Neuroprotective Effects of Esculetin against Hydrogen Peroxide-Induced Oxidative Stress in Human Neuroblastoma Cells

Sudhakar R Subramaniam, Elizabeth M Ellis. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.

Esculetin (6, 7-dihydroxycoumarin), a phenolic compound found in various plant products, is known to be a potent antioxidant and lipoxygenase inhibitor. In the present study, we investigated the protective effects of esculetin against hydrogen peroxide-induced oxidative stress in SH-SY5Y neuroblastoma cell lines. To evaluate the neuroprotective effect of esculetin, the cells were pre-treated with sub-toxic concentrations of esculetin (10-50 μM) and quercetin (40 μM) for 8h and various biological markers of cellular oxidative stress were determined. The effect of esculetin and hydrogen peroxide on cell growth was determined by MTT assay and the IC50 were found to be 0.38 ± 0.02 mM and 0.86 ± 0.07 mM respectively. The MTT assay showed that hydrogen peroxide exposure is toxic to SH-SY5Y cells and the pre-treatment with esculetin (25-50 μM) increased cell survival in a dose-dependent manner. To determine the antioxidant properties of esculetin, generation of reactive oxygen species (ROS) was measured using a fluorescent probe and the results showed that 8h pre-treatment with esculetin (25-50 μM) effectively scavenged the formation of intracellular ROS. Moreover, the redox status of the cells was measured by estimating the total cellular glutathione levels and esculetin (25 μM) treatment showed a marked increase in total glutathione levels. In addition, the coumarin also inhibited the decrease in total glutathione levels after hydrogen peroxide exposure. Furthermore, the expression of the antioxidant enzyme copper/zinc superoxide dismutase (SOD1) at mRNA and protein levels were measured by real-time quantitative PCR and immunoblotting respectively. The results showed an increase in SOD1 expression by 6-fold at protein levels and 30-fold at mRNA levels.

These findings confirm that esculetin, a naturally occurring plant coumarin protects neuroblastoma cells from hydrogen peroxide-induced oxidative stress. The results also suggest that esculetin may protect the neuronal cells by mechanisms including scavenging intracellular ROS generation, regulating the redox status of the cell and inducing the expression of the antioxidant enzyme SOD1.