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Title: The effects of flavonoids in reducing trophoblast oxidative stress and apoptosis
Author: Ebegboni, Vernon Justice
ISNI:       0000 0004 7959 9385
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2018
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Pregnancy is a complex state characterised by changes in maternal physiology to favour the development and growth of the developing foetus. This is mainly achieved by the placenta, a pregnancy specific temporary organ. In human's, the placenta is a highly specialised organ that acts as an interface for the exchange of nutrients, gaseous products, wastes and hormones between the mother and the foetus. Most importantly, the fate of human pregnancy relies on the successful invasion of early placental precursor cells called extravillous trophoblasts. These highly invasive cells, form a "plug" into material spiral arteries. This transforms the placental circulation from high-resistant low flow to low-resistant high flow state. Inadequate remodelling of the maternal spiral arteries can cause pregnancy complications such as pre-eclampsia, miscarriage and foetal/maternal death. The main cause for the incomplete transformation of spiral arteries is believed to be placental oxidative stress during early gestation. Therefore, measures to reduced early trophoblast oxidative stress have taken precedence in placental research. It has been hypothesised that the introduction of non-enzymatic dietary antioxidants such as flavonoids, metal chelates and vitamins may prevent this oxidative stress, especially during early pregnancy. Flavonoids are phenolic compounds derived from fruits and vegetable and possess antioxidant properties. In fact, normal pregnant mothers usually "crave" for certain plant products (such as fruits) which apparently have high flavonoids concentrations. Although, their antioxidant properties have been well documented in myocardial and neuronal cells, their role in the prevention of trophoblast oxidative stress is unknown. Thus, exploring the antioxidative oxidative effects of selected flavonoids, their metabolites alone or in combination is deemed essential. This study aims to understand the antioxidative properties of selected flavonoids (and their metabolite combinations) on a transformed early extravillous trophoblast cell line HTR-8/SVneo. It also explores the cytoprotective effects of these flavonoids during in vitro trophoblast invasion. To achieve this aim, optimisation of the non-toxic concentrations of flavonoids (quercetin, morin, naringin and hesperidin)/metabolites (Q3G, naringenin and hesperetin) on HTR-8/SVneo cells was carried out using MTT and CellTox™ green cytotoxicity assays. Afterwards, oxidative stress model for HTR-8/SVneo cells was established by hypoxia reoxygenation (HR). The effects of 24 h pre-treatment with flavonoids prior to HR-induced oxidative stress on HTR-8/SVneo cell viability was assessed using MTT and CellTiter-Glo® assays. Further investigation on the antioxidative effects of flavonoids was carried out by assessing their effects on glutathione levels, nicotinamide adenine dinucleotide phosphate system, H2O2 scavenging and apoptosis. Flavonoid involvement in the modulation of protein kinases (ERK1/2, PKB, JNK/SAPK and p38 MAPK), trophoblast invasion and migration as well as spheroid formation/invasion was also investigated. Flavonoids/metabolites below 10 μM were well tolerated by HTR-8/SVneo cells and pre-treatment with 3 μM flavonoid or 1 μM metabolite shown significant protection against H/R insult. Further investigation revealed that flavonoid cytoprotective effects against H/R insult were associated with their ability to interact with other antioxidant systems such as increasing GSH and decreasing GSSG levels, redox balancing (restoring NADP/NADPH ratio), inhibition of apoptosis by decreasing caspase 3/7 activity, and scavenging of H2O2. The data from this study also indicates that flavonoid pre-treatment significantly inhibited HR-induced protein kinase activation/phosphorylation (p < 0.0001), enhanced HTR-8/SVneo invasive capacity (p < 0.0001) and increased spheroid formation and invasion (p < 0.001). In summary, the study has shown for the first time that 24 h pre-treatment with flavonoids, their metabolites or in combination on HTR-8/SVneo cells significantly protected against HR-induced oxidative stress. Most importantly these combinations have enhanced trophoblast cell line invasion by reducing oxidative stress. These findings suggest that the ingestion of flavonoid rich foods during pregnancy may benefit placental development and health.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available