Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund’s adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human post-mortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ipsilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions

Effects of soluble guanylyl cyclase activation by BAY 60-2770 on murine pulmonary allergic inflammation and chemotactic activity of isolated human eosinophils

Orientador: Edson AntunesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O recrutamento de eosinófilos para o pulmão inflamado é uma característica central da asma alérgica. Há relatos de que a ativação da guanilil ciclase solúvel (GCs) exerce atividade anti-inflamatória por inibir o recrutamento de leucócitos, e que o aumento de GMPc intracelular inibe a migração in vitro de eosinófilos humanos. A GCs é estimulada pelo NO, seu agonista fisiológico. Outros compostos são capazes de estimular a GCs de forma independente do NO, como as moléculas da família dos estimuladores NO-independentes e heme-dependentes, e, mais recentemente, por compostos estimuladores NO- e heme-independentes, chamados de ativadores da GCs. Neste contexto, o objetivo deste trabalho foi avaliar os efeitos do BAY 60-2770 (ativador NO- e heme-independente da GCs) em modelo murino de asma alérgica e na quimiotaxia in vitro de eosinófilos humanos induzida pela eotaxina. Para tanto, camundongos machos C57Bl6 (25 g, 6 semanas de idade) foram sensibilizados (2 injeções s.c. de 100 ?g de ovalbumina, nos dias 0 e 7) e submetidos ao desafio intranasal com OVA (10 ?g; duas vezes por dia, com intervalos de 6 horas entre cada desafio) nos dia 14 e 15, na presença e/ou ausência (veículo) do tratamento crônico com BAY 60-2770 (1mg/Kg/gavage; 14 dias). Os animais foram anestesiados e sacrificados 48 horas após o primeiro desafio, e as amostras biológicas colhidas para análise. O perfil inflamatório alérgico foi avaliado através da contagem total e diferencial de leucócitos no lavado broncoalveolar (LBA), sangue periférico, perfusato de medula óssea (MO) e em corte histológico. Foram dosados os níveis de IgE sérica específica a OVA, citocinas Th1 (IFN-?), Th2 (IL-4, IL-5, IL-10), TNF-? e eotaxina no LBA. A expressão proteica da GCs (subunidades ?2 e ?1) e da iNOS foi quantificada no parênquima pulmonar. O desenho experimental foi dividido em quatro grupos, a saber: grupo tratado com Veículo e instilado com Salina (VS); grupo tratado com Veículo e desafiado com OVA (VO); grupo tratado com BAY 60-2770 e instilado com Salina (BS); grupo tratado com o BAY 60-2770 e desafiado com OVA (BO). Nossos resultados mostraram níveis elevados (p<0,05) de IgE em soro de animais sensibilizados comparados com animais naive, confirmando a eficácia da sensibilização, que não foi alterada pelo tratamento com BAY 60-2770. O desafio alérgico à OVA (grupo VO), como esperado, gerou uma resposta inflamatória alérgica pulmonar, caracterizada pela presença marcante de eosinófilos no pulmão e de níveis elevados de IL-4 no LBA, assim como eosinofilia sanguínea e eosinofilopoese na medula óssea. Esta resposta foi significativamente acompanhada da redução da expressão da GCs (subunidades ?1 e ?1) e aumento da expressão protéica da iNOS no parênquima pulmonar, comparada a animais não desafiados (grupo VS). Não foram observadas diferenças nos níveis de TNF-? e eotaxina no LBA entre os grupos. As citocinas IFN-? e IL-10 não foram detectadas no LBA em nenhum grupo experimental. O tratamento crônico com BAY 60-2770 (grupo BO) reduziu significativamente os níveis de IL-4 e IL-5 no LBA, inibiu a eosinofilia pulmonar (LBA), sanguínea e peri-bronquiolar, e suprimiu a eosinofilopoiese na medula óssea, os quais foram acompanhados da restauração da expressão da GCs (?1 e ?1) e da iNOS no parênquima pulmonar, comparado com o grupo desafiado tratado com veículo (grupo VO). Em experimentos separados, eosinófilos humanos de indivíduos saudáveis foram submetidos aos ensaios de quimiotaxia induzida por eotaxina (300 ng/mL) na presença do BAY 60-2770 individualmente e/ou co-incubado com inibidor da GCs, ODQ (10 ?M). Nas mesmas condições, foram determinados os níveis de GMPc intracelular, e realizado o teste de viabilidade celular por redução do MTT. A resposta quimiotática induzida por eotaxina foi significativamente inibida pelo BAY 60-2770 (10 ?M), de modo dependente de GMPc. O efeito inibitório do BAY 60-2770 foi potencializado pela prévia co-incubação com ODQ, que elevou marcantemente os níveis de GMPc intracelulares e inibiu significativamente a migração de eosinófilos frente à eotaxina nas concentrações de 1, 3 e 5 ?M de BAY 60-2770. A viabilidade celular não foi afetada pelo BAY 60-2770. Em conjunto, nossos resultados mostram que o BAY 60-2770 é capaz de reduzir a inflamação alérgica pulmonar murina, e de inibir de forma dependente de GMPc a migração de eosinófilos humanos induzida por eotaxina. Concluímos que o BAY 60-2770 apresenta potencial valor terapêutico na asma alérgicaAbstract: Eosinophil recruitment to inflamed lung is a hallmark of allergic asthma. The literature has shown that soluble guanylyl cyclase (sGC) plays a key antiinflammatory role by inhibiting leukocyte recruitment, and that increased cGMP intracellular levels inhibit human eosinophil chemotaxis in vitro. The soluble guanylyl cyclase is stimulated by NO, its physiological agonist. Other compounds are able to NO-independently stimulate sGC, such as the family of NO-independent and heme-dependent stimulators, and the NO- and heme-independent stimulators, known as sGC activators. Therefore, this study aimed to investigate the effects of BAY 60-2770 (NO- and haem-independent sGC activator) in pulmonary allergic inflammation in mice and human eosinophil chemotaxis in vitro induced by eotaxin. Part I: Male C57BL6 mice were sensitized (100 ?g of ovalbumin, s.c.; day 0 and 7) and intranasally challenged with OVA (10 ?g; twice a day, 6 h between challenges) at days 14 and 15, with BAY 60-2770 (1 mg/Kg/gavage; 14 days) and/or its vehicle. Mice were anaesthetized and exsanguinated at 48 hours post first-OVA challenge, and biological samples were collected. The inflammatory profile was evaluated by total and differential cell counts in bronchoalveolar lavage (BAL) fluids, peripheral blood, bone marrow perfusate and histological slides. Serum OVA-specific IgE levels were measured and Th1 (IFN-?), Th2 (IL-4, IL-5, IL-10) cytokines, TNF-? and eotaxin levels were quantified in BAL fluids. Soluble guanylyl cyclase subunits (?1 and ?1) and iNOS protein expression were determined in lung parenchyma. The experimental design was divided in four groups, namely: Vehicle treated-instilled with Saline (VS group); Vehicle treated-challenged with OVA (VO group); BAY 60-2770 treated-instilled with Saline (BS group) and BAY 60-2770 treated-challenged with OVA (BO group). Our results showed seric elevated IgE levels (p<0.05) in OVA-sensitized mice compared with naive animals, confirming the efficiency of sensitization procedure, which was not affected under BAY 60-2770 treatment. As expected, the OVA-challenge in VO group triggered a pulmonary allergic inflammatory response, characterized by markedly eosinophil migration to lung, elevated IL-4 levels in BAL fluid, as well as blood eosinophilia and marrow eosinophilopoiesis in bone marrow. This response was significantly accompanied by reduced GCs (subunits ?1 and ?1) and increased iNOS expression in lung parenchyma, compared with non-challenged animals (VS group). No differences on TNF-? and eotaxin levels in BAL fluids were observed among studied groups. Levels of IFN-? and IL-10 were not detected in BAL fluids of any groups. Chronic oral treatment with BAY 60-2770 (BO group) significantly reduced IL-4 and IL-5 levels in BAL fluids, inhibited pulmonary (BAL), blood and peri-bronchiolar eosinophilia, and supressed bone marrow eosinophilopoiesis, which was accompanied by restoration of sGC and iNOS expression in lung parenchyma, compared with vehicle treated group (VO). In separate experiments, isolated human eosinophils from healthy volunteers were individually incubated with BAY 60-2770 and/or previously co-incubated with the sGC inhibitor, ODQ (10 ?M), and allowed to migrate toward eotaxin (300 ng/ml; 1 h, 37°C) in chemotaxis chamber. In the same conditions, intracellular cGMP levels were determinated and cell viability test were performed by MTT reduction. Our data showed that human eosinophil migration to eotaxin was significantly inhibited by BAY 60-2770 (10 ?M) in a cGMP-dependent manner. Inhibitory effect of BAY 60-2770 was potentiated by previous co-incubation with ODQ, which markedly elevated cGMP intracellular levels and inhibited eosinophils migration induced by eotaxin at 1, 3 and 5 ?M BAY 60-2770 concentrations. The cell viability was not affected by BAY 60-2770. Together, our results show that BAY 60-2770 is able to reduce murine pulmonary allergic inflammation, and to inhibit human eosinophil migration toward eotaxin in cGMP-dependent manner. In conclusion, the compound BAY 60-2770 presents potential therapeutic value in allergic asthmaDoutoradoFarmacologiaDoutor em Farmacologi

Evaluation Of The Relaxant Effect Of The Nitric Oxide-independent Soluble Guanylyl Cyclase Stimulator Bay 41-2272 In Isolated Detrusor Smooth Muscle.

The nitric oxide (NO)-independent soluble guanylyl cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001-100 microM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3+/-6.6%, 95.1+/-9.9% and 91.7+/-5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 microM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca(2+) in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca(2+) influx blockade independently of cGMP production appears to be involved.637171-

Quercetin As An Inhibitor Of Snake Venom Secretory Phospholipase A2.

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.1899-1