatórios em comparação ao exercício de 1 min. Este estudo demonstra que a atividade física regular pode contribuir consistentemente para melhorar a funcionalidade articular e também apresentar um efeito anti-inflamatório.

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Rheumatoid arthritis (RA) is a chronic, multisystem inflammatory disease with autoimmune features evident, expressing a cellular response of Th1. Characterized primarily by chronic synovitis, erosive and symmetrical, preferably peripheral joints, where there is an intense inflammatory process. The purinergic signaling system plays an important role in the modulation of inflammatory and immune responses through extracellular biomolecules such as nucleotides adenine and adenosine derivative nucleodise therefore, which are essential to providing the trigger and maintain the trigger inflammatory response. The effects of these molecules are promoted by the action of purinergic receptors specific and controlled by ectoenzymes, in cell surface. Based on these principles, this study investigated the effect of cinnamaldehyde in thermal hyperalgesia, , arthritis score, paw edema and thermal hyperalgesia as well histolgical parameters beyond the activity of the E-NTPDase and E-ADA in lymphocytes in rats adjuvant arthritis. The rats were divided in four groups, of wich two were adjuvant induced arthritis in the other two control groups. The animal received the compound at a concentration of 2,1% orally for a period of 15 days. Not differences were observed among analysis the arthritis score, paw edema it is however noted differences in thermal hyperalgesia in about 60% and in group induced arthritic and treated with cinnamaldehyde. Compared histological analysis it was noticed a slight reduction the lynphocitytic inflammatory infiltration in rats induced arthritis and treated with cinnamaldehyde. We found the increased the ATP hydrolysis in about 94,14% in arthritis induced when compared with control group and 20,58% when compared with groups treated with cinnmaldehyde . However, E-NTPDase activity when used ADP as substrate rised in 152,56% in relation the control group and 122,76% in relation the control group treated with cinnamaldehyde. In E-ADA activity was observed the increased in about 151,84%%in group arthritis induced when compared with control groups and rise 69,7% when compared with group arthritis induced treated with cinnamakdehyde. In conclusion, the data indicate that cinnamaldehyde was able to reduce thermal hyperalgesia and alterations histological in rats induced arthritis, as well as decreased the ectonucleotidase cascade, once we observed a activity gradual decrease in lymphocytes in group induced arthritis and treated with cynnamaldehyde in relation with all others groups. Therefore cinnamaldehyde was act to skewing some effects inflammatory of induced arthritis. Although requiring further study, cinnamaldehyde could be used as a complementary fot the benefit of people with rheumatoid arthritis.
Artrite reumatoide (AR) é uma doença inflamatória crônica e multisistêmica, com características evidentes de autoimunidade, que expressa uma resposta celular do tipo Th1. Caracteriza-se basicamente por sinovite crônica, simétrica e erosiva, preferencialmente de articulações periféricas, onde existe um intenso processo inflamatório. O sistema de sinalização purinérgica desenvolve um papel importante na modulação das respostas inflamatórias e imunes, através de biomoléculas extracelulares, como os nucleotídeos de adenina e seu derivado nuclesídeo adenosina, os quais são indispensáveis para a iniciação e manutenção de respostas inflamatórias. Os efeitos de tais moléculas são promovidos pela ação dos receptores purinérgicos específicos e controlados por ectoenzimas, na superfície das células. Com base nestes princípios esta pesquisa avaliou os efeitos do cinamaldeído no escore de artrite, edema de pata e hiperalgesia termal bem como em análises histológicas além da atividade de E-NTPDae e E-ADA em linfócitos de ratos com artritre induzida por adjuvante. Os ratos foram divididos em quatro grupos, dois grupos com artrite induzida por adjuvante e dois grupos controles. Estes animais receberam o composto na concentração de 2,1%, via oral por um período de 15 dias. Não se observou diferenças entre análises de escore de artrite e edema de pata, porém notou-se-se alterações em hiperalgesia termal em cerca de 60% no grupo com artrite induzida tratado com cinamaldeído. Em relação às análises histológicas foi notada uma leve redução do infiltrado inflamatório linfocitico em ratos com artrite induzida e tratados com cinamaldeído. Mostrou-se um aumento da hidrólise do ATP em 94,14% no grupo com artrite induzida quando comparado com o grupo controle e em 20,58% quando comparado com o grupo tratado com cinamaldeído. Contudo, a atividade da E-NTPDase quando utilizado ADP como substrato obteve um aumento 152,56% no grupo artrite induzida quando comparado com o grupo controle e 122,76% quando comparado com grupo tratado com cinamaldeído. Na atividade da EADA foi observado um aumento em cerca de 151,84% no grupo artrite induzida em relação ao grupo controle e em 69,7% quando comparado com a grupo artrite induzida e tratado com cinamaldeído. Em conclusão, os dados indicam que cinamadeído foi capaz de reduzir hiperalgesia termal e alterações histológicas, como também diminui a cascata das ectonucleotidases, uma vez que observamos uma queda gradativa das atividades nos linfócitos no grupo com artrite induzida por adjuvante tratados com cinamaldeído quando comparado com os demais grupos. Desta forma cinamaldeído foi capaz de exercer seus efeitos desfavorecendo alguns eventos inflamatórios característico da artrite induzida por adjuvante. Embora careça de maiores estudos, o cinamaldeído poderia ser utilizado como alvo terapêutico complementar para a artrite reumatoide.

Orientador: Marcos Renato de Assis
Resumo: A expectativa de vida diminui com artrite reumatoide (AR) devido ao aumento da mortalidade por doenças cardiovasculares. Isto se deve, possivelmente, à disfunção endotelial resultante da intensa atividade inflamatória relacionada à AR. Evidências tem apontado para um possível envolvimento do sistema renina-angiotensina (SRA) nos danos cardiovasculares inerentes à AR. Acredita-se que a participação do SRA agrave o comprometimento endotelial decorrente de inflamações sistêmicas através da ativação do receptor de angiotensina II tipo 1 (AT1) pela angiotensina II (Ang II). Por outro lado, a literatura também reporta a influência dos hormônios andrógenos, principalmente da testosterona, nas ações da Ang II sobre os tecidos vasculares. Assim, o objetivo do presente estudo é investigar os efeitos da artrite induzida por adjuvante (AIA) sobre o equilíbrio redox, a função endotelial e as respostas da aorta de ratos à Ang II, bem como, verificar se esses efeitos podem ser influenciados pela redução dos níveis circulantes de testosterona. Para isto, ratos Wistar machos adultos foram submetidos à falsa-castração e falsa-imunização (Controles), castração seguida de falsa-imunização (ORX), falsa-castração seguida de imunização (ORX) e castração seguida de imunização (ORX+AIA). Ao final do experimento, segmentos de aorta torácica foram desafiadas em cubas de órgãos isolados com acetilcolina (ACh), Ang II, KCl e nitroprussiato de sódio e, das curvas concentração resposta obtidas, calculou-se... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Life expectancy of patients with rheumatoid arthritis (RA) is lower due to increased mortality in consequence of cardiovascular diseases. Presumably, this occurs due to endothelial dysfunction resulting from severe inflammatory activity related to RA. Evidence has demonstrated a possible involvement of the renin-angiotensin system (RAS) in the cardiovascular injury inherent to RA. The RAS involvement is considered to worsen the endothelial impairment due to systemic inflammation through angiotensin II type 1 receptor (AT1) activation of Ang II. (Ang II). Additionally, previous studies also observed that androgen hormones, mainly the testosterone, modulate the Ang II actions in cardiovascular tissues. Thus, the aim of the present study is to investigate the effects of adjuvant-induced arthritis (AIA) on systemic redox balance, endothelial function and rat aorta responses to Ang II, as well as, whether these effects may be influenced by the reduction of circulating levels of testosterone. For this, adult male Wistar rats were submitted to false-castration and false-immunization (Controls), castration followed by false-immunization (ORX), false-castration followed by immunization (ORX) and castration followed by immunization (ORX + AIA) . At the end of the experiment, thoracic aorta segments were challenged in isolated organ bath with acetylcholine (ACh), Ang II, KCl and sodium nitroprusside and, from the concentrantion-response curves obtained were calculated pEC50 and maximal ... (Complete abstract click electronic access below)
Doutor

Introdução: A vacinação contra a influenza pandêmica A/H1N1 resultou em soroproteção em mais de 85% dos indivíduos saudáveis. Entretanto, dados em pacientes com artrite reumatoide (AR) são escassos. Objetivos: O objetivo deste estudo é avaliar a imunogenicidade e a segurança em curto prazo da vacina contra influenza pandêmica A/H1N1 em pacientes com AR e a influência da atividade da doença e da medicação nesta resposta. Métodos: Trezentos e quarenta pacientes adultos com AR em seguimento e tratamento regular e 234 controles saudáveis foram examinados antes e 21 dias após receber uma dose da vacina sem adjuvante contra influenza A/California/7/2009. A atividade da doença (DAS28), o tratamento em uso e os títulos de anticorpos também foram avaliados. As taxas de soroproteção (títulos de anticorpos >= 1:40) e soroconversão (percentagem de pacientes com aumento de título de anticorpos maior ou igual a 4, se o título pré- vacinal fosse maior ou igual a 1:10; ou título pós-vacinal de pelo menos 1:40, se o título pré-vacinal era menor que 1:10), as médias geométricas dos títulos (MGT) e o fator de incremento das médias geométricas (FI-MGT) foram calculados. Os eventos adversos foram também registrados. Resultados: Os pacientes com AR e os controles tinham taxas pré-vacinais de soroproteção (10,8% vs. 11,5%) e MGT (8,0 vs. 9,3) comparáveis (p>0,05). Após a vacinação, foi observada redução significativa na resposta dos pacientes com AR versus controles (p<0,001) em todos os desfechos sorológicos: taxas de soroproteção (60,0 vs. 82,9%) e soroconversão (53,2% vs. 76,9%), MGT (57,5 vs. 122,9) e FI-MGT (7,2 vs. 13,2). A atividade de doença não prejudicou a soroproteção ou a soroconversão e se manteve estável em 97,4% dos pacientes. O metotrexato e o abatacepte foram associados à redução da resposta vacinal. A vacinação foi bem tolerada, com poucos efeitos adversos. Conclusão: Os dados confirmaram tanto a segurança em curto prazo como, diferente da maioria dos trabalhos com influenza sazonal, a redução da soroproteção em pacientes com AR, não relacionada à atividade de doença e à maioria das medicações em uso (com exceção do metotrexato e do abatacepte). A extrapolação da resposta imunológica de uma vacina para outra pode não ser possível e estratégias específicas de imunização (possivelmente em duas doses) podem ser necessárias
Background: Pandemic influenza A/H1N1 vaccination yielded seroprotection in more than 85% of healthy individuals. However, similar data are scarce in rheumatoid arthritis (RA) patients. Objectives: The objective of this study is to evaluate the immunogenicity and the short-term safety of anti- pandemic influenza A/H1N1 vaccine in RA patients, and the influence of disease activity and medication to the response. Methods: Three hundred and forty adult RA patients in regular follow-up and treatment, and 234 healthy controls were assessed before and 21 days after adjuvant-free influenza A/California/7/2009 vaccine. Disease activity (DAS28), current treatment and anti-pandemic influenza A/H1N1 antibody titres were also evaluated. Seroprotection (antibody titre >=1:40) and seroconversion (the percentage of patients with a fourfold or greater increase in antibody titre, if prevaccination titre was 1:10 or greater, or a postvaccination titre of 1:40 or greater, if prevaccination titre was less than 1:10) rates, geometric mean titres (GMT) and factor increase in geometric mean titre (FI-GMT) were calculated and adverse events registered. Results: RA patients and controls showed similar (p>0.05) prevaccination seroprotection (10.8% vs. 11.5%) and GMT (8.0 vs. 9.3). After vaccination a significant reduction (p<0.001) was observed in all endpoints in RA patients versus controls: seroprotection (60.0 vs. 82.9%; p<0.0001) and seroconversion (53.2% vs. 76.9%) rates, GMT (57.5 vs. 122.9) and FI-GMT (7.2 vs. 13.2). Disease activity did not preclude seroprotection or seroconversion and remained unchanged in 97.4% of patients. Methotrexate and abatacept were associated with reduced responses. Vaccination was well tolerated with minimal adverse events. Conclusions: The data confirmed both short-term anti-pandemic A/H1N1 vaccine safety and, different from most studies with seasonal influenza, reduced seroprotection in RA patients, unrelated to disease activity and to most medications (except methotrexate and abatacept). Extrapolation of xii immune responses from one vaccine to another may therefore not be possible and specific immunization strategies (possibly booster) may be needed

Mycolic acids, the characteristic, abundant waxes of the cell wall of Mycobacteria were purified by Counter Current Distribution (CCD) from alkaline methanolytic crude extracts of bacteria, aiming at investigating their role in eliciting immune responses. Crude mycobacterial cell-wall extracts were first made by saponification in potassium hydroxide methanol solution. Purification was then performed with CCD using a bi-phasic tricomponent system, consisting of double distilled deionized water (dddH2O), chloroform and methanol. Emulsions were formed in this system which in turn extended the purification time. The addition of a preliminary funnel extraction step, to reduce the saponified fatty acids in the crude extract, before CCD and the addition of NaCI as an emulsions breaker in the CCD solvent system, produced a high yield of pure mycolic acids. The purity of these mycolic acids were assessed using reversed-phase HPLC-analysis. This method proved not only to be applicable to purify mycolic acids from M. tuberculosis but was also applicable in purifying mycolic acids from other sources, such as M. vaccae. The immunogenic properties of the purified mycolic acids were confirmed in experiments in which they induced the formation of antibodies in Sprague-Dawley rats when immunized in Marcol 52 oil. The antibody response was monitored by ELISA after 3 months of repeated immunization every second week. A dose-related response was observed for the induction of antibodies specific for mycolic acids, immobilized on the ELISA plates. Mycolic acids also appeared to influence adjuvant arthritis. Pure mycolic acids, suspended in mineral oil were administered intradermally into Lewis rats one week before the intradermal administration of an arthritis-inducing dose of lyophilized M. tuberculosis H37Ra. Animals receiving Mycobacteria, but no mycolic acids treatment, developed severe symptoms of arthritis within two weeks after bacterial challenge. No arthritis symptoms were apparent in mycolic acids treated rats. Mycolic acids treatment alone, did not produce arthritis. Mycolic acids pre-treatment of M. tuberculosis H37Rv-infected mice, rendered tuberculosis susceptible Balb/c mice more resistant. This resistance was equivalent to that observed in tuberculosis resistant C57Bl/6 mice. Post-infection treatment of M. tuberculosis H37Rv-infected mice with MA had no effect. Resistance of C57Bl/6 mice is commonly associated with the expression of IL-12 and IFN-ã. The effect of mycolic acids in the spleens of M. tuberculosis-infected Balb/c mice was investigated. It was observed that there was no significant change on the THI and TH2 cytokines. The absence of mycolic acids-induced THI/TH2 cytokine bias implied that protection was not provided by the expression of IL-12 and IFN-ã in the spleen. These results support the hypothesis that mycolic acids are immunogenic in respect of being able to induce specific antibodies, to provide resistance against tuberculosis and to prevent the development of adjuvant arthritis. The mechanism by which mycolic acids perform these tasks is unknown, particularly in these rodent models, which differ from humans, in that they do not have the CD1b that presents mycolic acids in humans. Unravelling this mechanism, can possibly aid the development of a pharmaceutical formulation that introduces MA into the body to enhance resistance to TB and prevent arthritis as an associated side-reaction.
Dissertation (MSc)--University of Pretoria, 2010.
Biochemistry
unrestricted

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune inflammatory disease that mainly affects the joints, and is characterized by active inflammation as well as bone and cartilage destruction. Since structural joint damage is irreversible, early recognition and treatment are currently being emphasized, with the goal of inducing remission of the disease. Current RA therapies fail or produce only partial responses in most patients and have adverse toxicological effects, so there is still an unmet need for a drug that can offer an effective and safe treatment of RA. Celastrol, is a compound extracted from an herb used in Chinese medicine, which was previously identified by our work group as a potential candidate for the development of a new therapeutical drug for inflammatory diseases, such as RA. Therefore, the main goal of this project was to evaluate the efficacy and toxicity of the oral administration of a range of Celastrol dosages, using an adjuvant-induced arthritis (AIA) rat model. In order to achieve this, we treated AIA rats with dosages of Celastrol of 1 μg/g, 2.5 μg/g, 12.5 μg/g and 25 μg/g, from day 8 post disease induction until day 22, when rats where sacrificed. Blood and paw samples were collected for quantification of bone turnover and degradation serum markers, histological and immunohistochemical evaluation, as well as for quantification of toxicological blood parameters. Our work showed that an orally administered dosage of 2.5 μg/g of celastrol in the rat AIA model effectively reduces inflammation, infiltration and proliferation of synovial cells, suppresses bone erosion, reduces the number of osteoclasts and osteoblasts and reduces the number of synovial CD68+ cells, thus suggesting this treatment as effective. Moreover, we also showed that this treatment has no adverse toxicological effects at dosages of 1 μg/g and 2.5 μg/g, and that dosages of 25 μg/g and 12.5 μg/g can be considered lethal dose (LD) and LD50, respectively.

A major difficulty in understanding the etiology and pathogenic mechanisms of rheumatoid arthritis has been the lack of a suitable animal model. Based on the MRL-lpr spontaneous arthritis model, a new murine arthritis model was developed, that is reliable and practical for therapeutic evaluations. The MRL-lpr mouse strain develops an early autoimmune disease. After the characterization of our colony, it was concluded that while the strain did not exhibit differences in the lupus-like syndromes, the spontaneous arthritis became less severe than originally described. To enhance the spontaneous arthritis of MRL-lpr mice, the effect of complete Freund’s adjuvant (CFA) was investigated. In contrast to the low percentage observed in control animals, 67-82% of mice showed clinical evidence of arthritis. Similarly, the histopathological and immunohistological analyses of the CFA injected mice indicated a significantly higher frequency of inflammation, cartilage erosion and pannus formation, with marked infiltration of activated inflammatory cells, including lymphocytes. The requirement of the lpr gene and background MRL genes was then examined. It was found that while both 7 month old MRL-+ and 3 month old MRL-lpr mice developed arthritis, B6, B6-lpr, and 3 month old MRL-+ did not display the condition after CFA-treatment. These observations suggest that while the lpr gene causes a more severe early effect, background genes other than the lpr are also involved in the disease. The effect of pregnancy, as another possible enhancing factor, was also investigated. Sixty-eight percent of female MRL-lpr mice developed a post partum exacerbation of their mild spontaneous arthritis. Post-partum or post adjuvant-injection administration of estradiol prevented the enhancement of arthritis. The effectiveness of a recently developed photodynamic therapy (PDT) was also assessed and compared with conventional experimental therapies in the treatment of adjuvant arthritis. PDT inhibited the development of adjuvant enhanced arthritis with similar effectiveness as the conventional treatments, but without their negative side effects. These results illustrate that CFA-enhancement results in a reproducible model of murine arthritis, which is useful in evaluating experimental therapeutic regimes, and demonstrating effectiveness of PDT arthritis therapy.

Wong Hei Lui.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 192-226).
Abstracts in English and Chinese.
Publications Based On The Work In This Thesis --- p.i
Abstract --- p.ii
Acknowledgements --- p.vii
Abbreviations --- p.viii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Normal joint --- p.1
Chapter 1.11 --- Biology of joint --- p.1
Chapter 1.12 --- Structure of synovial joint --- p.1
Chapter 1.13 --- Components of the mature synovial joint --- p.3
Chapter 1.131 --- Articular cartilage --- p.3
Chapter 1.1311 --- Water --- p.4
Chapter 1.1312 --- Cartilage matrix --- p.4
Chapter 1.1313 --- Chondrocyte --- p.5
Chapter 1.132 --- Synovium --- p.5
Chapter 1.1321 --- Synovium vasculature --- p.6
Chapter 1.1322 --- Synovial blood flow --- p.7
Chapter 1.133 --- Synovial fluid --- p.8
Chapter 1.134 --- Bone --- p.9
Chapter 1.2 --- Pathological processes of arthritis --- p.11
Chapter 1.21 --- Activation of immune cells in arthritis --- p.11
Chapter 1.22 --- Synovial proliferation --- p.13
Chapter 1.221 --- Synovial lining cell activation --- p.13
Chapter 1.222 --- Pannus invasion --- p.14
Chapter 1.23 --- Cartilage and bone degradation --- p.14
Chapter 1.231 --- Depletion of proteoglycan (GAG) --- p.15
Chapter 1.232 --- Collagen denature --- p.15
Chapter 1.3 --- Tachykinins (TKs) --- p.17
Chapter 1.31 --- History --- p.17
Chapter 1.32 --- "Synthesis, storage and release of TKs" --- p.17
Chapter 1.33 --- Tachykinin receptors --- p.18
Chapter 1.331 --- Characterization of NK1 receptor --- p.19
Chapter 1.332 --- Characterization of NK2 receptor --- p.19
Chapter 1.333 --- Characterization of NK3 receptor --- p.20
Chapter 1.34 --- Effector systems of TKs --- p.21
Chapter 1.35 --- Termination of TK signals --- p.21
Chapter 1.351 --- Enzymatic breakdown --- p.21
Chapter 1.352 --- Receptor desensitization --- p.22
Chapter 1.353 --- Receptor endocytosis --- p.22
Chapter 1.36 --- TK receptor antagonists --- p.23
Chapter 1.361 --- Selective NK1 receptor antagonists --- p.23
Chapter 1.362 --- Selective NK2 receptor antagonists --- p.24
Chapter 1.363 --- Selective NK3 receptor antagonists --- p.25
Chapter 1.4 --- Roles of tachykinins in arthritis --- p.28
Chapter 1.41 --- Correlation between tachykinins and joint inflammation --- p.28
Chapter 1.42 --- Roles of tachykinins in immune cell activation --- p.30
Chapter 1.43 --- Roles of tachykinins in synovial proliferation --- p.31
Chapter 1.44 --- Roles of tachykinins in cartilage degradation --- p.32
Chapter 1.5 --- Animal model of arthritis --- p.33
Chapter 1.51 --- Instability model --- p.33
Chapter 1.52 --- Immobilization model --- p.34
Chapter 1.53 --- Noxious agent-induced model --- p.34
Chapter 1.531 --- Collagen-induced erosive arthritis --- p.34
Chapter 1.532 --- Cartilage oligometric matrix protein-induced arthritis --- p.35
Chapter 1.533 --- Oil-induced arthritis --- p.35
Chapter 1.534 --- Streptococcal cell wall-induced arthritis --- p.35
Chapter 1.535 --- Adjuvant-induced arthritis --- p.36
Chapter 1.536 --- Pristane-induced arthritis --- p.36
Chapter 1.6 --- Current anti-arthritic therapies --- p.39
Chapter 1.61 --- Non steroid anti-inflammatory drugs --- p.39
Chapter 1.62 --- Glucocorticoid --- p.44
Chapter 1.63 --- Second-line treatment --- p.46
Chapter 1.631 --- Sulfasalazine --- p.46
Chapter 1.632 --- Gold salts --- p.47
Chapter 1 633 --- D-penicillamine --- p.48
Chapter 1.634 --- Antimalarial --- p.49
Chapter 1 .635 --- Methotrexate --- p.51
Chapter 1.64 --- New trends for treatment of arthritis --- p.53
Chapter 1.641 --- Anti-cytokine therapy --- p.53
Chapter 1.642 --- Anti-angiogenesis therapy --- p.54
Chapter 1.7 --- Aims of study --- p.57
Chapter Chapter 2 --- Material and drugs --- p.62
Chapter Chapter 3 --- Methodology --- p.62
Chapter 3.1 --- Animals used and anaesthetization --- p.62
Chapter 3.2 --- Measurement of plasma protein extravasation --- p.63
Chapter 3.3 --- Measurement of knee joint sizes --- p.64
Chapter 3.4 --- Measurement of knee joint blood flow --- p.65
Chapter 3.5 --- Measurement of histological changes --- p.65
Chapter 3.51 --- Dissection and fixation --- p.65
Chapter 3.52 --- Decalcification --- p.66
Chapter 3.53 --- Processing --- p.66
Chapter 3.54 --- Embedding --- p.67
Chapter 3.55 --- Sectioning --- p.67
Chapter 3.56 --- Staining --- p.69
Chapter 3.6 --- Data analysis --- p.69
Chapter 3.61 --- Scoring systems --- p.72
Chapter Chapter 4 --- A model of monoarthritis in rats --- p.72
Chapter 4.1 --- Introduction --- p.72
Chapter 4.2 --- Method --- p.73
Chapter 4.3 --- Results --- p.73
Chapter 4.31 --- Lewis rats --- p.73
Chapter 4.32 --- Sprague-Dawley (SD) rats --- p.74
Chapter 4.33 --- Comparison of FCA-induced changes in Lewis and SD rats --- p.74
Chapter 4.34 --- Histological studies on arthritic SD rats --- p.75
Chapter 4.4 --- Discussion --- p.93
Chapter 4.5 --- Conclusions --- p.95
Chapter Chapter 5 --- Effect of Substance P on adjuvant-induced arthritis --- p.96
Chapter 5.1 --- Introduction --- p.96
Chapter 5.2 --- Method --- p.98
Chapter 5.3 --- Results --- p.99
Chapter 5.31 --- Evans blue extravasation --- p.99
Chapter 5.32 --- Joint size --- p.100
Chapter 5.33 --- Knee joint blood flow --- p.101
Chapter 5.34 --- Histology results --- p.102
Chapter 5.341 --- Infiltration of immune cells in synovial tissue --- p.102
Chapter 5.342 --- Synovial tissue proliferation --- p.102
Chapter 5.343 --- Cartilage degradation --- p.103
Chapter 5.344 --- Bone degradation --- p.103
Chapter 5.4 --- Discussion --- p.120
Chapter 5.5 --- Conclusions --- p.125
Chapter Chapter 6 --- Effects of tachykinin receptor antagonists on FCA-induced arthritis
Chapter 6.1 --- Introduction --- p.126
Chapter 6.2 --- Method --- p.128
Chapter 6. 21 --- Intravenous NK1 receptor antagonists on FCA-induced arthritis --- p.128
Chapter 6. 22 --- Intraperitoneal TK receptor antagonists on FCA-induced arthritis --- p.128
Chapter 6.3 --- Results --- p.129
Chapter 6.31 --- Intravenous NK1 227}0اreceptor antagonists on FCA-induced arthritis Evans blue extravasation and joint swelling --- p.129
Chapter 6.32 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced arthritis Evans blue extravasation and joint swelling --- p.129
Chapter 6.33 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced immune cell accumulation --- p.130
Chapter 6.34 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced synovial tissue proliferation --- p.131
Chapter 6.35 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced cartilage degration and bone erosion --- p.131
Chapter 6.4 --- Discussion --- p.159
Chapter 6.5 --- Conclusions --- p.162
Chapter Chapter 7 --- Individual and combined effects of dexamethasone and TK receptor antagonists on FCA-induced arthritis --- p.163
Chapter 7.1 --- Introduction --- p.163
Chapter 7.2 --- Method --- p.166
Chapter 7.3 --- Results --- p.167
Chapter 7.31 --- Evans blue extravasation --- p.167
Chapter 7.32 --- Knee joint size --- p.167
Chapter 7.33 --- Body weight --- p.168
Chapter 7.34 --- Cellular infiltration --- p.168
Chapter 7.35 --- Synovial tissue proliferation --- p.168
Chapter 7.36 --- Cartilage degradation --- p.169
Chapter 7.4 --- Discussion --- p.184
Chapter 7.5 --- Conclusions --- p.187
Chapter Chapter 8 --- General discussions and conclusions --- p.188
References --- p.192

碩士
國立中興大學
獸醫學系暨研究所
101
Acupuncture which originated in China has been used as a therapeutic intervention for the treatment of various diseases and symptoms for more than two thousand years. Acupuncture uses filiform needles to stimulate specific acupoints to achieve therapeutic effects, and is most widely studied in pain relief. Arthritis is one of the most common chronic diseases in the world, which involves the damage of the joints and surrounding tissues. The major complaints by individuals who have arthritis are joint pain and mobility-related functional limitations. The aims of this study were to evaluate the effects of acupuncture on experimental rheumatoid-like arthritis in rats and to compare the efficacy of dry needle, aquapuncture and electroacupuncture. In this study, the animal model was established by intra-articular injection of complete Freund''s adjuvant (CFA). After four times of CFA induction, acupuncture was applied to bilateral Yang Ling Quan (GB34) for 15 minutes, twice a week. Arthritis assessments include circumference and width of the knee, radiological features of arthritis and histological characteristics of knee joint. All of the acupuncture groups had significantly reduced the swelling around the joints and deterioration of arthritis without influence on body weight. Based on the present results, electroacupuncture was more effective than aquapuncture and dry needle in alleviating CFA-induced joint swelling and bone destruction. Aquapuncture was easy and convenient to manipulate and it may have an important role in veterinary clinical practice.

Ko, Wai Man.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 243-257).
Abstracts in English and Chinese.
Publications Based On The Work In This Thesis --- p.i
Abstract --- p.ii
Acknowledgements --- p.ix
Abbreviations --- p.x
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Prevalence of arthritis --- p.1
Chapter 1.2 --- Pathogenesis of arthritis --- p.4
Chapter 1.2.1 --- Histological changes --- p.6
Chapter 1.2.1.1 --- Synovium changes --- p.6
Chapter 1.2.1.2 --- Articular cartilage degradation --- p.8
Chapter 1.2.1.3 --- Bone erosions --- p.10
Chapter 1.3 --- Western medicines for arthritis --- p.14
Chapter 1.3.1 --- Nonsteroidal anti-inflammatory drugs (NSAIDs) --- p.15
Chapter 1.3.2 --- Glucocorticoids (GCs) --- p.18
Chapter 1.3.3 --- Disease modifying antirheumatic drugs (DMARDs) --- p.20
Chapter 1.3.4 --- Biological therapies --- p.22
Chapter 1.4 --- Traditional Chinese medicines for arthritis --- p.24
Chapter 1.4.1 --- Ganoderma lucidum (靈芝））) --- p.26
Chapter 1.4.1.1 --- Major chemical constituents --- p.27
Chapter 1.4.1.2 --- Functions --- p.27
Chapter 1.4.2 --- Cortex Phellodendri (黃柏） --- p.28
Chapter 1.4.2.1 --- Major chemical constituents --- p.29
Chapter 1.4.2.2 --- Traditional description --- p.29
Chapter 1.4.2.3 --- Functions --- p.30
Chapter 1.4.3 --- Atractylodisa Rhizoma (蒼术) --- p.31
Chapter 1.4.3.1 --- Major chemical constituents --- p.31
Chapter 1.4.3.2 --- Traditional description --- p.32
Chapter 1.4.3.3 --- Functions --- p.32
Chapter 1.4.4 --- Radix Achyranthis Bidentatae (牛膝) --- p.33
Chapter 1.4.4.1 --- Major chemical constituents --- p.34
Chapter 1.4.4.2 --- Traditional description --- p.34
Chapter 1.4.4.3 --- Functions --- p.34
Chapter 1.5 --- Animal models of arthritis --- p.36
Chapter 1.5.1 --- Adjuvant-induced arthritis --- p.37
Chapter 1.6 --- Aims of study --- p.42
Chapter Chapter 2 --- Materials and Drugs --- p.44
Chapter Chapter 3 --- Methodology --- p.49
Chapter 3.1 --- Induction of anaesthesia --- p.49
Chapter 3.2 --- Induction of monoarthritis --- p.49
Chapter 3.3 --- Measurements of knee extension angles --- p.50
Chapter 3.4 --- Measurements of knee joint sizes --- p.51
Chapter 3.5 --- Assessment of changes in articular blood flow --- p.52
Chapter 3.6 --- Assessment of morphological changes --- p.53
Chapter 3.6.1 --- Fixation --- p.53
Chapter 3.6.2 --- Decalcification --- p.53
Chapter 3.6.3 --- Processing --- p.54
Chapter 3.6.4 --- Embedding --- p.54
Chapter 3.6.5 --- Sectioning --- p.55
Chapter 3.6.6 --- Staining --- p.55
Chapter 3.6.7 --- Scoring --- p.56
Chapter 3.7 --- Statistical analysis --- p.57
Chapter Chapter 4 --- Adjuvant-induced Monoarthritic Rats
Chapter 4.1 --- Adjuvant-induced monoarthritic rats (1 week) --- p.58
Chapter 4.1.1 --- Method --- p.58
Chapter 4.1.2 --- Results --- p.59
Chapter 4.1.2.1 --- Body weight --- p.59
Chapter 4.1.2.2 --- Knee joint sizes --- p.59
Chapter 4.1.2.3 --- Knee extension angles --- p.59
Chapter 4.1.2.4 --- Knee joint blood flow --- p.60
Chapter 4.1.2.5 --- Histological evaluation --- p.60
Chapter 4.1.2.5.1 --- Cell infiltration --- p.60
Chapter 4.1.2.5.2 --- Synovial tissue proliferation --- p.61
Chapter 4.1.2.5.3 --- Cartilage degradation --- p.61
Chapter 4.2 --- Adjuvant-induced monoarthritic rats (2 weeks) --- p.68
Chapter 4.2.1 --- Method --- p.68
Chapter 4.2.2 --- Results --- p.69
Chapter 4.2.2.1 --- Body weight --- p.69
Chapter 4.2.2.2 --- Knee joint sizes --- p.69
Chapter 4.2.2.3 --- Knee extension angles --- p.69
Chapter 4.2.2.4 --- Knee joint blood flow --- p.70
Chapter 4.2.2.5 --- Histological evaluation --- p.70
Chapter 4.2.2.5.1 --- Cell infiltration --- p.70
Chapter 4.2.2.5.2 --- Synovial tissue proliferation --- p.71
Chapter 4.2.2.5.3 --- Cartilage degradation --- p.71
Chapter 4.3 --- Discussions --- p.78
Chapter Chapter 5 --- Effects of intra-articular injection of LS in adjuvant-induced monoarthritic rats --- p.82
Chapter 5.1 --- Method --- p.82
Chapter 5.2 --- Results --- p.83
Chapter 5.2.1 --- Body weight --- p.83
Chapter 5.2.2 --- Knee joint sizes --- p.83
Chapter 5.2.3 --- Knee extension angles --- p.85
Chapter 5.2.4 --- Knee joint blood flow --- p.87
Chapter 5.3 --- Discussions --- p.98
Chapter Chapter 6 --- Effects of oral administration of LS in adjuvant-induced monoarthritic rats --- p.102
Chapter 6.1 --- Oral administration of LS for 6 days after induction of arthritis --- p.102
Chapter 6.1.1 --- Method --- p.102
Chapter 6.1.2 --- Results --- p.103
Chapter 6.1.2.1 --- Body weight --- p.103
Chapter 6.1.2.2 --- Knee joint sizes --- p.103
Chapter 6.1.2.3 --- Knee extension angles --- p.105
Chapter 6.1.2.4 --- Knee joint blood flow --- p.106
Chapter 6.1.2.5 --- Histological evaluation --- p.107
Chapter 6.1.2.5.1 --- Cell infiltration --- p.107
Chapter 6.1.2.5.2 --- Synovial tissue proliferation --- p.107
Chapter 6.1.2.5.3 --- Cartilage degradation --- p.108
Chapter 6.2 --- Oral administration of LS for 7 days before and 7 days after induction of arthritis --- p.131
Chapter 6.2.1 --- Method --- p.131
Chapter 6.2.2 --- Results --- p.132
Chapter 6.2.2.1 --- Body weight --- p.132
Chapter 6.2.2.2 --- Knee joint sizes --- p.132
Chapter 6.2.2.3 --- Knee extension angles --- p.134
Chapter 6.2.2.4 --- Knee joint blood flow --- p.137
Chapter 6.2.2.5 --- Histological evaluation --- p.137
Chapter 6.2.2.5.1 --- Cell infiltration --- p.137
Chapter 6.2.2.5.2 --- Synovial tissue proliferation --- p.138
Chapter 6.2.2.5.3 --- Cartilage degradation --- p.138
Chapter 6.3 --- Oral administration of LS for 13 days after induction of arthritis --- p.165
Chapter 6.3.1 --- Method --- p.165
Chapter 6.3.2 --- Results --- p.166
Chapter 6.3.2.1 --- Body weight --- p.166
Chapter 6.3.2.2 --- Knee joint sizes --- p.166
Chapter 6.3.2.3 --- Knee extension angles --- p.168
Chapter 6.3.2.4 --- Knee joint blood flow --- p.169
Chapter 6.3.2.5 --- Histological evaluation --- p.170
Chapter 6.3.2.5.1 --- Cell infiltration --- p.170
Chapter 6.3.2.5.2 --- Synovial tissue proliferation --- p.170
Chapter 6.3.2.5.3 --- Cartilage degradation --- p.171
Chapter 6.4 --- Discussions --- p.194
Chapter Chapter 7 --- Effects of intra-peritoneal administration of LS in adjuvant-induced monoarthritic rats --- p.203
Chapter 7.1 --- Method --- p.203
Chapter 7.2 --- Results --- p.204
Chapter 7.2.1 --- Body weight --- p.204
Chapter 7.2.2 --- Knee joint sizes --- p.205
Chapter 7.2.3 --- Knee extension angles --- p.207
Chapter 7.2.4 --- Knee joint blood flow --- p.209
Chapter 7.2.5 --- Histological evaulation --- p.209
Chapter 7.2.5.1 --- Cell infiltration --- p.209
Chapter 7.2.5.2 --- Synovial tissue proliferation --- p.210
Chapter 7.2.5.3 --- Cartilage degradation --- p.210
Chapter 7.3 --- Discussions --- p.237
Chapter Chapter 8 --- Conclusions --- p.239
References --- p.243

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
A Artrite Reumatóide (AR) é uma doença auto-imune, progressiva e crónica que se caracteriza por uma acentuada inflamação nas articulações com consequente reabsorção do osso e da cartilagem. Não existe cura e a atual estratégia terapêutica implica o diagnóstico e início de tratamento o mais cedo possível com vista à eventual remissão. Os sintomas iniciais incluem rigidez, inchaço e dor nas pequenas articulações que posteriormente alastram para as articulações maiores. Apesar da etiologia da AR não ser inteiramente conhecida, sabe-se que a perda de tolerância imunológica está associada à predisposição genética do individuo associada à exposição a fatores de risco como o tabagismo ou à possível ocorrência de um episódio estocástico que leve ao surgimento de auto anticorpos. A inflamação articular desenvolve-se devido à anormal infiltração de células do sistema imunitário na membrana sinovial levando à libertação de citocinas pro-inflamatórias, quimiocinas e moléculas de adesão celular que sustêm e perpetuam a inflamação. O microambiente rico em mediadores inflamatórios ativa os osteoclastos, células responsáveis pela reabsorção do osso, desequilibrando desta forma a homeostase óssea a favor da reabsorção. Apesar da recente evolução das estratégias de tratamento e do surgimento de novas terapêuticas, estas não são completamente eficazes e induzem efeitos adversos provocando a descontinuidade do tratamento em cerca de 30% dos doentes. Assim, continua a ser necessário o desenvolvimento de uma terapia eficaz, bem tolerada, e disponível para a generalidade da população. Com base nesta problemática o nosso grupo tem investigado um composto bioactivo da planta Trypterigium wilfordii Hook F., o celastrol. Este composto para além de revelar propriedades anti-inflamatórias, reduz e a reabsorção do osso e da cartilagem. Com o intuito de dar continuidade à investigação desenvolvida pelo nosso laboratório, o presente estudo tem como principal objetivo explorar a eficácia anti-inflamatória e de protecção óssea do composto quando administrado oralmente assim como investigar possíveis efeitos adversos. Para tal, foi utilizado o modelo de artrite induzida por adjuvante (AIA) em rato. Os animais artríticos foram divididos em grupos sendo que a um dos grupos foi administrado por lavagem (intragastricamente) 5μg/g e a outro 7.5 μg/g diariamente de celastrol. Foi ainda utilizado como controlos um grupo de animais artríticos e um de ratos saudáveis, da mesma espécie e idade, que receberam veículo ou água, respectivamente. O estudo durou 22 dias sendo que o dia 0 corresponde ao dia da indução da doença, e o dia 8, fase em que se inicia o período agudo da artrite, foi o primeiro dia de tratamento (modelo terapêutico). De forma a avaliar o etanol como veículo foi administrado etanol a 17% em PEG400 a um grupo de animais artríticos e comparado com outro grupo de animais artríticos sem administração de veículo. Todos os animais foram pesados ao longo do periodo experimental assim como medido o diâmetro da pata traseira esquerda e atribuído um score de acordo com a severidade da inflamação. O etanol não provocou diferenças significativas nos parâmetros acima indicados. A análise histopatológica feita às patas traseiras esquerdas destes animais revelou que o etanol não altera a progressão da doença característica destes animais e não provocou uma diminuição do peso. Desta forma, foi possível utilizar o etanol como solvente do celastrol. Ambas as doses de celastrol administradas diminuíram significativamente a inflamação visível em todas as patas assim como o inchaço da pata traseira esquerda. Este resultado foi comprovado pela análise histopatológica. Ambas as doses de celastrol diminuíram significativamente a infiltração e proliferação celular assim como as erosões ósseas. De modo a explorar a ação do celastrol na proliferação de células na sinovia e nas células de rearbsorção e formação óssea, foram marcados por imunohistoquímica o ki67 (marcador de células proliferativas na sinovia), osteocalcina (marcador de osteoblastos) e catepsina K (marcador de osteoclastos em reabsorção). Todos os biomarcadores foram significativamente reduzidos nos animais em que o composto foi administrado em comparação com os animais artríticos não tratados. Desta forma, não só foi possível validar os efeitos anti-proliferativos do celastrol como também concluir que o celastrol atua no osso diminuindo as células de remodelação óssea presentes na articulação. Foi ainda avaliada a presença de macrófagos CD68+ na sinovia. A redução destas células na sinóvia funciona como biomarcador da resposta de novas terapêuticas para o tratamento da AR, quer em humanos quer em modelos animais. A sua diminuição significativa nos animais tratados permitiu validar o celastrol como candidato ao tratamento da artrite. Os efeitos do celastrol no osso foram validados pela análise no soro de CTX-I e P1NP, marcadores de reabsorção e de formação óssea, respectivamente. Os níveis de P1NP foram significativamente reduzidos em ambas as doses em comparação com os animais doentes não tratados e o CTX-I, apesar de não ter resultados significativos, segue a mesma tendência. Mais ainda, a quantificação no soro do TRAcP 5b, marcador preditivo do número de osteoclastos totais, levou a concluir que o celastrol diminui o número destas células, tal como observado na análise histopatológica e por imunohistoquímica. Os animais foram pesados ao longo do período experimental de forma a averiguar uma possível redução do peso corporal provocada pela administração do composto. Os ratos administrados com 5 μg/g/dia de celastrol não apresentaram redução de peso porém, o grupo que recebeu a dose mais alta do composto (7.5 μg/g/dia) sofreu uma diminuição no peso significativa no dia 11 e 13 em comparação ao 4º e ao 7º da experiência. A redução de 20% do peso em dois animais neste grupo comparativamente ao dia 4 provocou a sua eutanásia antecipada. Conduto, é de salientar que não existiram diferenças significativas no peso dos animais deste grupo nos dias em que receberam o tratamento. A avaliação dos biomarcadores de toxicidade no soro dos animais mostrou que o celastrol não provoca toxicidade nos rins e no fígado. Contudo, o enzima lactato desidrogenase (marcador de dano celular), aumentou significativamente nos animais tratados com 7.5 μg/g/dia de celastrol em comparação com os outros grupos e o enzima creatina cinase (marcador de dano muscular/cardíaco) revelou uma tendência a aumentar nos grupos tratados com celastrol em comparação aos ratos saudáveis. A análise ao marcador pro-ANP descartou efeitos adversos do celastrol no miocárdio. Por fim, foi ainda estudada a percentagem de leucócitos no sangue no fim do estudo. Nenhum dos grupos tratados com celastrol teve efeitos tóxicos nos leucócitos. Os resultados sugerem que o celastrol administrado oralmente a 5 e 7.5 μg/g/dia é eficaz a diminuir a inflamação assim como a reduzir as erosões ósseas. No entanto, os efeitos tóxicos significativos observados no grupo tratado com celastrol a 7.5 μg/g/dia excluem possíveis usos do composto em ratos Wistar acima desta dose. A outra dose utilizada neste estudo (5 μg/g/dia) está a ser considerada para futuros estudos pré-clínicos mais detalhados.
Rheumatoid arthritis (RA) is an uncurable auto-immune disease characterized by inflammation and tissue destruction in several joints. An early and adequate diagnosis are important to prevent the main symptoms, which are painful and swollen joints, morning stiffness, edema and progressive disability. Despite the improvement in therapeutic options, around 30% of patients discontinue treatment due to a weak clinical response, adverse effects or incapability to afford such expensive therapies. Therefore, an effective and safe therapy is desirable. Celastrol, a compound from the Chinese plant Trypterigium wilfordii Hook F., has revealed a remarkable outcome in the treatment of several inflammatory diseases. Recently, our group has proven its efficacy in the treatment of inflammatory signs and bone damage, with no short-term toxicity, when the compound is intraperitoneally administered in an early and late phases of arthritis in the adjuvantinduced artritis (AIA) rat model. These data thus support the hypothesis that celastrol is a promising candidate for drug development for the treatment of RA. In the present study, our group focused on exploring the efficacy of orally administrated celastrol, in two different doses, and studying inherent toxicity in the same rat model of arthritis. The study intent to provide a rationale for setting dose levels for further preclinical studies. In order to evaluate ethanol as a suitable vehicle for the oral administration of celastrol in this setup, a group of AIA rats received ethanol 17% in PEG400 by gavage and was compared with arthtiric animals that did not receive ethanol. The absence of body weight loss in the animals that received ethanol and the lack of differences on disease course between the two groups allowed the use of ethanol as a solvent for celastrol. Hence, two groups of AIA rats were treated with celastrol orally at two different doses (5 or 7.5 μg/g/day) starting 8 days after disease induction. Both celastrol-treated groups were compared with a group of arthritic-untreated and healthy rats which received vehicle (17% in PEG400) and water, respectively. All the animals were euthanized 22 days after disease induction and samples were collected to futher analysis. Our results showed that orally administered celastrol halts inflammation and decreases bone erosions in both dosages. Furthermore, our results also showed a reduction on the number of osteoclasts in celastrol-treated groups. However, significant toxic effects were observed in celastrol at 7.5 μg/g/day treated group. The other dose used in this study (5 μg/g/day) will be considered in future pre-clinial assessments conducted by our group.

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatológicas), Universidade de Lisboa, Faculdade de Medicina, 2017
Rheumatoid arthritis (RA) is a chronic, systemic and immune-mediated inflammatory disease that mainly affects the synovial membrane of multiple small joints. As a consequence, RA results in cartilage and bone damage, leading to functional impairment and an increase in morbidity and mortality. Early diagnosis and adequate treatment are critical to prevent RA progression, as joint destruction can occur immediately after its onset. The most characteristic feature of RA is synovial hyperplasia, which is mediated by several immune cells, such as T-cells, B-cells, neutrophils, macrophages and by a complex cytokine network, especially interleukin (IL)-1β, tumor necrosis factor (TNF) and IL-6. RA inflammatory environment induces osteoclastogenesis, promoting disturbances in skeletal bone remodeling, which ultimately leads to the development of secondary osteoporosis and consequent bone fragility. An opportunity for a more effective treatment intervention was identified in early RA, when permanent damage can be prevented and a higher number of patients can achieve remission. Early treatment intervention might also interfere systemically with bone biology preventing bone micro and nano architectural damage. The development of therapeutic strategies able to control both inflammation and bone degradation, with a high rate of disease remission, low incidence of side effects and low production costs is still an unmet medical need in RA. Our hypothesis is that the impact of inflammation on bone micro and nano properties (intrinsic bone tissue properties, independent of the overall bone architecture and directly dependent on the way bone cells, collagen and calcium crystals interact) occurs almost immediately, upon first symptoms, and that these effects can be prevented by early intervention with drugs able to control inflammation and capable of interfering also with bone metabolism. This thesis characterizes the early events of bone damage in RA and explores the effect of novel treatment interventions in this context. Accordingly, in the first part of this thesis, we used an adjuvant induced arthritis (AIA) rat model and observed a synovial sublining layer infiltration, increased lining layer cells, bone erosions and cartilage surface damage present since the early stages of arthritis, as well as increased levels of IL-6. This inflammatory environment promotes osteoclastogenesis, which is related to the observed local bone erosion and may interfere systemically with bone skeletal remodeling. Indeed, AIA animals showed an increased bone turnover, as depicted by increased CTX-I (Carboxy-terminal telopeptide of type I collagen) and P1NP (amino terminal propeptides of type I collagen) levels since the early stages of arthritis. Bone histology was consistent with this early onset spur of bone remodeling. Arthritic animals showed concentric lamellas in secondary osteons (SO), which are the consequence of intense bone remodeling. On the contrary, healthy animals presented more parallel-lamellae (PL) structures than SO structures and these PL structures are 10% harder than SO structures, representing the mature bone structure (normal bone remodeling). Thus, arthritic bone tissue was composed of a larger number of younger, less mineralized and less hard structures, explaining the reduced hardness that we have observed by nanoindentation. Moreover, an increased area occupied by osteocyte lacunae was detected early on in the arthritis process. This apparent change of osteocyte morphology might be related to bone necrosis, leading to mineral loss, decreased hardness and possibly mechanical weakness. In addition, we have also demonstrated that arthritis induces mineral and collagen loss in trabecular bone since the early phase of arthritis development. At a higher organizational level data, micro computed tomography (micro-CT) revealed in arthritic animals a lower fraction of cortical and trabecular bone volume with reduced trabecular thickness together with a higher trabecular separation, in comparison with controls. Results also demonstrated cortical differences in polar moment of inertia, suggesting mechanical weakness in arthritic groups since the early phase of arthritis. Furthermore, cortical and trabecular porosity were increased in the arthritic groups compared to healthy controls. We also confirmed these observations by classic histomorphometry, which demonstrated a decreased structural integrity in arthritic animals. Coherent with these structural defects, our results also showed that in very early arthritis bone has low mechanical competence. Altogether, these results revealed that inflammation promotes bone nano and micro structural disturbances, leading to bone fragility since the early stages of arthritis. In addition, we also provided the basis for using the AIA animal model of arthritis as an adequate model for studying the impact of inflammation on bone and for assessing candidate compounds for the control of arthritis and its associated bone damage. The quest for new RA treatments, more effective at inflammation and bone damage control, safer and less expensive is still a major need. Previously, we had demonstrated that celastrol, acts by downregulating IL1β and TNF production, was a promising RA therapeutic candidate. Herein we have demonstrated that celastrol was able to reduce the number of synovial B and T-cells as well as fibroblasts and CD68 macrophages. Accordingly, we showed that celastrol protects cartilage and bone from inflammation-induced focal damage. At a systemic level, we observed a reduction in bone turnover together with preservation of bone structural and mechanical properties. Moreover, celastrol therapy showed superior effects if administrated in an early phase of arthritis development, which highlights the importance of an early treatment to limit inflammation-induced bone damage. Tofacitinib was also tested in order to assess the effects on micro and nano structural and mechanical properties of bone in an AIA rat model of arthritis. Tofacitinib is a selective inhibitor of janus kinase 1 (JAK1) and janus kinase 3 (JAK 3). Results showed significant reduced arthritis manifestations, synovial tissue inflammation and bone erosions, accompanied by a reduced bone turnover rate and a predominance of parallel structures on bone tissue. At tissue level, measurements performed by nanoindentation showed that tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and 3-point bending tests revealed that tofacitinib did not revert the effects of arthritis on cortical and trabecular bone structure and mechanical properties. This effect on bone might be related to the mechanism of action of tofacitinib which has complex and conflictual molecular interactions with bone. We suggest that these interactions have an overall negative effect not totally compensated by the benefits resulting from the control of inflammation. On the other hand, tofacitinib may require more exposure time to have an impact on bone quality. Overall, the results of the present thesis support the hypothesis that the impact of inflammation on bone micro and nano properties occurs almost immediately, upon the appearance of first symptoms. Moreover, these observed effects can be prevented by very early intervention with drugs able to control inflammation and capable of interfering with bone metabolism.
Pfizer – ASPIRE 2013 Prize; ECTS/AMGEN Bone Biology 2014 Prize

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2015
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, which mainly affects the joints. RA is characterized by pronounced joint swelling, with progressive destruction of articular cartilage and bone, leading to functional impairment and increased morbidity and mortality. It is very important to have an early and accurate diagnose and treatment in order to prevent severe articular lesions. The pathogenesis of RA involves several immune cells and complex cytokine networks which perpetuate the inflammatory process and promote bone resorption and damage. Despite the existence of several drugs available for the treatment of RA, disease remission is still an unmet medical need for the great majority of patients. X, a Chinese herbal compound, has significant anti-inflammatory properties in some auto-inflammatory diseases, namely colitis and multiple sclerosis as well as in cancer. Our lab has previously shown significant anti-inflammatory effects of X in an adjuvant-induced arthritis (AIA) rat model. In the present work we hypothesized that X is a promising candidate for the safe treatment of inflammation and bone damage in arthritis. Thus, our main objective was to evaluate the anti-inflammatory and bone protective effects of X in vivo using the AIA rat model of arthritis. We treated AIA rats with X intraperitoneally at a dose of 1μg/g daily, in two different groups of treatment: one group initiated X treatment in the early phase of arthritis (4 days after disease induction) and the other group initiated the treatment in a later phase of disease progression (11 days after disease induction). After 22 days of disease duration, animals were sacrificed and samples were collected to access X efficacy, toxicity, anti-inflammatory and bone protective properties, locally (joint articular tissues) and systemically (serum). Our results have shown that X treatment was able to safely diminish the inflammatory signs, even when treatment was initiated in a later phase of arthritis progression. X was able to decrease the systemic levels of IL-6 and bone turnover markers. Moreover, locally, X treatment decreased the infiltration and proliferation of immune cells as well as bone erosions in the joints of treated animals. Bone mechanical properties were recovered in X early-treated rats. In conclusion, this work gives an insight in the synovial homeostasis and bone protective properties of X treatment in the AIA rat model of arthritis. Future work will focus in X bone quality protection. Altogether, we expect that our results give support to a preclinical trial.
A artrite reumatóide (AR) é uma doença crónica inflamatória autoimune, que afeta principalmente a membrana sinovial das pequenas articulações e que pode levar à destruição da cartilagem e osso subjacentes, causando um elevado grau de incapacidade, morbilidade e redução da esperança média de vida nos doentes. Numa fase inicial, manifesta-se através de inchaço, rigidez e dor nas pequenas articulações que progressivamente se alastra para as articulações maiores. É de salientar a importância de um diagnóstico e tratamento precoce e adequado, de modo prevenir o aparecimento de lesões mais graves. No processo inflamatório característico da AR estão envolvidos tanto o sistema imunitário inato como adaptativo, que atuam num processo contínuo de produção de citocinas e de recrutamento de células inflamatórias para a membrana sinovial, que reveste as articulações, tornando-a hiperplásica. Células T auto-ativadas desencadeiam a ativação de monócitos, macrófagos e fibroblastos, que por sua vez produzem citocinas pró-inflamatórias que perpetuam o sinal inflamatório, mediadores de atividade óssea e enzimas com capacidade para reabsover cartilagem e osso. O processo de remodelação óssea envolve a destruição e formação de osso, de uma forma controlada e que envolve vários tipos celulares: osteoclastos, osteoblastos e osteócitos. Existem também vários mediadores celulares, nomeadamente RANKL e OPG, que induzem e inibem a formação ou destruição de osso. Alguns dos mediadores e produtos destes processos podem servir como marcadores de turnover ósseo e da cartilagem, permitindo perceber o balanço da remodelação óssea. Apesar de ainda não existir cura para a AR, são já aplicadas várias abordagens terapêuticas, que incluem numa fase inicial de tratamento os glucocorticoides, que por terem efeitos secundários adversos em doses elevadas, são substituídos, numa segunda fase por DMARD’s (agentes antirreumáticos modificadores de doença), que incluem o metotrexato, o fármaco mais usado no tratamento destes doentes. No entanto, uma grande percentagem de doentes é não-respondedora ou deixa de responder ao tratamento, tendo por isso surgido uma nova categoria de fármacos, os DMARD’s biológicos, que são geralmente anticorpos que atuam sobre, por exemplo, TNF, IL-1, células B e T. Para alguns doentes, nenhuma destas terapias é eficaz, sendo por isso necessário continuar a investir na descoberta de novo medicamentos com melhores resultados e poucos efeitos secundários. O X, um composto extraído da planta Y, muito usado na medicina chinesa, mostrou ter efeitos anti-inflamatórios em várias doenças autoimunes, como a colite crónica a esclerose múltipla, e também no cancro. O nosso laboratório reportou também que este composto tem propriedades anti-inflamatórias num modelo animal de artrite (AIA) em rato. No presente trabalho o objetivo principal foi testar o X no tratamento da artrite, usando um modelo de artrite AIA. As suas propriedades anti-inflamatórias, antiproliferativas e protetoras do osso foram avaliadas. Os animais foram tratados intraperitonealmente com X (1μg/g diariamente) e divididos em dois grupos, com diferentes dias de início da administração: um dos grupos iniciou o tratamento ao dia 4 e outro iniciou o tratamento ao dia 11 após a indução da doença. Ao fim de 22 dias de duração da doença os animais foram eutanaziados e foram colhidas amostras de soro, órgãos (fígado, rim e baço) e ossos (fémures, tíbias e vértebras). A avaliação do score inflamatório e do diâmetro da pata dos animais permitiram perceber que a administração de X foi significativamente eficaz no tratamento dos sinais inflamatórios da artrite, tanto no grupo de animais que iniciou o tratamento numa fase inicial do desenvolvimento da doença como no grupo que começou o tratamento numa fase mais tardia. O peso, parâmetros bioquímicos de toxicidade e a histologia dos órgãos indicaram também que a administração de X intraperitonealmente nesta concentração não causou quaisquer efeitos secundários nos animais. Do painel de citocinas avaliadas (IL-1β, IL-6, IL-17 e TNF) apenas a citocina IL- 6 se observou significativamente aumentada no soro dos animais doentes em relação aos animais saudáveis. O tratamento com X diminuiu significativamente os níveis de IL- 6 em ambos os grupos de tratamento, comparativamente aos animais não-tratados. Foram ainda analisados no soro mediadores pro-inflamatórios envolvidos na osteoclastogénese, OPG e RANKL, e indicadores de atividade óssea, CTX-I, P1NP e TRACP-5b, e da cartilagem, CTX-II. Não se encontraram diferenças significativas no rácio RANKL/OPG entre animais saudáveis e doentes não-tratados. No entanto, foram observados níveis aumentados de CTX-I, P1NP e CTX-II nos animais doentes, em comparação com os animais saudáveis. O tratamento com X diminuiu significativamente os níveis de P1NP, CTX-II e TRACP-5b em ambos os grupos de tratamento, em relação aos animais doentes não-tratados. Não se encontraram diferenças entre os grupos de tratamento e os animais doentes não-tratados para o CTX-I. Realizou-se também uma avaliação histológica das patas dos animais tendo em conta os níveis de infiltração e proliferação da membrana sinovial, erosão óssea e impacto global da doença na estrutura articular. Observou-se que ambos os animais em ambos os grupos de tratamento com X têm níveis de infiltração e proliferação da membrana sinovial significativamente diminuídos em relação aos animais não-tratados, apresentando um fenótipo semelhante aos animais saudáveis. Observou-se também que as erosões ósseas aparecem desde uma fase muito inicial do desenvolvimento da doença, e que o tratamento com X é capaz de reduzir significativamente o aparecimento de novas erosões ósseas na articulação. As patas foram ainda analisadas por imunohistoquímica, usando marcadores de células imunes inflamatórias. Os animais tratados com X tinham significativamente menos células sinoviais em proliferação e uma redução do número de células T, B e macrófagos presentes na membrana sinovial, quando comparados com os animais doentes não-tratados. As propriedades mecânicas do osso foram avaliadas através de 3-point bending nos fémures dos animais. Verificou-se que os animais não-tratados têm uma menor fase elástica e plástica em comparação com os animais saudáveis, sendo que o tratamento com X foi capaz de preservar as propriedades mecânicas do osso no grupo de animais que começou o tratamento no dia 4. Os animais com início do tratamento ao dia 11 após indução da artrite não conseguiram recuperar estas propriedades mecânicas ósseas. Estes resultados apontam o X como uma terapêutica promissora para o tratamento da artrite reumatoide dadas as suas características anti-inflamatórias e protetoras do dano ósseo.