Efeito modulador da progesterona, vitamina D e silibinina sobre inflamassomas em monócitos de gestantes portadoras de pré-eclâmpsia
Carregando...
Arquivos
Data
2019-02-28
Autores
Matias, Mariana Leticia
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A pré-eclâmpsia (PE) é uma síndrome específica da gravidez, associada com inflamação sistêmica exacerbada, o que poderia ser dependente da deficiência de fatores reguladores, como a silibinina (SB), progesterona (PG) e vitamina D (VD), que são capazes de modular essa resposta inflamatória. O objetivo do trabalho foi avaliar, in vitro, o efeito modulador da SB, PG e VD sobre os inflamassomas NLRP1 e NLRP3 e sobre a via de sinalização dependente de TLR4-MyD88 em monócitos de gestantes portadoras de PE e em linhagem celular de monócitos humanos THP-1. Monócitos obtidos do sangue periférico de 20 gestantes com PE, 20 gestantes normotensas e células THP-1 foram cultivados na presença ou ausência de urato monossódico (MSU), hialurona (HA), SB, PG ou VD por diferentes períodos: a) por 30 min para determinação da fosforilação de NF-κB e para expressão dos fatores intra-citoplasmáticos NF-κB e IκBα por citometria de fluxo, b) por 4h para a expressão gênica de TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18, TNF e IL10 analisada por RT-qPCR e c) por 18h para a determinação da concentração das citocinas IL-1β, IL-18, TNF-α e IL-10 por ELISA e para expressão do receptor de superfície TLR4 por citometria de fluxo. Foram determinados os níveis plasmáticos de ácido úrico, PG e VD em gestantes pré-eclâmpticas e normotensas. Os resultados foram analisados por meio de testes paramétricos e não-paramétricos com nível de significância de 5%. Os níveis plasmáticos de PG e VD foram significativamente menores, enquanto os de ácido úrico foram maiores em gestantes com PE quando comparados com gestantes normotensas. Monócitos de gestantes com PE apresentaram maior ativação endógena dos genes inflamatórios (TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18 e TNF) e menor de IL10 em comparação com as gestantes normotensas. A expressão de TLR4 e NF-κB foi maior, enquanto IκBα foi menor no grupo de gestantes com PE quando comparada à de gestantes normotensas. A maior atividade basal de NF-κB fosforilado observada em monócitos de gestantes com PE apresentou-se menor após o tratamento dessas células com SB, PG e VD. O estímulo com MSU e HA aumentou a expressão dos genes e proteínas relacionados à inflamação e diminuiu a de IL-10 em gestantes com PE, enquanto o tratamento com SB, PG e VD teve efeito imunomodulador, diminuindo essa expressão. No grupo PE, os estímulos com MSU e HA aumentaram a expressão de NF-κB e diminuiram a de IκBα. Por outro lado, o tratamento dos monócitos com SB, PG e VD induziu diminuição de TLR4 e NF-κB e aumento de IκBα. Células THP-1 apresentaram perfil de resposta imunológica semelhante aos monócitos de gestantes pré-eclâmpticas, após estímulo com MSU e HA e tratamento com SB, PG e VD. Os resultados sugerem a participação de MSU e de HA na resposta inflamatória sistêmica da PE e que o tratamento com SB, PG ou VD é capaz de modular essa inflamação, demostrando o papel imunomodulador dessas substâncias na regulação da resposta inflamatória na PE.
Preeclampsia (PE) is a specific pregnancy syndrome, associated with exacerbated systemic inflammation, which could be dependent on the deficiency of regulatory factors, such as silibinin (SB), progesterone (PG), and vitamin D (VD), which are able to modulate this inflammatory response. The aim of this study was to evaluate the modulating effect of SB, PG and VD on the NLRP1 and NLRP3 inflammasomes and on the TLR4-MyD88-dependent signaling pathway in monocytes from pregnant women with PE and human monocyte cell line THP-1. Monocytes obtained from the peripheral blood of 20 pregnant women with PE, 20 normotensive pregnant women and THP-1 cells were cultured in the presence or absence of monosodium urate (MSU), hyaluronan (HA), SB, PG or VD for different periods: a) for 30 min for the determination of NF-κB phosphorylation and for the expression of the intra-cytoplasmic factors NF-κB and IκBα by flow cytometry, b) for 4 h for the gene expression of TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18, TNF and IL10, analyzed by qPCR and c) for 18h for the determination of IL-1β, IL-18, TNF-α and IL-10 cytokines concentration by ELISA and for expression of the surface receptor TLR4 by flow cytometry. Plasma levels of uric acid, PG and VD in preeclamptic and normotensive pregnant women were determined. The results were analyzed using parametric and non-parametric tests with a significance level of 5%. Plasma levels of PG and VD were significantly lower, whereas those of uric acid were higher in pregnant women with PE when compared to normotensive pregnant women. Monocytes from pregnant women with PE showed greater endogenous activation of the inflammatory genes (TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18 and TNF) and lower IL10 when compared to normotensive pregnant women. The expression of TLR4 and NF-κB was higher, whereas IκBα was lower in the group of pregnant women with PE than in normotensive pregnant women. The higher basal activity of phosphorylated NF-κB observed in monocytes of pregnant women with PE was lower after the treatment of these cells with SB, PG and VD. The stimulation with MSU and HA increased the expression of genes and proteins related to inflammation and decreased IL-10 in pregnant women with PE, whereas treatment with SB, PG and VD had an immunomodulatory effect, decreasing this expression. In PE group, MSU and HA stimuli increased NF-κB expression and decreased IκBα expression. On the other hand, treatment of monocytes with SB, PG and VD induced a decrease of TLR4 and NF-κB and increase of IκBα. THP-1 cells presented a similar immune response profile to monocytes from preeclamptic women after MSU and HA stimulation and SB, PG and VD treatment. The results suggest the participation of MSU and HA in the systemic inflammatory response in PE and that treatment with SB, PG or VD is able to modulate this inflammation, demonstrating the immunomodulatory role of these substances in the regulation of the inflammatory response in PE.
Preeclampsia (PE) is a specific pregnancy syndrome, associated with exacerbated systemic inflammation, which could be dependent on the deficiency of regulatory factors, such as silibinin (SB), progesterone (PG), and vitamin D (VD), which are able to modulate this inflammatory response. The aim of this study was to evaluate the modulating effect of SB, PG and VD on the NLRP1 and NLRP3 inflammasomes and on the TLR4-MyD88-dependent signaling pathway in monocytes from pregnant women with PE and human monocyte cell line THP-1. Monocytes obtained from the peripheral blood of 20 pregnant women with PE, 20 normotensive pregnant women and THP-1 cells were cultured in the presence or absence of monosodium urate (MSU), hyaluronan (HA), SB, PG or VD for different periods: a) for 30 min for the determination of NF-κB phosphorylation and for the expression of the intra-cytoplasmic factors NF-κB and IκBα by flow cytometry, b) for 4 h for the gene expression of TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18, TNF and IL10, analyzed by qPCR and c) for 18h for the determination of IL-1β, IL-18, TNF-α and IL-10 cytokines concentration by ELISA and for expression of the surface receptor TLR4 by flow cytometry. Plasma levels of uric acid, PG and VD in preeclamptic and normotensive pregnant women were determined. The results were analyzed using parametric and non-parametric tests with a significance level of 5%. Plasma levels of PG and VD were significantly lower, whereas those of uric acid were higher in pregnant women with PE when compared to normotensive pregnant women. Monocytes from pregnant women with PE showed greater endogenous activation of the inflammatory genes (TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18 and TNF) and lower IL10 when compared to normotensive pregnant women. The expression of TLR4 and NF-κB was higher, whereas IκBα was lower in the group of pregnant women with PE than in normotensive pregnant women. The higher basal activity of phosphorylated NF-κB observed in monocytes of pregnant women with PE was lower after the treatment of these cells with SB, PG and VD. The stimulation with MSU and HA increased the expression of genes and proteins related to inflammation and decreased IL-10 in pregnant women with PE, whereas treatment with SB, PG and VD had an immunomodulatory effect, decreasing this expression. In PE group, MSU and HA stimuli increased NF-κB expression and decreased IκBα expression. On the other hand, treatment of monocytes with SB, PG and VD induced a decrease of TLR4 and NF-κB and increase of IκBα. THP-1 cells presented a similar immune response profile to monocytes from preeclamptic women after MSU and HA stimulation and SB, PG and VD treatment. The results suggest the participation of MSU and HA in the systemic inflammatory response in PE and that treatment with SB, PG or VD is able to modulate this inflammation, demonstrating the immunomodulatory role of these substances in the regulation of the inflammatory response in PE.
Descrição
Palavras-chave
pré-eclâmpsia, monócitos, inflamassomas, citocinas, silibinina, progesterona, vitamina D