Desreguladores endócrinos versus Ginsenosídeos: modulação da via não genômica ativada por GPR30 e estresse oxidativo em células de Sertoli humanas (HSeC)
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Data
2019-02-25
Autores
Freitas, André Teves Aquino Gonçalves de
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Universidade Estadual Paulista (Unesp)
Resumo
Citocinas e proteínas quinases são fundamentais para o controle do processo espermatogênico, estando diretamente envolvidas na dinâmica da barreira hematotesticular. Diferentes mecanismos de controle são modulados por receptores como o GPR30, que ativa rapidamente diferentes vias de sinalização, responsáveis pelos processos de proliferação, sobrevivência e morte celular. Os desreguladores endócrinos (DEs) possuem grande afinidade pelo GPR30, além de potencial para ativar vias de estresse oxidativo e a abertura da barreira. Antagonistas funcionais dos DEs, como o Panax ginseng, podem ser protetores contra seus efeitos. Considerando a importância das vias de sinalização que regulam a espermatogênese e a constante exposição ambiental aos DEs a que estamos submetidos, este trabalho objetiva estudar a possível modulação da via não genômica ativada por GPR30 e do estresse oxidativo em células de Sertoli expostas a baixas doses do DE Monobutil Ftalato (MBP) bem como o potencial papel citoprotetor do GIM-1 (metabólito do P. ginseng) sobre essas células. Para tal, as células de Sertoli humanas (HSec) foram mantidas sobre matriz artificial, simulando o ambiente in vivo. A exposição ao MBP e ao GIM-1 foi realizada nos tempos de 30min, 1, 12, e 48 horas, em doses pré-estabelecidas pelo ensaio do MTT (teste de toxicidade) em 4 grupos: controle, MBP, GIM-1 e MBP + GIM-1. A morfologia celular foi avaliada pela coloração com Hematoxilina e Eosina, evidenciando efeitos deletérios do MBP sobre a distribuição celular e adesão na membrana basal; O grupo GIM-1 foi semelhante ao controle e o MBP+GIM-1 apresentou um aspecto intermediário. Para avaliar o estresse oxidativo, os marcadores enzimáticos glutationa peroxidase, superóxido dismutase e catalase foram analisados por método colorimétrico, sendo o MBP capaz de reduzir a atividade das três enzimas e o GIM-1 de aumentá-las e atenuar os efeitos do MBP no grupo MBP+GIM-1. A quantificação de proteínas por Western Blot mostrou que o MBP inibiu a expressão de NRF2 e aumentou a de caspase 3 clivada, ativou o receptor GPR30, PKA, Src, EGFR e a via da ERK1/2, enquanto o GIM-1 aumentou a expressão de SIRT1 e NRF2 e inibiu PKA, Src e as vias da ERK1/2 e AKT. O MBP também aumentou a expressão da Cofilina, diminuindo a intensidade de actina-F na superfície celular. A exposição combinada evidenciou o antagonismo entre os compostos. Nossos resultados mostram efeitos deletérios do MBP na linhagem HSec, através do estresse oxidativo e da via do GPR30, evidenciando o importante papel citoprotetor da GIM-1 sobre eles.
Cytokines and kinases protein are essential to control the spermatogenic process, being directly involved in the blood-testis barrier control. Activation of these mechanisms is modulated by receptors such as GPR30, which rapidly activates different signaling pathways responsible for proliferation and cell death processes. Endocrine Disruptors (EDs) have high affinity for GPR30, causing oxidative stress and possible barrier rupture. Functional antagonists of EDs, such as Panax ginseng, may be protective against their effects. Considering the importance of the signaling pathways that regulate spermatogenesis and the constant environmental exposure to the EDs to which we are subject, this work aims to study the possible modulation of the non-genomic pathway activated by GPR30 and oxidative stress in Sertoli cells exposed to low doses of the ED Monobutyl Phthalate (MBP) and the possible cytoprotective role of GIM-1 (P. ginseng metabolite) on these pathways. To this end, HSec human lineage cells were maintained on artificial matrix, simulating in vivo environment. Exposure to MBP and GIM-1 was performed at 30 min, 1, 12 and 48 hours at pre-set MTT (toxicity assay) levels in 4 groups: control, MBP, GIM-1 and MBP + GIM-1. Morphology and cell adhesion were evaluated by staining with Hematoxylin and Eosin, evidencing deleterious effects of MBP above cell distribution and adhesion in basement membrane; GIM-1 group was similar to Control and MBP+GIM-1 showed an intermediate aspect. In order to evaluate oxidative stress, the enzymatic markers glutathione peroxidase, superoxide dismutase and catalase were analyzed by colorimetric method, being MBP able to reduce all enzymatic activity and GIM-1 to promote it and attenuate MBP effects in MBP+GIM1. Quantification of proteins by Western Blot showed that MBP inhibited NRF2 expression and increased Cleaved-caspase3, activated the GPR30 receptor, PKA, Src, EGFR and the ERK1/2 pathway, while GIM-1 enhanced SIRT1 and NRF2 expression and inhibited PKA, Src, ERK1/2 and AKT pathways. MBP also enhances Cofilin expression, decreasing F-actin intensity in cell surface. The combined exposure evidenced the antagonism between the compounds. Our results show deleterious effects of MBP on the HSec line, through oxidative stress and GPR30 pathway, evidencing the important cytoprotective role of GIM-1 on them
Cytokines and kinases protein are essential to control the spermatogenic process, being directly involved in the blood-testis barrier control. Activation of these mechanisms is modulated by receptors such as GPR30, which rapidly activates different signaling pathways responsible for proliferation and cell death processes. Endocrine Disruptors (EDs) have high affinity for GPR30, causing oxidative stress and possible barrier rupture. Functional antagonists of EDs, such as Panax ginseng, may be protective against their effects. Considering the importance of the signaling pathways that regulate spermatogenesis and the constant environmental exposure to the EDs to which we are subject, this work aims to study the possible modulation of the non-genomic pathway activated by GPR30 and oxidative stress in Sertoli cells exposed to low doses of the ED Monobutyl Phthalate (MBP) and the possible cytoprotective role of GIM-1 (P. ginseng metabolite) on these pathways. To this end, HSec human lineage cells were maintained on artificial matrix, simulating in vivo environment. Exposure to MBP and GIM-1 was performed at 30 min, 1, 12 and 48 hours at pre-set MTT (toxicity assay) levels in 4 groups: control, MBP, GIM-1 and MBP + GIM-1. Morphology and cell adhesion were evaluated by staining with Hematoxylin and Eosin, evidencing deleterious effects of MBP above cell distribution and adhesion in basement membrane; GIM-1 group was similar to Control and MBP+GIM-1 showed an intermediate aspect. In order to evaluate oxidative stress, the enzymatic markers glutathione peroxidase, superoxide dismutase and catalase were analyzed by colorimetric method, being MBP able to reduce all enzymatic activity and GIM-1 to promote it and attenuate MBP effects in MBP+GIM1. Quantification of proteins by Western Blot showed that MBP inhibited NRF2 expression and increased Cleaved-caspase3, activated the GPR30 receptor, PKA, Src, EGFR and the ERK1/2 pathway, while GIM-1 enhanced SIRT1 and NRF2 expression and inhibited PKA, Src, ERK1/2 and AKT pathways. MBP also enhances Cofilin expression, decreasing F-actin intensity in cell surface. The combined exposure evidenced the antagonism between the compounds. Our results show deleterious effects of MBP on the HSec line, through oxidative stress and GPR30 pathway, evidencing the important cytoprotective role of GIM-1 on them
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Palavras-chave
Panax ginseng, GIM-1, HSeC, GPR30, estresse oxidativo, Células de Sertoli, Barreira hematotesticular, Desreguladores endócrinos, Monobutil ftalato, Sertoli cells, Blood-testis barrier, Endocrine disruptors, Monobutyl phthalate, Panax ginseng, GIM-1, HSec, GPR30, Oxidative stress