The Neuroprotection Of SIRT1 On NMDA-induced Excitotoxicity In Cultured Cortical Cell

Glutamate is an important neurotransmitter in central nervous system, mediating a variety of excitatory synaptic transmissions. It plays a critical role in the neuronal development, in various excitatory synaptic transmissions in adult brain, and in synaptic plasticity. However, excessive accumulation of glutamate at synaptic clefts would cause neuronal damage even cell death, which is called excitotoxicity. Excitotoxicity has been known to be involved in a variety of pathological processes, such as cerebral ischemic injuries and many neurodegenerative diseases. Studies have shown that during cerebral ischemia and brain trauma, the concentration f glutamate within the brain tissue increases rapidly. Excessive accumulation of glutamate causes overactivation of glutamate receptors on postsynaptic membranes, especially for the excitotoxicity model is the most commonly used (generally recognized) and the most oxic (inducing severe damage even neuronal death) nerve injury model, with the most complicated injury mechanism (involving Ca2+overload, oxidative stress, mitochondrialys function, cell necrosis and apoptosis, etc.) and the most clinical significance (excitotoxicity is involved in almost all neuropathological processes, such as cerebral ischemia, a variety of eurodegenerative disease).Silent Information Regulator 1 (SIRT1), an NAD+-dependent deacetylase, the Sir2 homolog, exist in mammalian cytoplasm and nucleus, is in the most in-depth study as member of SIRTUINS family. SIRT1 can act on histone and non-histone acetylation involved in the regulation of a variety of biological processes including: gene transcription, metabolism, insulin secretion, angiogenesis, neural protection, and aging. Recent studies have showed that SIRT1 possesses neuroprotective effects, however, it is still unknown whether it protects neurons from NMDA-mediated neurotoxicity.In this study CCK-8, lactate dehydrogenase (LDH) test, Calcein-AM and PI double staining and Western Blot were used to detect cells injury in the NMDA (100μmol/L, 2 h) induced excitotoxicity, at the same time, we using Resveratrol ( RSV) as the activator of SIRT1 and Sirtinol as the inhibitor to observe the role of SIRT1 in the NMDA-mediated toxicity, and further define the role of its downstream signaling molecule p53 in this process.The findings showed that: (1) Cell viability assay showed that NMDA decreases the cell viability (CCK-8 assay) obviously. However, Resveratrol ( RSV)(25μmol/L) elicited inhibitory effects on the decrease of NMDA-induced cell viability and reversed to 31.92%(P<0.05), which can be prevented by SIRT1 antagonist SIRTINOL(10μmol/L). (2) LDH assay showed that administration of NMDA increceased NMDA-induced LDH release by about 43.12%(P<0.05), whereas Resveratrol ( RSV)(25μmol/L)and can reduced NMDA-induced LDH release by 27.34%(P<0.05)and SIRTINOL(10μmol/L)can inhibit the release either ;(3) Calcein-AM and PI double staining, NMDA can significantly reduced the number of living cells, the Calcein-AM staining positive cells (live cells) decreased, PI staining positive cells (dead cells) increased. Resveratrol (25μmol/L) increase the number of living cells 18.99% (P<0.05), while inhibitorsSirtinolL (10μmol/L) inhibited the role of resveratrol in increase the number of cells; (4) Western Blot showed that NMDA-mediated neurotoxicity does not affect SIRT1 expression, whereas significantly increase the, downstream signaling molecule Ace-p53 levels(P<0.05). Resveratrol (25μmol/L) reduce Ace-p53 levels by 31.49% , while SIRTINOL also effectively blocked the resveratrol reduced role of Ace-p53 levels (P<0.05).These results show that, the activation of SIRT1 play a protective role in the NMDA-induced neurotoxicity, the inhibition SIRT1 contributes to cellular damage. The activation of SITR1 reduced the Ace-p53 and p53 levels possibly through its deacetylation function from non-histone to histone, inhibit the apoptosis induced by the p53, and plays a neuroprotective role .Conclusions: (1)The activation of SITR1 play a protective role in NMDA-induced neurotoxicity; (2)The activation of SIRT1 may inhibit NMDA-mediated nerve injury by reducing the level of p53 and Ace-p53.