Abstract
Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.
Highlights
Prolonged exposure to hypobaric hypoxia at high altitude is known to cause hippocampal neurodegeneration leading to loss of memory and higher order brain dysfunctions [1,2]
We investigated the effect of withanolide A on hippocampal glutathione biosynthesis during hypoxic exposure and its correlation with hypoxia induced neurodegeneration and memory dysfunction
Exposure to hypobaric hypoxia significantly elevated the level of reactive oxygen species generation (F (3, 20) = 22.3, p#0.05) and lipid peroxidation (F (3, 20) = 18.1, p#0.05) along with significant reduction in GSH level (F (3, 20) = 21.8, p#0.05) in hippocampus compared to normoxic group
Summary
Prolonged exposure to hypobaric hypoxia at high altitude is known to cause hippocampal neurodegeneration leading to loss of memory and higher order brain dysfunctions [1,2]. Hypoxic exposure weakens the antioxidant defense mechanisms by causing alterations in activity of antioxidant enzymes like glutathione reductase and glutathione peroxidase [4,5]. The cumulative effect of impaired antioxidant system and increased free radical generation leads to lipid peroxidation, membrane damage, protein oxidation, DNA damage [6] and altered gene expression [7] that may culminate in cell death. The brain is vulnerable to oxidative stress because of its high demand for oxygen, abundant fatty acids that are targets of lipid peroxidation, and lower antioxidant enzyme activities compared to other organs. Administration of free radical quenchers like quercetin or antioxidant precursors such as N-acetyl cysteine has been reported to enhance cell viability in hypoxic stress [9,10]
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