Natural LXRβ agonist stigmasterol confers protection against excitotoxicity after hypoxia- reoxygenation (H/R) injury via regulation of mitophagy in primary hippocampal neurons

Abstract

Ischemic brain injury represents insufficient oxygen supply to the brain and further damage occurs upon reoxygenation due to elevated intracellular levels excitatory neurotransmitter glutamate and subsequent production of reactive oxygen species (ROS) which has long been related to neuronal cell death of hippocampus brain region. Previously, using cell biological assay and transcriptomics analysis we reported that naturally occurring phytosterol Stigmasterol (ST) promotes brain development and function through the enhancement of neuronal cytoarchitectural complexity and functional maturation in rat hippocampal neurons by induction of immediate early genes (IEGs). In the present study we investigated the STs role in neuroprotection and found that ST also dose-dependently increased the neuronal viability in hypoxia reoxygenation (H/R) induced injury at hippocampal culture. ST, at an optimal concentration of 20 μM, significantly reduced the transport of vesicular glutamate (VGULT1), synaptic vesicle pool size, expression of GluN2B, rate of ROS formation (DCFDA) but restore mitochondrial membrane potential (JC1) and DNA fragmentation (H2AX) against H/R induced injury. More interestingly, ST also significantly induces the expression of autophagy marker protein LC3BII and the adaptor protein P62 but not HSC70 which indicates STs capability of induction of chaperon independent autophagy at H/R treated cultures. Furthermore densitometric analysis reveals ST also significantly increases PINK1 (PTEN induced protein kinase 1) expression therefore, indicates its role in mitophagy. In addition, molecular dynamic simulations study indicates that ST bind to LXRβ and forms hydrogen bonds with ASN239, GLU281, ARG319, THR316, SER278, ASN239 and SER278 residues at high occupancy with GLU281(20.21%) and ARG319 (21.04%,) residues, which is necessary for sterol binding to the LXRβ. Taken together these findings suggest that neuroprotective effect of ST might be associated with anti-excitatory and anti-oxidative actions on CNS neurons and could be a promising drug candidate for the treatment or prevention of ischemic stroke related neurological disorders.