The Neuroprotective Effects of SIRT1 on NMDA-Induced Excitotoxicity

Abstract

Silent information regulator 1 (SIRT1), an NAD⁺-dependent deacetylase, is involved in the regulation of gene transcription, energy metabolism, and cellular aging and has become an important therapeutic target across a range of diseases. Recent research has demonstrated that SIRT1 possesses neuroprotective effects; however, it is unknown whether it protects neurons from NMDA-mediated neurotoxicity. In the present study, by activation of SIRT1 using resveratrol (RSV) in cultured cortical neurons or by overexpression of SIRT1 in SH-SY5Y cell, we aimed to evaluate the roles of SIRT1 in NMDA-induced excitotoxicity. Our results showed that RSV or overexpression of SIRT1 elicited inhibitory effects on NMDA-induced excitotoxicity including a decrease in cell viability, an increase in lactate dehydrogenase (LDH) release, and a decrease in the number of living cells as measured by CCK-8 assay, LDH test, and Calcein-AM and PI double staining. RSV or overexpression of SIRT1 significantly improved SIRT1 deacetylase activity in the excitotoxicity model. Further study suggests that overexpression of SIRT1 partly suppressed an NMDA-induced increase in p53 acetylation. These results indicate that SIRT1 activation by either RSV or overexpression of SIRT1 can exert neuroprotective effects partly by inhibiting p53 acetylation in NMDA-induced neurotoxicity.

Figure 1

Effects of RSV on NMDA-induced decrease in cell viability in primary neurons. (a) Pretreatment of RSV (10 μM, 25 μM, 50 μM, and 75 μM) improved cell viability compared with the NMDA treatment group (P < 0.05). (b) RSV (25 μM) significantly reversed NMDA-induced decrease in cell viability (P < 0.05), and Sirtinol (10 μM) inhibited the effect of RSV (P < 0.05). RSV: resveratrol; MK: MK-801. Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group, ^&P < 0.05 versus the RSV + NMDA group.

Figure 2

Effects of RSV on NMDA-induced LDH release in primary neurons. RSV (25 μM) reduced NMDA-induced LDH release (P < 0.05), and Sirtinol (10 μM) abolished the role of RSV (P < 0.05). RSV: resveratrol; MK: MK-801. Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group. ^&P < 0.05 versus the RSV + NMDA group.

Figure 3

Effects of RSV on NMDA-induced decrease in the number of living cells in primary neurons. (a) Representative micrographs showing the suppression of RSV (25 μM) on NMDA-induced decrease of living cells (P < 0.05), which was abolished by Sirtinol (10 μM) (P < 0.05). Living cells were stained by Calcein-AM (green), and dead cells were stained by PI (red). (b) Bar graph of mean of living cells. RSV: resveratrol; MK: MK-801. Each value represents the mean ± S.E.M. of three independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group. ^&P < 0.05 versus the RSV + NMDA group.

Figure 4

Effects of RSV on NMDA-induced decrease of SIRT1 deacetylase activity in primary neurons. RSV (25 μM) significantly ameliorated SIRT1 activity reduced by NMDA (P < 0.05), and Sirtinol (10 μM) abolished the effect of RSV (P < 0.05). MK: MK-801. Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group, ^&P < 0.05 versus the RSV + NMDA group.

Figure 5

Effects of different concentrations (10–1000 μM) of NMDA on cell viability in SH-SY5Y cell. NMDA (100 μM, 500 μM, and 1000 μM) decreased cell viability at 6 h, 12 h, and 24 h after NMDA exposure for 2 h (P < 0.05). Only at 12 h and 24 h after NMDA (10 μM), treatment was significant (P < 0.05). Numbers represent the percentage of the living cells normalized to the control. Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group.

Figure 6

Overexpression of SIRT1 (WT-SIRT1 or DN-SIRT1) increased the levels of SIRT1 mRNA and protein reduced by NMDA (500 μM) in SH-SY5Y cell (P < 0.05). (a) Quantitative representations of SIRT1 mRNA by bar graph. (b) Western blot probed with antibodies against SIRT1 and β-actin (upper panel) and quantitative representations of SIRT1 protein expression by bar graph (lower panel). Each value represents the mean ± S.E.M. of three independent experiments. ^∗P < 0.05 versus the control group. ^#P < 0.05 versus the vector + NMDA group, ^&P < 0.05 versus the WT-SIRT1 + NMDA group, ^▲P < 0.05 versus the DN-SIRT1 + NMDA group.

Figure 7

Effects of SIRT1 overexpression on the deacetylase activity in NMDA-induced excitotoxicity of SH-SY5Y cell. WT-SIRT1 overexpression significantly reversed the deacetylase activity decreased by NMDA (P < 0.05), and DN-SIRT1 overexpression had no effect (P > 0.05). Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group. ^#P < 0.05 versus the vector + NMDA group, ^&P < 0.05 versus the WT-SIRT1 + NMDA group.

Figure 8

Effects of SIRT1 overexpression on p53 acetylation in NMDA-induced excitotoxicity of SH-SY5Y cell. WT-SIRT1 overexpression partially inhibited NMDA-stimulated p53 acetylation (P < 0.05), and DN-SIRT1 overexpression had no effect (P > 0.05). (a) Western blot probed with antibodies against p53 and Ace-p53. (b) Quantitative representations of Ace-p53 by bar graph. Each value represents the mean ± S.E.M. of three independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group.

Figure 9

Effects of SIRT1 overexpression on cell viability reduced by NMDA in the SH-SY5Y cell line. WT-SIRT1 overexpression reversed NMDA-induced decrease in cell viability (P < 0.05), and DN-SIRT1 overexpression did not affect cell viability in the NMDA group (P > 0.05). Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group, ^&P < 0.05 versus the WT-SIRT1 + NMDA group.

Figure 10

Effects of SIRT1 overexpression on NMDA-induced the amount of LDH release the in SH-SY5Y cell line. WT-SIRT1 overexpression reduced NMDA-induced LDH release (P < 0.05), and DN-SIRT1 overexpression did not protect against NMDA-mediated LDH release (P > 0.05). Each value represents the mean ± S.E.M. of six independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group, ^&P < 0.05 versus the WT-SIRT1 + NMDA group.

Figure 11

Effects of SIRT1 overexpression on the number of living cells reduced by NMDA in the SH-SY5Y cell line. (a) Representative micrographs showing the suppression of WT-SIRT1 overexpression on NMDA-induced decrease of living cells (P < 0.05) and no effect of DN-SIRT1 overexpression on the number of survival cells in the NMDA group (P > 0.05). Living cells were stained by Calcein-AM (green), and dead cells were stained by PI (red). (b) Bar graph of mean of living cells. Each value represents the mean ± S.E.M. of three independent experiments. ^∗P < 0.05 versus the control group, ^#P < 0.05 versus the NMDA group, ^&P < 0.05 versus the WT-SIRT1 + NMDA group.