Investigation of the effect of 3-hydroxybutyrate on hydrogen peroxide toxicity in SH SY-5Y cell line

Introduction and aim: Alzheimer's disease (AD) is a disease characterized by its progressive nature and the advance of irreversible neurodegeneration that eventually leads to memory loss. Despite amyloid beta (Aß) toxicity being one of the main mechanisms in the pathogenesis of AD, many other pathophysiological processes such as oxidative stress and mitochondria dysfunction also occur. Ketogenic diet has been used as a therapeutic option for some neurological diseases. Although the mechanism of action of ketogenic diet is not fully understood, strong evidence suggests that it works by improving mitochondrial function. On the other hand, neurosteroids, whose synthesis starts in cell mitochondria, are suggested to have various effects in the areas related to learning, memory, mood and cognitive ability of the brain. This can be attributed to them being neuroprotective, as well as having an active role as neurotransmitters. The aim of this study was to determine the effects of the ketone body 3-Hydroxybutyrate (3-OHB) administration (addition of 3-OHB at the determined concentrations to cell culture medium) on cell viability in Hydrogen Peroxide (H2O2) induced toxicity in SH SY-5Y cells in a concentration and time-dependent manner. In addition, we intended to evaluate the effects of 3-OHB on the levels of the first speed-limiting enzyme in steroid hormone synthesis; cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme as well as the levels of the neurosteroids dehydroepiandrosteron (DHEA) and dehydroepiandrosteron sulfate (DHEAS) which are presumed to have positive effects on cognitive functions. Methods: İn the presence of different glucose concentrations, we measured the effect of pretreating SH SY-5Y cells for 48 hours with 1 mM and 4 mM 3-OHB on cell viability in H2O2 toxicity using 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) test. Additionally, we quantified P450scc enzyme, DHEA and DHEAS levels using Enzyme-Linked ImmunoSorbent Assay (ELISA) technique. Results: In the toxicity model induced by H2O2, a dose of 200 μM H2O2 has managed to reduce cell viability by ~ 40-50% compared to the control group. We found that in all three glucose concentrations (17.5 mM, 5 mM and 1 mM) conditions, pretreatment with 1 mM 3-OHB for 48 hours caused no change in cell viability compared to control group (cells treated with 200 μM H2O2 only). Additionally, under the same conditions, pretreatment with 1 mM 3-OHB for 48 hours caused an increase in P450scc enzyme, DHEA and DHEAS levels compared to control group (cells treated with 200 μM H2O2 only) in all three glucose conditions. However, this increase was statistically significant only in the presence of 5 mM glucose in the case of P450scc enzyme (p= 0.043) and DHEAS (p=0.029) Conclusion: In conclusion, this study has shown that 3-OHB triggers the enzyme involved in the synthesis of neurosteroids and also causes an increase in DHEA and DHEAS levels, which are emphasized to have possible protective effects against oxidative damage. Moreover, we obtained important in vitro data showing that 3-OHB exerts its possible neuroprotective effect only in a normoglycemic condition and that this effect is associated with neurosteroids. Understanding the relationship between ketone bodies and hormones with neuronal steroid structure will provide important clues in preventing neurodegenerative changes and understanding the mechanisms of neurodegenerative diseases.