NEUROPROTECTIVE EFFECT OF MITOQUINONE ON HYDROGEN PEROXIDE-INDUCED NEUROTOXICITY IN SH-SY5Y NEUROBLASTOMA CELLS

Abstract

Several studies have investigated the major role that oxidative stress plays in the pathogenesis of traumatic brain injury (TBI). In fact, reactive oxygen species (ROS) accumulation, one major constituent of the secondary injury of TBI, has been shown to contribute to the processes of neuroinflammation, apoptosis, autophagy, and mitochondrial dysfunction, all of which are common characteristics of brain injury profile. The Nrf2-Keap1-ARE pathway is one particular pathway that has been of specific interest to researchers with several studies revealing its implication in TBI pathology. The possibility of targeting this pathway, using antioxidant compounds, has been proposed as a potential neurotherapy for TBI. Among these compounds is mitoquinone (MitoQ), an antioxidant that has been shown to exhibit a neuroprotective effect on both closed and open head injury models of TBI in vivo.

The goal of this study is to investigate the potential effect of MitoQ, an antioxidant drug that targets the mitochondria, on neurotoxicity induced by hydrogen peroxide (H2O2) in humans neuroblastoma SH-SY5Y cells as an in vitro model of oxidative stress.

The neuroprotective effect of mitoquinone on SH-SY5Y cells was assessed by MTT assay (for cell viability), SRB assay (for cell growth inhibition), and propidium iodide stain (for cell cycle) using two different concentrations of the drug (0.03 µg/mL and 0.05 µg/mL) as a pre-treatment and post-treatment for H2O2-induced stress. Oxidative stress was evaluated by NBT assay and DHE staining while mitochondrial integrity was studied using MitotrackerGreen Fluorescent dye. Finally, the gene expression profile of selected antioxidant genes (Nrf2, SOD, HMOX1, and CAT genes) and inflammatory genes (COX-2 and NFκB) were investigated via RT-qPCR or immunofluorescence imaging. Our results showed that pre-treatment with the drug MitoQ protects SH-SY5Y cells by increasing cell viability, decreasing cell growth inhibition, preserving cell morphology and cell cycle integrity and attenuating oxidative stress progression. Moreover, mitochondrial phenotype was preserved in cells treated with MitoQ prior to H2O2 stress-induction. The Nrf2-Keap-ARE was shown to be contributing to the protective effects of MitoQ with an upregulation of the antioxidant genes Nrf2 and HOX1 and a normalization of SOD1 gene expression. Also, the decrease in COX-2 levels indicates the anti-inflammatory effect of the antioxidant which supports even more the use of MitoQ as treatment for neurotoxicity.