abcArquivos Brasileiros de CardiologiaArq. Bras. Cardiol.0066-782X1678-4170Sociedade Brasileira de Cardiologia - SBCSão Paulo, SP, BrazilJH6TKRvNqKgbsMWYX6cFgZFS0066-782X2012000200006S0066-782X(12)0980020610.1590/S0066-782X2012005000006Artigos OriginaisResveratrol provoca efeitos antiaterogênicos em um modelo animal de ateroscleroseMatosRossane SerafimBaronciniLis Andréa VillelaPrécomaLeonardo BrandãoWinterGuilhermeLambachPedro HenriqueCaronKaiberFláviaPrécomaDalton BertolimCuritibaParanáPontifícia Universidade Católica do ParanáBrazil022012982136142260520110908201120072011This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
FUNDAMENTO: O resveratrol protege o sistema cardiovascular por meio de uma série de mecanismos, incluindo atividades antioxidantes e antiplaquetárias. OBJETIVO: Avaliar os possíveis efeitos anti-inflamatórios e antiaterogênicos do resveratrol, utilizando coelhos alimentados com uma dieta hipercolesterolêmica (1% de colesterol). MÉTODOS: Vinte coelhos brancos adultos do sexo masculino foram selecionados e divididos em dois grupos: grupo controle (GC), 10 coelhos; e grupo resveratrol (GR), 10 coelhos. Os animais foram alimentados com uma dieta hipercolesterolêmica por 56 dias. Para a dieta do GR, o resveratrol (2mg/kg peso/dia) foi adicionado do 33º ao 56º dia. RESULTADOS: Não houve diferença significativa entre os grupos no colesterol sérico total, no colesterol HDL, no colesterol LDL e nos triglicerídeos. No GC, 70% apresentaram lesões ateroscleróticas avançadas da aorta (tipos III, IV, V ou VI). Todos os animais do GR apresentaram lesões ateroscleróticas leves da aorta (tipos I ou II) ou não apresentaram lesões. A razão entre a área intimal e a área da camada intimal/medial mostrou-se significativamente menor no GR quando comparada ao GC (p < 0,001). Áreas positivas para moléculas de adesão celular vascular-1 (VCAM-1) foram menores no GR (p = 0,007). As concentrações de proteína quimiotática de monócitos-1 (MCP-1) e de interleucina-6 (IL-6) mostraram-se significativamente menores no GR do que no GC (p = 0,039 e p = 0,015, respectivamente). CONCLUSÃO: O Resveratrol apresentou importantes efeitos antiaterogênicos e anti-inflamatórios em um modelo animal com coelhos alimentados com uma dieta hipercolesterolêmica.
Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brasil
Correspondência
RESUMO
FUNDAMENTO: O resveratrol protege o sistema cardiovascular por meio de uma série de mecanismos, incluindo atividades antioxidantes e antiplaquetárias.
OBJETIVO: Avaliar os possíveis efeitos anti-inflamatórios e antiaterogênicos do resveratrol, utilizando coelhos alimentados com uma dieta hipercolesterolêmica (1% de colesterol).
MÉTODOS: Vinte coelhos brancos adultos do sexo masculino foram selecionados e divididos em dois grupos: grupo controle (GC), 10 coelhos; e grupo resveratrol (GR), 10 coelhos. Os animais foram alimentados com uma dieta hipercolesterolêmica por 56 dias. Para a dieta do GR, o resveratrol (2mg/kg peso/dia) foi adicionado do 33º ao 56º dia.
RESULTADOS: Não houve diferença significativa entre os grupos no colesterol sérico total, no colesterol HDL, no colesterol LDL e nos triglicerídeos. No GC, 70% apresentaram lesões ateroscleróticas avançadas da aorta (tipos III, IV, V ou VI). Todos os animais do GR apresentaram lesões ateroscleróticas leves da aorta (tipos I ou II) ou não apresentaram lesões. A razão entre a área intimal e a área da camada intimal/medial mostrou-se significativamente menor no GR quando comparada ao GC (p < 0,001). Áreas positivas para moléculas de adesão celular vascular-1 (VCAM-1) foram menores no GR (p = 0,007). As concentrações de proteína quimiotática de monócitos-1 (MCP-1) e de interleucina-6 (IL-6) mostraram-se significativamente menores no GR do que no GC (p = 0,039 e p = 0,015, respectivamente).
CONCLUSÃO: O Resveratrol apresentou importantes efeitos antiaterogênicos e anti-inflamatórios em um modelo animal com coelhos alimentados com uma dieta hipercolesterolêmica.
BACKGROUND: Resveratrol protects the cardiovascular system by a number of mechanisms, including antioxidant and anti-platelet activities.
OBJECTIVE: To assess the potential anti-inflammatory and antiatherogenic effects of resveratrol using rabbits fed a hypercholesterolemic diet (1% cholesterol).
METHODS: Twenty white male rabbits were selected and divided into two groups: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. The animals were fed a hypercholesterolemic diet for 56 days. For the RG diet, resveratrol (2mg/kg weight/day) was added from days 33 - 56.
RESULTS: There was no significant difference in the total serum cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides between the groups. Of the CG, 70% had advanced aortic atherosclerotic lesions (types III, IV, V, or VI). All animals from the RG had mild aortic atherosclerotic lesions (types I or II, or no lesions). The intima area and the intima/media layer area ratio was significantly lower in the RG as compared to the CG (p<0.001). Positive areas for VCAM-1 molecules were lower in the RG (p=0.007). The MCP-1 and IL-6 concentrations were lower in the RG than the CG (p=0.039 and p=0.015, respectively).
CONCLUSION: Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model with rabbits fed a hypercholesterolemic diet (1% cholesterol).
O transresveratrol (resveratrol) é um composto polifenólico encontrado em uvas frescas, suco de uva e vinho. O resveratrol é produzido pela pele das uvas em resposta à exposição fúngica. O resveratrol tem sido implicado em muitos estudos, mas não em todos, como um mediador da proteção cardiovascular independente de álcool que seria conferida pela ingestão de vinho tinto1. O resveratrol protege o sistema cardiovascular por uma série de mecanismos, incluindo: a inibição da oxidação de baixa densidade da lipoproteína mediada pelo resveratrol; a inibição da agregação plaquetária; a síntese de eicosanoides pró-aterogênicos; a inibição da proliferação celular; e a elevação do vasorrelaxamento. Estudos recentes também demonstraram que o resveratrol suprime a indução do fator tecidual pró-coagulante, um dos principais componentes responsáveis pela alta mortalidade por doença cardiovascular2-5. O objetivo deste estudo foi avaliar os possíveis efeitos anti-inflamatórios e antiaterogênicos do resveratrol em modelo animal experimental, com coelhos alimentados com uma dieta hipercolesterolêmica (1% de colesterol).
Métodos
Animais
Selecionaram-se 20 coelhos brancos adultos do sexo masculino, provenientes da Nova Zelândia e com idade média de 30 dias. Os animais foram tratados de acordo com os princípios norteadores sobre o cuidado e uso de animais. A aprovação do protocolo foi obtida a partir do Comitê de Pesquisa Animal da Pontifícia Universidade Católica. Os animais foram divididos em dois grupos: grupo controle (CG), 10 coelhos; e grupo resveratrol (GR), 10 coelhos. Durante os 56 dias do estudo, os animais foram alimentados com uma dieta específica para a espécie (Nuvilab®), com mais 1% de colesterol de ovos liofilizados. Do 29º ao 56º dia, o resveratrol (Resveratrol®, Laboratório Galena®) foi adicionado à dieta do GR (2 mg/kg/dia) e administrado por gavagem oral. No 56º dia, os animais foram submetidos à dissecção do arco aórtico e da aorta descendente. A anestesia foi induzida com quetamina (3,5 mg/kg, Vetanarcolâ, König) e xilazina intramuscular (5 mg/kg, Coopazineâ, Coopers). Após o procedimento, os coelhos foram sacrificados por dose letal de barbitúricos. O tamanho da amostra foi calculado com base no estudo de Zou e cols.6.
Química sanguínea
Amostras de sangue foram obtidas no primeiro dia do experimento e imediatamente antes do sacrifício por punção cardíaca. A avaliação clínico-laboratorial incluiu colesterol total (CT), colesterol da lipoproteína de alta densidade (HDL-C), colesterol da lipoproteína de baixa densidade (LDL-C) e triglicerídeos (TGC). As medidas foram aferidas por meio de sistema automatizado (Abbott Architect ci8200, Abbott Laboratories, Abbott Park, IL, EUA).
Análise histológica
As artérias foram retiradas e lavadas com formaldeído tamponado a 10% com fosfato (ph = 7,6) e fixadas em parafina. Para a avaliação desse modelo experimental, foram adotadas medidas histológicas qualitativas e quantitativas. Os cortes corados por hematoxilina e eosina (HE) foram analisados cegamente em um microscópio de cinco cabeças da marca Olympusâ BX 40. As lesões ateroscleróticas foram classificadas de 0 a VI, de acordo com os critérios qualitativos propostos por Stary e cols.7-9. A análise morfométrica foi realizada em orceína corada (elástica) para determinar a área intimal e as camadas mediais do arco aórtico e da aorta descendente, bem como para determinar a razão entre as áreas intimal e medial (RIM - área da camada intimal dividida pela área da camada medial). Para essa avaliação, o segmento mais lesado ou o segmento em estágio mais avançado de aterosclerose foi previamente selecionado no corte corado por HE. As análises foram realizadas microscopicamente em conjunto com o software Image Pro-plus® 4.5 (Media Cybernetics Inc., Silver Spring, MD, EUA).
Imuno-histoquímica
Avaliamos a concentração da molécula de adesão celular vascular (VCAM-1), da proteína quimiotática de monócitos-1 (MCP-1) e da interleucina-6 (IL-6) na íntima das artérias principais, utilizando anticorpos monoclonais. A leitura foi realizada com um microscópio Olympus® BX50, com objetiva de 20 vezes.
Análise estatística
As variáveis categóricas foram expressas em porcentagens, enquanto as variáveis contínuas foram expressas como a média ± DP e medianas. O teste de Shapiro-Wilks foi utilizado para testar a normalidade da amostra. Para as condições de simetria, algumas variáveis foram submetidas à transformação logarítmica. Em relação às variáveis nominais dicotômicas, o teste exato de Fisher foi empregado para comparar os grupos. No que se refere aos parâmetros quantitativos, o teste t de Student e o teste não paramétrico de Mann-Whitney foram utilizados para a comparação entre o GC e o GR. A significância estatística foi estabelecida em p < 0,05.
Resultados
Dois animais do GC morreram no meio do experimento e foram retirados da análise final. O peso corpóreo inicial médio dos animais do GC (1,78 kg) e do GR (1,82 kg) foi semelhante (p = 0,274). No final do estudo, os animais do GC apresentaram peso corpóreo médio de 2,58 kg, enquanto os animais do GR tinham peso corpóreo médio de 2,39 kg (p = 0,537).
Perfil lipídico
Os níveis basais de CT, HDL-C, LDL-C e TG sérico mostraram-se relativamente equivalentes em todos os grupos antes do início da dieta. No final do experimento, uma análise desses níveis não indicou diferença significativa entre os grupos (Tabela 1).
Análise histológica
No final dos experimentos, ficou demonstrado que houve uma diferença estatisticamente significativa entre os grupos de animais em relação à probabilidade de lesões dos tipos I, II, III, IV, V, VI ou ausência de lesões. Dos animais presentes no GC, 70% apresentaram lesões ateroscleróticas avançadas (tipos III, IV e V) no arco aórtico e na aorta descendente, enquanto 100% dos animais presentes no GR apresentaram lesões ateroscleróticas leves (tipos I e II) ou não apresentaram nenhuma lesão no arco aórtico e na aorta descendente (Figura 1).
Análise morfométrica
A área íntima apresentou-se significativamente menor no GR do que no GC (p = 0,001, Figura 1). Houve uma redução significativamente maior na razão entre as áreas intimal e medial (RIM) no GR do que no GC (p = 0,001). Não houve diferença estatisticamente significativa entre a área medial dos grupos no arco aórtico e na aorta descendente (Tabela 2).
Imuno-histoquímica
Uma análise das áreas positivas para a VCAM-1 revelou uma diferença significativa entre os grupos, com maior concentração no GC (p = 0,007). A análise da MCP-1 e da IL-6 mostrou valores significativamente maiores no GC em comparação ao GR (p = 0,039 e p = 0,015, respectivamente; Tabela 3, Figura 2).
Discussão
O resveratrol, presente no suco de uva e no vinho, é considerado o principal polifenol responsável por propiciar benefícios cardiovasculares devido às propriedades antioxidantes e atividades antiplaquetárias10,11. O resveratrol possui propriedades anti-inflamatórias que se manifestam como a inibição da expressão de ICAM-1 e VCAM, a adesão de monócitos às células endoteliais, a inibição da síntese de TNF-ae IL-1-be induzida por lipopolissacarídeos (LPS) e a liberação de IL-6 dos monócitos12. O resveratrol também inibe a migração e a proliferação de células musculares lisas vasculares (CMLVs) para a camada íntima, que é considerada a condição sine qua non da aterogênese13,14. Coletivamente, esses efeitos seriam responsáveis pela diminuição da incidência de doenças cardíacas na população francesa, processo denominado "paradoxo francês" (taxa de mortalidade por doenças cardiovasculares muito baixa, apesar de uma dieta rica em gordura), devido à preferência da população pelo vinho tinto15. Neste estudo, foram induzidas lesões ateroscleróticas com a adição de ovo liofilizado na dieta normal de coelhos, durante quatro semanas. Esse modelo experimental de indução de aterosclerose com o uso de ovo liofilizado é raramente descrito na literatura, embora seja comprovadamente eficiente e menos oneroso, quando comparado ao emprego de colesterol industrializado. O resveratrol foi adicionado na dieta do GR após quatro semanas, para testar sua capacidade de interferir em lesões ateroscleróticas estabelecidas. O estudo demonstrou que o resveratrol inibe a progressão de lesões ateroscleróticas e reduz a área íntima e a RIM, de modo que os animais não apresentaram lesões avançadas no arco aórtico e na aorta descendente no GR. Em estudo semelhante que também utilizou coelhos, Castro e cols.16 concluíram que o resveratrol funcionou como agente preventivo no desenvolvimento de lesões ateroscleróticas, tendo em vista a observação de um baixo grau de invasão das células de espuma na túnica média dos animais tratados com resveratrol. Os mecanismos pelos quais o resveratrol inibe a formação de lesões ateroscleróticas avançadas parecem decorrer do efeito inibitório sobre a oxidação do LDL, a agregação plaquetária e a proliferação vascular de células musculares lisas. A oxidação do LDL é a principal causa de lesão endotelial e indução da expressão de moléculas pró-inflamatórias em células endoteliais. Estudos anteriores relatam que o resveratrol protege os lipídios da degradação peroxidativa e interrompe a captação de LDL oxidado na parede vascular de forma dependente da concentração17-19. No entanto, existem resultados conflitantes sobre os efeitos do resveratrol nos níveis de lipídios, pois alguns estudos não demonstraram redução nos níveis de lipídios plasmáticos induzidos por essa substância20. O presente estudo não revelou qualquer evidência de uma diferença significativa no perfil lipídico entre os grupos, o que pode ter refletido o curto período do experimento. Daugherty e cols.21, em um estudo semelhante em coelhos hipercolesterolêmicos, mostraram que a redução nas lesões ateroscleróticas secundárias à redução do LDL-C é um processo que leva meses ou até mesmo anos. Em contrapartida, no presente estudo, demonstramos que uma dieta hipercolesterolêmica faz com que o desenvolvimento de lesões ateroscleróticas associadas a um processo inflamatório, juntamente com o resveratrol, reduziu a concentração de VCAM-1, MCP-1 e IL-6 na camada íntima de coelhos hipercolesterolêmicos. Sabe-se que a inflamação media todas as fases da aterosclerose desde a iniciação até a progressão e, finalmente, a ruptura da placa17. De fato, a VCAM-1, a MCP-1 e a IL-6 facilitam a aderência dos monócitos à superfície endotelial e a migração transendotelial de leucócitos22-25. Esse processo é exacerbado quando se elevam os níveis de LDL no sangue devido a maus hábitos alimentares ou predisposição genética e quando os sinais do endotélio aumentam a expressão de moléculas de adesão e potencializam a resposta inflamatória26. Embora não correlacionemos os níveis de lipídeos no sangue ao processo inflamatório, acreditamos que essa possível relação deve ser objeto de estudos futuros. Além disso, não analisamos a concentração de monócitos e de macrófagos, que parecem desempenhar um papel crucial no desenvolvimento da aterosclerose. Essa célula parece estar envolvida em todas as fases do desenvolvimento da placa aterosclerótica e é cada vez mais considerada uma candidata para a intervenção terapêutica e um biomarcador da progressão da doença e da resposta à terapia27-29.
Conclusão
O resveratrol apresentou importantes efeitos antiaterogênicos e anti-inflamatórios em modelo animal, utilizando-se coelhos alimentados com uma dieta hipercolesterolêmica (1% de colesterol).
Liz Andréa Villela Baroncini
Rua Buenos Aires, 764 / 601, Batel - 80250-070
Curitiba, PR, Brasil
E-mail:
lizavb@cardiol.br
lizandreabaroncini@hotmail.com
Artigo recebido em 26/05/11, revisado recebido em 20/07/11; aceito em 09/08/11.
1. Turner RT, Evans GL, Zhang M, Maran A, Sibonga JD. Is resveratrol an estrogen agonist in growing rats? Endocrinology. 1999;140(1):50-4.Is resveratrol an estrogen agonist in growing rats?Endocrinology19995041140TurnerRTEvansGLZhangMMaranASibongaJD2. Pendurthi US, Rao LVM. Resveratrol suppresses agonist-induced monocyte adhesion to cultured human endothelial cells. Thromb Res. 2002;106(4-5):243-8.Resveratrol suppresses agonist-induced monocyte adhesion to cultured human endothelial cellsThromb Res200224384-5106PendurthiUSRaoLVM3. Wilson T, Knight TJ, Beitz DC, Lewis DS, Engen RL. Resveratrol promotes atherosclerosis in hypercholesterolemic rabbits. Life Sci. 1996;59(1):PL15-21.Resveratrol promotes atherosclerosis in hypercholesterolemic rabbitsLife Sci1996PL1521159WilsonTKnightTJBeitzDCLewisDSEngenRL4. Pace-Asciak CR, Rounova O, Hahn SE, Diamandis EP, Goldberb DM. Wines and grape juices as modulators of platelet aggregation in healthy human subjects. Clin Chim Acta. 1996;246(1-2):163-82.Wines and grape juices as modulators of platelet aggregation in healthy human subjectsClin Chim Acta1996163821-2246Pace-AsciakCRRounovaOHahnSEDiamandisEPGoldberbDM5. Pace-Asciak CR, Hahn S, Diamandis EP, Soleas G, Goldberg DM. The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta. 1995;235(2):207-19.The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart diseaseClin Chim Acta1995207192235Pace-AsciakCRHahnSDiamandisEPSoleasGGoldbergDM6. Zou JG, Wang ZR, Huanhg YZ, Cao KJ, Wu JM. Effect of red wine and wine polyphenol resveratrol on endothelial function in hypercholesterolemic rabbits. Int J Mol Med. 2003;11(3):317-20.Effect of red wine and wine polyphenol resveratrol on endothelial function in hypercholesterolemic rabbitsInt J Mol Med200331720311ZouJGWangZRHuanhgYZCaoKJWuJM7. Stary HC, Blankenhorn DH, Chandler AB, Glagov S, Insull W Jr, Richardson M, et al. A definition of the intima of human arteries and of its atherosclerosis- prone regions: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1992;85(1):391-405.A definition of the intima of human arteries and of its atherosclerosis- prone regions: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart AssociationCirculation1992391405185StaryHCBlankenhornDHChandlerABGlagovSInsull WJrRichardsonM8. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME, et al. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994;89(5):2462-78.A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart AssociationCirculation1994246278589StaryHCChandlerABGlagovSGuytonJRInsull WJrRosenfeldME9. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arterisoclerosis, American Heart Association. Circulation. 1995;92(5):355-74.A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arterisoclerosis, American Heart AssociationCirculation199535574592StaryHCChandlerABDinsmoreREFusterVGlagovSInsull WJr10. Fulgenzi A, Bertelli AAE, Magni E, Ferrero E, Ferrero ME. In vivo inhibition of TNFa-induced vascular permeability by resveratrol. Transplant Proc. 2001;33(3):2341-3.In vivo inhibition of TNFa-induced vascular permeability by resveratrolTransplant Proc200123413333FulgenziABertelliAAEMagniEFerreroEFerreroME11. Olas B, Wachowicz B. Resveratrol and vitamin C as antioxidants in blood platelets. Thromb Res. 2002;106(2):143-8.Resveratrol and vitamin C as antioxidants in blood plateletsThromb Res200214382106OlasBWachowiczB12. Slater SJ, Seiz JL, Cook AC, Stagliano BA, Buzas CJ. Inhibition of protein kinase C by resveratrol. Biochim Biophys Acta. 2003;1637(1):59-69.Inhibition of protein kinase C by resveratrolBiochim Biophys Acta2003596911637SlaterSJSeizJLCookACStaglianoBABuzasCJ13. Poussier B, Cordova AC, Becquemin JP, Sumpio BE. Resveratrol inhibits vascular smooth muscle cell proliferation and induces apoptosis. J Vasc Surg. 2005;42(6):1190-7.Resveratrol inhibits vascular smooth muscle cell proliferation and induces apoptosisJ Vasc Surg200511907642PoussierBCordovaACBecqueminJPSumpioBE14. Chao HH, Juan SH, Liu JC, Yang HY, Yang E, Cheng TH, et al. Resveratrol inhibits angiotensin II-induced endothelin-1 gene expression and subsequent proliferation in rat aortic smooth muscle cells. Eur J Pharmacol. 2005;515(1-3):1-9.Resveratrol inhibits angiotensin II-induced endothelin-1 gene expression and subsequent proliferation in rat aortic smooth muscle cellsEur J Pharmacol2005191-3515ChaoHHJuanSHLiuJCYangHYYangEChengTH15. Rocha KK, Souza GA, Ebaid GX, Seiva FR, Cataneo AC, Novelli EL. Resveratrol toxicity: effects on risk factors for atherosclerosis and hepatic oxidative stress in standard and high-fat diets. Food Chem Toxicol. 2009;47(6):1362-7.Resveratrol toxicity: effects on risk factors for atherosclerosis and hepatic oxidative stress in standard and high-fat dietsFood Chem Toxicol200913627647RochaKKSouzaGAEbaidGXSeivaFRCataneoACNovelliEL16. Castro M, Pacheco MMR, Machado MRF. Morphology of aortic arch in rabbits with atherosclerosis treated with resveratrol. Intern J Appl Res Vet Med. 2009;7(4):190-5.Morphology of aortic arch in rabbits with atherosclerosis treated with resveratrolIntern J Appl Res Vet Med2009190547CastroMPachecoMMRMachadoMRF17. Fan E, Zhang L, Jiang S, Bai Y. Beneficial effects of resveratrol on atherosclerosis. J Med Food. 2008;11(4):610-4.Beneficial effects of resveratrol on atherosclerosisJ Med Food20086104411FanEZhangLJiangSBaiY18. Nigdikar SV, Williams NR, Griffin BA, Howard AN. Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nutr. 1998;68(2):258-65.Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivoAm J Clin Nutr199825865268NigdikarSVWilliamsNRGriffinBAHowardAN19. Shanmuganayagam D, Warner TF, Krueger CG, Reed JD, Folts JD. Concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbits. Atherosclerosis. 2007;190(1):135-42.Concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbitsAtherosclerosis2007135421190ShanmuganayagamDWarnerTFKruegerCGReedJDFoltsJD20. Castro M, Veiga APM, Pacheco MR. Plasma lipid profile of experimentally induced hyperlipidemic New Zealand white rabbits is not affected by resveratrol. J Appl Res. 2009;9(1):18-22.Plasma lipid profile of experimentally induced hyperlipidemic New Zealand white rabbits is not affected by resveratrolJ Appl Res2009182219CastroMVeigaAPMPachecoMR21. Daugherty A, Schonfeld G, Sobel BE, Lange LG. Metabolism of very low density lipoproteins after cessation of cholesterol feeding in rabbits: a factor potentially contributing to the slow regression of atheromatous plaques. J Clin Invest. 1986;77(4):1108-15.Metabolism of very low density lipoproteins after cessation of cholesterol feeding in rabbits: a factor potentially contributing to the slow regression of atheromatous plaquesJ Clin Invest1986110815477DaughertyASchonfeldGSobelBELangeLG22. Tang W, Pankow JS, Carr JJ, Tracy RP, Bielinski SJ, North KE, et al. Association of sICAM-1 and MCP-1 with coronary artery calcification in families enriched for coronary heart disease or hypertension: the NHLBI Family Heart Study. BMC Cardiovasc Disord. 2007;7:30.Association of sICAM-1 and MCP-1 with coronary artery calcification in families enriched for coronary heart disease or hypertension: the NHLBI Family Heart StudyBMC Cardiovasc Disord2007307TangWPankowJSCarrJJTracyRPBielinskiSJNorthKE23. Ferrero ME, Bertelli AE, Fulgenzi A, Pellegatta F, Corsi MM, Bonfrate M, et al. Activity in vitro of resveratrol on granulocyte and monocyte adhesion to endothelium Am J Clin Nutr. 1998;68(6):1208-14.Activity in vitro of resveratrol on granulocyte and monocyte adhesion to endotheliumAm J Clin Nutr1998120814668FerreroMEBertelliAEFulgenziAPellegattaFCorsiMMBonfrateM24. Carluccio MA, Siculella L, Ancora MA, Massaro M, Scoditti E, Storelli C, et al. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of mediterranean diet phytochemicals. Arterioscler Thromb Vasc Biol. 2003;23(4):622-9.Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of mediterranean diet phytochemicalsArterioscler Thromb Vasc Biol20036229423CarluccioMASiculellaLAncoraMAMassaroMScodittiEStorelliC25. Wung BS, Hsu MC, Wu CC, Hsieh CW. Resveratrol suppreses IL-6-induced ICAM-1 gene expression in endothelial cells: effects on the inhibition of STAT3 phosphorylation. Life Sci. 2005;78(4):389-97.Resveratrol suppreses IL-6-induced ICAM-1 gene expression in endothelial cells: effects on the inhibition of STAT3 phosphorylationLife Sci200538997478WungBSHsuMCWuCCHsiehCW26. McLaren JE, Ramji DP. Interferon gamma: a master regulator of atherosclerosis. Cytokine Growth Factor Rev. 2009;20(2):125-35.Interferon gamma: a master regulator of atherosclerosisCytokine Growth Factor Rev200912535220McLarenJERamjiDP27. Saha P, Modarai B, Humphries J, Mattock K, Waltham M, Burnand KG, et al. The monocyte/macrophage as a therapeutic target in atherosclerosis. Curr Opin Pharmacol. 2009;9(2):109-18.The monocyte/macrophage as a therapeutic target in atherosclerosisCurr Opin Pharmacol20091091829SahaPModaraiBHumphriesJMattockKWalthamMBurnandKG28. Ghosh S, Zhao B, Bie J, Song J. Macrophage cholesteryl ester mobilization and atherosclerosis. Vascul Pharmacol. 2010;52(1-2):1-10.Macrophage cholesteryl ester mobilization and atherosclerosisVascul Pharmacol20101101-252GhoshSZhaoBBieJSongJ29. Prieur X, Rõszer T, Ricote M. Lipotoxicity in macrophages: evidence from diseases associated with the metabolic syndrome. Biochim Biophys Acta. 2010;1801(3):327-37.Lipotoxicity in macrophages: evidence from diseases associated with the metabolic syndromeBiochim Biophys Acta20103273731801PrieurXRõszerTRicoteMOriginal ArticlesResveratrol causes antiatherogenic effects in an animal model of atherosclerosis
BACKGROUND: Resveratrol protects the cardiovascular system by a number of mechanisms, including antioxidant and anti-platelet activities. OBJECTIVE: To assess the potential anti-inflammatory and antiatherogenic effects of resveratrol using rabbits fed a hypercholesterolemic diet (1% cholesterol). METHODS: Twenty white male rabbits were selected and divided into two groups: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. The animals were fed a hypercholesterolemic diet for 56 days. For the RG diet, resveratrol (2mg/kg weight/day) was added from days 33 - 56. RESULTS: There was no significant difference in the total serum cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides between the groups. Of the CG, 70% had advanced aortic atherosclerotic lesions (types III, IV, V, or VI). All animals from the RG had mild aortic atherosclerotic lesions (types I or II, or no lesions). The intima area and the intima/media layer area ratio was significantly lower in the RG as compared to the CG (p<0.001). Positive areas for VCAM-1 molecules were lower in the RG (p=0.007). The MCP-1 and IL-6 concentrations were lower in the RG than the CG (p=0.039 and p=0.015, respectively). CONCLUSION: Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model with rabbits fed a hypercholesterolemic diet (1% cholesterol).
Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil
Mailing Address
ABSTRACT
BACKGROUND: Resveratrol protects the cardiovascular system by a number of mechanisms, including antioxidant and anti-platelet activities.
OBJECTIVE: To assess the potential anti-inflammatory and antiatherogenic effects of resveratrol using rabbits fed a hypercholesterolemic diet (1% cholesterol).
METHODS: Twenty white male rabbits were selected and divided into two groups: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. The animals were fed a hypercholesterolemic diet for 56 days. For the RG diet, resveratrol (2mg/kg weight/day) was added from days 33 - 56.
RESULTS: There was no significant difference in the total serum cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides between the groups. Of the CG, 70% had advanced aortic atherosclerotic lesions (types III, IV, V, or VI). All animals from the RG had mild aortic atherosclerotic lesions (types I or II, or no lesions). The intima area and the intima/media layer area ratio was significantly lower in the RG as compared to the CG (p<0.001). Positive areas for VCAM-1 molecules were lower in the RG (p=0.007). The MCP-1 and IL-6 concentrations were lower in the RG than the CG (p=0.039 and p=0.015, respectively).
CONCLUSION: Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model with rabbits fed a hypercholesterolemic diet (1% cholesterol).
Trans-resveratrol (resveratrol) is a polyphenolic compound found in fresh grapes, grape juice, and wine. Resveratrol is produced by the skin of grapes in response to fungal exposure. Resveratrol has been implicated in many, but not all, studies as a mediator of alcohol-independent cardiovascular protection that is allegedly conferred by drinking red wine1. Resveratrol protects the cardiovascular system by a number of mechanisms, including resveratrol-mediated inhibition of low-density lipoprotein oxidation, inhibition of platelet aggregation, synthesis of pro-atherogenic eicosanoids, inhibition of cell proliferation, and increased vasorelaxation. Recent studies have also shown that resveratrol suppresses the induction of procoagulant tissue factor, one of the key components thought to be responsible for high mortality from cardiovascular disease2-5. The objective of this study was to assess the potential anti-inflammatory and anti-atherogenic effects of resveratrol using an experimental animal model with rabbits fed a hypercholesterolemic diet (1% cholesterol).
Methods
Animals
Twenty white adult New Zealand male rabbits at a mean age of 30 days were selected. Animals were handled in compliance with the Guiding Principles in the Care and Use of Animals. Protocol approval was obtained from the Animal Research Committee of the Pontifícia Universidade Católica. The animals were divided into 2 groups, as follows: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. During the 56 days of the study, the animals were fed a specific diet for the species (Nuvilab®) plus 1% cholesterol from lyophilized eggs. From days 29 - 56, resveratol (Resveratrol®; extracted from the Galena® Laboratory) was added to the RG diet (2mg/kg/day) and administered by oral gavage. On day 56, the animals underwent dissection of the aortic arch and descending aorta. Anesthesia was induced with ketamine (Vetanarcol®, 3.5 mg/kg; König) and intramuscular xylazine (Coopazine®, 5 mg/kg; Coopers). After the procedure, the rabbits were sacrificed by a lethal dose of barbiturate. The sample size was calculated based on the study by Zou et al6.
Blood chemistry
Blood samples were obtained on the first day of the experiment and immediately before sacrifice by cardiac puncture. Clinical laboratory assessment included total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGC). Measurements were taken using an automated system (Abbott Architect ci8200; Abbott Laboratories, Abbott Park, IL, USA).
Histologic analysis
The arteries were removed and washed with 10% formaldehyde buffered with phosphate (ph=7.6) and waxed. For evaluation of this experimental model, qualitative and quantitative histologic measurements were adopted. The hematoxylin and eosin (HE) stained slices were analyzed blindly in a microscopy jellyfish five-head Olympus® BX 40. The atherosclerotic lesions were graded from 0 - VI according to the qualitative criteria proposed by Stary et al7-9. The morphometric analysis was performed on stained orcein (elastic) to determine the area of the intima and the medial layers of the aortic arch and descending aorta, as well as to determine the intima/media layer area ratio (IMR - the area of the intimal layer divided by the area of the medial layer). For this assessment, the more injured segment or the segment with more advanced stage of atherosclerosis was previously selected in the HE-stained slice. The analyses were performed microscopically in conjunction with Image Pro-plus® 4.5 software (Media Cybernetics Inc., Silver Spring, MD, USA).
Immunohistochemistry
We evaluated the concentration of the vascular cell adhesion molecule (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and interleucin-6 (IL-6) in the intima of the arteries using primary monoclonal antibodies. The reading was performed with an Olympus® BX50 microscope, with an objective of 20 times.
Statistical analysis
Categorical variables were expressed as percentages and continuous variables were expressed as the mean ± SD and medians. The Shapiro-Wilks test was used for testing sample normality. For symmetry conditions, some variables were submitted to logarithmic transformation. For dichotomic nominal variables, the Fisher exact test was used to compare the groups. For quantitative parameters, Student's t-test and Mann-Whitney non-parametric test were used for the comparison between CG and RG. Statistical significance was indicated by p < 0.05.
Results
Two animals from the CG died in the middle of the experiment and were removed from the final analysis. The initial mean body weight of animals in the CG (1.78 kg) and RG (1.82 kg) were similar (p=0.274). At the end of the study, CG animals exhibited a mean body weight of 2.58 kg, while RG animals had a mean body weight of 2.39 kg (p=0.537).
Lipid profiles
Baseline TC, HDL-C, LDL-C, and TGC levels were relatively equivalent in all groups before initiation of the diet. At the end of the experiment, an analysis of TC, HDL-C, LDL-C, and serum TG indicated no significant difference between the groups (Table 1).
Histologic analysis
At the end of the experiments, it was shown that there was a statistically significant difference between the animal groups with respect to the probability of type I, II, III, IV, V, and VI lesions or absence of lesions. Of those animals in the CG, 70% had advanced atherosclerotic lesions (types III, IV, and V) in the aortic arch and descending aorta, while 100% of the RG animals had mild atherosclerotic lesions (types I and II, or no lesions) in the aortic arch and descending aorta (Figure 1).
Morphometric analysis
The intimal area was significantly lower in the RG than the CG (p = 0.001; Figure 1). There was a significantly greater reduction in the intima/media layer area ratio (IMR) in the RG than in the CG (p = 0.001). There was no statistically significant difference between the medial layer area between the groups in the aortic arch and descending aorta (Table 2).
Immunohistochemistry
An analysis of positive areas for VCAM-1 revealed a significant difference between groups, with a higher concentration in CG (p=0.007). Analysis of MCP-1 and IL-6 showed significantly higher values in the CG compared with the RG (p=0.039 and p=0.015, respectively; Table 3; Figure 2).
Discussion
Resveratrol, present in the grape juice and wine, is considered the major polyphenol responsible for cardiovascular benefits because of its antioxidant and anti-platelet activities10,11. Resveratrol has anti-inflammatory properties which are manifested as inhibition of ICAM-1 and VCAM-1expression and the attachment of monocytes to endothelial cells, inhibition of lipopolysaccharide (LPS)-induced synthesis of TNF-α and IL-1-β, and release of IL-6 from monocytes12. Resveratrol also inhibits the migration and proliferation of vascular smooth muscle cells (SMCs) to the intima layer, which is considered the sine qua non of atherogenesis13,14. Collectively, these effects are thought to be responsible for decreased incidence of heart disease in the French population, and is thus termed the French Paradox (a very low mortality rate due to cardiovascular disease, despite a high-fat diet), because of the population's preference for red wine15. In this study, we induced atherosclerotic lesions with the addition of lyophilized egg to the normal diet of rabbits during 4 weeks. This experimental model of inducing atherosclerosis using lyophilized egg is rarely described in the literature, though it is proven to be efficient and less costly when compared to the use of industrialized cholesterol. Resveratrol was added in the diet of RG after 4 weeks to test its capacity to interfere in established atherosclerotic lesions. We demonstrated that resveratrol inhibits the progression of atherosclerotic lesions by reducing the intima area and the IMR, and showing no advanced lesions in the aortic arch and descending aorta in the RG. In a similar study using rabbits, Castro et al16 concluded that resveratrol worked as a preventive agent in the development of atherosclerotic lesions as they observed a low-degree of foam cell invasion in the tunica media of animals treated with resveratrol. The mechanisms by which resveratrol inhibits the formation of advanced atherosclerotic lesions appears to be through its inhibitory effect on LDL oxidation, platelet aggregation, and vascular proliferation of smooth muscle cells. LDL oxidation is a main cause of endothelial injury and induction of the expression of pro-inflammatory molecules in endothelial cells. Resveratrol has been shown to protect lipids from peroxidative degradation and to stop the uptake of oxidized LDLs in the vascular wall in a concentration-dependent manner17-19. However, there are conflicting results regarding the effects of resveratrol on lipid levels, because some studies have failed to show a reduction in plasma lipids levels induced by such a substance20. In this study, we did not uncover any evidence of a significant difference in lipid profiles between groups, which may have reflected the short period of the experiment. Daugherty et al21, in similar a study in hypercholesterolemic rabbits, showed that the reduction in atherosclerotic lesions secondary to the reduction in LDL-C levels is a process that takes months, or even years. In contrast, in this study, we showed that a hyphercholesterolemic diet causes the development of atherosclerotic lesions associated with an inflammatory process, and resveratrol reduced the concentration of VCAM-1, MCP-1, and IL-6 in the intima layer of hypercholesterolemic rabbits. It is known that inflammation mediates all stages of atherosclerosis from initiation to progression, and eventually plaque rupture17. Indeed, VCAM-1, MCP-1, and IL-6 facilitate the adherence of monocytes to the endothelial surface and facilitate leukocyte transendothelial migration22-25. This process is exacerbated when blood LDL levels are high from poor dietary habits or an inherited genetic pre-disposition, and signals the endothelium to increase adhesion molecule expression and potentiate the inflammatory response26. Even though we did not make the correlation between blood lipid levels and the inflammatory process, we believe that such a possible correlation should be the subject of future studies. Also, we did not analyze the monocyte/macrophage concentration that appears to have a crucial role in the development of atherosclerosis. This cell appears to be involved in all stages of atherosclerotic plaque development and is increasingly regarded as a candidate for therapeutic intervention and as a potential biomarker of disease progression and response to therapy27-29.
Conclusions
Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model of rabbits fed a hypercholesterolemic diet (1% cholesterol).