Estudo proteômico do tecido hepático e muscular de frangos de corte suplementados com óleo da semente de maracujá
Data
2022-08-01
Autores
Assunção, Andrey Sávio de Almeida
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Entre as alternativas estudadas para substituir o uso de antibióticos na avicultura industrial, destacam-se os produtos compostos por óleos essenciais e/ou extratos vegetais, denominados de aditivos fitogênicos. Esses produtos possuem potencial pois são naturais e livre de resíduos em comparação aos antibióticos. Dentre os aditivos naturais com potencial de serem utilizados na prática industrial, encontra-se o óleo da semente de maracujá (OSM). No entanto, não há relatos na literatura científica sobre a utilização de abordagens proteômicas para elucidar o efeito do OSM sobre o proteoma hepático e muscular de frangos de corte expostos ao estresse cíclico por calor. A proteômica permite avaliar as mudanças na expressão de proteínas de tecidos e fluídos, auxiliando dessa forma, na compreensão dos mecanismos moleculares envolvidos na saúde, bem-estar, estresse e estados patológicos dos animais. Neste sentido, a tese foi dividida em dois capítulos principais correspondendo aos artigos científicos. Artigo 1 - O objetivo com o presente estudo foi investigar o efeito da suplementação do OSM sobre o perfil diferencial de proteínas expressas no tecido hepático de frangos de corte submetidos ao estresse cíclico por calor. Para isso, 225 pintainhos machos de 1 dia de idade foram alojados em gaiolas dispostas em câmara climática onde foram mantidos sob estresse cíclico por calor durante oito horas por dia. Foram formuladas cinco dietas experimentais a base de milho e farelo de soja, sendo uma dieta controle (sem adição de OSM) e quatro dietas nas quais foi realizada a inclusão de diferentes níveis de OSM (0,30; 0,50; 0,70 e 0,90%). Aos 36 dias de idade, nove aves de cada tratamento foram abatidas para coleta das amostras de fígado. A partir de pools das amostras de fígado, foi realizado o fracionamento do proteoma hepático por eletroforese bidimensional (2D-PAGE) e caracterização das proteínas por espectrometria de massas em sequência acoplada com cromatografia líquida (LC-MS/MS). A suplementação de 0,90% de OSM aumentou a expressão de sete proteínas de choque térmico, enquanto foi observado menor expressão de seis proteínas antioxidantes. Proteínas envolvidas no metabolismo lipídico e de carboidratos também apresentaram alterações na expressão. Estes resultados sugerem que a suplementação de OSM influencia a ativação de mecanismos de reparação dos danos provocados pela exposição cíclica ao calor por meio do aumento da expressão das chaperonas moleculares no tecido hepático. Artigo 2 - O objetivo com este estudo foi caracterizar as proteínas diferencialmente expressas no músculo Pectoralis major de frangos de corte suplementados com OSM em condições de estresse cíclico por calor. Para isso, 90 pintainhos machos de um dia de idade foram alojados em gaiolas dispostas em câmara climática onde foram mantidos sob estresse cíclico por calor durante oito horas por dia. As aves foram distribuídas em dois grupos experimentais, sendo um grupo suplementado com 0,90% de OSM e um grupo controle (CON) sem a suplementação de OSM. Aos 36 dias de idade, 18 aves foram abatidas para coleta das amostras de músculo. A partir de pools das amostras de filé de peito de cada grupo, foi realizado a clivagem proteolítica dos extratos proteicos e posteriormente a análise dos peptídeos por LC-MS/MS. A suplementação de 0,90% de OSM revelou a modulação de 57 proteínas no músculo Pectoralis major de frangos de corte expostos ao estresse cíclico por calor. Dentre elas, quatro proteínas foram reguladas positivamente e 46 proteínas foram reguladas negativamente. Além disso, sete proteínas foram expressas unicamente no grupo CON. Estes resultados sugerem que a suplementação de OSM demonstrou mecanismo adaptativo para aumentar a tolerância ao calor, reduzindo o estresse oxidativo. Além disso, promoveu ativação de mecanismos de neuroproteção, proteção celular contra ocorrência de apoptose celular, diminuição das respostas inflamatórias e regulação do metabolismo energético.
Among the alternatives studied to replace the use of antibiotics in industrial poultry, products composed of essential oils and/or plant extracts, called phytogenic additives, stand out. These products have potential because they are natural and residue-free compared to antibiotics. Among the natural additives with the potential to be used in industrial practice, there is passion fruit seed oil (OSM). However, there are no reports in the scientific literature on the use of proteomic approaches to elucidate the effect of OSM on the liver and muscle proteome of broilers exposed to cyclic heat stress. Proteomics allows the evaluation of changes in the expression of proteins in tissues and fluids, thus helping to understand the molecular mechanisms involved in the health, welfare, stress, and pathological states of animals. Thus, the thesis was divided into two main chapters corresponding to the papers. Paper 1 - The aim of the present study was to investigate the effect of OSM supplementation on the differential profile of proteins expressed in the liver tissue of broilers subjected to cyclic heat stress. For this, 225 1-day-old male chicks were housed in cages in a climatic chamber where they were kept under cyclic heat stress for eight hours a day. Five experimental diets based on corn and soybean meal were formulated, being a control diet (without addition of OSM) and 4 diets in which different levels of OSM were included (0.30; 0.50; 0.70 and 0.90%). At 36 days of age, nine birds from each treatment were slaughtered to collect liver samples. From pools of liver samples from each treatment, fractionation of the liver proteome was performed by twodimensional electrophoresis (2D-PAGE) and characterization of proteins by sequence mass spectrometry coupled with liquid chromatography (LC-MS/MS). Supplementation of 0.90% OSM increased the expression of seven heat shock proteins, while lower expression of six antioxidant proteins was observed. Proteins involved in lipid and carbohydrate metabolism also showed changes in expression. These results suggest that OSM supplementation influences the activation of damage repair mechanisms caused by cyclic exposure to heat by increasing the expression of molecular chaperones in liver tissue. Paper 2 - The aim of this study is to characterize the proteins differentially expressed in the Pectoralis major muscle of broilers, supplemented with OSM under cyclic heat stress conditions. For this, 90 one-day-old male chicks were housed in cages in a climatic chamber, where they were kept under cyclic heat stress for eight hours a day. The birds were divided into two experimental groups, one group supplemented with 0.90% OSM and a control group (CON) without OSM supplementation. At 36 days of age, 18 birds were slaughtered to collect muscle samples. From pools of breast fillet samples (Pectoralis major muscle) of each group, the proteolytic cleavage of the protein extracts was performed and later the analysis of the peptides by LC-MS/MS. Supplementation of 0.90% OSM revealed the modulation of 57 proteins in the Pectoralis major muscle of broilers exposed to cyclic heat stress. Among them, four proteins were upregulated and 46 proteins were down-regulated. In addition, seven proteins were expressed only in the CON group. These results suggest that OSM supplementation demonstrated an adaptive mechanism to increase heat tolerance, reducing oxidative stress. In addition, it promoted activation neuroprotection mechanisms, cellular protection against the occurrence of cellular apoptosis decreased inflammatory responses and regulation of energy metabolism.
Among the alternatives studied to replace the use of antibiotics in industrial poultry, products composed of essential oils and/or plant extracts, called phytogenic additives, stand out. These products have potential because they are natural and residue-free compared to antibiotics. Among the natural additives with the potential to be used in industrial practice, there is passion fruit seed oil (OSM). However, there are no reports in the scientific literature on the use of proteomic approaches to elucidate the effect of OSM on the liver and muscle proteome of broilers exposed to cyclic heat stress. Proteomics allows the evaluation of changes in the expression of proteins in tissues and fluids, thus helping to understand the molecular mechanisms involved in the health, welfare, stress, and pathological states of animals. Thus, the thesis was divided into two main chapters corresponding to the papers. Paper 1 - The aim of the present study was to investigate the effect of OSM supplementation on the differential profile of proteins expressed in the liver tissue of broilers subjected to cyclic heat stress. For this, 225 1-day-old male chicks were housed in cages in a climatic chamber where they were kept under cyclic heat stress for eight hours a day. Five experimental diets based on corn and soybean meal were formulated, being a control diet (without addition of OSM) and 4 diets in which different levels of OSM were included (0.30; 0.50; 0.70 and 0.90%). At 36 days of age, nine birds from each treatment were slaughtered to collect liver samples. From pools of liver samples from each treatment, fractionation of the liver proteome was performed by twodimensional electrophoresis (2D-PAGE) and characterization of proteins by sequence mass spectrometry coupled with liquid chromatography (LC-MS/MS). Supplementation of 0.90% OSM increased the expression of seven heat shock proteins, while lower expression of six antioxidant proteins was observed. Proteins involved in lipid and carbohydrate metabolism also showed changes in expression. These results suggest that OSM supplementation influences the activation of damage repair mechanisms caused by cyclic exposure to heat by increasing the expression of molecular chaperones in liver tissue. Paper 2 - The aim of this study is to characterize the proteins differentially expressed in the Pectoralis major muscle of broilers, supplemented with OSM under cyclic heat stress conditions. For this, 90 one-day-old male chicks were housed in cages in a climatic chamber, where they were kept under cyclic heat stress for eight hours a day. The birds were divided into two experimental groups, one group supplemented with 0.90% OSM and a control group (CON) without OSM supplementation. At 36 days of age, 18 birds were slaughtered to collect muscle samples. From pools of breast fillet samples (Pectoralis major muscle) of each group, the proteolytic cleavage of the protein extracts was performed and later the analysis of the peptides by LC-MS/MS. Supplementation of 0.90% OSM revealed the modulation of 57 proteins in the Pectoralis major muscle of broilers exposed to cyclic heat stress. Among them, four proteins were upregulated and 46 proteins were down-regulated. In addition, seven proteins were expressed only in the CON group. These results suggest that OSM supplementation demonstrated an adaptive mechanism to increase heat tolerance, reducing oxidative stress. In addition, it promoted activation neuroprotection mechanisms, cellular protection against the occurrence of cellular apoptosis decreased inflammatory responses and regulation of energy metabolism.
Descrição
Palavras-chave
2D-PAGE, Gel free, Estresse cíclico por calor, Estresse oxidativo, Proteínas de choque térmico, Cyclic heat stress, Oxidative stress, Heat shock proteins