Resposta inflamatória pulmonar induzida por LPS na geração F2 de ratos desnutridos intrauterinamente
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Data
2019
Autores
Rodrigues, Larissa Gonçalves Aguiar [UNIFESP]
Orientadores
Landgraf, Richardt Gama [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A restrição alimentar global durante todo o período gestacional provoca alterações no crescimento
do feto e prejudica na sua resposta imunológica pós natal. Em trabalhos anteriores do nosso grupo
foi observado redução da resposta inflamatória, indicando que a deficiência materna em nutrientes
acarreta em alterações no metabolismo e sistema imunológico do feto na vida adulta. O presente
trabalho teve como objetivo avaliar os possíveis mecanismos envolvidos na redução da resposta
inflamatória pulmonar induzida por LPS na geração F2 de ratos desnutridos intrauterinamente,
com 12 semanas de idade. Foram utilizados ratos machos Wistar com peso normal ao nascer
(PNN), provenientes de mães que receberam dieta ad libitum na gestação e com baixo peso ao
nascer (BPN), provenientes de mães que sofreram desnutrição intrauterina, devido restrição
alimentar materna de 50% durante o período gestacional. Seis horas após instilação de LPS
(100μl/750 μg/animal) nos animais experimentais e salina nos controles, foi realizado o lavado
broncoalveolar (LBA) para verificar o infiltrado celular e posteriormente retirado o pulmão para
analise histológica da região peribronquial onde observados menor infiltrado celular no pulmão
dos animais BPN inflamados. A análise proteica do tecido pulmonar das enzimas COX-1, COX2, 5-LO, TLR-4 e GR foram realizadas pela técnica de Western blotting, enquanto que a
quantificação das citocinas no tecido pulmonar e no soro, da corticosterona, ACTH e melatonina
no soro por kit Multiplex, a concentração de LTB4 e PGE2, enzimas HDAC e HDAC-1 no tecido
pulmonar foram realizados por kit EIA, e a metilação global do DNA por kit DNA methylation
no tecido pulmonar. Verificamos que o grupo BPN-LPS apresentou baixos níveis de COX-2, já a
COX-1 apresentou maior expressão em níveis basais (BPN controle) e diminuição da expressão
após estímulo no grupo BPN-LPS. O estímulo com LPS não apresentou diferença entre os níveis
de PGE2. De outro lado, a enzima 5-LO e a produção de LTB4 apresentaram perfil semelhante
quanto ao grupo nutrido (PNN) com aumento apenas no grupo estimulado PNN-LPS. Ratos
estimulados com LPS apresentaram diminuição da expressão de TLR-4 apenas no grupo BPNLPS. Foi observado que o GR apresentou diminuição da sua expressão nos animais PNN-LPS e
aumento da sua expressão nos ratos BPN-LPS quando comparado com os ratos PNN também
estimulados; este mesmo perfil pode ser observado no ACTH. Diferentemente da corticosterona
onde ratos PNN apresentaram aumento desse hormônio após estímulo com LPS (PNN-LPS) e
níveis basais aumentados no grupo BPN em relação ao PNN. Já a melatonina apresentou perfil
semelhante em ambos os grupos, sendo que após estímulo inflamatório houve aumento da
concentração desse hormônio em relação ao seu controle. Foi observado desregulação na
produção de citocinas inflamatórias tanto no tecido pulmonar quanto no soro. No tecido pulmonar
todas as citocinas IL-1β, IL-6, TNFα, IL-10, IFNγ e leptina não apresentaram aumento após
estímulo inflamatório no grupo BPN-LPS. Quando essas mesmas citocinas foram avaliadas no
soro, apenas a IL-6 apresentou aumento após estímulo com LPS no grupo BPN-LPS. No soro, o
IFNγ não apresentou diferença entre os grupos e o TNFα falhou em aumentar em ambos os grupos
(PNN-LPS e BPN-LPS) após estímulo inflamatório. De acordo com os dados obtidos, podemos
observar que a redução da resposta inflamatória permanece na geração F2. Realizamos ainda a
avaliação da metilação global do DNA e as enzimas HDAC e HDAC-1 no tecido pulmonar para
verificar possíveis alterações epigenéticas envolvidas e observamos que ratos do grupo BPNcontrole já apresentam níveis de metilação elevados mesmo antes do estímulo inflamatório. Na
atividade da HDAC, observamos aumento dessa enzima após estímulo inflamatório no grupo
BPN-LPS. Além disso aumento do inibidor da HDAC-1 no grupo estimulado PNN-LPS. Esses
dados sugerem que, a redução da resposta inflamatória observada na geração F2 pode estar
relacionada às alterações epigenéticas observadas através desses mecanismos.
Food restriction throughout the gestational period causes changes in fetal growth and impairs in its immune response in adult life. These changes were observed in previous studies of our group and a reduction in the inflammatory response was observed, indicating that maternal nutrient deficiency causes alterations in the metabolism and immune system of the fetus in adult life. The present study aimed objective to evaluate the possible mechanisms involved in the reduction of lung inflammatory response induced by LPS in the F2 generation of 12-week-old intrauterine malnourished rats. Male Wistar rats with normal birth weight (NBW) from mothers who received ad libitum diet during pregnancy and with low birth weight (LBW) from mothers who suffered intrauterine malnutrition due to maternal food restriction of 50% during the gestational period. Six hours after instillation of LPS (100μl/750 μg/animal) in the experimental and saline animals at the controls, bronchoalveolar lavage (BAL) was performed to verify the cellular infiltrate and later the lung was removed for histological analysis of the peribronchial region where the smallest cellular infiltrate was observed in lung of inflamed LBW animals. Protein analysis of the lung tissue of the COX-1, COX-2, 5-LO, TLR-4 and GR enzymes were performed by the Western blotting technique, whereas the quantification of the cytokines in lung tissue and serum, corticosterone, ACTH and melatonin in the serum by multiplex kit, the concentration of LTB4 and PGE2 lung tissue were performed by EIA kit, methylation of DNA by DNA methylation kit and enzymes HDAC and HDAC-1 in lung tissue were performed by EIA kit in lung tissue. We found that the LBW-LPS group presented low levels of COX-2, whereas COX-1 presented higher expression at basal levels (LBW control) and decreased expression after stimulation in the LBWLPS group. LPS stimulation showed no difference between PGE2 levels. On the other hand, the 5-LO enzyme and the LTB4 production presented a similar profile for the nourished group (NBW) with increase only in the NBW-LPS stimulated group. Rats stimulated with LPS showed decreased TLR-4 expression only in the LBW-LPS group. It was observed that GR showed a decrease in expression in NBW-LPS animals and an increase in their expression in LBW-LPS rats when compared to NBW rats also stimulated, this same profile can be observed in ACTH. Differently in corticosterone where, NBW rats showed an increase in this hormone after LPS stimulation (NBW-LPS) and increased baseline levels in the LBW group in relation to the NBW. Melatonin presented a similar profile in both groups, and after an inflammatory stimulus there was an increase in the concentration of this hormone in relation to its control. Deregulation was observed in the production of inflammatory cytokines in both lung tissue and serum. In lung tissue all cytokines IL-1β, IL-6, TNFα, IL-10, IFNγ and leptin did not show increase after inflammatory stimulus in the LBW-LPS group. When these same cytokines were evaluated in the serum, only IL-6 showed increase after stimulation with LPS in the LBW-LPS group. In the serum, IFNγ showed no difference between groups and TNFα failed to increase in both groups (NBW-LPS and LBW-LPS) after inflammatory stimulation. According to the data obtained, we can observe that the reduction of the inflammatory response remains in the F2 generation. We also evaluated global DNA methylation and the HDAC and HDAC-1 enzymes in lung tissue to check for possible epigenetic alterations involved, and we observed that rats LBW-control group already had elevated levels of methylation even before the inflammatory stimulus. In HDAC activity, we observed an increase of this enzyme after inflammatory stimulus in the LBW-LPS group. In addition to the increase of the HDAC-1 inhibitor in the NBW-LPS stimulated group. These data suggest that the reduction of the inflammatory response observed in generation F2 may be related to the epigenetic alterations observed through these mechanisms.
Food restriction throughout the gestational period causes changes in fetal growth and impairs in its immune response in adult life. These changes were observed in previous studies of our group and a reduction in the inflammatory response was observed, indicating that maternal nutrient deficiency causes alterations in the metabolism and immune system of the fetus in adult life. The present study aimed objective to evaluate the possible mechanisms involved in the reduction of lung inflammatory response induced by LPS in the F2 generation of 12-week-old intrauterine malnourished rats. Male Wistar rats with normal birth weight (NBW) from mothers who received ad libitum diet during pregnancy and with low birth weight (LBW) from mothers who suffered intrauterine malnutrition due to maternal food restriction of 50% during the gestational period. Six hours after instillation of LPS (100μl/750 μg/animal) in the experimental and saline animals at the controls, bronchoalveolar lavage (BAL) was performed to verify the cellular infiltrate and later the lung was removed for histological analysis of the peribronchial region where the smallest cellular infiltrate was observed in lung of inflamed LBW animals. Protein analysis of the lung tissue of the COX-1, COX-2, 5-LO, TLR-4 and GR enzymes were performed by the Western blotting technique, whereas the quantification of the cytokines in lung tissue and serum, corticosterone, ACTH and melatonin in the serum by multiplex kit, the concentration of LTB4 and PGE2 lung tissue were performed by EIA kit, methylation of DNA by DNA methylation kit and enzymes HDAC and HDAC-1 in lung tissue were performed by EIA kit in lung tissue. We found that the LBW-LPS group presented low levels of COX-2, whereas COX-1 presented higher expression at basal levels (LBW control) and decreased expression after stimulation in the LBWLPS group. LPS stimulation showed no difference between PGE2 levels. On the other hand, the 5-LO enzyme and the LTB4 production presented a similar profile for the nourished group (NBW) with increase only in the NBW-LPS stimulated group. Rats stimulated with LPS showed decreased TLR-4 expression only in the LBW-LPS group. It was observed that GR showed a decrease in expression in NBW-LPS animals and an increase in their expression in LBW-LPS rats when compared to NBW rats also stimulated, this same profile can be observed in ACTH. Differently in corticosterone where, NBW rats showed an increase in this hormone after LPS stimulation (NBW-LPS) and increased baseline levels in the LBW group in relation to the NBW. Melatonin presented a similar profile in both groups, and after an inflammatory stimulus there was an increase in the concentration of this hormone in relation to its control. Deregulation was observed in the production of inflammatory cytokines in both lung tissue and serum. In lung tissue all cytokines IL-1β, IL-6, TNFα, IL-10, IFNγ and leptin did not show increase after inflammatory stimulus in the LBW-LPS group. When these same cytokines were evaluated in the serum, only IL-6 showed increase after stimulation with LPS in the LBW-LPS group. In the serum, IFNγ showed no difference between groups and TNFα failed to increase in both groups (NBW-LPS and LBW-LPS) after inflammatory stimulation. According to the data obtained, we can observe that the reduction of the inflammatory response remains in the F2 generation. We also evaluated global DNA methylation and the HDAC and HDAC-1 enzymes in lung tissue to check for possible epigenetic alterations involved, and we observed that rats LBW-control group already had elevated levels of methylation even before the inflammatory stimulus. In HDAC activity, we observed an increase of this enzyme after inflammatory stimulus in the LBW-LPS group. In addition to the increase of the HDAC-1 inhibitor in the NBW-LPS stimulated group. These data suggest that the reduction of the inflammatory response observed in generation F2 may be related to the epigenetic alterations observed through these mechanisms.