Abstract
BackgroundGlutamate, the major excitatory neurotransmitter of CNS acts as a neurotoxin at higher concentrations. Prolonged activation of glutamate receptors results in progressive neuronal damage by aggravating calcium influx, inducing mitochondrial damage and oxidative stress. Excitotoxic cell death is associated with the pathogenesis of various neurodegenerative disorders such as trauma, brain injury and neurodegenerative diseases. The current study was designed to investigate the neuroprotective and neuroregenerative potential of Tinospora cordifolia against glutamate-induced excitotoxicity using primary cerebellar neuronal cultures as a model system.MethodsMonosodium salt of glutamate was used to induce neurotoxic injury in primary cerebellar neurons. Four extracts including Hexane extract, Chloroform extract, Ethyl acetate, and Butanol extract were obtained from fractionation of previously reported aqueous ethanolic extract of T. cordifolia and tested for neuroprotective activity. Out of the four fractions, Butanol extract of T. cordifolia (B-TCE) exhibited neuroprotective potential by preventing degeneration of neurons induced by glutamate. Expression of different neuronal, apoptotic, inflammatory, cell cycle regulatory and plasticity markers was studied by immunostaining and Western blotting. Neurite outgrowth and migration were also studied using primary explant cultures, wound scratch and gelatin zymogram assay.ResultsAt molecular level, B-TCE pretreatment of glutamate-treated cultures normalized the stress-induced downregulation in the expression of neuronal markers (MAP-2, GAP-43, NF200) and anti-apoptotic marker (Bcl-xL). Further, cells exposed to glutamate showed enhanced expression of inflammatory (NF-κB, AP-1) and senescence markers (HSP70, Mortalin) as well as the extent of mitochondrial damage. However, B-TCE pretreatment prevented this increase and inhibited glutamate-induced onset of inflammation, stress and mitochondrial membrane damage. Furthermore, B-TCE was observed to promote regeneration, migration and plasticity of cerebellar neurons, which was otherwise significantly inhibited by glutamate treatment.ConclusionThese results suggest that B-TCE may have neuroprotective and neuroregenerative potential against catastrophic consequences of glutamate-mediated excitotoxicity and could be a potential therapeutic candidate for neurodegenerative diseases.
Highlights
Glutamate, the major excitatory neurotransmitter of CNS acts as a neurotoxin at higher concentrations
Butanol extract of T. cordifolia (B-TCE) modulated the effect of glutamate on cellular and nuclear morphology Effect of glutamate and B-TCE pretreatment was initially studied by phase contrast microscopy followed by confocal imaging for α- tubulin immunostaining
As the cultures treated with B-TCE alone showed cellular and nuclear morphology similar to control, it may be suggested that B-TCE did not have any adverse effects on primary cerebellar neurons
Summary
The major excitatory neurotransmitter of CNS acts as a neurotoxin at higher concentrations. L-glutamic acid is a major excitatory amino acid in the CNS, which plays a major role in neurotransmission and is responsible for performing fundamental brain functions such as neuronal circuit formations and synaptic plasticity underlying memory and cognition [2]. Glutamate acts through both inotropic as well as metabotropic receptors and increased extracellular levels of glutamate lead to overactivation of glutamate receptors resulting in neuronal damage [3]. The ethanolic extract of T. cordifolia was reported to exhibit neuroprotective activity against 6-OHDA induced Parkinsonism [15]. Glycosides and aliphatic compounds are broadly considered responsible for immune modulatory and neuroprotective properties of this herb [17,18,19]. n-Butanol fraction of T. cordifolia extract has been reported to have tinocordifolioside A and tinocordiside as active compounds [20, 21]
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