Edaravone alleviates cell apoptosis and mitochondrial injury in ischemia-reperfusion-induced kidney injury via the JAK/STAT pathway

BACKGROUND: Kidney ischemia-reperfusion injury is a common pathophysiological phenomenon in the clinic. A large number of studies have found that the tyrosine protein kinase/signal transducer and activator of transcription (JAK/STAT) pathway is involved in the development of a variety of kidney diseases and renal protection associated with multiple drugs. Edaravone (EDA) is an effective free radical scavenger that has been used clinically for the treatment of postischemic neuronal injury. This study aimed to identify whether EDA improved kidney function in rats with ischemia-reperfusion injury by regulating the JAK/STAT pathway and clarify the underlying mechanism. METHODS: Histomorphological analysis was used to assess pathological kidney injury, and mitochondrial damage was observed by transmission electron microscopy. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) staining was performed to detect tubular epithelial cell apoptosis. The expression of JAK2, P-JAK2, STAT3, P-STAT3, STAT1, P-STAT1, BAX and Bcl-2 was assessed by western blotting. Mitochondrial function in the kidney was assessed by mitochondrial membrane potential (ΔψM) measurement. RESULTS: The results showed that EDA inhibited the expression of p-JAK2, p-STAT3 and p-STAT1, accompanied by downregulation of the expression of Bax and caspase-3, and significantly ameliorated kidney damage caused by ischemia-reperfusion injury (IRI). Furthermore, the JC-1 dye assay showed that edaravone attenuated ischemia-reperfusion-induced loss of kidney (ΔψM). CONCLUSION: Our findings indicate that EDA protects against kidney damage caused by ischemia-reperfusion through JAK/STAT signaling, inhibiting apoptosis and improving mitochondrial injury.

Perfil de expressão dos fatores de transcrição STAT-1, STAT-4 e T-bet no carcinoma escamoso de pênis / Expression profile of STAT-1, STAT-4 and T-bet transcription factors in squamous carcinoma of the penile

Introdução: O câncer de pênis (CaPe) é uma doença rara em países desenvolvidos, porém representa um problema de saúde pública em países em desenvolvimento. O Brasil tem a maior incidência de CaPe do mundo, chegando a atingir até 6,8 casos por 100.000 homens, principalmente na região norte/nordeste, onde o índice de desenvolvimento humano é o pior do país. Seu diagnóstico ocorre geralmente de forma tardia, retardando o tratamento e piorando o prognóstico. As células Th1 atuam na imunidade celular e expressam fatores de transcrição como transdutor de sinal e ativador de transcrição 1 (STAT1) e 4 (STAT4). Os fatores STAT1 e STAT4 estão relacionados a diversas funções do sistema imune e na oncogênese, além de promover o início do processo de diferenciação das células T "naive" em T efetoras do tipo Th1. A desregulação da via JAK/STAT está associada com a formação de tumores primários, assim como com a supressão da resposta imune, o que leva ao aumento da sobrevivência tumoral e da angiogênese. Objetivo: O objetivo geral deste estudo foi avaliar a expressão de STAT1, STAT4 e T-bet nos linfócitos T circulantes pacientes com carcinoma escamoso de pênis. Métodos: Foi realizado um estudo de corte transversal, com 30 pacientes com CaPe atendidos no ambulatório de Urologia do Hospital de Câncer de Pernambuco e 15 controles saudáveis do sexo masculino. As análises laboratoriais foram realizadas no Laboratório de Pesquisa Translacional do IMIP. A análise da expressão de STAT1, STAT4 e T-bet foi realizada por citometria de fluxo. Para análise estatística das variáveis numéricas foi aplicado o teste de normalidade de Shapiro-Wilk. Foi utilizado teste não paramétricos de Mann-Whitney e o de correlação de Spearman. Foi adotado o nível de significância estatística de p<0.05. Para análise de correlação foi realizado o teste de Spearman. Toda a análise estatística foi realizada através do GraphPadPrism v6.0 (GraphPad Software, San Diego, CA). Resultados: Foi observado redução dos níveis percentuais de linfócitos B no sangue periférico de pacientes com CaPe quando comparados aos controles (p=0.0002). Os níveis percentuais de linfócitos T com expressão de STAT1 e STAT4 foram elevados (p=0,004 e p=0,008, respectivamente) em comparação ao grupo controle. Os níveis de linfócitos T com expressão de T-bet nos pacientes foram inferiores quando comparados aos dos controles (p=0,02). Observou-se que pacientes com tamanho tumoral <3,5cm apresentaram níveis percentuais elevados de células T CD8+ quando comparados aos com tamanho ≥3,5 cm (p= 0,04). Verificou-se que os pacientes com IPN negativa obtiveram níveis percentuais elevados de linfócitos T CD8+ (p=0,04) e de linfócitos T com expressão de STAT1, STAT4 e T-bet quando comparados aos pacientes IPN positiva (p=0,0004, p=0,02 e p=0,0001, respectivamente). Foi observada também a correlação entre T-bet e STAT4 nos pacientes com CaPe (r=0,58; p=0.002). Conclusão: Os fatores de transcrição STAT1, STAT4 e T-bet estão associados a doença avançada, e podem estar diretamente relacionados as alterações dos níveis de linfócitos B e TCD8+ nos pacientes com câncer de pênis

Introduction: Penile cancer (CaPe) is a rare disease in developed countries, but it represents a public health problem in developing countries. Brazil has the highest incidence of CaPe in the world, reaching up to 6.8 cases per 100,000 men, mainly in the north / northeast region, where the human development index is the worst in the country. Its diagnosis usually occurs late, delaying treatment and worsening the prognosis. Th1 cells act on cellular immunity and express transcription factors such as signal transducer and transcription activator 1 (STAT1) and 4 (STAT4). The factors STAT1 and STAT4 are related to several functions of the immune system and oncogenesis, in addition to promoting the differentiation of "naive" T cells into effector T-type Th1 cells. Deregulation of the JAK / STAT pathway is associated with the formation of primary tumors, as well as with the suppression of the immune response, which leads to increased tumor survival and angiogenesis. Objective: The general objective of this study was to evaluate the expression of STAT1, STAT4 and T-bet in circulating T lymphocytes in patients with squamous carcinoma of the penis. Methods: A cross-sectional study was carried out with 30 CaPe patients seen at the Urology outpatient clinic of the Hospital de Câncer de Pernambuco and 15 healthy male controls. Laboratory analyzes were performed at the Translational Research Laboratory at IMIP. The analysis of the expression of STAT1, STAT4 and T-bet was performed by flow cytometry. For statistical analysis of numerical variables, the Shapiro-Wilk normality test was applied. Mann-Whitney non-parametric tests and Spearman's correlation tests were used. The level of statistical significance was set at p <0.05. For correlation analysis, the Spearman test was performed. All statistical analysis was performed using GraphPadPrism v6.0 (GraphPad Software, San Diego, CA). Results: A reduction in the percentage levels of B lymphocytes in the peripheral blood of patients with CaPe was observed when compared to controls (p = 0.0002). The percentage levels of T lymphocytes with STAT1 and STAT4 expression were high (p = 0.004 and p = 0.008, respectively) compared to the control group. The levels of T lymphocytes with T-bet expression in patients were lower when compared to controls (p = 0.02). It was observed that patients with tumor size <3.5 cm had high percentage levels of CD8 + T cells when compared to those with size ≥3.5 cm (p = 0.04). It was found that patients with negative IPN obtained high percentage levels of CD8 + T lymphocytes (p = 0.04) and T lymphocytes with expression of STAT1, STAT4 and T-bet when compared to patients with positive IPN (p=0.0004, p = 0.02 and p = 0.0001, respectively). The correlation between T-bet and STAT4 was also observed in patients with CaPe (r = 0.58; p = 0.002). Conclusion: The transcription factors STAT1, STAT4 and T-bet are associated with advanced disease and may be directly related to changes in the levels of B lymphocytes and TCD8 + in patients with penile cancer

Myeloproliferative neoplasms and the JAK/STAT signaling pathway: an overview

ABSTRACTMyeloproliferative neoplasms are caused by a clonal proliferation of a hematopoietic progenitor. First described in 1951 as 'Myeloproliferative Diseases' and reevaluated by the World Health Organization classification system in 2011, myeloproliferative neoplasms include polycythemia vera, essential thrombocythemia and primary myelofibrosis in a subgroup called breakpoint cluster region-Abelson fusion oncogene-negative neoplasms. According to World Health Organization regarding diagnosis criteria for myeloproliferative neoplasms, the presence of the JAK2 V617F mutation is considered the most important criterion in the diagnosis of breakpoint cluster region-Abelson fusion oncogene-negative neoplasms and is thus used as a clonal marker. The V617F mutation in the Janus kinase 2(JAK2) gene produces an altered protein that constitutively activates the Janus kinase/signal transducers and activators of transcription pathway and other pathways downstream as a result of signal transducers and activators of transcription which are subsequently phosphorylated. This affects the expression of genes involved in the regulation of apoptosis and regulatory proteins and modifies the proliferation rate of hematopoietic stem cells.

Estudo da mutação da STAT5B em Criciúma-Santa Catarina: frequência e caracterização fenotípica de indivíduos heterozigotos / Study of STAT5B mutation in Criciúma - Santa Catarina: frequency and phenotypic characterization of heterozygous individuals

Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois...

Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous...

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.

Estudo da participação de reguladores negativos endógenos da atividade de STAT1 e STAT3 (SOCS1 e SOCS3) na doença periodontal experimental / Role of endogenous negative regulators of STATI and STAT3 (SOCS1 and SOCS3 during experimental periodontal disease

A expressão de citocinas inflamatórias é um processo estritamente regulado por mecanismos variados, incluindo o controle da sinalização intracelular e da atividade transcricional por inibidores endógenos, os quais são pouco estudados e compreendidos. Três grupos de proteínas: SHP, PIAS e SOCS inibem de maneira distinta e específica a transdução de sinais pela via JAK/STAT, bem como a atividade dos fatores de transcrição, eventos que modulam a expressão de diversas citocinas. As doenças periodontais estão associadas à inflamação persistente, com elevados níveis de citocinas proinflamatórias, no entanto praticamente não existem informações sobre a participação destes mecanismos de regulação nas diferentes condições clínicas periodontais. Os objetivos deste projeto incluíram avaliar a cinética de expressão das proteínas SOCS1 e SOCS3 e suas proteínas-alvo, STAT1 e STAT3, respectivamente, durante a evolução da doença periodontal. Foram utilizados 36 ratos Wistar divididos em 2 grupos: DP - doença periodontal induzida por 2 métodos: ligaduras ao redor dos 1os molares inferiores e injeções de 60 µg de LPS de E. coli no tecido gengival palatino dos molares superiores, 3x/semana; Grupo controle negativo - recebeu apenas injeções de PBS (veículo). Os ratos foram sacrificados 7, 15 e 30 dias após a indução da doença periodontal para avaliação histológica e análise macroscópica da perda óssea alveolar. A expressão de SOCS1 e SOCS3 e a ativação de STAT1 e STAT3 foram avaliadas nas biópsias gengivais por PCR em tempo real e Western blot. Ambos os modelos apresentaram significante e progressiva perda óssea dos 7 aos 30 dias. A inflamação foi evidente já no período de 7 dias em ambos os modelos, porém enquanto manteve-se similar nos demais períodos no modelo de indução por LPS, apresentou uma diminuição na severidade da inflamação no modelo de ligadura aos 30 dias. Houve um significante (p<0.05) aumento na expressão gênica de SOCS1 e SOCS3 no grupo DP comparado com o grupo controle aos 15 dias (ambos os modelos) e aos 30 (modelo de indução por ligadura). A ativação de STAT1 e -3 foram aumentadas nos períodos iniciais no modelo por ligadura e nos períodos mais tardios no modelo de LPS. Estes resultados sugerem que SOCS1 e -3 participam na regulação do processo inflamatório responsável pela destruição periodontal

Inflammatory cytokine gene expression is a process strictly regulated by various mechanisms, including the negative regulation of signaling of cytokine receptors and of the activity of transcription factors such as STATs. These mechanisms involve endogenous proteins and are largely unknown, especially in periodontal diseases. Three groups of proteins, SHP, PIAS and SOCS modulate in a fairly specific manner JAK/STAT signaling and/or STAT activity. Periodontal diseases are infectious-inflammatory conditions of the supporting tissues of the teeth associated with increased levels of proinflammatory cytokines, but there are no information regarding the role of these endogenous mediators of JAK/STAT during its course. The aims of this study included the evaluation of the expression kinetics of inducible negative regulators and their target proteins during the course of experimentally induced periodontal disease. 36 Wistar rats were divided into two groups: PD - experimental periodontal disease induced by two methods: ligature placement around the first mandibular molars and E. coli lipopolysaccharide (LPS) injections into the palatal gingival tissues of the maxillary molars, 3x/week, and Negative Control group. Rats were sacrificed 07, 15 and 30 days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western Blot. Both disease models presented significant progressive bone loss from 7 to 30 days. Inflammation was evident and similar for all the periods in LPS injected sites; however, a decrease on severity at the end of the experimental period was observed in the ligature model. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control group at 15 (both disease models) and 30 days (ligatureinduced model). The activation of STAT1 and STAT3 were increased in earlier periods in the ligature model and in later periods in the LPS model. This study suggests that SOCS 1 and 3 participate in the regulatory mechanism of the inflammatory process responsible for the periodontal disease destruction