MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE EFEITOS HEPATOPROTETORES DO CARVEDILOL EM MODELO DE ESTEATO- HEPATITE ALCOÓLICA INDUZIDA EM RATOS WISTAR VINÍCIUS BARRETO GARCIA NATAL/RN 2017 VINÍCIUS BARRETO GARCIA EFEITOS HEPATOPROTETORES DO CARVEDILOL EM MODELO DE ESTEATO- HEPATITE ALCOÓLICA INDUZIDA EM RATOS WISTAR Dissertação apresentada ao programa de Pós- Graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito para a obtenção do título de Mestre em Ciências da Saúde. Orientador: Prof. Dr. Raimundo Fernandes de Araújo Júnior NATAL/RN 2017 I Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publica ção na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde – CCS Garcia, Vinícius Barreto. Efeitos hepatoprotetores do carvedilol em modelo de esteato- hepatite alcoólica induzida em ratos wistar / Vinícius Barreto Garcia. - Natal, 2017. 72f.: il. Dissertação (Mestrado)-Programa de Pós-Graduação em Ciências da Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte. Orientador: Prof. Dr. Raimundo Fernandes de Araújo Júnior. Coorientador: Profa. Dra. Aurigena Antunes de Araújo. 1. Esteato-hepatite alcoólica - Dissertação. 2. Carvedilol - Dissertação. 3. Inflamação - Dissertação. I. Araújo Júnior, Raimundo Fernandes de. II. Araújo, Aurigena Antunes de. III. Título. RN/UF/BSCCS CDU 616.36 II MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE Coordenador do Programa de Pós-Graduação em Ciências da Saúde Prof Dr. Eryvaldo Sócrates Tabosa do Egito III VINÍCIUS BARRETO GARCIA EFEITOS HEPATOPROTETORES DO CARVEDILOL EM MODELO DE ESTEATO- HEPATITE ALCOÓLICA INDUZIDA EM RATOS WISTAR Aprovada em ___ /___ /___ Banca examinadora: Presidente da banca: Prof. Dr. Raimundo Fernandes de Araújo Júnior Membros da banca: Profª. Drª, Gerlane Coelho Bernardo Guerra Prof. Dr. Jeymesson Raphael Cardoso Vieira Prof. Dr. Sergio Adriane Bezerra de Moura Profª. Drª. Caroline Addison Carvalho Xavier de Medeiros IV DEDICATÓRIA Aos meus pais, por sempre estarem ao meu lado, me aconselhando quando estou perdido, me repreendendo quando mereço e me amando sempre. Vocês são os meus mestres e o meu orgulho. V AGRADECIMENTOS A Deus, por sua infinita, maravilhosa, eficaz e irresistível Graça; Aos meus pais, por todo o zelo que empregaram na minha criação e educação. A eles eu devo a minha existência e cada uma das minhas realizações, pois não apenas sonharam comigo, mas me ajudaram a tornar meus sonhos em realidade; À Nayana Luíza, por seu amor tão presente, paciente e leal; Ao Prof. Dr. Raimundo Fernandes, meu orientador e pai científico, por ter acreditado em mim, ainda na monitoria de histologia no segundo semestre da graduação, e ter investido tempo e muita paciência na minha formação enquanto profissional; À Prof. Dra. Aurigena Antunes, que sempre me impressionou com o seu grande conhecimento científico e didática, e que divide a bancada com os alunos, como uma mãe que se faz presente enquanto o filho aprende a andar; À Maria de Lourdes, a ―Lourdinha‖, por todas as técnicas histológicas que me ensinou, sempre com muito bom humor e vigor; À Dona Neida, por ter me ensinado a trabalhar com animais de laboratório; Aos alunos de iniciação científica Marcel Setúbal, Sara Ester e Paula Emília, pois sem eles eu não conseguiria lidar com tantos experimentos em tão pouco tempo; A todos aqueles que contribuíram, direta ou indiretamente, para a realização desse trabalho e, consequentemente, para a minha formação, o meu mais sincero obrigado! VI RESUMO A Doença Hepática Alcoólica (DHA) corresponde a diversas patologias hepáticas reversíveis e irreversíveis que ocorrem em resposta à ingestão do etanol, dentre elas a esteato-hepatite alcoólica que, embora reversível, ainda não possui terapia farmacológica específica. O objetivo deste estudo foi avaliar os efeitos hepatoprotetores do carvedilol (CARV) em ratos com esteato-hepatite alcoólica. Para isso, ratos Wistar foram divididos em 5 grupos: controle negativo, controle positivo, CARV 1mg, CARV 3mg e CARV 5mg (5 animais por grupo – sendo os grupos CARV duplicados). Durante 28 dias consecutivos os animais foram submetidos à gavagem oral de solução salina (NaCl 0,9% - controle negativo) ou solução alcoólica a 30%, 7g/kg (grupo controle positivo e grupos CARV). Os grupos CARV recebiam a dose respectiva do fármaco por gavagem 1h antes da solução alcoólica. O sangue dos animais foi coletado via punção cardíaca para dosagem de triglicerídeos (TG) e transaminases hepáticas (AST e ALT) e as amostras hepáticas foram submetidas à análise colorimétrica do malonaldeído (MDA), mieloperoxidase (MPO) e glutationa reduzida (GSH), à análise imuno-enzimática (ELISA) das citocinas Interleucina 1 beta (IL-1β), fator de necrose tumoral alfa (TNF-α) e interleucina 10 (IL-10), à PCR quantitativa em tempo real (RT-qPCR) dos genes pró- colágenos I e III, Fator Nuclear κB (NF- κB) e TNF-α e à análise histopatológica por Hematoxilina e Eosina, Picro-Sirius, Imuno-histoquímica para COX-2, RANK, RANKL, IBA-1, ICAM-1, SOCS-1, SOD-1 e GPx-1 e imunofluorescência para IL-1β e NF- κB. Todas as técnicas utilizadas demonstraram que o efeito hepatoprotetor do carvedilol se dá por meio da regulação que ele desempenha sobre as Células de Kupffer e Células Estreladas, levando a respostas anti-inflamatórias, antioxidantes e anti-fibróticas. Palavras-chave: Esteato-hepatite alcoólica; Carvedilol; inflamação; VII ABSTRACT Alcoholic liver disease (DHA) corresponds to several reversible and irreversible liver diseases that occur in response to ethanol intake, among them alcoholic steatohepatitis which, although reversible, does not have specific pharmacological therapy yet. The aim of this study was to evaluate the hepatoprotective effects of carvedilol (CARV) in rats with alcoholic steatohepatitis. Therefore, Wistar rats were divided into 5 groups: negative control, positive control, CARV 1mg, CARV 3mg and CARV 5mg (5 animals per group - the CARV groups being duplicated). During 28 consecutive days the animals were submitted to oral gavage of saline solution (NaCl 0.9% - negative control) or alcohol solution at 30%, 7g / kg (positive control group and CARV groups). The CARV groups received a more adequate dose to the drug by gavage 1h before the alcoholic solution. The blood of the animals was collected via cardiac puncture for the determination of triglycerides (TG) and hepatic transaminases (AST and ALT), and as hepatic samples were submitted to colorimetric analysis of malonaldehyde (MDA), myeloperoxidase (MPO) and reduced glutathione (GSH), immunoenzymatic analysis (ELISA) of the cytokines Interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), real-time quantitative PCR (RT-qPCR) of Pro-collagen I and III genes, Nuclear Factor κB (NF- κB) and TNF-α and hertopathological analysis by Hematoxylin and Eosin, Picro- Sirius, Immunohistochemistry for COX-2, RANK, RANKL, IBA-1, ICAM -1, SOCS-1, SOD-1 and GPx-1 and immunofluorescence for IL-1β and NF-κB. All techniques demonstrated that the hepatoprotective effect of carvedilol occurs through the regulation it plays on as Kupffer Cells and Star Cells, leading to anti-inflammatory, antioxidant and anti-fibrotic responses. Key words: alcoholic steatohepatitis; carvedilol; inflammation; VIII LISTA DE ABREVIATURAS E SIGLAS DHA: Doença Hepática Alcoólica TNF-α: Fator de Necrose Tumoral alfa IL-10: Interleucina 10 TGO: Transaminase Glutâmica-Oxalacética TGP: Transaminase Glutâmica-Pirúvica γ-GT:Gama glutamil-transferase CK: Células de Kupffer CEH: Células Estreladas Hepáticas LPS: Lipopolissacarídeo NF-κB: Fator Nuclear Kappa Beta IL-1b: Interleucina 1 beta PCI: Pró-colágeno I PCIII: Pró-colágeno III ERO: Espécies Reativas de Oxigênio AASLD:Associação Americana para o Estudo de Doenças Hepáticas AST: Aspartato Amino-Transferase ALT: Alanina Amino-Transferase MPO: Mieloperoxidase MDA: Malonaldeído GSH: Glutationa Reduzida RT-qPCR: Reação em Cadeia de Polimerase quantitativa COX-2: Cicloxigenase 2 RANK: Receptor Ativador do Fator Nuclear kappa RANKL: Ligante doReceptor Ativador do Fator Nuclear kappa SOCS-1:Supressor de Sinaliazação de Citocinas 1 IX CARV: Carvedilol EDTA: Ácido Etilenodiamino Tetra-Acético DTNB: Acido 2,2'-dinitro-5,5'-ditiodibenzoico TCA: Ácido Tricloroacético ELISA:Ensaio de Imunoabsorção Enzimática H&E: Hematoxilina e Eosina PBS: Tampãofosfato-salino mRNA: RNA mensageiro cDNA:DNA complementar GAPDH:Gliceraldeído-3-fosfato Desidrogenase ICAM-1:Molécula de Adesão Intercelular 1 SOD-1:Superóxido Dismutase 1 GPx-1:Glutationa Peroxidase 1 X Sumário 1. INTRODUÇÃO .................................................................................................... 12 2. JUSTIFICATIVA .................................................................................................. 15 3. OBJETIVOS ........................................................................................................ 16 3.1. Objetivo geral ............................................................................................... 16 3.2. Objetivos específicos ................................................................................... 16 4. METODOLOGIA ................................................................................................. 17 4.1. Preparação e administração de etanol ......................................................... 17 4.2. Manejo dos animais ..................................................................................... 17 4.3. Indução da lesão hepática por etanol........................................................... 17 Mieloperoxidase (MPO) ......................................................................................... 19 4.4. Malonaldeído (MDA) .................................................................................... 19 4.5. Glutationa Reduzida (GSH) .......................................................................... 20 4.6. IL-1β, IL-10, e ensaio de TNF-α ................................................................... 20 4.7. Estudos de microscopia óptica e análise bioquímica ................................... 20 4.8. Marcação imuno-histoquímica para COX-2, RANK, RANKL, IBA-1, ICAM-1, SOCS-1, SOD-1 e GPx-1. ...................................................................................... 22 4.9. Análise de imunofluorescência confocal ...................................................... 23 4.10. PCR quantitativa em tempo real para TNFα, PCI, PCIII e NF-κB ............. 23 4.11. Estatística ................................................................................................. 24 5. PUBLICAÇÕES .................................................................................................. 25 6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES .................................................... 61 REFERÊNCIAS ......................................................................................................... 64 ANEXOS ................................................................................................................... 67 XI 12 1. INTRODUÇÃO Segundo a Organização Mundial da Saúde, o álcool é uma substância psicoativa com potencial de desenvolver dependência química em seus usuários. O consumo do álcool varia em todo o mundo, levando a diferentes consequências, mas as doenças e mortes relacionadas a ele ainda representam uma grande preocupação à maioria dos países. Seu uso crônico está entre os 5 principais fatores de risco para doenças, invalidez e morte em todo o mundo, sendo o responsável por aproximadamente 3,3 milhões de mortes anualmente, que correspondem a 5,9% de todas as mortes registradas 1, 2. No Brasil, o consumo de álcool total estimado é equivalente a 8,7L por pessoa, quantidade superior à média mundial (6,2L). Estima-se que homens consumam 13,6L por ano, e as mulheres, 4,2L por ano. Quando são considerados apenas os indivíduos que consomem álcool, esta média sobe para 15,1L de álcool puro por pessoa (sendo mulheres: 8,9L e homens: 19,6L)1. Estudos recentes mostram uma estreita relação causal entre o consumo crônico do álcool e a incidência de doenças infecciosas como a tuberculose e a AIDS3, 4, bem como sua influência deletéria sobre o curso da AIDS5, 6. A doença hepática alcoólica (DHA) representa um conjunto de doenças e alterações morfológicas que variam desde degeneração gordurosa nos hepatócitos (esteatose), levando à inflamação com fibrose e necrose (esteato-hepatite alcoólica) e, finalmente, à fibrose progressiva (cirrose alcoólica)7. Já foi comprovado que o consumo excessivo de álcool aumenta significativamente a morbidade e mortalidade de doenças infecciosas e o risco de desenvolvimento de doenças sistêmicas envolvendo órgãos como o cérebro, o pâncreas, os rins e o sistema cardiovascular 7, além de contribuir para a progressão da hepatite viral crônica, sendo, também, um fator predisponente ao desenvolvimento do hepatocarcinoma8. Polimorfismos de genes que codificam citocinas pró-inflamatórias (TNF-α) e anti-inflamatórias (IL-10) têm sido associados ao aumento do risco de desenvolvimento da DHA9 e sabe-se que o consumo de álcool acima de 40g/d - quantidade equivalente à 2,6 latas de cerveja, 4 taças de vinho ou 4 doses de cachaça - é capaz de induzir a esteatose hepática alcoólica em humanos10. A ingestão excessiva de álcool gera alterações no metabolismo da glicose, 13 ácidos graxos e lipoproteínas, levando à degeneração gordurosa dos hepatócitos, que corresponde ao acúmulo de lipídeos em vacúolos citoplasmáticos11, e essas alterações no metabolismo podem ser observadas por meio de marcadores bioquímicos como a glicose, o colesterol, triglicerídeos, as transaminases glucâmica- oxalacética (TGO) e glutâmico pirúvica (TGP), albumina, proteínas totais e gama glutamil transferase (γ-GT). Quando a degeneração gordurosa atinge mais de 5% dos hepatócitos, é diagnosticado o quadro de esteatose hepática 12. De acordo com Tannapfel et al. (2011), o acúmulo de lipídios tem um padrão microvesicular no início da doença, comprometendo os hepatócitos da zona 1 (ou periportal) do lóbulo hepático, mas tende a evoluir a um padrão macrovesicular em resposta à contínua β-oxidação dos ácidos graxos, resultando em hepatócidos balonizados na zona 3 (ou centrolobular) do lóbulo nos estágios mais avançados da doença. Na esteato-hepatite, além dos vacúolos lipídicos, ocorre infiltrado inflamatório, que frequentemente encontra-se próximo aos hepatócitos balonizados. Fibrose periportal com consequente obliteração e necrose vascular com degeneração hialina também são achados comuns na esteato-hepatite alcoólica em seus estágios iniciais, e esse último achado é caracterizado por inclusões citoplasmáticas eosinofílicas originados de proteínas como a queratina, chaperonas e outras proteínas relacionadas à maquinaria de degradação proteica, criando estruturas conhecidas como ―corpos de Mallory-Denk‖13. As Células de Kupffer (CK), juntamente com as células estreladas hepáticas (CEH),desempenham um papel central na esteato-hepatite alcoólica. O álcool aumenta a permeabilidade intestinal, levando a um quadro de endotoxemia decorrente do lipopolissacarídio (LPS) da microbiota14. O LPS estimula a produção de citocinas pró-inflamatórias por meio da interação com o receptor CD14 das CK e ativação do Fator Nuclear-κB (NF-κB) 15. Dentre as citocinas produzidas pelas CK em resposta ao LPS estão o fator de necrose tumoral-α (TNF-α) e a interleucina 1β (IL-1β) 15, 16. Essas citocinas atuam sobre as CEH, que também respondem ao LPS por meio da interação deste com o receptor TLR4. Nas CEH, a ligação do LPS ao TLR4 leva também à ativação do NF-κB, que inibe a expressão do pseudoreceptor BAMBI que, em circunstâncias normais, regula a atividade do receptor TGF-βR1. A ativação do NF-κB também leva à liberação de quimiocinas por parte das CEH, que atraem as CK que, em resposta, liberam o fator de crescimento transformador beta (TGF-β), que induz uma alteração no fenótimo das CEH, fanzendo com que elas 14 tornem-se ativas e passem a produzir colágenos dos tipos I e III (PC-I e PC-III)16. Dessa forma, o processo inflamatório hepático desencadeado pelo álcool está diretamente relacionado à fibrose, que progride enquanto a exposição ao álcool durar17, 18. O Carvedilol é um bloqueador não seletivo dos receptores α1,β1 e β2 que tem se mostrado melhor do que beta-bloqueadores tradicionais em virtude de suas ações antioxidantes e anti-fibróticas em estudos envolvendo doenças cardiovasculares19-22. O grupo carbazol presente na molécula de Carvedilol confere a ele a sua ação antioxidante, capaz de neutralizar radicais de oxigênio, protegendo a membrana das células cardíacas contra a peroxidação lipídica23. Além de doenças cardiovasculares, o Carvedilol tem mostrado efeitos imuno- reguladores e antioxidantes em vários modelos de doenças inflamatórias. Em miocardite viral induzida em ratos, o Carvedilol diminuiu os níveis de citocinas pró- inflamatórias e aumentou a expressão de citocinas anti-inflamatórias24. Em um modelo de artrite, o Caverdilol diminuiu o estresse oxidativo e a peroxidação lipídica, além de suprimir citocinas pró-inflamatórias (TNF- α e IL-6)25. O Carvedilol também demonstrou atividades anti-fibróticas e antioxidantes em modelos de injúria hepática por tetracloreto de carbono 26, e é capaz de reduzir os efeitos de mediadores de células estreladas responsáveis pela lipogênese e fibrogênese27, tendo sido eficaz, também, em diminuir a resistência à insulina e a fibrose induzidas pelo álcool ao regular a atividade sináptica de fibras simpáticas 28- 30. Por conta disso, decidimos usar esse fármaco para estudar os mecanismos pelos quais ele atua para desempenhar sua ação hepatoprotetora em ratos submetidos ao modelo crônico de DHA. 15 2. JUSTIFICATIVA Entre as drogas de abuso, o álcool é a substância usada mais largamente, e seu uso já corresponde a 95% dos resultados de morbidez e mortalidade relatados31, 32. Assim, a ingestão excessiva de álcool também é considerada um grande problema de saúde pública, pois não somente adultos, como também adolescentes, têm sido cada vez mais afetados pelo uso da substância, causando danos físicos e mentais a esses usuários32. Além de suas consequências relacionadas à DHA, o alcoolismo crônico é considerado a segunda principal causa de demência, estando atrás apenas da doença de Alzheimer, e cerca de 50-75% dos consumidores crônicos dessa droga podem apresentar déficits colinérgicos, cognitivos e motores33, 34. De acordo com a Associação Americana para o Estudo de Doenças Hepáticas (AASLD), a pentoxifilina pode ser uma alternativa ao uso de corticoesteróides, já que tem se mostrado efetiva em pacientes com DHA e disfunção hepato-renal. Entretanto, biofármacos anti-TNFs, por exemplo, não têm demonstrado os resultados confiáveis35. A Naltrexona e o Acamprosato têm sido eficazes em diminuir a ingestão de álcool em etilistas crônicos36. O Disulfiram, embora seus reais efeitos sejam controversos37, tem sido usado no tratamento do alcoolismo e, Segundo alguns estudos, esse fármaco, sob administração supervisionada, pode favorecer a recuperação de pacientes etilistas38. O Topiramato tem se mostrado seguro e eficaz em diversos ensaios clínicos ao diminuir tanto o anseio quanto os sintomas de abstinência, melhorando a qualidade de vida de indivíduos etilistas crônicos39. Além desses medicamentos, o baclofeno tem se mostrado eficaz em promover a abstinência em pacientes com cirrose hepática alcoólica40. Apesar dos avanços na compreensão da patogênese da DHA e da descoberta de fármacos que possam auxiliar no tratamento, não há terapia farmacológica específica registrada em órgãos reguladores de saúde, e mesmo os efeitos dos fármacos aqui citados são controversos. Dessa forma, entender os mecanismos por trás dos efeitos pleiotrópicos de fármacos potencialmente benéficos para pacientes com DHA pode ser o primeiro passo na direção de novas abordagens e tecnologias visando a melhor recuperação de pacientes etilistas. 16 3. OBJETIVOS 3.1. Objetivo geral Caracterizar, por meio da análise de biomarcadores, a atividade anti- inflamatória, antioxidante e anti-fibrótica do Carvedilol em modelo de doença hepática alcoólica induzida em ratos. 3.2. Objetivos específicos 3.1.1 Determinar as concentrações séricas de AST, ALT e Triglicerídeos pelo método colorimétrico; 3.1.2 Determinar o grau de infiltração leucocitária no tecido hepático por meio da análise colorimétrica da mieloperoxidase (MPO); 3.1.3 Determinar o grau de peroxidação lipídica por meio da dosagem de malonaldeído (MDA) no tecido hepático; 3.1.4 Avaliar a ação antioxidante do carvedilol e o efeito oxidativo do álcool por meio da dosagem colorimétrica de glutationa reduzida (GSH); 3.1.5 Avaliar a ação anti-inflamatória do carvedilol por meio da análise por ELISA de IL-1β, TNF-α e IL-10 no tecido hepático; 3.1.6 Avaliar,por meio da análise histológica com H&E, a capacidade do carvedilol em reverter a esteatose; 3.1.7 Avaliar, por meio das técnicas de Picro-Sírius e RT-qPCR para os genes Pró-Colágeno I e III, o efeito anti-fibrótico do carvedilol; 3.1.8 Descrever, utilizando as técnicas de imuno-histoquímica, imunofluorescência e RT-qPCR, o papel das Células de Kupffer e das Células Estreladas na fisiopatologia da esteato-hepatite alcoólica e como essas células são moduladas pelo Carvedilol. 17 4. METODOLOGIA 4.1. Preparação e administração de etanol Para as gavagens, foi feita uma solução alcoólica a 30% v/v (7g/kg). Foram dissolvidos 30g de etanol P.A. em água destilada até completarem-se 100 mL. Foram administrados, via gavagem, 6,2mL da solução alcoólica preparada para cada animal diariamente. 4.2. Manejo dos animais Os experimentos foram realizados em ratos Wistar albinos que pesavam entre 250 e 300g. Os animais foram alojados num ambiente de temperatura e humidade controladas sob um ciclo claro/escuro de 12h (luzes acesas às 06:00). A comida e água estavam disponíveis ad libitum. Os animais usados nas experiências originavam do Departamento de Biofísica e farmacologia e foram alojados individualmente em gaiolas de polipropileno de 41x34x16cm e autoclavadas. Vários sinais da saúde dos animais foram monitorados, incluindo: condição de pelagem, resposta a estímulos, fezes e urina. A dieta padrão (Composição Básica: farinha de soja, dextrina, cascas de arroz, farelo de trigo, farelo de arroz, farinha de carne, farinha de peixe, cloreto de sódio, óxido de magnésio, sulfato de ferro, sulfato de cobre, monóxido de manganês, óxido de zinco, iodato de cálcio, sulfato de cobalto, selenito de sódio, vitamina A, vitamin D3, vitamina E, vitaminK3, vitamina B1, vitaminB2, niacina,ácido pantoténico, vitamina B6, o ácido fólico, biotina, vitamina B12, cloreto de colina, lisina, metionina, ácido propiónico, Agrobacterium tumefaciens, e Bacillus thuringiensis; Presence/ Evialis do Brasil Nutrição animal LTDA, São Paulo) e fonte de água (engarrafada). 4.3. Indução da lesão hepática por etanol Para esse estudo, foram separados 5 grupos de animais: controle negativo (n=5), controle positivo (n=5), grupo Carvedilol 1mg/kg (n=10), Carvedilol 3mg/kg 18 (n=10) e Carvedilol 5mg/kg (n=10), de tal forma que quarenta ratos* machos com 7 semanas de idade foram divididos aleatoriamente entre os 5 grupos. O grupo controle negativo recebeu solução salina (0,9% NaCl) por gavagem oral durante 28 dias. Nesse grupo, eram realizadas duas gavagens diárias a fim de emular o tratamento com carvedilol e a administração do álcool dos outros grupos. Dessa forma, 1mL da solução salina (referente ao tratamento com carvedilol) era administrado diariamente sempre 1h antes da segunda gavagem, de 6,1mL de solução salina, referente à administração do álcool. O grupo controle positivo recebeu 1mL de solução salina por gavagem oral e, após 1h, 6,2mL de etanol (7g/kg) também por gavagem durante os 28 dias. Os grupos tratados com Carvedilol (1mg/kg, 3mg/kg e 5mg/kg) receberam, cada um, 1mL da suspensão de carvedilol em água destilada na concentração respectiva a cada grupo. Para a preparação dessa suspensão, foram utilizados comprimidos de 6,25mg de Carvedilol , que foram triturados e suspendidos em água destilada. Essa mistura era preparada diariamente e agitada antes de cada gavagem, uma vez que o carvedilol não é dissolvido em água (Figura 1). A eutanásia dos animais foi feita no vigésimo nono dia com tiopental a 2% (80mg/kg, i.p.) (Cristália, São Paulo, Brasil). Enquanto os animais estavam anestesiados, uma punção cardíaca foi realizada e amostras de sangue foram coletadas para leucograma e análises bioquímicas. Os fígados dos ratos foram congelados em freezer -80°C para análise dos níveis de mieloperoxidase (MPO), malonaldeído (MDA), glutationa reduzida (GSH) e citocinas. Para o exame histopatológico, fragmentos do fígado extraído de cada animal foram imersos em formalina tamponada a 10%. Todas as metodologias desse estudo foram submetidas à análise da Comissão de Ética no Uso de Animais e foram aprovadas (número de aprovação: 053/2013). Os animais foram monitorizados após a indução da lesão hepática induzida por etanol uma vez por semana durante 28 dias. Não havia nenhuma mortalidade acidental como resultado de lesão hepática. * Após a solicitação da revista para a execução da qPCR, outros animais tiveram que ser usados, uma vez que não tínhamos amostras suficientes para a análise requisitada. Por isso, no artigo registramos que, ao todo, foram usados 80 animais. 19 Figura 1 - Divisão dos grupos experimentais e suas respectivas gavagens ao longo dos 28 dias. Mieloperoxidase (MPO) A extensão da acumulação de neutrófilos nas amostras de fígado foi medida pelo ensaio de atividade de MPO. As amostras de fígado foram colhidas tal como descrito acima e armazenados a -70 ° C até serem utilizadas para o ensaio. Após a homogeneização e centrifugação (2000 x g durante 20 min), a atividade de MPO foi determinada por um método colorimétrico descrito por Souza et al. 2003. Os resultados são apresentados como unidades de MPO por grama de tecido41. A curva padrão foi calculada tendo y = 0,0011x - 0,0005 e R² = 0,9996. 4.4. Malonaldeído (MDA) O Malonaldeído (MDA) é um produto final da peroxidação lipídica. Para quantificar o aumento de radicais livres na amostra de fígado, o teor de MDA foi medido através do ensaio descrito por Esterbauer e Cheeseman (1990). As amostras de fígado foram suspensas em tampão Tris HCl 1:5 (w/v) e picadas com tesouras por 15 segundos sobre uma placa com gelo. A suspensão resultante foi homogeneizada durante 2 minutos com um homogeneizador Potter automático e centrifugada a 2500 x g a 4°C durante 10 min42. Os sobrenadantes 20 foram testados para determinar o teor de MDA. Os resultados são expressos como nM de MDA/grama de tecido. 4.5. Glutationa Reduzida (GSH) Os níveis de GSH nos tecidos hepáticos foram medidos para se avaliar a ação antioxidante. A quantidade de GSH foi medida através do ensaio descrito por Costa et al. 2006. As amostras de fígado foram armazenadas em -70°C até a sua utilização. Para esse ensaio, os homogenatos de tecido hepático (0,25mL de uma solução de tecido a 5% preparada em EDTA 0,02 M) foram adicionados a 320mL de água destilada e 80mL de TCA a 50%. As amostras foram centrifugadas a 3000 rpm durante 15 minutos a 4°C. O sobrenadante (400 mL) foi adicionado a 800 ml de tampão 0,4 MTris a pH 8,9 e 20 uL de DTNB 0,01M. A absorbância de cada amostra foi medida a 420 nm, e os resultados foram registados como unidades de GSH por miligrama de tecido43. 4.6. IL-1β, IL-10, e ensaio de TNF-α As amostras de fígado (três amostras por grupo) foram armazenadas em freezer -70°C até a sua utilização. O tecido foi homogeneizado e processado tal como descrito por Safieh-Garabedian et al. 1995. Os níveis de IL-1β, IL-10 e TNF-α nas amostras de fígado foram determinadas com um kit comercial de ELISA (Sistemas R&D, Minneapolis, MN, EUA). Todas as amostras estavam dentro do comprimento de onda utilizado no espectrofotômetro de UV-VIS (absorbância medida a 490nm) 44. O intervalo de detecção para IL-1β, IL-10 e TNF-α foi 62,5- 4000pg/ml e os limites de detecção foram 12,5ng/ml para IL-1β e IL-10 e 50ng/mL para TNF-α. 4.7. Estudos de microscopia óptica e análise bioquímica Os fígados foram retirados rapidamente e lavados com solução salina isotônica. Cada seguimento hepático foi pesado e cortado longitudinalmente. Três segmentos do fígado foram analisados (cinco animais por grupo). Os espécimes foram fixados em formol tamponado a 10%, desidratados e emblocados em parafina. Cortes de 5µm de espessura foram realizados para a 21 coloração com hematoxilina e eosina (H&E) e examinados por microscopia óptica (40x, Olympus BX50, Departamento de Morfologia/UFRN). A análise histopatológica foi feita de acordo com os seguintes escores: esteatose (a porcentagem de hepatócitos com vacúolos lipídicos): <25% = 1, <50% = 2, <75% = 3, >75% = 4; inflamação e necrose: 1 para um foco por campo; 2 para dois ou mais focos. A análise histopatológica foi feita de forma duplo-cega por um dos autores e por um pesquisador externo. O número de neutrófilos em cada corte hepático foi determinado ao se fazer a contagem de células em três campos por lâmina em grande aumento (400x). O acúmulo de lipídios causa a balonização dos hepatócitos e o estreitamento do espaço sinusoidal. Isso pode afetar o número de hepatócitos e espaços sinusoidais por campo; assim, o número de hepatócitos também foi contado e o número de neutrófilos foi expresso por neutrófilos/100 hepatócitos. A média dos valores foi utilizada para a análise estatística45, 46. Os cortes histológicos foram corados utilizando hematoxilina e eosina (EasyPath) e com o kit Picro-Sirius Red (1% de Sirius Red em ácido pícrico saturado; EasyPath, Indaiatuba, Brasil) por 1h, e examinadas sob microscopia de luz (Nikon Eclipse 2000 equipado com Nikon DS-Fi2; Nikon Corporation, Tóquio, Japão). Para fins de análise quantitativa do colágeno, duzentas imagens de microscopia óptica (200X) foram aleatoriamente selecionadas por amostra de fígado, incluindo áreas de grandes veias centrolobulares e grandes extensões portais (≥150mm). Cerca de 20 imagens usando microscopia de luz polarizada no microscópio Olympus BX60 (Olympus, Tóquio, Japão) (200X) foram capturadas por cada espécime e analisadas utilizando um sistema de detecção de cores com limiar desenvolvido no Image J (National Institutes of Health). Controles positivos e negativos conhecidos foram incluídos em cada lote de amostras. A reação tecidual em todos os grupos foi avaliada. Os valores foram expressos como percentagem da área positiva. As medições de índice de contraste foram obtidas a partir da área x 100/área total selecionada posicionada entre as regiões de interesse (três amostras por animal). A fibrose hepática foi quantificada utilizando o escore Ishak: Nível 1 indicando ausência de fibrose; nível 2 indicando alargamento da área portal; nível 3 foi atribuído à expansão da fibrose para a maioria dos espaços porta; nível 4 foi 22 atribuído a lóbulos com expansão fibrótica na maioria das áreas porta com ocasional ligação fibrótica entre espaços porta; nível 5 foi atribuído a lóbulos com expansão fibrótica na maior parte dos espaços porta com ponte fibrótica acentuada (espaço porta-espaço porta e espaço porta-veia centrolobular); nível 6 foi designado para lóbulos com pontes fibróticas acentuadas (espaço porta- espaço porta e espaço porta-veia centrolobular) e com formação de nódulos fibróticos ocasionais (cirrose incompleta); nível 7 caracteriza a Cirrose nos lóbulos47. Para as análises bioquímicas, foram feitos testes para os marcadores de lesão hepática alanina aminotransferase (ALT/GPT), aspartato aminotransferase (AST / GOT) e triglicerídeos (TG), todos feitos em kits de ensaio da marca Gold Analisa Diagnóstica Ltta, seguindo as recomendações do fabricante. As amostras foram centrifugadas a 2.500 rpm durante 10 minutos a temperatura ambiente para a separação do soro. Todos os testes bioquímicos foram realizados usando o analisador bioquímico semi-automático BIOPLUS BIO-2000. 4.8. Marcação imuno-histoquímica para COX-2, RANK, RANKL, IBA-1, ICAM- 1, SOCS-1, SOD-1 e GPx-1. Cortes finos de fígado (4 uM) de cada grupo foram feitos com um micrótomo e transferidos para lâminas silanizadas. Cada corte de tecido foi então desparafinizado e reidratado. Os cortes de tecido de fígado em lâminas silanizadas foram lavados com Triton X-100 0,3%, tampão de fosfato (PBS) e com peróxido de hidrogênio a 3% para bloqueio da peroxidase endógena. Os cortes foram incubados overnight a 4ºC com os anticorpos primários (Santa Cruz Biotechnology, INTERPRISE, Brazil) anti-COX-2, RANK, RANKL, SOCS-1,SOD- 1, GPx-1 e anticorpos primários (Spring-ABCAM, USA) anti-IBA-1 e anti-ICAM- 1.Testes de diluição foram realizados (três diluições) e as diluições de 1:600, 1:800, 1:1000, 1:500, 1:800, 1:600,1:300 e 1:500 foram consideradas as mais apropriadas para COX-2, RANK, RANKL, SOCS-1,SOD-1, GPx-1, IBA-1 e ICAM-1,respectivamente. Os cortes foram lavados com PBS e incubados com o anticorpo secundário conjugado a estreptoavidina/HRP (Biocare Medical, Concord, CA, USA) por 30 minutos. Os cortes foram contra-corados com hematoxilina. Controles positivos conhecidos e controles negativos para a marcação imuno-histoquímica foram incluídos na análise. 23 A microscopia com planimetria (NikonE200LED, Departamento de Morfologia/ UFRN) com um objetiva de maior aumento (40x) foi utilizada para determinar a intensidade da imunomarcação celular: 1 = ausência de células positivas; 2 = número reduzido de células positivas ou células isoladas; 3 = número moderado de células positivas; e 4 = grande número de células positivas. A intensidade da marcação foi avaliada por dois examinadores previamente treinados e de forma duplo-cega. Foram avaliados cinco cortes de tecido por animal (três animais por grupo). 4.9. Análise de imunofluorescência confocal Os cortes de tecido foram desparafinados com xilol e lavados em várias concentrações de etanol e em PBS. A recuperação antigênica foi feita com solução de citrato de sódio 10mM e 0,05% de Tween 20 durante 40 minutos a 95º C. O Sudan-Black a 0,1% foi diluído em etanol a 70% durante 40 minutos a temperatura ambiente (TA) e foi utilizado para reduzir a autofluorescência do tecido. Os cortes foram incubados durante a noite com o anticorpo primário de coelho anti-IL-1 (1:200 diluído em solução bloqueadora Dako, Brasil EUA) e, em seguida, lavados três vezes em PBS e Triton X-100 a 0,2% durante 5 minutos para finalmente incubar o anticorpo secundário de cabra anti-coelho conjugado com Alexa Fluor 488 (1: 700 em solução bloqueadora, Dako, Brasil EUA). Finalmente, as lamínulas foram aplicadas utilizando meio de montagem Vectashield com DAPI. As imagens da imunofluorescência foram obtidas em um microscópio Carl Zeiss Laser Scanning (LSM 710, objetiva 20x, Carl Zeiss, Jena, Alemanha). Controles positivos e negativos conhecidos para a marcação imunofluorescente foram incluídos. A reatividade do tecido em todos os grupos foi avaliada por análise de densitometria computadorizada das imagens digitais captadas com o referido microscópio de imunofluorescência confocal. A média dos valores densitométricos foram calculadas no software Imagem J (http://rsb.info.nih.gov/ij/). As medições do índice de contraste foram feitas a partir da fórmula (área selecionada x 100)/área total do fundo após a remoção de regiões de interesse (três amostras por animal). 4.10. PCR quantitativa em tempo real para TNFα, PCI, PCIII e NF-κB O RNA total foi extraído do tecido hepático utilizando o reagente TRIzol (Invitrogen Co. USA) e o Sistema de isolamento de RNA total (Promega, Madison, 24 WI). A fita de cDNA foi sintetizada a partir de 1μg do RNA total com o ―ImProm-IITM Reverse Transcriptase System‖ para qPCR (Promega) seguindo as recomendações do fabricante. A qPCR dos mRNAs de TNF-α, PCI, PCIII e GAPDH foram realizadas com SYBR Green Mix no Applied Biosystems® 7500 FAST system (Applied Biosystems, Foster City, CA), de acordo com o protocolo padrão dos seguintes primers: GAPDH (senso: 5’ AAC TTT GGC ATC GTG GAA GG 3’; anti-senso: 5’ GTG GAT GCA GGG ATG ATG TTC 3’, temperatura de anelamento do primer: 60ºC), TNF-α (senso: 5’ AGT CCG GGC AGG TCT ACT TT3’;anti-senso: 5’ TTC AGC GTC TCG TGT GTT TC 3’, temperatura de anelamento do primer: 56.5°C), PCI (senso:5’ CAG GGA GTA AGG GAC ACG AA 3’;anti-senso: 5’TCC CAC AGC AGT TAG GAA CC 3’, temperatura de anelamento do primer: 56.8°C), PCIII (senso: 5’ ATG GTG GCT TTC AGT TCA GC 3’;anti-senso: 5’ TGG GGT TTC AGA GAG TTT GG 3’, temperatura de anelamento do primer: 55.2°C), and NF-κB (senso: 5’ TCT GCT TCC AGG TGA CAG TG 3;anti-senso: 5’ ATC TTG AGC TCG GCA GTG TT 3’, temperatura de anelamento do primer: 55.2°C. As condições padrões para a PCR foram: 50°C por 2 min e 95°C por 10 min, seguidos por quarenta ciclos de 30s a 94°C, temperatura de anelamento variável do primer por 30s, e 72°C por 1 min. Os experimentos foram realizados em triplicata e repetidos três vezes. Os valores CT da media foram usados para se calcular a níveis de expressão dos genes alvos relativos aos grupos experimentais e ao controle negativo; os dados de expressão relativos ao gene GAPDH foram normalizados através da fórmula 2–ΔΔCt. 4.11. Estatística Para as análises paramétricas foi utilizada a Anlálise de Variância (ANOVA) com post-hoc Bonferroni e, para os escores, foi feito o teste de Kruskal-Wallis com post-hoc de Dunn para comparar medianas no softwareGraphPad Prism 5.0 (La Jolla, CA, USA). Valores de p menores que 0,05 foram considerados estatisticamente significativos. 25 5. PUBLICAÇÕES O artigo ―Carvedilol Improves Inflammatory Response, Oxidative Stress and Fibrosis in the Alcohol-Induced Liver Injury in Rats by Regulating Kuppfer Cells and Hepatic Stellate Cells‖foi aceito para publicação no periodic PLos One que possui fator de impacto 3,41e é classificado no sistema Qualis-Capes como A2 para a área de Medicina II.DOI:10.1371/journal.pone.0148868 26 Carvedilol Improves Inflammatory Response, Oxidative Stress and Fibrosis in the Alcohol-Induced Liver Injury in Rats by Regulating Kuppfer Cells and Hepatic Stellate Cells Raimundo Fernandes de Araújo Júnior1,2,+*, Vinícius Barreto Garcia1,+, Renata Ferreira de Carvalho Leitão3, Gerly Anne de Castro Brito3, Emilio de Castro Miguel4, Paulo Marcos Matta Guedes5, Aurigena Antunes de Araújo6 1 Postgraduate Program in Health Science, UFRN, Natal, RN, Brazil; 2 Postgraduate Program in Functional and Structural Biology/Department of Morphology/UFRN, Natal, RN, Brazil; 3 Department of Morphology/ Postgraduate Program in Morphology/UFC, Fortaleza, CE, Brazil; 4 Department of Physical/Analytical Center/UFC, Fortaleza, CE, Brazil; 5 Department of Microbiology and Parasitology, UFRN, Natal, RN, Brazil; 6 Department of Biophysics and Pharmacology, UFRN, Postgraduate Programs in Public Health and Pharmaceutical Science, Natal, RN, Brazil; + Both authors contributed equally to the preparation of the manuscript. *Corresponding author: Raimundo Fernandes de Araújo Jr. Departamento de Morfologia, Centro de Biociências/UFRN, Av. Senador Salgado Filho, S/N, Campus Universitário, Lagoa Nova, 59072-970, Natal, RN, Brazil. E-mail: araujojr@cb.ufrn.br 27 ABSTRACT Aim:To evaluate the anti-inflammatory, anti-oxidant and antifibrotic effects of carvedilol (CARV) in rats with ethanol-induced liver injury.Methods:Liver injury was induced by gavage administration of alcohol (7 g/kg) for 28 consecutive days. Eighty Wistar rats were pretreated with oral CARV at 1, 3, or 5 mg/kg or with saline 1 h before exposure to alcohol. Liver homogenates were assayed for interleukin (IL)-1β, IL-10, and tumor necrosis factor (TNF)-α level as well as for myeloperoxidase (MPO) activity and malonyldialdehyde (MDA) and glutathione (GSH) levels. Serum aspartate aminotransferase (AST) activity and liver triglyceride (TG) levels were also assayed. Immunohistochemical analyses of cyclooxygenase 2 (COX-2), receptor activator of nuclear factor kappa-B/ ligand (RANK/RANKL), suppressor of cytokine signalling (SOCS1), the Kupffer cell marker IBA-1 (ionized calcium-binding adaptor molecule 1), intercellular adhesion molecule 1 (ICAM-1), superoxide dismutase (SOD-1), and glutathione peroxidase (GPx-1) expression were performed. Confocal microscopy analysis of IL-1β and NF-κB expression and real-time quantitative PCR analysis for TNFα, PCI, PCIII, and NF-κB were performed.Results:CARV treatment (5 mg/kg) during the alcohol exposure protocol was associated with reduced steatosis, hepatic cord degeneration, fibrosis and necrosis, as well as reduced levels of AST (p < 0.01), ALT (p < 0.01), TG (p < 0.001), MPO (p < 0.001), MDA (p < 0.05), and proinflammatory cytokines (IL-1β and TNF-α, both p < 0.05), and increased levels of the anti-inflammatory cytokine IL-10 (p < 0.001) and GSH (p < 0.05), compared to the alcohol-only group. Treatment with CARV 5 mg/kg also reduced expression levels of COX-2, RANK, RANKL, IBA-1, and ICAM-1 (all p < 0.05), while increasing expression of SOCS1, SOD-1, and GPx-1 (all p < 0.05) and decreasing expression of IL-1β and NF-κB (both, p <0.05). Real-time quantitative PCR analysis showed that mRNA production of TNF-α, procollagen type I (PCI), procollagen type III (PCIII), and NF-κB were decreased in the alcohol-CARV 5 mg/kg group relative to the alcohol- only group.Conclusions:CARV can reduce the stress oxidative, inflammatory response and fibrosis in ethanol-induced liver injury in a rat model by downregulating signalling of Kuppfer cells and hepatic stellate cells (HSCs) through suppression of inflammatory cytokines. Key words:ethanol-induced liver injury model; carvedilol; inflammation; 28 INTRODUCTION Alcohol-induced liver disease (ALD) has a wide range of presentations, ranging from simple steatosis to cirrhosis and hepatocellular carcinoma. ALD continues to be a major health issue worldwide [1]. Chronic ethanol administration to rodents leads to numerous hepatic changes, including steatosis, hepatocellular necrosis, inflammatory cell infiltration, terminal hepatic venular sclerosis, proliferation of the smooth endoplasmic reticulum, and mitochondrial aberrations. All of these changes also occur during the early phases of human ALD, demonstrating the relevance of rodent ALD models [2]. Many histological abnormalities associated with ethanol-induced steatosis are particularly prevalent in the perivenular region of the liver lobules, due to the lower oxygen concentration in this region and the increased susceptibility of perivenous cells to ischemic necrosis. Low-oxygen conditions that are sufficiently hypoxic to damage hepatocytes may develop in this area after alcohol consumption [3]. Another factor that may contribute to ethanol-induced injury in the perivenular region is the high regional expression of CYP2E1, which has been associated with the production of potentially harmful oxygen radicals [4]. Furthermore, hepatocytes in the perivenous area contain relatively low levels of antioxidant factors [e.g., glutathione (GSH)] and antioxidant enzymes [e.g., glutathione peroxidase (GPx-1)] [5]. The most convincing data indicating that oxidative stress contributes to alcohol-induced liver injury come from studies using the intragastric infusion model of alcohol administration. In these studies, alcohol-induced liver injury was associated with enhanced lipid peroxidation, protein carbonyl formation, formation of the 1-hydroxyl ethyl radical, formation of lipid radicals, and decreases in hepatic antioxidant defences, especially GSH. Furthermore, the small Kupffer cells in the perivenous area produce high levels of cytotoxicity-associated cytokines [6]. Alcohol intake increases intestinal permeability to various substances, including bacterial endotoxins, such as lipopolysaccharide [7]. Lipopolysaccharide sensitizes Kupffer cells by binding to the CD14 receptor, thereby activating nuclear factor (NF)-κB, which increases the transcription of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and transforming growth factor (TGF)-β. TNF-α and IL-6 play important roles in cholestasis and the synthesis of 29 acute-phase proteins. Meanwhile, TGF-β plays a central role in fibrogenesis through the activation of hepatic stellate cells (HSCs), a process of fibrosis that is associated with necro-inflammation and apoptosis, and which leads to the progression of liver disease and, ultimately, cirrhosis. The suppressor of cytokine signalling (SOCS) family consists of SOCS1–7 and cytokine-inducible SH2-containing protein. Members of this protein family mediate negative feedback regulation of the cytokine-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal transduction pathway [8]. SOCS1 has been reported to inhibit inflammation [9], and cytokines whose expression is induced by inflammation have been shown to upregulate SOCS1 expression [10]. Under normal conditions, HSCs are quiescent and produce only small amounts of extracellular membrane (ECM) constituents, such as laminin and collagen type IV, which are essential components of basement membranes [11]. Upon exposure to soluble factors from damaged hepatocytes or activated Kupffer cells, HSCs reduce their lipid content (retinyl palmitate), and undergo a morphological transition to myofibroblast-like cells (11). Activated HSCs then pro- duce large amounts of ECM components, including collagen I, in an accelerated fashion, triggering a fibrogenic response [12]. Carvedilol (CARV) blocks sympathetic neural activation via antagonism of β1- , β2-, and α1-adrenoreceptors. CARV has been shown to provide greater cardiovascular benefits thantraditional β-blockers in both humans and animals, and these benefits have been attributed to its antioxidant, anti-inflammatory, and antifibrotic properties [13,14]. CARV has been shown to contribute to the attenuation of alcoholic fatty liver disease development in rats and appears to improve ethanol- induced liver injury by modifying the interaction between oxidative stress and sympathetic hyperactivity [15]. The antifibrotic effects of CARV have been associated with the amelioration of oxidative stress in carbon tetrachloride-induced hepatotoxicity model [16] and suppression of HSC-derived lipogenesis and fibrogenesis-related mediators [17]. The goal of this study was to characterize the anti-inflammatory activity of CARV in a rat model of ethanol-induced liver injury through an analysis of markers of the alcohol-induced inflammatory process. We also examined procollagen type I 30 (PCI) and procollagen type III (PCIII) levels in ethanol-injured livers, with or without CARV, because HSC cells undergo an activation process in response to liver injury, in which they produce a fibrotic matrix rich in PCI and PCIII. MATERIALS AND METHODS Chemicals Ethanol was purchased from LIBBs Farmacêutica Ltda, São Paulo, Brazil. Carvedilol (Ictus 6,25mg, Biolab Sanus Farmacêutica ltda, São Paulo, Brazil), O- Dianisine Sigma (São Paulo, Brazil), antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil): COX-2; RANK; RANKL; SOCS-1, Streptavidin-HRP- conjugated secondary antibody (Biocare Medical, Concord, CA, USA).TrekAvidin- HRP Label + Kit from Biocare Medical, Dako, USA.IL-1β, IL-10, TNF-α ELISA kit (R&D Systems, Minneapolis, MN, USA). Preparation and administration of ethanol 7g per kg body weight of 30% v/v ethanol solution was used as chronic dose in this experiment. 30g absolute ethanol was dissolved in distilled water and made up to 100ml. 6,2ml of the solution was daily administered for four weeks to each rat treated with ethanol. Animal preparation Experiments were performed on male Wistar albino rats weighing between 250 and 300 g, purchased from Bioterio Department of Biophysical and Pharmacology. Animals were housed in a temperature and humidity controlled environment under a 12-h light/dark cycle (lights on at 6 AM). Food and water were available ad libitum. The animals used in the experiments originated from the Department of Biophysics and Pharmacology. The animals were housed individually in polypropylene cages measuring 41 x 34 x 16 cm Autoclaved. Various signals of the health of the animals were monitored, including: coat condition, response to stimuli, faeces and urine. Only those animals in perfect health were kept in the experiment. Standard diet (Basic Composition: Soybean meal, dextrin, rice husks, wheat bran, rice bran, meat meal, fish meal, sodium chloride, magnesium oxide, iron sulfate, copper sulfate, manganese 31 monoxide, zinc oxide, calcium iodate, cobalt sulphate, sodium selenite, vitamin A, vitaminD3, vitamin E, vitaminK3, vitamin B1, vitaminB2, niacin, pantothenic acid, vitaminB6, folic acid, biotin, vita-min B12, choline chloride, lysine, methionine, propionic acid, Agrobacteriumtumefaciens, and Bacillusthuringiensis; Presence/Evialis do Brasil Nutrição Animal LTDA, São Paulo) and watersource (bottled). The National Institutes of Health Guidelines for the Care and Use of Laboratory Animals were followed. All efforts were made to minimize the number of animals used and their suffering degree. Animals were sacrificied on by an overdose of anaesthesia with 2% thiopental (80 mg/kg, i.p.). After death a cardiac puncture was performed. The sacrificed animals were kept in -20°C freezer. Animals were monitored after induction of ethanol induced liver injury for once week once week during for 28 days. There was not any incidental mortality as a result of liver injury. This research and methods used in this investigation were approved by by the Animal Ethics Committee–CEUA- of the Universidade Federal do Rio Grande do Norte (approval number: 053/2013). Induction of ethanol-induced liver injury Eighty rats were randomly divided into eight groups (five animals per group, duplicate groups). The vehicle control group received normal saline orally by gastric gavage and i.p saline (0.9% NaCl) for 28 days. The positive control group received saline orally by gastric gavage and after 01 hour ethanol (7 g/kg) by gastric gavage too for 28 days. Three groups received oral CARV at 1, 3 and 5 mg/kg, respectively, by gastric gavage and after 01 hour saline (0.9% NaCl) by gastric gavage too for 28 days. Three additional groups received oral CARV at 1, 3 and 5 mg/kg by gastric gavage too, respectively, by gastric gavage and after 01 hour ethanol (7 g/kg) for 28 days. Animals were euthanized on the twenty-ninth day with (80 mg/kg, i.p.) 2% thiopental (Cristália, São Paulo, Brazil). Following euthanasia, a cardiac puncture was performed and blood samples were taken for leukogram and biochemical analyses. The livers of the rats were frozen at -80°C for analyses of cytokine and myeloperoxidase (MPO) levels. Livers were immersed in 10% buffered formalin for histopathological analysis. 32 Myeloperoxidase (MPO) Activity The extent of neutrophil accumulation in the Liver samples was measured by assaying MPO activity. Liver samples were harvested as described above and stored at −70°C until required for assay. After homogenisation and centrifugation (2000 × g for 20 min), MPO activity was determined by a previously described colorimetric method [18]. Results are reported as units of MPO per gram of tissue. Malonyldialdehyde (MDA) assay Malonyldialdehyde (MDA) is an end product of lipid peroxidation. To quantify the increase in free radicals in liver sample, MDA content was measured via the assay described by Esterbauer and Cheeseman [19]. Liver samples were suspended in buffer Tris HCl 1:5 (w/v) and minced with scissors for 15 sec on an ice- cold plate. The resulting suspension was homogenised for 2 min with an automatic Potter homogenizer and centrifuged at 2500 × g at 4°C for 10 min. The supernatants were assayed to determine MDA content. The results are expressed as nanomoles of MDA per gram of tissue. Glutathione (GSH) levels GSH levels in liver tissues were measured to antioxidant. GSH content was measured via the assay described by Costa et al [20]. Liver samples (5 per group) were stored at 70°C until use. Liver tissue homogenates (0.25 mL of a 5% tissue solution prepared in 0.02 M EDTA) were added to 320 mL of distilled water and 80 mL of 50% TCA. Samples were centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant (400 mL) was added to 800 mL of 0.4 M Tris buffer at pH 8.9 and 20 μL of 0.01 M DTNB. The absorbance of each sample was measured at 420 nm, and the results were reported as units of GSH per milligram of tissue. IL-1β, IL-10, and TNF-α assay Liver samples (three samples per group) were stored at −70°C until use. The tissue was homogenised and processed as described by Safieh-Garabedian, et al (1995) [21]. Levels of IL-1β (detection range: 62.5–4000 pg/mL; sensitivity or lower limit of detection [LLD]: 12.5 ng/mL of recombinant mouse IL-1β), IL-10 (detection range: 62.5–4000 pg/mL; sensitivity or LLD: 12.5 ng/mL of recombinant mouse IL-10) 33 and TNF-α (detection range: 62.5–4000 pg/mL; sensitivity or LLD: 50 ng/mL of recombinant mouse TNF-α) in the liver samples were determined with a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA). All samples were within the wavelength used in UV-VIS spectrophotometry (absorbance measured at 490 nm). Light microscopy studies and biochemical analysis The livers were excised quickly and washed with cold isotonic saline. Each segment was weighed and cut longitudinally. Three sections of liver (five animals per group) were analyzed. The specimens were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. Sections of 5 μm thickness were obtained for haematoxylin–eosin staining (H&E) and examined by light microscopy (40x, Olympus BX50, Morphology Department/UFRN). Liver pathology was scored as follows: steatosis (the percentage of liver cells containing fat):<25% = 1, < 50% = 2, <75% = 3, >75% = 4.; inflammation and necrosis: 1 focus per low-power field; 2 or more foci. Pathology was scored in a blinded manner by one of the authors and by an out-side expert in rodent liver pathology. Fat accumulation causes ballooning of hepatocytes and narrowing of the sinusoidal space. This could affect the number of hepatocytes and sinusoidal space in each field; therefore, the number of hepatocytes also was counted and the number of neutrophils was expressed per 100 hepatocytes [22,23]. The mean values were used for statistical analysis. Histological sections were stained using hematoxylin and eosin (Easypath) and picrosirius red staining kit (1% Sirius red in saturated picric acid; EasyPath, Indaiatuba, Brazil) for 1h, both examined under light microscopy (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan). For the purpose of Quantitative analysis the collagen content, randomly sampled two hundred light microscope images (200X) per liver specimen, including large centrilobular veins and large portal tracts (150 mm) were analyzed. About 20 polarized light microscopy images using an Olympus BX60 microscope (Olympus, Tokyo, Japan) (200X) per specimen were captured and analyzed using a color threshold detection system developed in ImageJ (National Institutes of Health). Known positive and negative controls were included in each batch of samples. Tissue reactivity in all groups (negative control, alcohol group and alcohol carv 5) was assessed. Values are expressed as percentage of positive area. Contrast index 34 measurements were obtained from selected area x 100/ total area positioned across the regions of interest (three samples per animal). Moreover, hepatic fibrosis was quantified using Ishak scoring system: level 1 indicating the absence of fibrosis; level 2 indicated enlargement of portal area; level 3 was assigned to fibrous expansion of most portal areas; level 4 was assigned to lobules with fibrous expansion of most portal areas with occasional portal to portal bridging; level 5 was assigned to lobules with fibrous expansion of most portal areas with marked bridging (portal to portal and portal to central); level 6 was assigned to lobules with marked bridging (portal to portal and portal to central) with occasional nodules (incomplete cirrhosis); level 7 was observed Cirrhosis in the lobules [24]. Blood samples were collected by cardiac puncture after the euthanasia on the 29th day of the experiment. For biochemical analysis, tests of serum markers of injury and hepatic functionality as alanine aminotransferase (ALT / GPT), aspartate aminotransferase (AST / GOT) and triglycerides (TG) were conducted in the assay kits from Gold Analisa Diagnóstica Ltta, following all the manufacturer's recommendations. The samples were centrifuged at 2,500 rpm for 10 minutes at room temperature to separate the serum. All biochemical tests were performed using the semi-automatic biochemical analyzer BIOPLUS BIO-2000. Immunohistochemical staining of COX-2, RANK, RANKL, IBA-1, ICAM-I, SOCS-1, SOD-1 and GPx-1 Thin sections of liver (4 μm) were obtained from each group (negative control, alcohol and alcohol CARV 5 mg/kg) with a microtome and transferred to gelatine- coated slides. Each tissue section was then deparaffinised and rehydrated. The liver tissue slices were washed with 0.3% Triton X-100 in phosphate buffer (PB) and quenched with endogenous peroxidase (3% hydro-gen peroxide). Tissue sections were incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil) against COX-2, RANK, RANKL, SOCS-1, SOD-1 GPx-1 and primary antibodies (Spring-ABCAM, USA) against IBA-1 and ICAM-I. Dilution tests (3 dilutions) were performed with all antibodies to identify the 1:600, 1:800, 1:1000, 1:500, 1:800, 1:600 and 1:300, 1:500, dilutions as appropriate, respectively. Slices were washed with phosphate buffer and incubated with a streptavidin/HRP-conjugated secondary antibody (Bio-care Medical, Concord, 35 CA, USA) for 30 minutes. Immunoreactivity to the various proteins was visualised with a colorimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA). Sections were counter-stained with hematoxylin. Known positive controls and negative controls were included in each set of samples. Planimetry microscopy (Nikon E200 LED, Morphology Department/UFRN) with a high-power objective (40×) was utilised to score the intensity of cell immunostaining: 1 = absence of positive cells; 2 = small number of positive cells or isolated cells; 3 = moderate number of positive cells; and 4 = large number of positive cells. Labelling intensity was evaluated by two previously trained examiners in a double-blind fashion. Three tissue sections per animal (five animals per group) were evaluated. Confocal immunofluorescence Three tissue sections from each animal (5 animals per group) were deparaffinized in xylene and washed in a series of concentrations of ethanol and PBS. Antigen retrieval was performed by placing the sections in a 10 mM sodium citrate with 0.05% Tween 20 for 40 minutes at 95°C. Autofluorescence background noise was reduced by incubating the sections in 0.1% Sudan black in 70% ethanol for 40 min at Room Temperature (RT). The sections were incu-bated overnight with rabbit anti-IL-1β and NF-κB primary antibody (1:200 and 1:100, respectively, in blocking solution/1% normal goat serum; ABCAM, USA and Santa Cruz Biotechnology, USA, respectively) at 4°C, washed three times in PBS/0.2% triton X- 100 for 5 min and incubated with Alexa Fluor 488- conjugated goat anti-rabbit secondary antibody (1:500 in BSA 1%) and DAPI nuclear counterstain (SIGMA, USA). Finally, the sections were mounted with Vectashield medium. Fluorescent images were obtained on a Carl Zeiss Laser Scanning Microscope (LSM 710, 20× objective, Carl Zeiss, Jena, Germany, Brazil). Known positive and negative controls were included in each batch of samples. Tissue reactivity in all groups (negative control, alcohol, and alcohol carv 5) was assessed by computerised densitometry analysis of digital images captured with the aforementioned confocal immunofluorescence microscope. Average densitometric values were calculated in Image J software (http://rsb.info.nih.gov/ij/). Contrast index 36 measurements were obtained from the formula (selected area × 100)/total area after removal of back-ground in regions of interest (three samples per animal). RT-PCR quantitation of TNFα, PCI, PCIII, and NF-κB gene expression Total RNA was extracted from liver tissue with TRIzol reagent (Invitrogen Co. USA) and the SV Total RNA Isolation System (Promega, Madison, WI). First-strand cDNA was synthesized from 1 μg of total RNA with the ImProm-IITM Reverse Transcriptase System for RT-PCR (Promega) according to the manufacturer’s protocol. Real-time quantitative PCR analyses of TNF-α, PCI, PCIII, and GAPDH mRNAs were performed with SYBR Green Mix in the Applied Biosystems1 7500 FAST system (Applied Biosystems, Foster City, CA), according to a standard protocol with the following primers: GAPDH (forward: 5’ AAC TTT GGC ATC GTG GAA GG 3’; reverse: 5’ GTG GAT GCA GGG ATG ATG TTC 3’, annealing primer temperature: 60°C), TNFα (forward: 5’ AGT CCG GGC AGG TCT ACT TT3’; reverse: 5’ TTC AGC GTC TCG TGT GTT TC 3’, annealing primer temperature: 56.5°C), PCI (forward:5’ CAG GGA GTA AGG GAC ACG AA 3’; reverse: 5’TCC CAC AGC AGT TAG GAA CC 3’, annealing primer temperature: 56.8°C), PCIII (forward: 5’ ATG GTG GCT TTC AGT TCA GC 3’; reverse: 5’ TGG GGT TTC AGA GAG TTT GG 3’, annealing primer temperature: 55.2°C), and NF-κB (forward: 5’ TCT GCT TCC AGG TGA CAG TG 3; reverse: 5’ ATC TTG AGC TCG GCA GTG TT 3’, annealing primer temperature: 55.2°C. The standard PCR conditions were as follow: 50°C for 2 min and 95°C for 10 min, followed by forty 30-s cycles at 94°C, a variable annealing primer temperature for 30 s, and 72°C for 1 min. The experiments were performed in triplicate and repeated at least three times. Mean Ct values were used to calculate the relative expression levels of the target genes for the experimental groups, relative to those in the negative control group; expression data were normalized relative to the housekeeping gene GAPDH using the 2– Ct formula. RESULTS Animals’ health status No animals died during the experiment; thus, the group numbers did not vary. Body weights of the animals were similar across the experimental groups (250–300 g throughout the experiment). Coat health and faeces control also did not differ 37 between the groups throughout the experimental period. The alcohol-administered groups showed growing impairments in ambulation and spontaneous motor activity in the open-field activity test (Insight, São Paulo, Brazil), relative to no-alcohol controls, at the end of each week (data not shown). Effects of CARV on MPO activity and on MDA and GSH levels Livers from the alcohol-only treatment group had significantly greater MPO activity than livers harvested from the negative control group (p< 0.001), and this increase was attenuated in the groups that received alcohol with CARV (1 mg/kg, 3 mg/Kg, or 5 mg/kg). The high-dose CARV treatment (5 mg/kg) increased GSH levels (p< 0.05 vs. alcohol-only; Fig 1). Levels of MDA were increased significantly in the alcohol-only group livers compared to levels in livers from the negative control group (p< 0.05), and this increase was blocked in livers from animals given high-dose CARV (5 mg/kg; p< 0.05 vs. alcohol-only; Fig 1). Effect of CARV treatment on inflammation and IL-1β, IL-10, and TNF-α level Alcohol administration elevated IL-1β (p< 0.001) and TNF-α (p< 0.01) levels, but reduced levels of IL-10 (p < 0.01), compared to levels in the negative control group. These alcohol-induced effects could be reversed with CARV treatment. Specifically, IL-1β and TNF-α level in the alcohol-CARV 5 mg/kg group were lower than in the alcohol only group (p< 0.05 and p <0.01, respectively). Furthermore, IL-10 levels were higher in all three CARV dose groups(1 mg/kg, 3 mg/kg, 5 mg/kg) than in the alcohol-only group (all p< 0.001, Fig 2). Histology There were no pathological changes observed after 28 days in the negative control animal livers, as indexed by a semi-quantitative scoring system. Meanwhile, the livers from rats that were given alcohol for 28 days exhibited fat accumulation, inflammation, and necrosis, resulting in a high pathology scores (Fig 3J). Additionally, the alcohol-only treatment group had significantly greater steatosis than the negative control group (p< 0.001). Conversely, alcohol-induced liver damage was reduced in the alcohol-CARV 3 mg/kg and alcohol-CARV 5 mg/kg groups (p < 0.05 and p < 0.001 vs. alcohol-only group, respectively; Fig 3F, 3I and 3J), but not the alcohol- CARV 1 mg/kg group (p> 0.05 vs. alcohol-only group, Fig 3C and 3J). Reduced 38 inflammation was most clearly observed in the alcohol-CARV 5 mg/kg group (Fig 3I1), which exhibited decreased areas of steatosis and reduced levels of necrosis relative to the alcohol-only group. Livers from the alcohol-only group were characterized by excessive Sirius red- stained collagen I fibres (p< 0.05 vs. negative controls, Fig 4B). The stained fibres appeared orange-red in colour and were observed under polarized light, due to their increased thickness. Sirius red-stained collagen fibres were decreased in the alcohol- CARV 5 mg/kg to relative to alcohol-only group (p> 0.05, Fig 4C, 4D and 4E). H&E staining showed fatty droplet accumulation around the central vein (Fig 3B, 3E and3H,) and the inflammatory infiltrate contains neutrophils and lymphocytes (Fig 3H1), with higher levels of AST (Fig 5A), ALT (Fig 5B) and hepatic triglycerides (TG) (Fig 5C) in livers from rats subjected to chronic alcohol exposure. However, CARV treatment modulated this alcohol-induced hepatosteatosis and liver injury (Fig 5). Immunohistochemistry and confocal immunofluorescence Compared to the alcohol-only group, the alcohol-CARV 5 mg/kg group exhibited reductions in levels of COX-2, RANK, RANK-L, IBA-1, and ICAM-1 (all p< 0.05; Fig 6C, 6F, 6I and 6L, Fig 7O and Fig 8A, 8B, 8C, 8D and 8E) and an increased levels of SOCS1, SOD-1, and GPx-1(all p< 0.05, Fig 7R, 7U and 7Z and Fig 8F, 8G and 8H). These changes were consistent with the normalization toward non-alcohol exposure levels. Cellular IL-1β and NF-κB labelling (green) were strong and diffuse in the alcohol group (Fig9B and 9E), weak in the alcohol-CARV 5 mg/kg group (Fig 9C and 9F), and absent in the negative control group (Fig 9A and 9D). Densitometric analysis confirmed that there were significantly increased IL-1α and NF-κB immunoreactivities in the alcohol-only group, relative to the negative control group, that were blocked in the alcohol-CARV 5 mg/kg group (Fig 9G and 9H). CARV treatment decreased mRNA expression of TNFα, PCI, PCIII andNF-κB TNFα mRNA expression was significantly decreased in the alcohol-CARV 5 mg/kg group rela-tive to levels in the alcohol-only group (p< 0.01, Fig 10A). The expression of PCI and PCIII mRNA was decreased in alcohol- CARV 5 mg/kg group compared to the alcohol-only group (p< 0.05 and p<0.001, respectively, Fig 10B and 39 10C). In addition to this, the expression of NF-κB mRNA was decreased in alcohol- CARV 5 mg/kg group compared to the alcohol-only group (p>0.05, Fig 10D). DISCUSSION In the present study, we observed severe fat accumulation, mild inflammation, necrosis and accumulation of PCI and PCIII around centrilobular hepatic vein and the hepatic triad, resulting in moderate steatosis, as well as elevated neutrophil, MPO, and pro-inflammatory cytokine levels in animals given alcohol (by gavage) for 4 weeks. Meanwhile no pathological changes, neutrophil infiltration, MPO, or cytokine level changes were observed over the same time period in saline control rats. Moreover, the alcohol-related changes were dampened significantly in animals in the alcohol-CARV 5 mg/kg group. Despite major advances in our understanding of the pathogenesis of alcohol- related liver injury, there are no Food and Drug Administration-approved treatments for ALD. Treatment of the underlying addiction, aggressive nutritional intervention, and ―off-label‖ use of various pharmacotherapies aimed at the underlying mechanisms of injury (e.g., cytokine dysregulation, endotoxin translocation, and oxidative stress) represent the primary approaches to treating ALD. Macrovesicular steatosis - the first and most common pathologic change associated with chronic alcohol ingestion - is observed in up to 90% of heavy alcohol users [25]. These changes are reversed by eliminating alcohol consumption. Hepatocyte apoptosis and areas of microvesicular steatosis can also be seen in livers with chronic alcohol exposure, although they are more common in steatohepatitis (a.k.a. alcoholic hepatitis). Ballooning degeneration of hepatocytes, infiltrating neutrophils, Mallory bodies, and fibrosis are pathologic findings indicative of steatohepatitis [26]. Many studies have implicated proinflammatory cytokines, especially TNF-α, in the development and progression of ALD. Several clinical manifestations of ALD resemble the biological effects observed after in vivo administration of proinflammatory cytokines, suggesting that cytokines contribute to clinical complications and liver injury [27]. Administration of TNF-αantibodies prevented liver necrosis and inflammation, but not steatosis, in rats [28] and knock-out mice lacking TNF receptor 1 were protected from the inflammatory responses associated 40 with chronic ethanol administration. These findings suggest strongly that TNF-α may be a proximal mediator of ethanol-induced liver damage. Additionally, the proinflammatory cytokines IL-1 and IL-6 and the chemokine IL-8 are important mediators of ALD. Each may enhance the effects of the others. For example, although IL-1 participates in the pathogenesis of ALD, it does not seem to cause liver injury alone; rather, it appears to act synergistically with TNF-α [29]. Our present findings showing that CARV treatment decreased alcohol-induced changes in IL-1β and TNF-α levels support the notion that these factors are involved in the pathogenesis of ALD. CARV’s anti-inflammatory influence has been associated with reductions in TNF-α and IL-1β levels coincident with increased IL-10 levels [30]. IL-10 is a potent anti-inflammatory molecule that has been shown to inhibit the production of TNF-α and IL-1 and to suppress the activation of NF-κB [31]. IL-10 reduces macrophage production of nitric oxide and reactive oxygen intermediates, and also reduces the expression of adhesion molecules and chemokines [32]. Our present observation of increased MPO activity, suggestive of neutrophil infiltration, in alcohol-injured livers is consistent with previous observations. For example, Jan Petrasek et al. (2010) demonstrated that alcoholic steatohepatitis is characterized by infiltration of various inflammatory cells, including monocytes, macrophages, neutrophils, and lymphocytes, as a consequence of the activation of inflammatory mediators induced by Toll-like receptors signal-ling [33]. Neutrophils that accumulate in the hepatic microvasculature (sinusoids and postsinusoidal venules) can extravasate/transmigrate into the hepatic parenchyma after receiving signals from distressed cells. Neutrophils adhere to the distressed hepatocytes through neutrophil β2 integrins and hepatocyte ICAM-1. ICAM-1—a member of the immunoglobulin super-family and critical adhesion molecule expressed on several cell types, including endothelial cells, epithelial cells, and fibroblasts—is induced by pro-inflammatory cytokines, including TNF-α and IL-1 [34]. Neutrophil contact with hepatocytes mediates the oxidative killing of hepatocytes by initiation of the respiratory burst and neutrophil degranulation, leading to hepatocellular oncotic necrosis. Neutrophil-mediated liver injury has been demonstrated in various diseases and chemical/drug-induced toxicities [31]. Our present findings that MDA levels were increased in the alcohol-only group, 41 and that this increase was blocked in the alcohol-CARV 5 mg/kg group, are consistent with prior research showing that increases in MDA are found in chronic- alcoholism model rats and human alcoholics [35]. The lipid peroxide MDA, a widely used indirect biomarker of oxidative stress, is generated when free radicals produced by chronic, excess ethanol exposure attack polyunsaturated fatty acids in membranes. In the intragastric ethanol infusion rodent model, liver damage is associated with enhanced lipid peroxidation, decreased formation of carbonyl in GSH, and formation of 1-hydroxyethyl radicals and lipid radicals [36]. Reactive aldehydes, such as acetaldehyde, are produced from ethanol metabolism and ethanol-induced oxidant stress. Ethanol administration can also generate ethanol-derived free radicals via at least two path-ways: production of hydroxyl radicals from endogenous H2O2 in a Fenton-type reaction; and CYP2E1-mediated radical generation. The resultant ethanol-derived free radicals can exacerbate reactive oxygen species (ROS)- mediated cellular damage [37]. The present findings suggest that CARV (5 mg/kg) can counter these effects by increasing levels of both enzymatic (i.e. SOD and GPx- 1) and non-enzymatic (i.e. GSH) antioxidant defences. These results are consistent with prior research showing that CARV protects cells against acetaminophen-induced oxidative injury and suggesting that CARV’s cytoprotective effects may be related the O -2 scavenging properties of CARV or its metabolites [38]. Hepatocytes subjected to inflammatory conditions release IL-1β and TNFα, which leads to NF-κB activation and inflammation [39]. Chemokines and cytokines are involved in the pathogenesis of alcoholic hepatitis and contribute to leukocyte migration into the liver during chronic ethanol intoxication. These compounds are associated with increased basal H2O2 formation and enhanced activation of NF-κB in Kupffer cells. Chronic alcoholic injury augments the activation of NF-κB and the production of proinflammatory cytokines, including TNF-α and IL-1 [40]. Our results showing that CARV at the 5 mg/kg dosage significantly inhibited alcohol-induced IL- 1β and TNFα levels and the mRNA expression of NF-κB supports the notion that reduces inflammatory conditions. COX-2–deficient mice are protected against the toxic effects of endotoxin [41]. Expression of COX-2 is increased in alcoholic liver injury, in association with necro- inflammatory changes and endotoxemia. Kupffer cells (recognized by their IBA-1 expression) are the primary source of COX-2 in the liver [42]. The present results 42 showing that alcohol-induced increases in COX-2, IBA-1, and ICAM-1 expression levels were countered by CARV treatment (5 mg/ml) suggest that CARV may block the activation of Kupffer cells, thereby preventing the subsequent production of TNF- α and COX-2. SOCS1 is an important suppressor of cytokine signalling and inflammation, suppressing the activation of many cytokines, including IL-2, IL-3, IL-4, IL-6, interferon (IFN)-α, IFN-β, IFN-γ, and TNF-α[43,44].SOCS1-mediated suppression of inflammation involves regulatoryeffects on innate immune cells and nonimmune cells. Selective hepatocyte depletion of SOCS1 in liver-specific SOCS1 knockout mice resulted in enhanced concanavalin A-induced hepatitis associated with pro-apoptotic signals (including STAT1 and JNK) [45]. Cytoplasmic SOCS1 has been hypothesized to regulate the transcription factor NF-κB [46,47]. Interestingly, in this study, the anti- inflammatory influence of CARV was accompanied by an increase in RANK/ RANKL expression and elevated SOCS1 expression levels, suggesting that CARV-induced increases in RANK/RANKL complexes may prevent the nuclear translocation of NF- κB and, thereby, reduce the production of proinflammatory cytokines [27]. Alcoholic liver fibrosis is characterized by excessive deposition of ECM proteins, especially collagen types I and III. Activated HSCs produce large amounts of ECM components rapidly, triggering a fibrogenic response [11]. Before liver fibrosis occurs, hepatocellular damage is initiated during the cross-talk between liver cell types mediated by cytokines, ROS, and other soluble factors. Our findings show that CARV (5 mg/kg) treatment inhibited the mRNA expression of PCI and PCIII significantly is consistent with the notion that CARV may counter alcohol-induced changes in HSC biosynthesis. On the basis of our findings, we hypothesise that the protective effects of CARV treatment against alcoholic liver injury are mediated through suppression of inflammatory cytokine and ROS induction. We suggest that CARV reduces inflammation and oxidative stress in the liver caused by alcohol, thereby reducing the activity of HSCs and Kupffer cells. The present work extends prior research demonstrating the antioxidant activity of CARV in alcohol-induced liver injury [15] and the preventive role of CARV on the development of hepatosteatosis [17] by providing information about molecular changes in inflammatory pathways. In summary, inhibition of the SOCS1 signalling pathway by alcohol was reversed by CARV treatment, resulting in liver recovery. Specifically, CARV treatment 43 reduced the expression levels of IL-1β and TNF-α, downregulated expression of COX-2, RANKL/RANK, IBA-1 and ICAM-1, and upregulated SOCS1, SOD-1 and GPx-1 expression. Additionally, CARV treatment inhibited hepatic expression of several pro-inflammatory mediators and ROS levels in ethanol-fed rats by modulating the activity of Kupffer cells and HSCs. The positive effect of CARV on liver architecture in alcoholic liver injury suggests that CARV treatment may aid in the recovery of hepatosteatosis, been sufficient to prevent liver fibrosis. ACKNOWLEDGMENTS This work was supported by CNPq (Conselho Nacional de Pesquisa) / Universal n. 47.6996/ 2013. RFAJ wishes to thank the study core in microscopy and image processing (NEMPI, UFC). AUTHOR CONTRIBUTIONS Conceived and designed the experiments: RFAJ. Performed the experiments: RFAJ, VBG and AAA. Analyzed the data: RFAJ, AAA, VBG. Contributed reagents/materials/analysis tools: RFAJ, AAA, RFCL, ECM, GACB, PMMG. Wrote the paper: RFAJ and VBG. 44 References 1. Frazier TH, Stocker AM, Kershner NA, Marsano LS, McClain CJ (2011) Treatment of alcoholic liver dis-ease. Therap Adv Gastroenterol 4: 63–81. doi: 10.1177/1756283X10378925 PMID: 21317995 2. Hall PD (1994) Pathological spectrum of alcoholic liver disease. Alcohol Alcohol Suppl 2: 303–313. PMID: 8974350 3. Mendez-Sanchez N, Almeda-Valdes P, Uribe M (2005) Alcoholic liver disease. An update. Ann Hepatol 4: 32–42. PMID: 15798659 4. Lieber CS (2005) Metabolism of alcohol. Clin Liver Dis 9: 1–35. PMID: 15763227 5. 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Maine GN, Mao X, Komarck CM, Burstein E (2007) COMMD1 promotes the ubiquitination of NF-kap-paB subunits through a cullin-containing ubiquitin ligase. EMBO J 26: 436–447. PMID: 17183367 47. de Araujo RF Jr, Reinaldo MP, Brito GA, Cavalcanti PdF, Freire MA, Medeiros CAX, et al. (2014) Olme-sartan decreased levels of IL-1beta and TNF-alpha, down-regulated MMP-2, MMP-9, COX-2, RANK/ RANKL and up-regulated SOCs-1 in an intestinal mucositis model. PLoS One 9: e114923. doi: 10.1371/journal.pone.0114923 PMID: 25531650 49 Figures legends Fig 1.CARV modulates MPO activity, MDA activity, and GSH levels in the livers of rats with alcohol-induced liver injury. Experiments were conducted in duplicate. *p<0.05; ***p<0.001. Fig 2.CARV counters alcohol-mediated effects on cytokine production in rats with alcohol-induced liver injury. Alcohol treatment elevated IL-1β (***p < 0.001) and TNF-α (**p < 0.01) levels, but reduced IL-10 levels (**p < 0.01), compared to the negative control group. CARV administration had a dose-dependent, alcohol- countering effect on IL-10 (5 mg/kg ***p < 0.001); only the 5 mg/kg dose of CARV had significant alcohol-countering effects on IL-1β (*p < 0.05) and TNF-α (5 mg/kg **p < 0.01) levels. Experiments were conducted in duplicate. Fig 3.Histological examination of liver specimens in rats with alcohol-induced liver injury.Five animals per group and three H&E sections per animal were analysed. Images and data from the CARV 1 mg/ml, 3 mg/ml, and 5 mg/ml groups are shown in panels A–C, D–F, and G–I, respectively (arrow, fatty changes within hepatocytes;triangle, necrosis area). Negative control group slides are shown in A, D, and G. Alcohol group slides are shown in panels B, E, and H (middle images). In the alcohol-only group, rat livers exhibited steatosis with various degrees of diffuse hepatic steatosis and intralobular, deranged hepatic cord, and necrotic areas. Liver injury (e.g., steatosis and inflammation) persisted in the alcohol-CARV 1 mg/kg group (C). (F, I) Reduced steatosis and hepatocyte regeneration associated with reduced necrosis were observed in the livers of animals in the alcohol-CARV 3 mg/kg and alcohol-CARV5 mg/kg groups. The neutrophils infiltrate the parenchyma, as seen here, and often surround ballooned hepatocytes (blue arrow: neutrophils and circle: lymphocytes and neutrophils infiltrate, Fig 3H1). Reduced neutrophilic infiltration was observed in the livers of animals in the alcohol-CARV 5 mg/kg groups (Fig 3I1). Magnification 400×, scale bar = 100 μm. (J) Representative samples from each CARV treatment group are shown with graphs summarizing the mean histopathological score of each group. (***p < 0.001 vs. negative control, #p > 0.05, *p < 0.05, and ***p < 0.001 vs. alcohol-only group). 50 Fig 4.Representative photomicrographs of liver sections stained with picrosirius red. Five animals per group and three H&E sections per animal were analysed. (A) Negative control group livers had weak staining limited to centrilobular veins (left panel) and the portal tract (right panel). (B) Liver sections from the alcohol- only group exhibited marked portal fibrosis (left panel) and staining between the hepatic cords (right panel). (C) The alcohol-CARV 5 mg/kg group livers had a weak fibrotic response limited to centrilobular veins (left panel) and portal tract (right panel). (D) Morphometric quantification of Sirius red stained areas demonstrated an attenuation of the fibrotic process in the alcohol-CARV 5 mg/kg group compared to the alcohol-only group. (E) The area fraction of total fibrosis, including fibrosis in the portal tract area, in rats with alcohol-induced liver injury in relation to the Ishak score. The alcohol-CARV 5 mg/kg group livers had an attenuation of the fibrotic process. PT, portal tract; CV, central vein; asterisk, hepatic cords. Magnification 400×, scale bar = 50 μm. (**p < 0.01, *p < 0.05, Kruskal-Wallis test followed by Dunn’s test). Fig 5.Biochemical analysis of blood AST (mg/dl), ALT (mg/dl), and triglyceride (mg/dl) levels in rats with alcohol-induced liver injury. CARV modified the ethanol-induced increases in levels of AST (A, n = 5, p < 0.01), ALT (B, n = 5, p < 0.01), and hepatic triglycerides (B, n = 5, p < 0.001). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the alcohol group and ***p < 0.001 for negative control vs. alcohol group (analysis of variance followed by Bonferroni’s correction). Fig 6.COX-2, RANK, RANK-L, and IBA-1 immunohistochemical findings.Generally, livers from alcoholic rats had greater COX-2 (B), RANK (E), RANK-L (H) and IBA-1 (K) immunoreactivity than livers from the negative control and alcohol-CARV 5 mg/kg groups. For each antigen, three immuno-labelled sections were analysed per animal (N = 5 animals per group). Asterisk: strong labelling; black narrow: labelling to Kupffer cells (IBA-1). Magnification 4000×, scale bar = 100 μm. Fig 7.ICAM-1, SOCS1, SOD-1, and GPx immunohistochemical findings. Livers from alcohol-administered rats had greater ICAM-1 (N) immunoreactivity than the saline control and alcohol-CARV 5 mg/kg group rats. Livers from alcohol- administered rats had reduced SOCS1 (Q), SOD-1 (T), and GPx (X) immunoreactivity levels than the negative control and alcohol-CARV 5 mg/kg group rats. For each antigen, three immuno-labelled sections were analysed per animal (N 51 = 5 animals per group). Asterisk: strong labelling. Magnification 4000×, scale bar = 100 μm. Fig 8.CARV effects on ethanol-induced liver injury in rats with alcohol-induced liver injury. Five immunohistochemistry sections from each animal in each group were analysed (N = 5 animals per group). Representative samples from the alcohol- CARV 5 mg/kg treatment group are shown with graphs summarizing the mean group scores for COX-2, RANK, RANK-L, IBA-1, ICAM-1, SOCs, SOD-1 and GPx-1 immunoreactivity. The alcohol-induced effects were reversed (i.e., normalized) in the alcohol-CARV 5 mg/kg livers. *p < 0.05 vs. alcohol group and ***p < 0.001 for negative control vs. alcohol group (Kruskal-Wallis test followed by Dunn’s test). Fig 9.CARV modulates IL-1β and NF-κB expression. Representative confocal photomicrographs of IL-1β and NF-κB immunoreactivity in liver specimens from each group (green) with DAPI nuclear counterstained (blue). (A,D) Negative control rat liver without IL-1β and NF-κB labelling, respectively. (B,E) IL-1β and NF-κB labelling (white narrow) was diffuse and strong in the alcohol-only group, respectively. Besides, the NF-κB labelling was more concentrated between the hepatic cell cords. (C,F) Weak IL-1β and NF-κB labelling (red arrows) was seen in the alcohol-CARV 5 mg/kg group, respectively. Scale bar, 50 mm. (G,H) Densitometric analysis confirmed significant increases in IL-1β and NF-κB immunoreactivity in the alcohol-only group that were blocked in the alcohol-CARV 5 mg/kg group. Five immunofluorescence sections from each animal in each group were analysed (N = 5 animals per group) (**p < 0.01, *p < 0.05, Kruskal-Wallis test followed by Dunn’s test). Fig 10.CARV effects on TNFα, PCI, PCIII, and NF-κB mRNA expression in rats with alcohol-induced liver injury. The expression of TNFα mRNA was increased in alcohol group (**p< 0.01, Fig 10A) and decreased in alcohol- CARV 5 mg/kg group (**p< 0.01, Fig 10A). The expression of PCI mRNA was decreased in alcohol- CARV 5 mg/kg group compared to the alcohol-only group (*p< 0.05, Fig 10B). PCIII mRNA levels appeared to be lower in the alcohol-CARV 5 mg/kg group compared to the alcohol-only group (***p<0.001, Fig 10C) and the expression of NF-κB mRNA was decreased in alcohol- CARV 5 mg/kg group compared to the alcohol-only group (*p>0.05, Fig 10D). (N = 5 animals per group; Kruskal-Wallis test followed by Dunn’s test). 52 Figure 2 53 Figure 3 54 Figure 4 55 Figure 5 56 Figura 6 57 Figure 7 58 Figure 8 59 Figure 9 60 Figure 10 61 Figure 11 6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES Inicialmente, o projeto intitulava-se ―Avaliação das atividades anti- inflamatórias e antioxidantes do anti-hipertensivo carvedilol e do selênio em ratos Wistar com doença hepática alcoólica (DHA)”. O nosso objetivo era investigar os níveis do estresse oxidativo em tecidos hepático e cerebral de ratos com DHA tratados com a associação do carvedilol e selênio em parâmetros (diferentes doses combinadas ou administradas separadamente); analisar o perfil de morte celular programada (apoptose) em tecidos hepático e cerebral desses ratos e determinar os efeitos da associação do carvedilol e selênio nas funções comportamentais e motoras. Além das metodologias apresentadas neste documento, o projeto inicial incluía a imuno-histoquímica do córtex pré-frontal, amígdala e hipocampo para avaliação dos danos teciduais por morte celular ou por alterações bioquímicas nessas áreas e buscava correlacionar esses achados ao comportamento motor dos 62 animais ao submetê-los ao campo aberto monitorado. Dentre os obstáculos que encontramos para a execução do projeto inicial estavam a quantidade de animais necessários para o estudo, a dificuldade de treinamento de alunos de iniciação científica para auxiliarem no manejo dos animais e gavagens e, finalmente, a má fixação dos cérebros em virtude do método utilizado para obtê-los: dissecação seguida de fixação, em vez de perfusão. Esse último problema foi o mais grave, pois nos impossibilitou de analisar os efeitos antioxidantes da droga até aquele momento testada (o carvedilol) e correlacionar esses achados ao monitoramento em campo aberto, cujos dados foram guardados para futuras análises. Inicialmente o projeto não incluía a qPCR para a avaliação dos efeitos anti- inflamatórios e anti-fibróticos do Carvedilol, então a revista nos solicitou essa análise. Por conta das repetições que precisamos fazer para as análises de MPO, MDA e GSH, não tínhamos mais amostras suficientes para a realização da qPCR e, as poucas que sobraram, não estavam em micro-tubos RNAse-free. Dessa forma, decidimos usar os animais que haviam sido separados para os testes com selênio, a fim de termos uma publicação mais sólida. Por isso acabamos por 80 animais. Nesse trabalho incluímos três técnicas que não estavam previstas no projeto original: qPCR, Picro-Sirius Red e Imunofluorescência. Técnicas estas que se mostraram fundamentais para mostrarmos a ação do Carvedilol sobre as Células de Kupffer e Células Estreladas. O trabalho com imunofluorescência também possibilitou a segunda produção acadêmica desse mestrado, que foi o depósito de pedido de patente de invenção para a utilização de nanopartículas de ouro como substituição do anticorpo secundário Alexa-Fluor 488, tornando a técnica mais barata e rápida. O mestrado pelo Programa de Pós-graduação em Ciências da Saúde amadureceu a minha maneira de estudar e de fazer ciência. Aprendi muitas técnicas novas e pude até mesmo aperfeiçoar alguns protocolos antigos usados em nossos laboratórios. Foi durante o mestrado que eu passei a realmente gostar de fazer pesquisa, e não apenas de ensinar. Pretendo fazer meu doutorado pelo mesmo programa de pós-graduação, me aprofundando nos conhecimentos de nanotecnologia aplicada a processos inflamatórios e neoplasias malignas. 63 Segue a produção científica gerada pelo projeto de pesquisa: -Apresentação em congresso: Garcia, V.B., Araújo Júnior, R.F., Leitão, R.F.C., Brito, G.A.C., Miguel, E.C., Guedes, P.M.M., Araújo, A.A. O carvedilol diminui a resposta inflamatória, o estresse oxidativo e a fibrose em modelo de injúria hepática induzida pelo álcool em ratos ao regular a ação das Células de Kupffer e Células Estreladas. XI Reunião Regional da Federação de Sociedades de Biologia Experimental – FeSBE. Centro de Biociências, UFRN, Natal, 12 a 14 de Maio de 2016. -Pedido de depósito de patente de invenção: ARAÚJO JÚNIOR, RAIMUNDO FERNANDES DE ;GARCIA, V. B. ; OLIVEIRA, A. L. C. S. L. ; GASPAROTTO, L. H. S. ; SILVA, H. F. O. ; ARAUJO, A. A. . SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO. 2016, Brasil.Patente: Privilégio de Inovação. Número do registro: BR10201602447, título: "SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO" , Instituição de registro: INPI - Instituto Nacional da Propriedade Industrial, Depositante (s): Universidade Federal do Rio Grande do Norte, Depósito: 20/10/2016 64 REFERÊNCIAS 1. WORLD HEALTH ORGANIZATION. Global status report on alcohol and health - 2014 ed. WORLD HEALTH ORGANIZATION. 2014, p. 376. 2. Lim SS, Vos T, Flaxman AD, et al. 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Journal of hepatology. 1995; 22: 696-9. 67 ANEXOS 68 Anexo 1 – Parecer consubstanciado final da avaliação do projeto pela Comissão de Ética no Uso de Animais - CEUA 69 Anexo 2 – Certificado de apresentação do trabalho na XI Reunião Regional da FeSBE 70 Anexo 3a – Certificado de ministração em Curso de Atualização Teórico-Prática de Métodos Analíticos Em Microscopia e em Estresse Oxidativo 71 Anexo 3b – Certificado de ministração em Curso de Atualização Teórico-Prática de Métodos Analíticos Em Microscopia e em Estresse Oxidativo 72 Anexo 4a - Patente de invenção “SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO” 73 Anexo 4b - Patente de invenção “SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO” 74 Anexo 4c - Patente de invenção “SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO”