Estudo metaloproteômico do tecido hepático e plasma de frangos de corte suplementados com cobre e manganês
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Data
2023-03-28
Autores
Martins, Renata Aparecida [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O cobre e o manganês são essenciais para o metabolismo normal de todos os animais. A ausência desses minerais nas dietas de frangos de corte prejudica vários processos bioquímicos e fisiológicos afetando substancialmente a saúde das aves. Portanto, a suplementação dietética de microminerais é fundamental para manter a produtividade na avicultura. Contudo, nem todas as fontes de suplementação disponíveis no mercado apresentam características adequadas que favoreçam a absorção de minerais. Este fato impulsiona a realização de numerosos estudos para avaliar quais fontes apresentam maior biodisponibilidade mineral. Porém, até o momento nenhum estudo avaliou os efeitos de diferentes fontes e níveis de cobre e manganês sobre a regulação de proteínas. Dessa forma, a presente tese foi dividida em dois capítulos principais (Artigo 1 e 2) correspondendo aos estudos proteômicos no tecido hepático e plasma de frangos corte. Artigo 1 – O objetivo com o estudo foi avaliar o proteoma hepático de frangos de corte suplementados com duas fontes (sulfato e hidroxicloreto) e dois níveis de cobre (15 e 150 mg kg-1) e manganês (80 e 120 mg kg-1) utilizando ferramentas metaloproteômicas. Foram considerados quatro tratamentos: S15-80 (15 mg kg-1 de CuSO4 e 80 mg kg-1 de MnSO4); S150-120 (150 mg kg-1 de CuSO4 e 120 mg kg-1 de MnSO4); H15-80 (15 mg kg-1 de Cu(OH)Cl e 80 mg kg-1 de Mn(OH)Cl); e H150-120 (150 mg kg-1 de Cu(OH)Cl e 120 mg kg-1de Mn(OH)Cl). Para o estudo foram feitos quatros pools a partir de amostras de fígado de 10 aves/tratamento. As proteínas foram fracionadas utilizando eletroforese bidimensional em gel de poliacrilamida (2D-PAGE) e a análise de expressão dos spots proteicos foi realizada por meio do software ImageMaster Platinum v.7.0. Os spots diferencialmente expressos foram excisados dos géis e digeridos para mapeamento do Cu e Mn por espectrometria de absorção atômica em forno de grafite (GFAAS). A quantificação dos minerais nas amostras de fígado e pellets proteicos foi realizada por espectrometria de absorção atômica em chama (FAAS). A identificação das proteínas dos spots associados ao Cu e Mn foi realizada por espectrometria de massas em sequência acoplada com cromatografia líquida (LC-MS/MS). 19 spots foram associados ao Cu, 10 spots ao Mn, e 5 spots ao Cu e Mn concomitante. As concentrações de Cu e Mn no fígado, pellets e spots proteicos sugerem maior biodisponibilidade das fontes hidroxicloretos em relação aos sulfatos. A análise proteômica demonstrou indução das proteínas de choque térmico (HSPs) e das glutationas-s-transferases (GSTs) em frangos de corte suplementados com níveis mais altos de Cu e Mn, sugerindo o envolvimento dessas proteínas na tolerância e estresse por metais. Artigo 2 - O objetivo foi avaliar os efeitos da suplementação de duas fontes (sulfatos e hidroxicloreto) e dois níveis de Cu (15 e 150 mg kg-1) e manganês (80 e 120 mg kg-1) sobre o proteoma plasmático de frangos de corte. Foram avaliados quatro grupos experimentais: S15-80; S150-120; H15-80; e H150-120. A partir de amostras de plasma obtida de cada ave do mesmo tratamento foram feitos quatro pools considerando 10 aves por grupo. O fracionamento do proteoma plasmático foi realizado por 2D-PAGE. A análise de expressão dos spots proteicos foi realizada utilizando o software ImageMaster Platinum v.7.0. Os spots diferencialmente expressos foram extraídos dos géis para identificação das proteínas por LC-MS/MS. As determinações de Cu e Mn nas amostras e pellets proteicos de plasma foi realizada por FAAS. Os grupos S150-120 e H150-120 apresentaram maiores concentrações de Cu e Mn nas amostras e pellets proteicos quando comparados aos grupos S15-80 e H15-80 (P<0.05). O grupo H15-80 apresentou maior concentração de ambos os minerais quando comparado ao grupo S15-80, sugerindo maior biodisponibilidade da fonte hidroxicloreto. Foram caracterizados 31 spots proteicos diferencialmente expressos entre os grupos, demonstrando que a suplementação de níveis elevados de Cu e Mn promovem mudanças no nível de expressão de proteínas envolvidas principalmente nas vias da homeostase.
Copper and manganese are essential for the normal metabolism of all animals. The absence of these minerals in broiler diets impairs several biochemical and physiological processes, substantially affecting the health of the birds. Therefore, dietary supplementation of microminerals is essential to maintain productivity in poultry. However, not all sources of supplementation available on the market have adequate characteristics that favor mineral absorption. This fact drives numerous studies to assess which sources have greater mineral bioavailability. However, to date, no study has evaluated the effects of different sources and levels of copper and manganese on protein regulation. Thus, this thesis was divided into two main chapters (Paper 1 and 2) corresponding to proteomic studies in liver tissue and plasma of broiler chickens. Paper 1 – The objective of the study was to evaluate the hepatic proteome of broilers supplemented with two sources (sulfate and hydroxychloride) and two levels of copper (15 and 150 mg kg-1) and manganese (80 and 120 mg kg-1) using metalloproteomic strategies. Four treatments were considered: S15-80 (15 mg kg-1 of CuSO4 and 80 mg kg-1 of MnSO4); S150-120 (150 mg kg-1 CuSO4 and 120 mg kg-1 MnSO4); H15-80 (15 mg kg-1 Cu(OH)Cl and 80 mg kg-1 Mn(OH)Cl); and H150-120 (150 mg kg-1 of Cu(OH)Cl and 120 mg kg-1 of Mn(OH)Cl). For the study, four pools were made from liver samples of 10 broilers/treatment. Proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and expression analysis was performed using ImageMaster Platinum v.7.0 software. The differentially expressed spots were excised from the gels and subjected to acid digestion for Cu and Mn mapping by graphite furnace atomic absorption spectrometry (GFAAS). Quantification of minerals in liver samples and protein pellets was performed by flame atomic absorption spectrometry (FAAS). The identification of spot proteins associated with Cu and Mn was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). 19 spots were associated with Cu, 10 spots with Mn, and 5 spots with Cu and Mn concomitantly. Cu and Mn concentrations in liver, protein pellets and spots suggest greater bioavailability of hydroxychloride sources compared to sulfates. Proteomic analysis demonstrated induction of heat shock proteins (HSPs) and glutathione-s-transferases (GSTs) in broiler chickens supplemented with higher levels of Cu and Mn, suggesting the involvement of these proteins in metal tolerance and stress. Paper 2 - The objective was to evaluate the effects of supplementation from two sources (sulfates and hydroxychloride) and two levels of Cu (15 and 150 mg kg-1) and manganese (80 and 120 mg kg-1) on the plasma proteome of broilers. Four experimental groups were evaluated: S15-80; S150-120; H15-80; and H150-120. From plasma samples obtained from each bird of the same treatment, four pools were made considering 10 broilers per group. Plasma proteome fractionation was performed by 2D-PAGE. Expression analysis of protein spots was performed using ImageMaster Platinum v.7.0 software. The differentially expressed spots were extracted from the gels for protein identification by LC-MS/MS. Cu and Mn determinations in plasma protein samples and pellets were performed by FAAS. Groups S150-120 and H150-120 showed higher concentrations of Cu and Mn in protein samples and pellets when compared to groups S15-80 and H15-80 (P<0.05). The H15-80 group had a higher concentration of both minerals when compared to the S15-80 group, suggesting greater bioavailability of the hydroxychloride source. Thirty-one protein spots differentially expressed between groups were characterized, demonstrating that supplementation with high levels of Cu and Mn promote changes in the expression level of proteins mainly involved in homeostasis pathways.
Copper and manganese are essential for the normal metabolism of all animals. The absence of these minerals in broiler diets impairs several biochemical and physiological processes, substantially affecting the health of the birds. Therefore, dietary supplementation of microminerals is essential to maintain productivity in poultry. However, not all sources of supplementation available on the market have adequate characteristics that favor mineral absorption. This fact drives numerous studies to assess which sources have greater mineral bioavailability. However, to date, no study has evaluated the effects of different sources and levels of copper and manganese on protein regulation. Thus, this thesis was divided into two main chapters (Paper 1 and 2) corresponding to proteomic studies in liver tissue and plasma of broiler chickens. Paper 1 – The objective of the study was to evaluate the hepatic proteome of broilers supplemented with two sources (sulfate and hydroxychloride) and two levels of copper (15 and 150 mg kg-1) and manganese (80 and 120 mg kg-1) using metalloproteomic strategies. Four treatments were considered: S15-80 (15 mg kg-1 of CuSO4 and 80 mg kg-1 of MnSO4); S150-120 (150 mg kg-1 CuSO4 and 120 mg kg-1 MnSO4); H15-80 (15 mg kg-1 Cu(OH)Cl and 80 mg kg-1 Mn(OH)Cl); and H150-120 (150 mg kg-1 of Cu(OH)Cl and 120 mg kg-1 of Mn(OH)Cl). For the study, four pools were made from liver samples of 10 broilers/treatment. Proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and expression analysis was performed using ImageMaster Platinum v.7.0 software. The differentially expressed spots were excised from the gels and subjected to acid digestion for Cu and Mn mapping by graphite furnace atomic absorption spectrometry (GFAAS). Quantification of minerals in liver samples and protein pellets was performed by flame atomic absorption spectrometry (FAAS). The identification of spot proteins associated with Cu and Mn was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). 19 spots were associated with Cu, 10 spots with Mn, and 5 spots with Cu and Mn concomitantly. Cu and Mn concentrations in liver, protein pellets and spots suggest greater bioavailability of hydroxychloride sources compared to sulfates. Proteomic analysis demonstrated induction of heat shock proteins (HSPs) and glutathione-s-transferases (GSTs) in broiler chickens supplemented with higher levels of Cu and Mn, suggesting the involvement of these proteins in metal tolerance and stress. Paper 2 - The objective was to evaluate the effects of supplementation from two sources (sulfates and hydroxychloride) and two levels of Cu (15 and 150 mg kg-1) and manganese (80 and 120 mg kg-1) on the plasma proteome of broilers. Four experimental groups were evaluated: S15-80; S150-120; H15-80; and H150-120. From plasma samples obtained from each bird of the same treatment, four pools were made considering 10 broilers per group. Plasma proteome fractionation was performed by 2D-PAGE. Expression analysis of protein spots was performed using ImageMaster Platinum v.7.0 software. The differentially expressed spots were extracted from the gels for protein identification by LC-MS/MS. Cu and Mn determinations in plasma protein samples and pellets were performed by FAAS. Groups S150-120 and H150-120 showed higher concentrations of Cu and Mn in protein samples and pellets when compared to groups S15-80 and H15-80 (P<0.05). The H15-80 group had a higher concentration of both minerals when compared to the S15-80 group, suggesting greater bioavailability of the hydroxychloride source. Thirty-one protein spots differentially expressed between groups were characterized, demonstrating that supplementation with high levels of Cu and Mn promote changes in the expression level of proteins mainly involved in homeostasis pathways.
Descrição
Palavras-chave
2D-PAGE, Frangos de corte, GFAAS, Hidroxicloretos, Sulfatos, Suplementação mineral, Broilers, Hydroxychlorides, Mineral supplementation, Sulfates