Sodium valproate ameliorates aluminum-induced oxidative stress and apoptosis of PC12 cells

Abstract

Objectives: According to recent studies, valproate shows some protection against oxidative stress (OS) induced by neurotoxins. Current investigation tried to determine the possible ameliorating effects of sodium valproate (SV) against aluminum (Al)-induced cell death, apoptosis, mitochondrial membrane potential (MMP), and OS in PC12 cells.

Materials and methods: In this in vitro study, PC12 cells were treated with different concentrations of aluminum maltolate (Almal) with and without SV (50-400 µM). Cell viability was assessed by MTT assay. To measure quantitatively the effects of SV on Al-induced apoptosis and reactive oxygen species (ROS), flowcytometry using 7AAD/annexin-V and 2', 7'-dichlorofluorescein diacetate staining were employed, respectively. MMP was monitored using the retention of rhodamine 123. Catalase (CAT) activity was assayed by the rate of decomposition of hydrogen peroxide.

Results: Exposure of PC12 cells for 48 hr to Almal (125-2000 µM) significantly reduced cell viability (IC₅₀=1090 μM), increased ROS generation and apoptosis, and reduced MMP and CAT activity. SV reduced the Almal-induced cell death and apoptosis. Furthermore, the effects of Almal on ROS generation, catalase activity, and MMP reduction were significantly diminished by SV.

Conclusion: Data from this study suggest that SV can inhibit Al-induced cell death and apoptosis of PC12 cells via ameliorating OS.

Keywords: Aluminum maltolate; Apoptosis; Histone deacetylase – inhibitor; Oxidative stress; Valproic acid.

Figure 1

The effects of Almal (A) and SV (B) on cell viability. Forty-eight hours after treatment of PC12 with Almal (0–2000 µM) or SV (0–1500 µM) the cell survival was determined using the MTT method. GraphPad Prism software was used for analysis of the data and calculation of IC₅₀ values

Figure 2

The effects of SV on cell death induced by Almal. MTT results revealed the protective effects of SV (50–600 μM) against Almal (1000 µM)-induced cell death. The data obtained from five independent MTT tests were analyzed using one-way ANOVA followed by the LSD post hoc test. The histogram represents mean±SD of viable cells. * and # show significant differences at P-value<0.05 compared with control and Almal-treated groups, respectively

Figure 3

The ameliorating effect of SV on the apoptosis of PC12 cells induced by Almal. The flow cytometric graphs illustrate the percentage of viable cells (Q4), apoptotic cells (early: Q3 + late: Q2), and dead cells (Q1) in the control group (A) and the PC12 cells treated with 1000 µM of Almal alone (B) or Almal (1000 µM) in the presence of 50 µM (C), 100 µM (D), 200 µM (E), and 400 µM of SV (F). The histogram reveals the mean±SD of total apoptotic cells in various experimental groups. One-way ANOVA and LSD post hoc test were used for analysis of the data

Figure 4

Flow cytometric analysis of intracellular ROS content in the PC12 cells treated with Almal and varying concentrations of SV. A: the representative spectra of fluorescent DCF in the cells treated with Almal, Almal+SV, and TBHP as a positive control. B: the comparative analysis of DCF fluorescence in various experimental groups. Each histogram represents mean ± SD of at least three ROS determination experiments. *, **, and *** show significant difference compared with the control cells at P-value<0.05, P-value< 0.01, and P-value<0.001, respectively

Figure 5

Evaluation of mitochondrial membrane potential of the PC12 cells using Rh123 fluorescence. The histograms represent mean±SD values of Rh123 fluorescence obtained from the PC12 cells co-treated with Almal (1000 μM) and SV (50–400 μM). ** shows significant difference at P-value<0.01 compared with the control untreated cells

Figure 6

Evaluation of CAT activity of PC12 cells co-treated with Almal (1000 µM) and SV (50–400 μM). The histograms represent mean±SD of CAT activity obtained from at least three experiments. One-way ANOVA and LSD post hoc test were used for the analysis of data. * shows significant difference at P-value<0.05 compared with the control group, ** and *** show significant difference at P-value<0.01 and P-value<0.001 compared with the Almal group, respectively