Análise dos mecanismos pró-inflamatórios do ácido úrico solúvel: efeito sobre proteínas sensíveis à modulação redox e sobre a polarização de células THP-1 / Analysis of the pro-inflammatory mechanisms of soluble uric acid: effect on redox sensitive proteins and THP-1 cell polarization

Vários estudos epidemiológicos estabelecem correlação positiva entre os níveis de ácido úrico sérico e o aumento do risco para doenças cardiovasculares. Fatores dietéticos e socioeconômicos, além da presença de comorbidades estão diretamente associados aos níveis séricos de ácido úrico. Países desenvolvidos apresentam maior incidência e prevalência da gota e alguns grupos étnicos são particularmente susceptíveis à hiperuricemia. Cristais de ácido úrico são descritos por iniciar e perpetuar resposta inflamatória, e sinalizar um padrão de resposta molecular associado ao dano (DAMP), permitindo a diferenciação de macrófagos para perfis pró-inflamatórios. Por outro lado, os efeitos do ácido úrico em sua forma solúvel ainda carecem de estudos. Macrófagos derivados de precursores monocíticos apresentam diferenciação específica e respondem a um conjunto de fatores extrínsecos, resultando em perfis distintos, um fenômeno conhecido como polarização. Assim, os macrófagos podem ser classicamente ativados para uma resposta Th1 (T helper 1) e polarizados a um perfil pró- inflamatório (M1, resposta Th1) ou a um perfil alternativo e oposto, um perfil de resolução da inflamação (M2, resposta Th2, T helper 2). Nesse sentindo, buscamos analisar os efeitos do ácido úrico solúvel sobre vias de modulação da polarização fenotípica de macrófagos e modificação redox. Utilizamos a linhagem monocítica humana THP-1, a qual foi diferenciada em macrófagossímile por acetato miristato de forbol (PMA; 5 ng.mL-1) por 48 h, seguidas da incubação com ácido úrico em meio ausente de tióis e soro fetal bovino por 8h ou 24h (0-1000 µM). A expressão de fatores de transcrição e marcadores de polarização foi realizada através de citometria de fluxo, western-blotting e por microscopia de fluorescência com alto conteúdo de imagens (HCI). Em concentrações fisiológicas, verificamos que o ácido úrico solúvel regulou positivamente a frequência de células para receptor manose CD206, um marcador clássico de perfil alternativo/M2 e regulou negativamente a expressão óxido nítrico sintase induzível (iNOS), um marcador M1, sugerindo inicialmente uma modulação para o perfil de polarização M2. Além disso, as proteínas redoxsensíveis, heme oxigenase-1 (HO-1) e tiorredoxina (Trx) tiveram sua expressão reduzida e aumentada, respectivamente, pelo tratamento com ácido úrico. Os fatores de transcrição Nrf2 e STAT3 tiveram regulação negativa após a exposição ao ácido úrico solúvel. Os resultados apresentados nesta tese sugerem uma função do urato no priming de macrófagos através da alteração da polarização destas células

Several epidemiological studies have established a positive correlation between high serum uric acid levels and increased risk for cardiovascular diseases. Developed countries have a higher incidence and prevalence of gout and some ethnic groups are particularly susceptible to hyperuricemia. Although hyperuricemia is a prevalent condition, it has still controversy biological consequences. Uric acid crystals are described as capable of initiating and perpetuating inflammatory responses, by activating the damage-associated molecular response pattern (DAMP) cascade, allowing macrophage differentiation to inflammatory profiles. In spite of that, biological response to soluble uric acid are not completely understood. Monocyte-derived macrophages respond to a set of extrinsic factors that result in different profiles and can be polarized to a proinflammatory (M1) or anti-inflammatory (M2) profile. In this thesis, we analyzed the effects of soluble uric acid on redox-modulated pathways and the phenotypic polarization of macrophages. We used human monocytic THP-1 cell line, differentiated into macrophage by phorbol myristate acetate (PMA; 5 ng.mL-1) for 48 h. After differentiation, cells were incubated with soluble uric acid in medium without thiols and fetal bovine serum for 8 h and 24 h (0-1000 µM). The expression of transcription factors and polarization markers were assessed by flow cytometry, western-blotting and fluorescence microscopy with high content imaging (HCI). At physiological concentrations, soluble uric acid positively regulated the frequency of cells for mannose receptor CD206, a classic marker of the anti-inflammatory M2 profile and negatively regulated the inducible nitric oxide synthase (iNOS) expression, a proinflammatory M1 marker, suggesting that the soluble uric acid changes the polarization profile to M2 profile. In addition, the redox-sensitive proteins heme oxygenase-1 (HO-1) and thioredoxin (Trx) had their expression decreased and increased, respectively, after exposure to urate. STAT3 and Nrf2 transcription factors were downregulated upon soluble uric acid exposure. The results presented in this thesis suggest a role of uric acid in macrophage priming through the alteration of cell polarization

Ethacrynic acid decreases expression of proinflammatory intestinal wall cytokines and ameliorates gastrointestinal stasis in murine postoperative ileus

OBJECTIVES: Several compounds characterized by an olefin linkage conjugated to a carbonyl group have anti-inflammatory properties. The diuretic ethacrynic acid (EA) is a compound of this type. Herein, we tested the hypothesis that ethacrynic acid can modulate the development of ileus after bowel manipulation. METHODS: Groups (n=9) of male C57Bl/6 mice underwent surgical manipulation of the small intestine using a pair of cotton-tipped applicators (MAN). Control animals (CONT) did not undergo any surgical intervention or receive treatment. MAN mice were pre- and post-treated with four intraperitoneal doses of phosphate buffered saline (PBS), EA1 (1mg/kg per dose), or EA10 (10mg/kg per dose). Gastrointestinal transit of non-absorbable FITC-labeled dextran was assessed by gavaging the mice with the tracer 24h after operation and assessing FD70 concentration 120 min later in the bowel contents from the stomach, 10 equally long segments of small intestine, cecum, and two equally long segments of colon. The geometric center for the tracer was calculated for each animal. Expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) transcripts in the ileal muscularis propria was assessed using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: In control animals, the mean (±SE) geometric center for the transit marker was 9.89±0.47, whereas it was 4.59±0.59 for PBS-treated animals (p<0.05 vs CONT). The geometric center for pre- post treatment with low (1mg/kg) and high (10mg/kg) doses of ethacrynic acid were 7.23±0.97 and 5.15±0.57, respectively. Compared to PBS, treatment with ethacrynic acid (1mg/kg) significantly decreased manipulation-induced IL-6 and iNOS mRNA expression in the wall of the small bowel. CONCLUSIONS: Pre- and post-treatment with ethacrynic acid ameliorates ileus and modulates inflammation in the gut wall induced by bowel manipulation.

Are there gender differences in left ventricular remodeling after myocardial infarction in rats? / Há diferenças entre os gêneros no remodelamento ventricular esquerdo após infarto do miocárdio em ratos?

Objective: An unclear issue is whether gender may influence at cardiac remodeling after myocardial infarction (MI). We evaluated left ventricle remodeling in female and male rats post-MI. Methods: Rats were submitted to anterior descending coronary occlusion. Echocardiographic evaluations were performed on the first and sixth week post-occlusion to determine myocardial infarction size and left ventricle systolic function (FAC, fractional area change). Pulsed Doppler was applied to analyze left ventricle diastolic function using the following parameters: E wave, A wave, E/A ratio. Two-way ANOVA was applied for comparisons, complemented by the Bonferroni test. A P≤=0.05 was considered significant. Results: There were no significant differences between genders for morphometric parameters on first (MI [Female (FE): 44.0±5.0 vs. Male (MA): 42.0±3.0%]; diastolic [FE: 0.04±0.003 vs. MA: 0.037±0.005, mm/g] and systolic [FE: 0.03±0.0004 vs. MA: 0.028±0.005, mm/g] diameters of left ventricle) and sixth (MI [FE: 44.0±5.0 vs. MA: 42.0±3.0, %]; diastolic [FE: 0.043±0.01 vs. MA: 0.034±0.005, mm/g] and systolic [FE: 0.035±0.01 vs. MA: 0.027±0.005, mm/g] of LV) week. Similar findings were reported for left ventricle functional parameters on first (FAC [FE: 34.0±6.0 vs. MA: 32.0±4.0, %]; wave E [FE: 70.0±18.0 vs. MA: 73.0±14.0, cm/s]; wave A [FE: 20.0±12.0 vs. MA: 28.0±13.0, cm/s]; E/A [FE: 4.9±3.4 vs. MA: 3.3±1.8]) and sixth (FAC [FE: 29.0±7.0 vs. MA: 31.0±7.0, %]; wave E [FE: 85.0±18.0 vs. MA: 87.0±20.0, cm/s]; wave A [FE: 20.0±11.0 vs. MA: 28.0±17.0, cm/s]; E/A [FE: 6.2±4.0 vs. MA: 4.6±3.4]) week. Conclusion: Gender does not influence left ventricle remodeling post-MI in rats. .

Objetivo: A influência do gênero no remodelamento cardíaco após o infarto do miocárdio é uma questão em intenso debate. Nós avaliamos o remodelamento ventricular esquerdo em ratos infartados de ambos os gêneros. Métodos: O infarto do miocárdio foi induzido por oclusão da artéria coronária descendente anterior (fêmeas [FM]; machos [MC]). A ecocardiografia foi realizada na primeira e sexta semana pós-oclusão para determinar o tamanho do infarto do miocárdio e a função sistólica do ventricular esquerdo (mudança na área fracional [FAC]). A função diastólica derivou dos seguintes parâmetros: onda E; onda A; razão E/A. ANOVA duas vias com pós-teste de Bonferroni foi aplicado nas comparações (P≤=0,05). Resultados: Todas variáveis morfométricas foram similares (P>0,05) entre os gêneros com uma (infarto do miocárdio [FM: 44,0±5,0 vs. MC: 42,0±3,0, %]; diâmetro diastólico [FM: 0,04±0,003 vs. MC: 0,037±0,005, mm/g] e sistólico [FM: 0,03±0,0004 vs. MC: 0,028±0,005, mm/g] do VE) e seis (IM [FM: 44,0±5,0 vs. MC: 42,0±3,0, %]; diâmetro diastólico [FM: 0,043±0,01 vs. MC: 0,034±0,005, mm/g] e sistólico [FM: 0,035±0,01 vs. MC: 0,027±0,005, mm/g] do ventricular esquerdo) semanas. Achado similar ocorreu para os dados funcionais com uma (FAC [FM: 34,0±6,0 vs. MC: 32,0±4,0, %]; onda E [FM: 70,0±18,0 vs. MC: 73,0±14,0, cm/s]; onda A [FM: 20,0±12,0 vs. MC: 28,0±13,0, cm/s]; E/A [FM: 4,9±3,4 vs. MC: 3,3±1,8]) e seis (FAC [FM: 29,0±7,0 vs. MC: 31,0±7,0, %]; onda E [FM: 85,0±18,0 vs. MC: 87,0±20,0, cm/s]; onda A [FM: 20,0±11,0 vs. MC: 28,0±17,0 cm/s]; E/A [FM: 6,2±4,0 vs. MC: 4,6±3,4]) semanas. Conclusão: O gênero não é determinante para o remodelamento ventricular esquerdo pós-infarto do miocárdio em ratos. .

Nitric oxide in liver fibrosis: The role of inducible nitric oxide synthase

The inducible form of nitric oxide synthase (iNOS) is expressed in hepatic cells in pathological conditions. Its induction is involved in the development of liver fibrosis, and thus iNOS could be a therapeutic target for liver fibrosis. This review summarizes the role of iNOS in liver fibrosis, focusing on 1) iNOS biology, 2) iNOS-expressing liver cells, 3) iNOS-related therapeutic strategies, and 4) future directions.

Electroacupuncture ameliorates experimental colitis induced by TNBS through activation of interleukin-10 and inhibition of iNOS in mice

PURPOSE: To study the anti-inflammatory actions of electroacupuncture (EAc) on an experimental colitis model in mice. METHODS: Thirty-eight male Swiss mice, divided in five groups, were subjected to induction of colitis by TNBS in 50% ethanol. Saline (SAL) and ethanol (ETNL) groups served as controls. TNBS+EAc and TNBS+ dexamethasone subgroups were treated with EAc 100Hz and dexamethasone (DEXA) 1 mg/Kg/day, respectively. After three days, a colon segment was obtained for quantification of myeloperoxidase (MPO) activity, immunohistochemistry for iNOS, malondialdehyde (MDA) and cytokines (IL-1β and IL-10). RESULTS: Neutrophilic activity, assayed as MPO activity, was significantly higher in the TNBS colitis group than that in the saline control group. TNBS+EAc group showed suppression of IL-10 in the colon. EAc treatment significantly reduced the concentration of MDA and the expression of iNOS, as compared to the other groups. CONCLUSION: Electroacupuncture 100Hz applied to acupoint ST-36 promotes an anti-inflammatory action on the TNBS-induced colitis, mediated by increase of IL-10 and decrease of iNOS expression. .

A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1v (Kier’s first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

Anti-inflammatory effects of Zingiber officinale roscoe involve suppression of nitric oxide and prostaglandin E2 production

Inflammation is a physiological response to injury and infection. However, chronic inflammation causes tissue damage and is a feature of most chronic diseases. Despite significant progress in developing therapies to target chronic inflammation over the years, almost all current therapies have serious side effects. The current investigation is to identify naturally-existing anti-inflammatory therapies with fewer side effects. The anti-inflammatory effects and mechanisms of action of extracts and fractions obtained using vacuum liquid chromatography [VLC] from ginger [Zingiber officinale] on the production of nitric oxide [NO] and prostaglandin E[2] [PGE[2]] were investigated. NO and PGE[2] production were induced by stimulating the mouse RAW264.7 monocyte/macrophage cell line with lipopolysaccharide [LPS]. Levels of NO and PGE[2] were deter-mined using the Griess method and enzyme linked sorbent assay [ELISA], respectively. Extracts of two Zingiber officinale species obtained with chloroform showed po-tent inhibitory effects on NO and PGE[2] production. The extracts had a higher potency than N[G]-nitro-L-arginine methyl ester [L-NAME], a known specific inducible nitric oxide synthase [iNOS] inhibitor and were comparable in their effects on PGE[2] with Indomethacin, a specific PGE[2] inhibitor. Further, we identified a fraction [F6] that had most potent inhibi-tory effects. The study shows that extract of Zingiber officinale have strong inhibitory effects on key pro-inflammatory mediators involved in chronic inflammation. Both the extracts and F6 had better inhibitory effects than established pharmaceutical inhibitors of NO and PGE[2]

Involvement of nitric oxide pathways in neurogenic pulmonary edema induced by vagotomy

OBJECTIVE: The objective of this study was to evaluate the involvement of peripheral nitric oxide (NO) in vagotomy-induced pulmonary edema by verifying whether the nitric oxide synthases (NOS), constitutive (cNOS) and inducible (iNOS), participate in this mechanism. INTRODUCTION: It has been proposed that vagotomy induces neurogenic pulmonary edema or intensifies the edema of other etiologies. METHODS: Control and vagotomized rats were pretreated with 0.3 mg/kg, 3.0 mg/kg or 39.0 mg/kg of L-NAME, or with 5.0 mg/kg, 10.0 mg/kg or 20.0 mg/kg of aminoguanidine. All animals were observed for 120 minutes. After the animals' death, the trachea was catheterized in order to observe tracheal fluid and to classify the severity of pulmonary edema. The lungs were removed and weighed to evaluate pulmonary weight gain and edema index. RESULTS: Vagotomy promoted pulmonary edema as edema was significantly higher than in the control. This effect was modified by treatment with L-NAME. The highest dose, 39.0 mg/kg, reduced the edema and prolonged the survival of the animals, while at the lowest dose, 0.3 mg/kg, the edema and reduced survival rates were maintained. Aminoguanidine, regardless of the dose inhibited the development of the edema. Its effect was similar to that observed when the highest dose of L-NAME was administered. It may be that the non-selective blockade of cNOS by the highest dose of L-NAME also inhibited the iNOS pathway. CONCLUSION: Our data suggest that iNOS could be directly involved in pulmonary edema induced by vagotomy and cNOS appears to participate as a protector mechanism.

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.

Efeito do óxido nítrico no transporte mucociliar / Effects of nitric oxide in mucociliary transport

As vias aéreas, constituídas por epitélio ciliado e secretor de muco, promovem ao trato respiratório mecanismo de defesa que livra esta superfície das partículas inaladas durante a respiração. É de fundamental importância o entendimento da fisiologia e dos mecanismos envolvidos com a atividade mucociliar. A literatura sugere que o NO, em especial o produzido pela expressão da iNOS, mantém a função mucociliar e a defesa imune da cavidade nasal. Objetivo: Avaliar o envolvimento do NO e das vias enzimáticas da produção do NO no transporte mucociliar, utilizando inibidores da NO sintase constitutiva e indutiva, L-NAME e aminoguanidina, respectivamente. Materiais e métodos: Preparações de palatos de rã foram imersos em soluções de ringer (controle), L-NAME ou aminoguanidina. Os palatos foram imersos nestas soluções por quatro períodos de 15 minutos. Medidas da velocidade do transporte mucociliar foram feitas antes e após cada exposição. Resultos: Palatos controles mantiveram estável a velocidade do transporte. O L-NAME aumentou, enquanto a aminoguanidina reduziu a velocidade de transporte do muco. Conclusão: O bloqueio inespecífico da cNOS com L-NAME e bloqueio relativamente específico da iNOS com aminoguanidina permitiu propor que dependendo da via o NO pode aumentar ou diminuir o transporte mucociliar em palatos de rã.

The airways are made up of ciliated epithelium which secretes mucous, protecting the respiratory tract from particles inhaled during breathing. Its is paramount to understand the physiology and the mechanisms involved in mucociliary activity. Literature suggests that Nitric oxide (NO), especially the one produced by iNOS expression, maintains the mucociliary function and the immune defense of the nasal cavity. AIM: to assess NO participation and the enzymatic pathways in the production of NO and mucociliary transport, using constructive and inductive NO synthetase inhibitors, L-NAME and aminoguanidine, respectively. Materials and methods: frog palates were prepared and immerse in ringer (control), L-NAME or aminoguanidine solutions. The palates were immerse in these solutions for four periods of 15 minutes. Mucociliary transport measures were carried out before and after each exposure. Results: control palates maintained stable their transportation speed. L-NAME increased, while aminoguanidine reduced mucous transportation velocity. Conclusion: unspecific cNOS block with L-NAME and relatively specific iNOS block with aminoguanidine results leads us to propose that depending on the pathway, the NO can increase or reduce mucociliary transport in frog palates.

Hidropsia endolinfática experimental sob ação de inibidor da óxido nítrico sintase tipo II: avaliação com emissões otoacústicas e eletrococleografia / Experimental endolymphatic hydrops under action of a type II nitric oxide synthase inhibitor: otoacoustic emissions evaluation and electrocochleography

No modelo experimental de hidropsia endolinfática há redução na amplitude das emissões otoacústicas produtos de distorção (EOAPD) e elevação nos limiares eletrofisiológicos na eletrococleografia. Estudos mostraram que há expressão da óxido nítrico sintase tipo II (ONS II) na cóclea com hidropsia, sugerindo a participação do óxido nítrico (ON) na patogênese desta doença. O objetivo deste trabalho foi avaliar a ação de um inibidor da ONS II nas EOAPD e eletrococleografia em cobaias com hidropisia endolinfática experimental. MATERIAL E MÉTODOS: Foram estudadas 16 cobaias nas quais se induziu hidropsia endolinfática experimental por obliteração do ducto e saco endolinfático na orelha direita durante 16 semanas, divididas em dois grupos: oito cobaias recebendo um inibidor da ONS II, a aminoguanidina, por via oral e um grupo de oito cobaias como controle. Comparamos as amplitudes das EOAPD nas médias geométricas de freqüências de 1062, 2187, 4375 e 7000Hz, os limiares eletrofisiológicos nas freqüências de 1000, 2000, 4000 e 6000Hz e a relação entre os potenciais de somação e de ação (PS/PA) entre os grupos. RESULTADOS: Não houve diferença significante nas EOAPD e na relação PS/PA entre os grupos. O grupo que recebeu a aminoguanidina apresentou menor elevação nos limiares eletrofisiológicos nas freqüências de 2000 (p<0,05) e 6000 Hz (p<0,05) na 12ª semana e nas freqüências de 1000 (p<0,05), 2000 (p<0,001), 4000 (p<0,001) e 6000Hz (p<0,001) na 16ª semana. CONCLUSÕES: O inibidor da ONS II reduziu a elevação dos limiares eletrofisiológicos na eletrococleografia na hidropsia endolinfática experimental.