Aims. To assess the possible effect of polyphenol-rich olive extracts on lipid metabolism in medaka fish by quantifying the expression of lipogenic and lipolytic genes. Materials and methods. Adult medaka fish were maintained in tanks for five days with five extracts at 0.01% in water, causing obesity through a diet rich in carbohydrates, with a control group maintained in water with a normal diet. The extracts contained polyphenols ranging between 7 and 116 mg/g (oleuropein, hydroxytyrosol) with an antioxidant power of 2–13 mmol of 2,4,6-tri(2-pyridyl)-1,3,5-triazine/100 g. After five days, the fish were sacrificed and the hepatic mRNA and its complementary DNA were extracted by reverse transcription. Complementary DNAs were quantified for three lipolytic and three lipogenic genes by real-time PCR. The relative gene expression was calculated from the amplification curves in reference to the control group. Results. The expression of genes involved in lipolysis, including peroxisome proliferator-activated receptor-±, acyl-CoA oxidase 1, and carnitine palmitoyltransferase 1, were clearly decreased in fish subjected to an obesogenic diet, and this situation could not be reversed in fish maintained with polyphenol-rich extracts. In contrast, lipogenic fatty acid synthase, acetyl-CoA carboxylase 1, and sterol regulatory element-binding protein 1 genes increased considerably with the obesogenic diet and reverted to the normal state with the olive extracts. The effect was not dependent on the total polyphenol content, the specific oleuropein or hydroxytyrosol concentration, or the antioxidant power, suggesting a synergistic effect. Conclusion. Olive polyphenols, acting as anti-lipogenic agents, have a positive effect on lipid metabolism, but their mechanism in each gene is different according to the extract, which supports synergistic mechanisms with the different proportions of polyphenols and accompanying phytochemicals in each extract.
ResumenObjetivo: evaluar la actividad protectora del extracto de un subproducto como son los huesos de aceitunas, mediante su capacidad de inhibir la apoptosis en la línea celular humana de neuroblastoma SH-SY5Y inducida con H 2 O 2 . Material y métodos: se han cultivado 20.000 cel/pocillo, iniciando diferenciación con ácido retinoico y, una vez diferenciadas las células, se ha inducido la apoptosis con H 2 O 2 con extracto y sin presencia del mismo. Finalmente se efectúa la extracción de cDNA y el análisis de los genes proapoptótico Bax y antiapoptótico Bcl-2. La cuantificación de la expresión génica se realiza frente al gen marcador GAPDH. Resultados: la viabilidad celular con el extracto es del 97,6% (SD 5,7) con 10 mg/l y 62,8% (SD 1,2) a 50 mg/l, utilizando 10 mg/l para el ensayo de biomarcadores. Las células de la línea SH-S diferenciadas con ácido retinoico (10 µM), muestran una clara apoptosis al ser tratadas con H 2 O 2 150 µM, con una relación Bax/Bcl2 de 3,75 (SD 0,80) frente a las células diferenciadas control y sometidas a H 2 O 2 y con extracto que tienen la misma relación de 1,02 (SD 0,01-0,03). Conclusión: el extracto de huesos de aceitunas presenta una actividad antiapoptótica frente a la provocación de la muerte celular por peróxido de hidrógeno, preservando a las células de neuroblastoma humano SH-SY5Y en su estado de normalidad, al defenderlas del estrés oxidativo que produce un significativo aumento de la relación de genes apoptóticos frente a antiapoptóticos (Bax/Bcl2).Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells AbstractObjective: We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H 2 0 2 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. Material and methods: To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H 2 O 2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. Results: Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 µM) shows a clear apoptosis when treated with H 2 O 2 150 µM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H 2 O 2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). Conclusion:The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2).
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