1 Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Ciências Farmacêuticas Efeito de compostos naturais bioativos sobre o metabolismo lipídico e a produção de estresse oxidativo durante a replicação do vírus da hepatite C em cultura celular – VHCcc Moema de Souza Santana Tese apresentada ao Programa de Pós-graduação em Alimentos e Nutrição para obtenção do título de Doutor em Alimentos e Nutrição. Área de Concentração: Ciências Nutricionais. Orientador: Prof. Dr. Paulo Inácio da Costa. Araraquara 2020 2 Efeito de compostos naturais bioativos sobre o metabolismo lipídico e a produção de estresse oxidativo durante a replicação do vírus da hepatite C em cultura celular – VHCcc Moema de Souza Santana Tese apresentada ao Programa de Pós-graduação em Alimentos e Nutrição para obtenção do título de Doutor em Alimentos e Nutrição. Área de Concentração: Ciências Nutricionais. Orientador: Prof. Dr. Paulo Inácio da Costa. Araraquara 2020 3 4 5 Agradecimentos Pessoais Agradeço a Deus pelo cuidado sem fim durante todo meu caminho em busca da realização dos meus sonhos. Aos meus pais Pedro Jackson e Geraldina Batista pelo dom da vida e pelos exemplos de caráter, dignidade e força. Ao meu orientador Prof. Dr. Paulo Inácio da Costa pelo acolhimento, generosidade e exemplo de honestidade, ética e amor pela ciência. Ao amor da minha vida Iuri Lima Aiura pelo companheirismo, carinho e apoio e à minha querida sogra pela força e alegria. Aos amigos fieis que cultivei em Araraquara em especial a Rute Lopes, Carla Cruz, Rafael Fulindi e Carolina Ribeiro pelo companheirismo, apoio e convívio amoroso. A todos os colegas de laboratório e de curso por todo apoio e companheirismo durante o desenvolvimento do projeto e cursada das disciplinas. Aos amigos e aos familiares que deixei na Bahia e que fazem presentes mesmo em distância, me dando a força necessária para prosseguir diante da certeza de apoio e de aconchego no retorno para casa. 6 Agradecimentos À Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (processo: 2017/04500-9) e a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Código de Financiamento 001; Processo: 250 - 10/2017) pelas bolsas concedidas. Ao meu orientador Prof. Dr. Paulo Inácio da Costa pela orientação no planejamento e execução desse trabalho. A minha colega Rute Lopes pelo apoio incondicional e acolhimento durante todas as etapas de realização desse trabalho. Aos docentes da Pós-graduação em Alimentos e Nutrição pelo ensino, dedicação e conhecimento compartilhado. À sessão de Pós-graduação em Alimentos e Nutrição pela disponibilidade, apoio e eficiência nas orientações. 7 “Toda caminhada começa no primeiro passo. A natureza não tem pressa segue seu compasso, inexoravelmente chega lá.” (Santanna. A natureza das coisas, 2007). 8 Resumo Introdução: As complicações relacionadas à cronicidade de hepatite C estão associadas com o aumento do estresse oxidativo promovido pelo Vírus da Hepatite C (VHC), que causa danos ao DNA, alterações na peroxidação lipídica e está associadada a morte celular. O resvetrarol (RVT) apresenta ampla ação antioxidante, e potencial de síntese de derivados. O f- resveratrol, derivado RVT com a adição de uma molécula de fluor apresenta atividade antioxidante promissora. A taurina (TAU) destaca-se pala ação que desempenha na estabilidade da membrama plasmatica, a reprodução, a imunidade. Objetivo: Avaliar o efeito do RVT, do F-RVT e da TAU sobre o perfil de morte celular e a replicação do VHC. Métodos: Trabalho desenvolvido utilizando células Huh-7.5 e células Huh-7.5 expressando estavelmente o replicon subgenômico SGR-JFH1. Os CNBs analisados foram o RVT, F-RVT e TAU. A viabilidade células foi analisada por meio do ensaio colorimétrico de MTT. O mecanismo de apoptose e necrose foi analizado por citometria de fluxo. A quantificação de RNA do VHC, nas células SGR-JFH1 sem tratamento e pós-tratamento, foi realizada por ensaio de PCR em tempo real. Aplicou-se o modelo de análise de variância. Foi aplicado d.m.s. do teste de Tukey a nível de 5% de probabilidade. Resultados: O controle positivo (H2O2) apresentou expressiva citotoxicidade, morte celular de aproximadamente 80% nas células SGR- JFH1 e de 95% nas células Huh-7.5. Os compostos apresentaram tendência de indução de apoptose precoce pelos tratamentos com os CNBs nas células Huh-7.5 e nas SGR-JFH1 para as concentrações testadas. Houve ausência de influencia dos tratamentos com os CNBs sob o padrão de necrose das células Huh-7.5 e nas SGR-JFH1 para as concentrações testadas. Verificou-se ainda redução da carga viral intracelular como efeitos dos tratamentos com RVT, F-RVT e TAU em 1 dia em tratamentos. Conclusão: Os compostos estudados apresentaram efeito sobre a indução de apoptose precose e sobre a redução da carga viral intracelular do VHC. Palavras-Chave: VHC, Hepatite C, estresse oxidativo e apoptose. 9 Abstract Introduction: Complications related to the chronicity of hepatitis C are associated with increased oxidative stress promoted by the Hepatitis C Virus (HCV), which causes DNA damage, changes in lipid peroxidation and is associated with cell death. Resvetrarol (RVT) has a broad antioxidant action and derivatives synthesis potential. The f-resveratrol derivative RVT with the addition of a fluoride molecule presents promising antioxidant activity. Taurine (TAU) stands out for the action it plays on the stability of the plasma membrane, reproduction, immunity. Objective: To evaluate the effect of RVT, F-RVT and TAU on cell death profile and HCV replication. Methods: The study was developed using Huh-7.5 cells and Huh-7.5 cells expressing the SGR-JFH1 subgenomic replicon. The viability analysis of the cells was analyzed by means of the MTT colorimetric assay. Apoptosis and necrosis mechanism analysis was performed by flow cytometry. The quantification of HCV RNA was performed in SGR-JFH1 cells without treatment and after treatment. The variance analysis model was applied. The contrast between means was done by the d.m.s. of the Tukey test at 5% probability level. Results: The positive control (H2O2) presented expressive cytotoxicity, cellular death approximately 80% in SGR-JFH1 cells and 95% in Huh-7.5 cells. The compounds presented the tendency to induction of early apoptosis by the treatments with NBCs in the Huh-7.5 cells and in the SGR-JFH1 for the concentrations tested. There was no influence of the treatments with the NBCs under the necrosis pattern in the Huh-7.5 cells and in the SGR-JFH1 for the concentrations tested. There was also a reduction of intracellular viral load as effects of treatments with RVT, F-RVT and TAU in 1 day in treatments and absence of influence on extracellular viral load in the same period. Conclusion: The compounds studied had an effect on the induction of apoptosis Precose in Huh-7.5 cells and SGR-JFH1 cells and on the reduction of intracellular viral load of HCV. Palavras-Chave: VHC, Hepatite C, estresse oxidativo e apoptose. 10 Lista de abreviaturas e sigla CCL-5 Ligante do Receptor de Quimiocina 5 CNBs Compostos Naturais Bioativos ERONs Espécies Reativas de Oxigênio e Nitrogênio F-RVT F-Resveratrol IL-8 Interleucina 8 HCC Carcinoma Hepatocelular Huh-7.5 Provenientes de Hepatocarcinoma Humano PPAR Proliferadores de Peroxisoma OMS Organização Mundial da Saúde NO Óxido Nítrico SREBP Proteína Reguladora do Elemento Regulador do Esterol RVT Resveratrol TAU taurina VHC Vírus da Hepatite C 11 Lista tabelas e quadros Introdução. Quadro 1. Combinações medicamentosas preconizadas para o tratamento da Hepatite C no Brasil. 21 Capitulo 1. Table 1. Natural bioactive compounds (NBCs) with biological or anti- hepatitis C virus activities. 44 Capitulo 2. Table 1. Summary of signaling pathways involved in hepatic lipid processing and resulting metabolic effects. 65 Capitulo 3. Tabela 1. Concentrações inibitórias médias de resveratrol e f- resveratrol para as linhagens celulares Huh-7.5 e SGR-JFH1. 93 Capitulo 4. Tabela 1. Concentrações inibitórias médias de taurina para as linhagens celulares Huh-7.5 e SGR-JFH1. 114 12 Lista de figuras Introdução Figura 1. Esquema visual do ciclo de replicação do Vírus da Hepatite C. 18 Figura 2. Estrutura molecular do resveratrol. 23 Figura 3. Estrutura molecular do f-resveratrol. 24 Figura 4. Estrutura molecular da taurina. 25 Capitulo 1. Figura 1. Viral and host chronicity factors involved in hepatitis C progression. 34 Capitulo 2. Figura 1. The role of cell signaling molecules regarding steatosis. 72 Figura 2. Cell signaling pathways involved in lipid metabolism. 76 Capitulo 3. Figura 1. Viabilidade celular das células Huh-7.5 e SGR-JFH1 após tratamento com H2O2 1 mmol L-1 (CP). 94 Figura 2. Ensaio de apoptose por citometria de fluxo. 95 Figura 3. Análise do índice de apoptose precoce em células Huh-7.5 e SGR-JFH1 após tratamento com resvetratrol e f-resveratrol. 96 Figura 4. Análise do índice de necrose em células Huh-7.5 e SGR- JFH1 após tratamento com resveratrol e f-resvaratrol. 97 Figura 5. Análise quantitativa por RT-qPCR da carga viral de RNA do VHC em sobrenadante de células SGR-JFH1. 98 Figura 6. Análise quantitativa por RT-qPCR da carga viral de RNA do VHC meio intracelular e no sobrenadante de células SGR-JFH1. 99 Capitulo 4. Figura 1. Análise quantitativa por RT-qPCR da carga viral de RNA do VHC meio intracelular e no sobrenadante de células SGR-JFH1 após tratamento com taurina. 115 Figura 2. Análise do índice de apoptose precoce em células Huh-7.5 e SGR-JFH1 após tratamento com taurina. 116 Figura 3. Análise do índice de necrose em células Huh-7.5 e SGR- JFH1 após tratamento com taurina. 117 13 Sumário Introdução. 14 Capítulos 27 Capítulo 1. Natural Bioactive Compounds As Adjuvant Therapy For Hepatitis C Infection. 28 Abstract 29 Graphical Abstract 29 Introduction 30 Methods 35 Results and discussion 35 Conclusion 46 Funding 46 References 46 Capítulo 2. Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review. 62 Abstract 63 Introduction 63 Methods 64 Lipids and cell signaling pathways 65 Conclusion 77 Acknowledgements 77 References 77 Capítulo 3. Efeito do resveratrol e derivado sobre a morte celular e replicação do Vírus da Hepatite C. 83 Resumo 84 Introdução 84 Metodos 84 Resultados 93 Discussão 100 Conclusão 102 Referências 102 Capítulo 4. Avaliação do potencial antioxidante da taurina sob a modulação da morte celular e replicação do vírus da hepatite C. 106 Resumo 107 Introdução 108 Metodos 108 Resultados 114 Discussão 118 Conclusão 119 Referências 119 Considerações Finais 120 14 Introdução 15 Hepatites virais: breve histórico A história das hepatites virais remonta vários milênios. Existem relatos de ocorrência de icterícia entre na população chinesa há mais de cinco mil anos. Além disso, a descrição de casos de icterícia de origem infecciosa relacionada a problemas hepáticos foi descrita por Hipócrates. No entanto, somente no século XVIII introduziu-se o termo hepatite para designação da sintomatologia destas doenças (REUBEN, 2002). A observação da sintomatologia e vias de transmissão das hepatites foi foco de interesse da ciência por anos, e sabe-se que a suspeita da transmissão destas por via parenteral foi descrita pela primeira vez em 1895, devido ao desenvolvimento de quadro de icterícia em 14,8% dos indivíduos vacinados contra a varíola por meio de vacina produzida a partir de linfa humana. A sintomatologia da doença manifestou-se de dois meses a oito meses após a aplicação da vacina, caracterizando-se por fadiga, anorexia, queixas digestivas, icterícia e por intenso prurido cutâneo. No entanto, o estabelecimento da transmissão das hepatites por inoculação parenteral, ocorreu apenas em 1937, com a ocorrência de casos de icterícia sintomáticos de dois a sete meses após a inoculação de vacina contra febre amarela preparada com adição de soro humano para fins de estabilização (REUBEN, 2002; ALTER, 2003). A ocorrência de quadro de icterícia a partir de outras vias de contaminação (via fecal-oral, através de água ou alimentos contaminados), com período de incubação mais curto (sete a 10 dias exposição) também foram descritas ao longo dos anos, corroborando com a hipótese de que a sintomatologia das hepatites variava de acordo com diferentes agentes etiológicos (REUBEN, 2002; ALTER, 2003). Dessa forma, esforços passaram a ser dispendidos no sentido de isolar os agentes infecciosos das hepatites. O vírus da hepatite B foi o primeiro a ser descoberto (BLUMBERG et al., 1965). Posteriormente o vírus da hepatite A foi isolado (FEINSTONE et al.,1973) e em 1989, a partir de estudos de biologia molecular, identificou-se o genoma do agente viral 16 responsável por 80 a 90% das hepatites pós-transfusionais não-A e não- B16. Tal agente foi denominado de vírus da hepatite C. (CHOO et al., 1989). Relêvancia epidemiológica e formas de infecção da hepatite C A Organização Mundial da Saúde (OMS) estima que aproximadamente 71 milhões de pessoas estejam infectadas pelo vírus da hepatite C (VHC) mundialmente, e que o número de mortes relacionadas a essa infecção alcance 400 mil pessoas por ano (WORLD HEALTH ORGANIZATION, 2018). Os dados epidemiológicos no Brasil para hepatites virais são escassos e muitas vezes divergentes, pois apesar de se tratar de uma doença de notificação compulsória, o sistema informacional conta como principal fonte notificadora os bancos de sangue e hemocentros que estão situados em áreas geográficas restritas, o que dificulta a estimativa precisa para a população geral (CARVALHO et al., 2014; MARTINS et al., 2010). No entanto, estima-se que cerca de 700 mil indivíduos são sorreagentes anti- VHC no Brasil (BENZAKEN et al., 2018). As principais formas de infecção com o VHC são a transfusão de sangue de indivíduos infectados, o compartilhamento de material para uso de drogas (seringas, agulhas, cachimbos, entre outros) e de higiene pessoal (lâminas de barbear e depilar, escovas de dente, alicates de unha ou outros objetos perfurocortantes), a confecção de tatuagem ou colocação de piercings com objetos contaminados, a transmissão vertical (transferência do vírus da mãe infectada para o filho durante a gestação e/ou parto) e sexo sem camisinha com indivíduo infectado (forma mais rara de infecção) (MARTINS et al., 2010). A infecção pelo VHC pode ser aguda ou crônica. A hepatite C aguda geralmente é assintomática e progride para a cura espontânea em 15 a 45% dos casos. No entanto, existem casos de persistência da infecção e de evolução para hepatite C crônica (55 a 85% dos casos). Os mecanismos responsáveis pela persistência da infecção pelo VHC não foram ainda completamente elucidados, mas, sabe-se que o dano hepático produzido na 17 cronicidade dessa doença pode ocasionar fibrose hepática, cirrose, falência hepática e/ou carcinoma hepatocelular (HCC) (BURKE & COX, 2010). Assim, a infecção pelo VHC constitui-se como a principal causa de hepatopatias crônicas (60%) e maior causa de indicação de transplante de fígado no mundo, visto que, de 5 a 20% dos indivíduos infectados pelo vírus desenvolvem cirrose em até 30 anos da infecção. Além disso, de 1 a 5% indivíduos infectados falecem em decorrência da doença, por cirrose ou em decorrência do HCC (CENTERS FOR DISEASE CONTROL AND PREVENTION, 2016; WORLD HEALTH ORGANIZATION, 2018). Heterogenineidade genética e ciclo do VHC O VHC pertence à família Flaviviridae (gênero Hepacivirus) e é constituído por RNA com genoma fita simples de polaridade positiva. O RNA viral codifica uma extensa poliproteína, composta por aproximadamente 3.000 aminoácidos, que distingue-se em proteínas estruturais, o capsídeo (core) e as glicoproteínas de envelope E1 e E2, e em proteínas não estruturais (p7, NS2, NS3, NS4A, NS4B, NS5A e NS5B) que são responsáveis pela replicação viral (CHAMPEIMONT et al., 2016; DUBUISSON & COSSET, 2014). O processo de replicação viral do VHC ocorre nos hepatócitos e permite a caracterização de 6 genótipos e 67 subtipos (identificados por letras minúsculas segundo ordem de descoberta) desse vírus, no entanto, durante o processo de replicação pode ocorrer variações na inclusão de nucleotídeos dentro de um mesmo genótipo e subtipo do VHC. Tais variações, denominadas quasispecies, ocorrem devido à replicação imperfeita do vírus e do surgimento de pequenas e constantes mutações espontâneas durante a replicação viral e contribuem para aabsorvância grande diversidade e variabilidade gênica do VHC (MESSINA et al., 2015; SMITH et al., 2014). A entrada do VHC na célula é um processo altamente orquestrado, que envolve fatores virais e da célula hospedeira. A primeira etapa do ciclo de vida do vírus é a ligação da partícula viral à célula hospedeira, para a 18 qual há uma interação específica entre receptores na superfície da célula hospedeira (Glicosaminoglicanos, Receptores Lipoproteína de Baixa Densidade, Tetraspanina - CD81, Scavenger Receptor – SR-BI, Claudin – CLDN e Occludin - OCLN) e glicoproteínas virais de adesão (E1 e E2 do envelope) que induz a endocitose e entrada de partículas de VHC através da membrana plasmática celular (WHIDBY et al., 2009). Após a entrada do vírus na célula hospedeira, há a liberação do RNA viral fita simples positiva no citoplasma. A fita de RNA positiva é replicada em fita negativa que é usada para a síntese de outras fitas positivas por intermediário replicativo. Assim, este genoma pode ser empregado em tradução de novas proteínas do vírus, replicação de maiores quantidades de RNA e montagem de novas partículas virais infecciosas (BARTENSCHLAGER et al., 2010; LINDENBACH & RICE, 2013). A figura 1 apresenta o esquema visual do ciclo de replicação do VHC. Figura 1. Esquema visual do ciclo de replicação do Vírus da Hepatite C. Legenda: Particulas virais ligam-se ao hepatócito por meio de interação especifica entre as glicoproteínas do envelope e os receptores da membrana célular. As partículas vírais são interiorizadas por meio de endocitose. No citoplasma ocorre liberação do genoma viral após 19 o desnudamento da partícula. A tradução ocorre no RER (retículo endoplasmático rugoso), assim uma lipoptroteina é clivada em proteínas estruturais e não estruturais. As proteínas não estruturais participam da replicação viral e as estruturais fazem parte da estrutura do capsídeo e compõem as glicoproteínas do envelope. Ocorre então a montagem e a maturação das partículas virais e posteriormente a liberação destas da célula hospedeira. Fonte: Oliveira, 2007, com adaptações. O ciclo de vida do VHC está intimamente relacionado ao metabolismo lipídico desde os processos de entrada e replicação viral até a montagem e liberação das partículas infecciosas. Assim, os processos relacionados com o aumento da lipogênese, da redução da degradação de lipídios no fígado e da redução da exportação desses metabólitos do tecido hepático para o organismo podem ser desencadeados pelo VHC, com o objetivo de aumentar a disponibilidade de constituintes necessarios à sua replicação e montagem (CHANG, 2016). Alguns autores destacam que a inibição da proteína reguladora do elemento regulador do esterol (SREBP), da enzima ácido graxo sintase, da síntese de triacilglicerídeos e da síntese de colesterol e de seus produtos (geranilgeranil) inibem a replicação do VHC (XIANG et al., 2015; SYED et al., 2011). A redução da degradação de lipídios no fígado pelo VHC relaciona-se com a redução da via de β-oxidação de ácidos graxos na mitocondria, induzida pelo vírus, o que resulta em baixa combustão lipídica e a inibição da atividade da proteína trifuncional mitocondrial (AMAKO et al., 2015). Além disso, a proteina de core do VHC tem o potencial de aumentar a atividade de receptores ativados por proliferadores de peroxisoma (PPAR), acentuando a desregulação na β-oxidação lipídica e a depossição de gordura no técido hepatico (CHANG, 2016). As partículas virais infecciosas, depois de formadas, são liberadas causando danos ao tecido hepático resultante de processo inflamatório caracterizado por um infiltrado inflamatório com diferentes populações de células imunes (WHIDBY et al., 2009). O processo inflamatório e os mecanismos da doença no tecido hepático, na hepatite C, ainda não estão bem definidos, mas sugere-se que exista a liberação de citocinas quimiotáticas pelas células endoteliais. Essas citocinas, entre as quais 20 destacam-se a Interleucina 8 (IL-8) ou ligante 8 da quimiocina e o ligante do receptor de quimiocina 5 (CCL-5), pelo grande potencial quimiotraente de neutrófilos e super expressão na infecção crônica pelo VHC, recrutam linfócitos específicos para o fígado e aumentam a inflamação através das interações entres as células endoteliais e os leucócitos (FIERRO et al., 2015). O processo inflamatório na hepatite C pode ainda sofrer influência da liberação de óxido nítrico (NO) por macrófagos, neutrófilos e células endoteliais. O NO é um importante mediador pleiotrópico da inflamação e pode levar o tecido a uma lesão inflamatória potencial agravada pela liberação das espécies reativas de oxigênio e nitrogênio (ERONs), os quais aumentam ainda mais a expressão de citocinas (ex. IL-8 e CCL-5) e moléculas de adesão endotelial, amplificando a cascata de inflamação e gerando o estresse oxidativo ao tecido hepático. Assim, o estresse oxidativo (marcado por um aumento de oxidantes e uma diminuição da capacidade antioxidante das células) surge como importante mecanismo bioquímico envolvido na patogênese hepática induzida pelo VHC, uma vez que o equilíbrio entre a geração de ERONs e das defesas antioxidantes é fundamental para a homeostase do hepatócito e um desequilíbrio entre esses agentes pode gerar dano aos hepatócitos, endotélio, células de Kupffer e células estreladas levando à inflamação, isquemia, apoptose, necrose e dano ao DNA, com consequente desenvolvimento do HCC (CAPONE et al., 2012; DIESEN & KUO, 2010; MING-JU, et al., 2011). O desequilíbrio entre os sistemas antioxidante e pró-oxidante, com predomínio da ação oxidante, promove também alterações na peroxidação lipídica. A peroxidação lipídica é definida como uma reação de oxirredução em cadeia dos ácidos graxos poliinsaturados das membranas celulares. Essa reação altera a permeabilidade, fluidez e integridade das membranas celulares e está associada com a formação de produtos citotóxicos e morte celular (FRANÇA et al., 2013). 21 Tratamento antiviral e mecanismos de ação No Brasil, a lista de medicamentos disponíveis para hepatite C foi ampliada nos últimos anos em decorrência dos avanços no tratamento dessa infecção, destacando atualmente as diferentes combinações terapêuticas antivirais com ação direta sobre a replicação viral para diferentes subtipos do VHC (MINISTÉRIO DE SAÚDE, 2019). Dessa forma, as alternativas terapêuticas para o tratamento da hepatite C, com registro no Brasil e incorporadas ao Sistema Único de Saúde (SUS), apresentam efetividade terapêutica mensurada pela resposta virológica sustentada. A resposta virológica sustentada pode então ser comparada entre os esquemas propostos em casos de situações clínicas semelhantes, permitindo a análise da oferta dos esquemas terapêuticos no SUS baseada na observação da relação custo-minimização, ou seja, priorização das alternativas que implicam um menor impacto financeiro ao sistema, sem deixar de garantir o acesso a terapias seguras e eficazes às pessoas com hepatite C. Esta estratégia permite a ampliação do acesso ao tratamento medicamentoso a todos os pacientes infectados pelo VHC (MINISTÉRIO DE SAÚDE, 2019). Entre os medicamentos de uso isolado disponíveis no país podemos citar o daclatasvir, um inibidor do complexo enzimático ns5a, o simeprevir, que apresenta ação inibitória de protease NS3/4A e o sofosbuvir, um análogo de nucleotídeo que inibe a polimerase do VHC. As combinações medicamentosas preconizadas para o tratamento da Hepatite C, os mecanismos de ação dos fármacos e os efeitos adversos potenciais que estas combinações apresentam estão apresentadas no quadro 1. Quadro 2. Combinações medicamentosas preconizadas para o tratamento da Hepatite C no Brasil. Medicamento Mecanismo de Ação Efeitos Adversos Ombitasvir Inibidor do complexo enzimático NS5A Insônia, náusea, prurido na pele, astenia, fadiga e anemia. Odasabuvir Inibidor não nucleosídico da polimerase NS5B 22 Veruprevir Inibidor de protease NS3/4ª Ritonavir Potencializador farmacocinético Ledipasvir Inibidor do complexo enzimático NS5A Náusea, diarreia, constipação, dispepsia, vômito, dor abdominal, dor abdominal superior, boca seca, fadiga, irritabilidade, astenia, diminuição do apetite, mialgia, cefaleia, tontura, distúrbios da atenção, problemas de memória, insônia, ansiedade, depressão, distúrbios do sono, purido e angioedema. Osofosbuvir Análogo de nucleotídeo que inibe a polimerase do VHC Elbasvi Inibidor do complexo enzimático NS5A Náusea, dor abdominal, dor no abdomen superior, constipação, diarreia, boca seca, vômito, astenia, redução do apetite, artralgia, fadiga, cefaleias, mialgia, tontura, ansiedade, depressão, insônia, irritabilidade, alopecia e prurido. Grazoprevir Inibidor de protease NS3/4ª Fonte: Ministério de Saúde, 2019. Nota-se, que apesar dos avanços no tratamento da hepatite C, nos últimos anos, os medicamentos disponíveis possuem tolerância relativa, apresentando uma ampla gama de efeitos adversos severos e eficácia variável, de acordo com os diferentes genótipos virais envolvidos na infecção (BUTT et al., 2016; KUMTHIP & MANEEKARN, 2015; LIU et al., 2016). Assim, diante da gravidade de hepatite C e do grande impacto dessa infecção à saúde pública, diversas pesquisas têm sido realizadas com a finalidade de conhecer os processos metabólicos relacionados à patogênese da hepatite C e de desenvolver produtos que possam exercer atividade no processo inflamatório (diretamente relacionado à doença induzida pelo VHC) apresentando alta eficiência, boa tolerância pelo organismo dos portadores dessa doença e resistência aos mecanismos de evasão por mutabilidade do vírus. Compostos naturais bioativos Nesse cenário, destaca-se o esforço aplicado no desenvolvimento de compostos naturais bioativos (CNBs), na forma de coadjuvantes terapêuticos, de forma a atuar sinergicamente com os tratamentos já estabelecidos, interferindo direta ou indiretamente em diferentes etapas da 23 inflamação para potencializar os resultados terapêuticos. Os CNBs podem ser definidos como uma classe de produtos naturais (puros ou misturados) derivados de alimentos que apresentam benefícios contra doenças, e podem ser alimentos funcionais, suplementos dietéticos, nutrientes isolados, produtos fitoterápicos ou alimentos processados (SILVA et al., 2015; LOVELACE & POLYAK, 2015). Entre os CNBs vale destacar o resveratrol (RVT), um polifenol natural com estrutura de estilbeno e peso molecular de 228,25g/mol (Figura 2). Este composto apresenta baixa estabilidade frente à oxidação, fotosensibilidade e a solubilidade aquosa reduzida, no entanto, possui efeito biológico amplo, apresentando efeitos anit-envelhecimento (promoção de longevidade e atenuação contra doenças neurodegenerativas como doença de Alzheimer), atividades antimutagênica/antitumorais, anticarcinogênica, antiagregante plaquetária, anti-inflamatórias, antioxidantes e antivirais, além de um importante papel na prevenção de doenças cardiovasculares (GAMBINI et al., 2015; KULKARNI & CANTÓ, 2015). Figura 2. Estrutura molecular do Resveratrol. Fonte: Elaborado pelo autor. A atividade biológica do RVT está relacionada à inibição da COX, e consequente promoção de efeitos de quimioprotetores para prevenção do câncer, redução do estresse oxidativo e regulação de mediadores inflamatórios. A inbição da clicoxigenase também suprime o NF-κB e inibe a replicação viral, pela redução da expressão gênica e da síntese de proteínas virais. Descata-se ainda o potencial de regulação de angiogênege do RVT e O H O H O H 24 possível relação deste potencial com a prevenção de câncer (GAMBINI et al., 2015; KULKARNI & CANTÓ, 2015). A inibição da formação da placa de ateroma, por sua vez, associa-se a atuação do RVT pela melhora que este composto produz no perfil lipídico e na função cardíaca, na modulação da inflamação (redução da produção de várias citocinas angiogênicas, incluindo IL-8) e no aumento da produção de oxido nítrico endotelial com consequente vasodilatação e efeito cardioprotetor, além disso, o RVT também regula positivamente o nível da glicose e o metabolismo do tecido adiposo (KULKARNI & CANTÓ, 2015; ABBA et al., 2015). Especificamente em relação à atividade do RVT na terapia contra VHC, os resultados são considerados ser inconsistentes, uma vez que, existem evidências indicando efeito inibidor do RVT na replicação viral pela supressão da expressão da proteína NS3 (LEE et al, 2016) e descrição de atividade do RVT no aumento da replicação do VHC (CARREÑO, 2014). Considerando a grande atividade biológica do RVT como agente oxidante e o potencial deste para síntese de derivados diversos. Torna-se oportuno citar o desenvolvimento de um composto derivado do RVT a partir da adição da molécula de flúor. Este composto com peso molecular de 299,13 g/mol, denominado f-resveratrol (F-RVT), foi desenvolvido pelo Laboratório de Pesquisa e Desenvolvimento de Fármacos (Lapdesf) da Faculdade de Ciências Farmacêuticas - UNESP/Araraquara, sob a coordenação do Prof. Dr. Jean Leandro dos Santos e da Profa. Dra. Chung Man Chin. A estrutura molecular do F-Resveratrol pode ser observada na figura 3. Figura 3. Estrutura molecular do f-resveratrol. Fonte: Elaborado pelo autor, apartir de informações do laboratório desenvolvedor. 25 Apesar de não ter sua atividade biologica completamente elucidada, acredita-se no efeito potencial do F-RVT sobre o VHC por sua capacidade antioxidante e efeito sobre PPAR. A taurina (TAU) ou ácido 2-aminoetanossulfônico é aminoácido condicionalmente essencial que contem enxofre e deriva-se dos aminiacidos cisteina e metionina. Este composto, de peso molecular de 125,15 g/mol, difere-se dos demais aminoácidos pela presença de um grupamento sulfônico (-SO3) ao invés do grupamento carboxílico (COOH) (IKUBO et al., 2011). Figura 4. Estrutura molecular da taurina. Fonte: Elaborado pelo autor. A TAU não participa da síntese proteica, no entanto, constitui-se como um dos aminoácidos mais abundantes do organismo humano, destacando- se pelo desempenho de funções fisiologicas importantes no metabolismo energético, no sistema nervoso central e na retina, nos músculos esqueléticos e nos ossos (modulaçao de cálcio), no coração e nos intestinos. Além disso, o composto apresenta ação sobre a estabilidade da membrama plasmatica, a reprodução, a imunidade (IKUBO et al., 2011; MENZIE et al., 2014; MURAKAMI, 2015). Entre efeitos da TAU destaca-se seu potencial antioxidante e anti- inflamatório. Esses efeitos estão relacionados à capacidade da TAU de estabilizar as biomembranas, reduzindo a peroxidação de lipídica das mesmas e eliminando espécies reativas de oxigênio (IKUBO et al., 2011). A 26 TAU apresenta também efeitos de neuromodulação, neuroproteção e modulação do metabolismo da glicose (MENZIE et al., 2014; MURAKAMI, 2015). Diante do potencial destes CNBs faz-se necessário desvendar e caracterizar detalhadamente os possíveis mecanismos de ação de tais CBNs como inibidores do ciclo de replicação viral e do processo inflamatório induzido pelo VHC. Desse modo, propõe-se a realização desse estudo com o objetivo de avaliar e a influência dos CNBs (RVT, F-RVT e TAU) sobre o estresse oxidativo em células provenientes de hepatocarcinoma humano (Huh-7.5), transfectadas ou não com o replicon subclone genômico do vírus da hepatite C, SGR-JFH1 (Japanese fulminant hepatitis-1, genótipo 2a). Para tanto, buscou-se i) determinar o perfil de citotoxicidade in vitro induzido pelos CNBs nas células Huh-7,5 e SGR-JFH1, ii) avaliar o potencial de inibição da carga viral induzidos pelos compostos bioativos e iii) avaliar o potencial de indução de apoptose e necrose dos CNBs nas células Huh-7,5 e SGR-JFH1 por citometria de fluxo. Esse trabalho será apresentado em quatro capítulos. Os dois primeiros capítulos são artigos de revisão, sendo o primeiro relacionado à utilização CNBs como possibilidade de adjuvantes terapeuticos na infecção por hepatite C e o segundo relacionado às vias de sinalização celular envolvidas na regulação do metabolismo lipídico no fígado. Os dois últimos artigos, por sua vez, buscam responder aos objetivos do trabalho (descritos no paragrafo anterior). Deste modo buscou-se sistematiza a pesquisa desenvolvida e os resultados encontrados segundo CNB de interesse. Assim, capitulo 3 estará relacionado aos CNBs RVT e F-RVT e o capitulo 4 a TAU. Vale salientar que as normas de formatação de cada um dos cápitulos seguem as orientações das revistas para as quais os artigos foram ou serão enviados. 27 Capítulos 28 Capítulo 1. Natural Bioactive Compounds As Adjuvant Therapy For Hepatitis C Infection Publicação em 8 de outubro de 2020 Current Nutrition and Food Sciences (ISNN: 2212-3881; QUALIS/CAPES - CIÊNCIAS ALIMENTOS: B1). 29 Natural Bioactive Compounds As Adjuvant Therapy For Hepatitis C Infection Moema S. Santana a , Rute Lopes b , Isabela H. Peron a , Carla R. Cruz, Ana M. M. Gaspar b and Paulo I. Costa a* a Food and Nutrition Department, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara-SP, Brazil b Department of Biotechnology, Institute of Chemistry, São Paulo State University (UNESP), Araraquara-SP, Brazil *Author for correspondence. E-mail: costapi.unesp@gmail.com Abstract Background: Hepatitis C virus infection is a significant global health liability, which causes acute or chronic hepatitis. Acute hepatitis C is generally asymptomatic and progresses to cure, while persistent infection can progress to chronic liver disease and extrahepatic manifestations. Standard treatment is expensive, poorly tolerated, and has variable sustained virologic responses amongst the different viral genotypes. New therapies involve direct acting antivirals; however, it is also costly and may not be accessible for all patients worldwide. In order to provide a complementary approach to the already existing therapies, natural bioactive compounds have been investigated as to their several biologic activities, such as direct antiviral properties against hepatitis C, and effects on mitigating chronic progression of the disease, which include hepatoprotective, antioxidant, anticarcinogenic and anti-inflammatory activities. Additionally, these compounds represent advantages, such as chemical diversity, low cost of production and milder or inexistent side effects. Objective: To present a broad perspective on hepatitis C infection, chronic disease, and natural compounds with promising anti-HCV activity. Methods: This review consists of a systematic review study about the natural bioactive compounds as a potential therapy for hepatitis C infection. Results: The quest for natural products has yielded compounds with biologic activity, including viral replication inhibition in vitro, demonstrating antiviral activity against hepatitis C. Conclusion: One of the greatest advantages of using natural molecules from plant extracts is the low cost of production, not requiring chemical synthesis, which can lead to less expensive therapies available to low and middle-income countries. Keywords: Hepatitis C virus, natural bioactive compounds, hepatitis, adjuvant therapy, viral replication, molecular pathways. Graphical Abstract 30 1. INTRODUCTION Hepatitis C virus (HCV) is a single-stranded, RNA virus in the Flaviviridae family of about 9.6 kb in length with positive polarity, enveloped in a lipid bilayer, which encodes a single polyprotein of approximately 3,000 amino acids. The RNA genome is composed of a single open reading frame flanked by 5’- and 3’- highly structured untranslated regions. The 5’- untranslated regions contain a type III internal ribosomal entry site that is critical for viral replication. The polyprotein is cleaved by virus and host proteins into three structural proteins, core, the envelope glycoproteins E1 and E2, and seven nonstructural proteins, p7, NS2, NS3, NS4A, NS4B, and NS5B[1,2] Hepatitis C, liver inflammation caused by the hepatitis C virus (HCV), is considered one of the major hepatic diseases due to its worldwide distribution and great impact on public health. HCV may cause acute or chronic infection. Acute infection is usually asymptomatic, and 15 to 45% of people respond spontaneously to cure, or viral clearance, up to 6 months after infection. However, persistent infection may lead to chronic liver disease in 55 to 85% of infected individuals.[3,4] The World Health Organization (WHO) estimates that in 2015 there were 1.75 million newly infected individuals and 71 million living with chronic liver disease, while 399,000 died from end-stage infection, not including the deceased from extrahepatic complications of HCV infection and background mortality from other causes.[5] Currently there are no HCV vaccines available, therefore prevention is necessary to reduce the burden of HCV infection, which includes HCV screening, management and antiviral therapy to the already infected, and activities to reduce or eliminate HCV transmission due to blood, blood components and plasma derivatives; as a result of high-risk activities, as unsafe administration of drug and unprotected sex with multiple partners; and transfusion of blood in health care settings, or in tattoo and body piercing establishments.[6] 31 The general goal of hepatitis C treatment is to maintain or improve the patients’ quality of life through a safe therapy that promotes a slower progress of liver injury and prevents cirrhosis complications, in order to reduce the risk of evolution to hepatocellular carcinoma.[7] Nevertheless, the standard treatment is frequently poorly tolerated, with severe adverse effects and variable efficacy,[8–10]; hence many types of research analyze natural products that may present antiviral activity against HCV, with good tolerability and high efficacy. Natural bioactive compounds (NBCs) are natural products derived from foods, dietary supplements, isolated nutrients or phytotherapeutic products that have been extracted from plants and used in medicine since ancient times. NBCs have generated substances with several biologic activities, including antiviral effects on hepatitis B and C, human immunodeficiency virus, herpes simplex virus and influenza virus; hepatoprotective, antioxidant, anticarcinogenic, and anti-inflammatory properties, and many other effects. The purpose of this article is to address HCV infection, its chronic implications, and discuss NBCs that have been linked to anti-HCV activity, presenting the promising possibilities of the use of NBCs as adjuvant therapeutics of hepatitis C. 1.1. Chronic Development of the Disease HCV infection is a significant global burden that presents a personal, social and economic impact and can cause both acute and chronic hepatitis. The persistent infection can progress to chronic hepatitis C. Chronic HCV infection causes chronic inflammatory disease, possibly evolving to liver fibrosis, cirrhosis, hepatocellular carcinoma and death. Hepatitis C is the leading cause of liver transplantation in the United States and Europe being a chronic liver disease.[4,6,11] The natural history of hepatitis C is difficult to characterize and it has not been completely elucidated due to inaccurate information about transmission patterns, the incidence of cases in the asymptomatic period, 32 long development course and individual differences in morbidity. It has been suggested the evolution course of the infection might be related to viral factors, as genotype, HCV RNA serum levels and quasispecies; host factors, as genetic components, age at infection (considering that people over 70 years old present increased rates of fibrosis progression), male gender, obesity, type 2 diabetes, insulin resistance, co-infection with hepatitis B or HIV, immunosuppression; and behavioral factors, as chronic alcoholism.[12] The co-infection with other viruses that present similar course of contamination, like HIV, may characterize a faster progression of the disease, compared to HIV negative patients,[13] in a similar fashion as to what is observed in hepatitis B co-infection.[14] As for individual aspects of hepatitis C, it is complex to provide a prognosis and determine the outcome of treatment because the progression of the disease is multi-factorial.[12] Considering genotype influence, people infected with genotype 3 exhibit a faster progression to fibrosis and higher risk of developing hepatic steatosis and hepatocellular carcinoma.[15–17] Also, genotype 3 patients are less responsive to direct acting antivirals treatment when compared to other genotypes.[18] In respect to quasispecies (spontaneous mutations with nucleotide inclusion variations in the same HCV genotype and subtype), there is a possibility of a heterogeneous ensemble of virus infections that would favor HCV evasion from the host immune system and the establishment of a chronic infection, furthermore influencing the resistance to treatment and making the design of a vaccine more challenging.[19] Concerning behavioral factors, alcoholic consumption effects on hepatitis C is thus far inconsistent. In some studies, intense alcoholic consumption is related to fibrosis aggravation and the progression of the disease chronicity, suggesting a direct connection between alcohol intake and hepatic lesions.[20,21] Alternatively, other researchers report that moderate alcohol intake by HCV carriers does not increase the risk of progression to serious liver disease[22]; nevertheless it should be noted that there is a debate on the subject of the safe limit of consumption.[21] 33 It should be emphasized that the hepatic damage in the course of HCV infection is associated with immune-mediated mechanisms. The quality of cellular immune system seems crucial for the elimination or persistence of HCV.[23,24] Therefore, CD4+ lymphocytes may present two distinct responses to HCV infection. In the first response, Th1 mediated, the secretion of interleukin (IL)-2 and interferon gamma (IFN-γ) stimulates the antiviral host response. In the second response, Th2 mediated, the synthesis of IL-4 and IL-10 stimulates antibody production and inhibits the Th1 response. In this sense, the imbalance between Th1 and Th2 mediated responses might be responsible for both inability to eliminate the HCV as well as the gravity of the hepatic injury. However, the elements that modulate immune response have not been fully elucidated.[23] When the persistence of the infection is related to the imbalance between Th1 and Th2 responses, an inflammatory infiltrate is formed with distinct immune cell populations.[24] These cells are recruited by chemotactic cytokines released by endothelial cells that mobilize specific lymphocytes to the liver and increase inflammation through interactions between endothelial cells and leukocytes.[23] The recruited cells (macrophages, neutrophils and endothelial cells), in the attempt to eliminate the virus, produce and release nitric oxide, which is an important pleiotropic mediator of inflammation and may cause a potential inflammatory injury to the tissue, that might be aggravated by the release of reactive oxygen and nitrogen species (RONS). The RONS increase even more the expression of cytokines (IL-8, CCL5) and other endothelial adhesion molecules, amplifying the inflammation cascade and generating oxidative stress to the hepatic tissue. Thus, oxidative stress (featured by an increase of oxidizing agents and diminished antioxidant ability in cells) occurs as an important biochemical mechanism involved with hepatic pathogenesis induced by HCV infection, since the equilibrium between RONS production and antioxidant defenses is crucial to physiological homeostasis, and an imbalance might cause injury to hepatocytes, endothelium, Kupffer cells, and hepatic stellate cells (HSC) This results in inflammation, ischemia, DNA damage, apoptosis and necrosis, which may lead to hepatocellular 34 carcinoma.[25,26] There is a possibility of direct induction of RONS and mitochondrial dysfunction caused by the virus, since the increase in RONS production and oxidative stress induction by the virus core protein, NS3 and NS5A in vitro and in vivo has been reported.[11] The critical role of host-virus interaction might be demonstrated by the capacity of viral proteins, especially core protein, to trigger an initiation signal of cellular proliferation, differentiation or apoptosis processes. In the normal liver, HSCs are quiescent, but when activated by cytokines in response to liver injury, HSCs transform proliferative, contractile myofibroblasts, producing other extracellular matrix (ECM) proteins and cytokines in excess and disrupting the balance between deposition and dissolution of ECM proteins. It eventually leads to fibrotic scarring, liver dysfunction, cirrhosis and its complications. HCV proteins modulate signaling and metabolic pathways, promoting an immune response that will cause chronic inflammation.[27,28] Figure 1 displays viral and host chronicity factors involved in hepatitis C progression. Figure 1. Viral and host chronicity factors involved in hepatitis C progression. Chronic hepatitis C and the inflammatory response are linked associated with extrahepatic manifestations including type 2 diabetes, 35 glomerulonephritis, thyroid disorders, porphyria cutaneous tarda, mixed cryoglobulinemia, lichen planus, B-cell non-Hodgkin lymphoma, Sjögren’s syndrome, atherosclerosis, cardiovascular and brain disease.[29–31] 2. METHODS This review consists of a systematic study about the natural bioactive compounds as therapeutic potential for hepatitis C infection. The databases Medline, Scopus and Web of Science were included in the search. The search was conducted using the descriptors: ("natural bioactive compounds") AND ("hepatitis C"). Studies were restricted to those published from 2014 to 2019 in English language. 3. RESULTS AND DISCUSSION 3.1. NBCs in hepatitis c treatment Currently, the standard treatment for hepatitis C consists of pegylated interferon alpha (PEG-IFN-α) with ribavirin, frequently poorly tolerated, thus causing severe side effects which causes discontinuation of the treatment, and it also shows variable efficacy according to viral genotype.[8–10] In the last few years, HCV therapy has been thoroughly improved after many direct acting antivirals were accepted to be used in the clinical practice. This new treatment has shown high sustained virologic response, reduction of both dosage and treatment duration, preferably of oral intake, and with fewer side effects. However, developing a flawless treatment is a challenge. Despite the advances already achieved, drug resistance and genotype specific efficacy are some issues to be considered.[32,33] Therefore, in view of hepatitis C gravity and the great impact this infection has on public health, several researches have been focusing on the development of potential products, amongst them are NBCs which are complementary therapeutics and in synergy to the already established treatment course, that could present high efficacy, displaying less or no side effects, and that could be resistant to the virus evasion mechanisms by mutagenesis. 36 NBCs can be defined as a class of natural products, pure compounds or mixtures, derived from foods that present health benefits, dietary supplements, isolated nutrients, phytotherapeutic products or processed foods.[34,35] The WHO estimates approximately 80% of the population worldwide predominantly depend on traditional medicine for their primary health care.[36] Throughout history, several natural compounds have been extracted from plants and used in medicine. Plants exhibit the ability to produce a wide variety of compounds, responsible for their essential functions related to growth and development. The quest for natural products has yielded compounds with biologic activity, including viral replication inhibition in vitro, with certain natural medicines demonstrating antiviral activity against hepatitis B and C, human immunodeficiency virus, herpes simplex virus and influenza virus. Also, several herbs are known for their hepatoprotective properties.[37] In this way, NBCs are important pharmacologically, being part of the composition of most modern medicines with prophylactic or therapeutic intervention mechanics. [37] When it comes specifically to hepatitis C, the important role that NBCs can play in reducing infection of new hepatocytes is highlighted, when NBCs act in reducing on viral entrey (interactions with specific receptors) and release (formation of lipid drop), reducing viral load, when the mechanisms of action relate with the reduction of viral replication and assembly (influences on viral proteins), and reduction of the inflammatory process and the advancement of the chronicity of the disease through interference of NCBs on virus-host-specific interactions (influence on the production of reactive oxygen and nitrogen species and chemotactic factors).[37,38]. Amidst these compounds, flavonoids, lignans, phytoalexins and tannins should be highlighted.[38] One of the greatest advantages of using natural molecules from plant extracts is the low cost of production, not requiring chemical synthesis, which can lead to less expensive therapies available to low and middle-income countries. 37 NBCs can be classified into 5 major groups, polyphenols, carotenoids, alkaloids, nitrogen-containing compounds, and organosulfur compounds. The most studied main groups are polyphenols and carotenoids.[39,40] The focus of this work is the NBCs with activity directly or indirectly linked to hepatitis C therapy. 3.1.1. Polyphenols Polyphenols have 4 subgroups, corresponding to the number of phenol rings they present: flavonoids, phenolic acids, stilbenes, and lignans. [39,40]. 3.1.1.1. Flavonoids Flavonoids comprise the largest group of natural metabolites with variable phenolic structures observed in vegetables, fruits, grains, bark, roots, stems, flowers, tea, wine and seeds, and they act on the defense against pathogens and insects. The flavonoids are classified into other subgroups: flavones, flavanones, isoflavones, flavanols or catechins, flavanonols, anthocyanins and chalcones. It is reported that some flavonoids have antiviral activity on HCV [37,40]. 3.1.1.1.1. Flavones Flavones constitute a major class in the flavonoids family and their antiviral activity has been known since the 1990’s. In flavones, the B ring is attached to C-2.[37,41] The Asteraceae family, one of the largest angiosperm families with more than 1,000 genera and 30,000 species, has been widely studied for its oral treatment of liver diseases. The compound apigenin, a flavone isolated from the family Asteraceae, has shown activity against RNA viruses, including HCV, reducing mature microRNA miR122, a liver-specific microRNA which positively regulates HCV replication.[37,42,43] Nobiletin (NOB) is a polymethoxylated flavone found in peels of citrus fruits such as Citrus depressa and C. sinensis that concomitantly with its 38 metabolites has displayed numerous physiologic properties, including anti- inflammatory, antiviral, antitumor, anti-dementia, antioxidant, anti-obesity, anti-metabolic syndrome, anti-hyperlipidemia, anti-diabetes, and neuroprotective activities.[44–47] These effects may be attributed to NOB and its metabolites ability to modulate cellular signaling pathways, mainly related to cyclic adenosine monophosphate (AMPc) and nuclear factor kappa B (NF-κB).[46,48,49] NOB supposedly regulates differentiation and lipolysis and improves insulin resistance, apparently directly affecting adipocyte functions[46]. Moreover, it modulates the expression of essential genes for learning and memory by the activation of the AMPc response element-binding protein signaling pathway.[49] The inhibition of NF-κB, allows the modulation of the inflammatory process and the response to oxidative stress in a coordinated fashion, whereas NOB and its metabolites may inhibit tumor necrosis factor alpha (TNF-α), IL-1, mitogen-activated protein kinase (MAPK), LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), and c-Jun N- terminal kinase (JNK), reducing pro-inflammatory signals related to cellular differentiation and apoptosis.[44,48] Regarding the interaction between NOB, its metabolites and HCV, there is evidence that the antiviral activity is mediated by the inhibition of viral protein NS3.[37] Additionally, a study investigating the inhibitory effect of NOB in vitro, on hepatocarcinoma cells, and in vivo, in mouse model demonstrated a promotion of cell cycle arrest, apoptosis, and regulation of the expression of proteins B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved caspase-3, and cyclooxygenase-2 (COX-2), interfering with apoptosis signaling pathways and the inflammatory process, and significantly reducing tumor growth.[50] 3.1.1.1.2. Flavanones Flavanones constitute a subclass of flavonoids glycosylated by a disaccharide at position 7, and, contrary to other more abundant classes of 39 flavonoids, flavanones are found almost exclusively in citrus fruits and, in a lower degree, in tomatoes and some aromatic herbs.[51] Naringenin, a citric flavanone, is a metabolite produced from naringin hydrolysis by enterobacteria. In a research performed by Assini and collaborators[52] in mice fed cholesterol-enriched diets, naringenin attenuated peripheral and systemic inflammation, resulting in protection from atherosclerosis, and also reduced fatty acids synthesis through induction of hepatic fatty acids oxidation, prevented hepatic steatosis, hepatic VLDL overproduction, and hyperlipidemia. The compound apparently presents COX-inhibitory activity potential for the treatment of inflammation, and could traverse the blood–brain barrier for neurodegenerative diseases treatment.[40] Regarding anti-HCV activity, naringenin inhibits the microsomal triglyceride transfer protein (MTTP) activity, the transcription of 3-hydroxy-3- methyl-glutaryl-coenzyme A (HMG-Coa) reductase and acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) in infected cells, and reduces HCV secretion in a dose dependent manner without affecting intracellular levels of the viral RNA or protein, by blocking the assembly of intracellular infectious viral particles.[53,54] 3.1.1.1.3. Isoflavones Isoflavones are simple isomers of flavonoids that display the B ring attached to C-3 of the heterocyclic ring and are naturally found as secondary plant metabolites of the class phytoestrogen.[41] Curcumin, a curcuminoid phenolic compound that exhibits a bright yellow color, is extracted from Curcuma longa L. (turmeric) rhizomes, and has applications as a food ingredient or condiment and in traditional medicine, as a therapeutic adjuvant for the treatment of asthma, dermatitis, Alzheimer’s disease, type 1 and 2 diabetes, rheumatoid arthritis, chronic psoriasis vulgaris, fibromyalgia, and many other diseases. This compound displays several properties, as anti-inflammatory, antiviral, antibacterial, antifungal, antioxidant, antiparasitic and antitumor activity.[55–57] 40 The curcumin plays a role in HCV infection evaluated in studies which aim to elucidate its molecular pathways and its effects over viral replication. [58–60] There are indications that curcumin reduces the intracellular expression of HCV RNA and viral proteins NS5A and NS5B in a dose dependent manner, the activity mediated by the increased expression of heme oxygenase-1 (HO-1) and the inhibition of the protein phosphorylation of ERK and protein kinase B (AKT).[58] Curcumin also inhibits the expression of NF-κB in the HCV infected cell; however, studies indicate that the compound activity reduces the HCV RNA expression by the suppression of AKT activation, not by NF-κB pathway.[58,59] 3.1.1.1.4. Flavanols or catechins Flavanols, flavan-3-ols, dihydroflavonols or catechins are a wide group of polyphenols that are 3-hydroxy derivatives of flavanones.[40] Epigallocatechin gallate (EGCG) is a polyphenol which is most abundant in green tea extract. This compound presents anti-inflammatory, antitumor, and anti-hepatic steatosis effects,[61–63] and its biologic activity is mainly related to the coordinated action of the metabolic activation pathways of adenosine 5'-monophosphate-activated protein kinase (AMPK) and the phosphorylation inhibition of AKT, which in turn enables inflammation regulation, blocking the production of inflammatory mediators such as inducible nitric oxide synthase (iNOS) and IL-6, additionally influencing cellular apoptosis.[64] Concerning the interaction with HCV, there is evidence that the compound interferes with viral cycle by suppressing viral entry into the cell in a dose dependent manner, decreasing RNA replication and obstructing the infectious particle release to the ECM.[65–67] Apparently, binding inhibition between virus and cell surface occurs through the direct action of EGCG over the viral particle, since there is no alteration on expression levels of cellular factors upon HCV entry (CD81, CLDN1, OCLN, SR-BI) when administering the compound. Hence, it is thought that EGCG interacts with envelope glycoproteins E1 and E2, suppressing viral binding to the target cell 41 surface.[65,66] Research further indicates that EGCG inhibits the expression of viral proteins NS3 and NS5B in in vitro assays, suggesting a relationship between this effect on RNA replication reduction and viral particle release.[67] 3.1.1.2. Phenolic Acids Phenolic acids may be regarded as promising antiviral candidates, especially caffeic acid and tannins as gallic acid, and their derivatives. [68] Coffee and green tea are rich in caffeine and polyphenols. However, only coffee contains chlorogenic acid in its composition, which, after its absorption, it is metabolized into caffeic and quinic acids. An in vitro study performed by Tanida and collaborators [69] demonstrated that the number of viral particles released by HCV infected cells decreased after treatment with caffeic acid, and NS3 protein levels also decreased. Furthermore, it was also observed that HCV propagation is inhibited when cell treatment with caffeic acid is continuous. Ajala and collaborators [70] point out the antiviral capacity on HVC of hydrolyzable tannins (ellagic acid and gallic acid) through the inhibitory action of NS3 and NS4A proteases, demonstrating the potential use of these compounds as broadspectrum antivirals. Liu and collaborators [71] demonstrated that tannic acid, a polymer of gallic acid and glucose, is a potent inhibitor of HCV entry into Huh7.5 cells at low concentrations and blocks the spread of HCV in infectious cell cultures without inhibiting HCV replication after infection. Tannic acid seems to act by preventing the initial anchoring of the HCV virus to the cell surface. In this study, gallic acid, a structural component of tannic acid, did not present anti- HCV activity at a concentration of up to 25 μm. However, the gallic acid and another phenolic compound has been researched for their anti-HCV in vitro properties by Hsu and collaborators [72], and they reported that the compound could inhibit HCV entry into primary human hepatocytes cell culture. 42 3.1.1.3. Stilbenes Resveratrol is a natural polyphenol with stilbene structure that demonstrates antitumor, anti-inflammatory, antioxidant and antiviral activities, apart from an important role in cardiovascular disease prevention.[73,74] The biologic activity of resveratrol is related to COX inhibition, producing effects on cancer prevention, oxidative stress reduction, and regulating inflammatory mediators by suppressing NF-κB and inhibiting viral replication, with decreased genic expression and viral proteins synthesis. There are pieces of evidence on the potential inhibition of atheroma plaque formation showing improvement of lipid profile and increased production of endothelial nitric oxide with resulting vasodilation and cardioprotective effect. [74,75] Specifically regarding resveratrol’s activity on HCV therapy, results may be inconsistent. The evidence indicates viral replication inhibition by the suppression of NS3 protein expression,[76] however, others report an increase in replication. [77] 3.1.1.4. Lignans Lignans are one of three groups of natural compounds classified as phytoestrogens, due to a diphenolic ring that makes them structurally similar to estrogens. They are isolated from woody portions of plants, seeds and grains, and they act predominantly as antioxidants.[78] Honokiol is a small active molecular compound extracted from magnolia (Magnolia officinalis), a flower used in traditional medicine, belonging to the lignans group.[37] In 2012, Lan and collaborators[79] reported that honokiol displays inhibitory effects on HCV infection in vitro showing promising effects on viral entry into the cell, in genotypes 1a, 1b and 2a, by suppressing the expression of OCLN and SR-BI, which are essential to viral entrance. Moreover, honokiol acted on the replication stage of genotypes 1a and 2a, reducing and even inhibiting, in a dose dependent manner, viral proteins expression, such as NS3, NS5A and NS5B. Finally, honokiol showed more accentuated inhibitory effects on HCV when combined with INF-α than when combined with both ribavirin and INF-α. 43 3-Hydroxy Caruilignan C (3-HCL-C) is a lignan isolated stems of Swietenia macrophylla, and it was described by Wu and collaborators[80] for its anti-HCV properties. This research demonstrated that 3-HCL-C reduced NS3 protein and RNA levels in vitro by the induction of an interferon response pathway (IFN signaling pathway), which is suppressed during HCV infection. Moreover, the combination of 3-HCL-C and IFN- α or telaprevir decreased viral RNA replication. 3.1.2. Carotenoids Carotenoids are a class of phytonutrients comprising over 600 natural pigments of intense color and high liposolubility, found in fruits, vegetables and some fish.[81] Although they exist in large quantity, there are only a few carotenoids that might be found in human tissues[82] and their anticarcinogenic activity is well documented. [83] These compounds may be employed as cancer biomarkers, since the amount of carotenoids in the organism is inversely proportional to the disease progression.[84] Lycopene, a carotenoid acyclic found in red grapefruits and in red color vegetables,[85] is known for its antiviral and anticarcinogenic effects.[86] A research by Sheriff [81] evaluated rats with induced hepatitis C, with lycopene, and demonstrated that this compound helps restore hepatic function due to its antioxidant properties. According to Seren and collaborators [86], lycopene molecule structure promotes the inactivation of free radicals and interfere with lipid peroxidation, therefore preventing possible hepatic tissue injuries. 3.1.3. Alkaloids Alkaloids are a highly diverse group of secondary compounds isolated from plants, less frequently from fungi or animals. Alkaloid compounds can be classified as true alkaloids, derived from amino acid and a heterocyclic ring with nitrogen; protoalkaloids, when the N atom derived from an amino acid is not a part of the heterocycle; and pseudoalkaloids, in which the basic carbon skeletons are not derived from 44 amino acids, whereas the definition criteria is based on structure. Alkaloid- containing plants have been used as a medication since early history, including a wide variety of applications, like morphine and codeine as analgesics, hyoscyamine as antispasmodic and anti-parkinson drugs, taxol to treat breast and ovary carcinomas, and many others.[87,88] Michellamine B, a naturally occurring alkaloid with antioxidant properties, has reduced virus particles that effect cell culture production in persistently infected cells without effecting intracellular RNA levels.[89] Jardim and collaborators[88] reported that APS, a natural alkaloid isolated from Maytrenus ilicifolia, had inhibitory effects over HCV replication decreased HCV replication in a dose dependent manner, using subgenomic replicons in vitro. The Myrioneuron alkaloids are a family of lysine-based metabolites synthesized by plants of the genus Myrioneuron R. Br. (Rubiaceae) that have a common promising anti-HCV activity, with low cytotoxicity.[91,92] Different NBCs groups, the main compounds described in this work, and their biological anti-HCV activities are summarized in Table 1. Table 1. Natural bioactive compounds (NBCs) with biological or anti-hepatitis C virus activities. Main group NBCs Biologic activity Effect on HCV References Nobiletin ↓AMPc phosphorylation ↓ stearoyl-CoA enzyme ↓ NF-κB ↓ TNF-α ↓ IL-1 ↓ MAPK, ↓ ERK and JNK phosphorylation ↓ NS3 Hepatocellular carcinoma ↑ Bcl-2 gene ↓ Bax (pro-apoptosis) ↑ Bcl-2 itself (anti- apoptosis) ↓ caspase-3 ↓ COX-2 [38,44-46, 48, 50] Flavonoids Naringenin Anti-inflammatory Prevents atherosclerosis ↓ FA ↓ COX ↓ VLDL production ↓ MTTP ↓ HMG-Coa ↓ ACAT2 ↓ HCV secretion [40, 52-54] Curcumin COX-2 ↓ iNOS ↓ JNK ↓ MAPK ↓ NF-κB, ↓ AP-1 ↓ PKC ↓ 5-LOX ↓ IL-6 ↓ IL-8 ↓ IL-1 ↑ Nrf2 ↓ VCAM-1 ↓ AKT ↓ Viral replication ↓ RNA expression ↓ NS5A ↓ NS5B ↑HO-1 ↓ ERK and AKT phosphorylation [55–59] 45 Nobiletin ↓AMPc phosphorylation ↓ stearoyl-CoA enzyme ↓ NF-κB ↓ TNF-α ↓ IL-1 ↓ MAPK, ↓ ERK and JNK phosphorylation ↓ NS3 Hepatocellular carcinoma ↑ Bcl-2 gene ↓ Bax (pro-apoptosis) ↑ Bcl-2 itself (anti- apoptosis) ↓ caspase-3 ↓ COX-2 [38,44-46, 48, 50] Phenolic acids EGCG ↑ AMPK ↓ AKT ↓ iNOS ↓ IL-6 ↓ Viral entry into the cell ↓ RNA replication ↓ Viral release reduction ↓ NS3 and NS5B [64–67] Caffeic acid - ↓ release of viral particles ↓ NS3 ↓ HCV propagation [69] Gallic acid - ↓ NS3 and NS4A ↓ Viral entry into the cell [70,72] Ellagic acid - ↓ NS3 and NS4A [70] Tannic acid - ↓ Viral entry into the cell [71] Stilbene Resveratrol - Inconsistent information: ↓ Viral replication [74] ↓ NS3 [74] ↑ Viral replication [75] [73–77] Lignans Honokiol Antioxidant activity ↓ OCLN and SR-BI ↓ NS3, NS5A and NS5B [38, 79] 3-HCL-C ↓ NS3 ↓ RNA levels ↑ IFN signaling pathway [38,80] Carotenoids Lycopene Antiviral and anticarcinogenic effects Antioxidant activity: ↓free radicals and interfere on lipid peroxidation [81,86] Alkaloids Michellamine B, APS, Myrioneuron Family Antioxidant activity Anti-HCV activity in vitro [90-92] Legend: AMPc: cyclic adenosine monophosphate; NF-κB: nuclear factor kappa B; IL (-6;-8;- 1): interleukin; MAPK: mitogen-activated protein kinase; ERK: extracellular signal–regulated kinases; JNK: c-Jun N-terminal kinase; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; COX-2: cyclooxygenase-2; FA: fatty acids; MTTP: microsomal triglyceride transfer protein; HMG-Coa: 3-hydroxy-3-methyl-glutaryl-coenzyme A; ACAT2: acyl-coenzyme A:cholesterol acyltransferase 2 enzyme; iNOS: inducible nitric oxide synthase; AP-1: activator protein 1; PKC: protein kinase C; 5-LOX: 5-lipoxygenase; Nrf2: nuclear factor erythroid 2 (NFE2)-related factor 2; VCAM-1: vascular cell adhesion protein 1; AKT: Protein 46 Kinase B; HO-1: heme oxygenase-1; EGCG: epigallocatechin gallate; AMPK: Adenosine 5'- monophosphate-activated protein kinase; OCLN: occluding; SR-BI: scavenger receptor class B member 1; IFN-γ: interferon gamma; TNF-α: tumor necrosis factor alpha; 3-HCL-C: 3-Hydroxy Caruilignan C. CONCLUSION Some NBCs compounds have antagonistic effects to HCV due to the ability to modulate cellular signaling pathways, the inflammatory process and the response to oxidative stress in a coordinated fashion, reducing pro- inflammatory signals related to cellular differentiation and apoptosis. In this way, the possibilities of using NCBs as adjuvants in hepatitis C therapy are very promising, but they need to be explored extensively in vivo and later in controlled and randomized clinical trials to define usage techniques and recommended doses to minimize adverse effects and enhance traditional therapy for the treatment of hepatitis C. FUNDING This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília - DF, Brazil, Finance Code 001. 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Short and Scalable Total Synthesis of Myrioneuron Alkaloids (±)-α,β-Myrifabral A and B. Org Lett 2016; 18(4): 669-71. doi:10.1021/acs.orglett.6b00005. 62 Capítulo 2. Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review Publicado em 31 de março de 2020 Acta Scientiarum Biological Sciences (ISNN: 1807-863X; QUALIS/CAPES - CIÊNCIAS ALIMENTOS: B5). 63 Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review Rute Lopes 1 , Moema Souza Santana 2 , Carla Rios da Cruz 2 , Rafael Bianchini Fulindi 2 , Ana Maria Minarelli Gaspar 1 and Paulo Inácio da Costa 2* 1 Department of Biotechnology, Institute of Chemistry, São Paulo State University (UNESP), Araraquara-SP, Brazil. 2 Food and Nutrition Department, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara-SP, Brazil. *Author for correspondence. E-mail: costapi.unesp@gmail.com ABSTRACT The liver is primarily responsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism is regulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic gene expression and cholesterol metabolism and act in the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential to various metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications. Keywords: Lipid Synthesis, Hepatic Molecular Routes, Fatty Acids, Steatosis. Introduction The liver consists largely of hepatocytes, the major scaffold for lipoprotein synthesis, but it also contains many other cell types (Aizawa, Seki, Nagano, & Abe, 2015; La Rosa Rodriguez & Kersten, 2017). The main function of lipid metabolism is to distribute lipids to peripheral tissues or to ensure their return to the liver to be reprocessed (Tang, 2016). The fatty acids in the liver originate from the diet in energy abundant states, from de 64 novo lipogenesis, or are derived from the adipose tissue, and enter the liver as free fatty acids (La Rosa Rodriguez & Kersten, 2017). The diseases or metabolic disorders related to lipid processing have a large public health impact because of both the increasing population affected worldwide and the potential of these disorders to advance to chronic complications that lead to poor quality of life, in which is associated with increased global expenditures directly connected to those health issues as well as high morbidity and mortality rates (Trogdon et al., 2015). Some of the most important pathological conditions associated with lipid metabolic disorders include type 2 diabetes mellitus, obstructive sleep apnea, coronary artery disease, non-alcoholic steatohepatitis (NASH) and some types of cancer (Tang, 2016). Furthermore, lipid metabolism has been demonstrated to have an important role in cancer cell survival and proliferation under hypoxic conditions (Chen & Li, 2016; Tang, 2016). The central cell signaling pathways related to lipid metabolism include AKT, AMPK, SREBP, PPAR and JNK. Understanding lipid metabolism and liver function as well as the main signaling pathways connected to these phenomena, is crucial to identify abnormalities and develop strategies to manage and regulate metabolic disorders and metabolic disorder-associated complications. Methods This review consists of a systematic review study about the major cell signaling pathways involved with the regulation of lipid metabolism in the liver. The databases Medline (https://www.ncbi.nlm.nih.gov/pubmed), Scopus (https://www.scopus.com) and Web of Science (https://apps.webofknowledge.com) were included in the search. The search was conducted using the descriptors: ("lipid metabolism") AND ("cell signaling" OR "pathway" OR "signaling") AND "steatosis". Studies were restricted to those published from 2014 to 2019, in English language. 65 Lipids and cell signaling pathways Cell metabolism is regulated by several molecules in many signaling pathways and consists of biochemical stimuli transmitted to effector molecules, by the inhibition or activation of downstream molecules, to elicit an intended response. Lipids, particularly fatty acids and cholesterol, constitute an important