Resveratrol attenuates hypoxia-induced neuronal cell death, inflammation and mitochondrial oxidative stress by modulation of TRPM2 channel

Abstract

Hypoxia (HYPX) induced-overload Ca²⁺ entry results in increase of mitochondrial oxidative stress, inflammation and apoptosis in several neurons. Ca²⁺ permeable TRPM2 channel was gated by ADP-ribose (ADPR) and reactive oxygen species (ROS), although its activity was modulated in HYPX-exposed neurons by resveratrol (RSV). The aim of this study was to evaluate if a therapy of RSV can modulate the effect of HYPX in the TRPM2 expressing SH-SY5Y neuronal and HEK293 (no expression of TRPM2) cell lines. The SH-SY5Y and HEK293 cells were divided into four groups as control, RSV (50 μM and 24 hours), and HYPX and RSV + HYPX. For induction of HYPX in the cells, CoCl₂ (200 μM and 24 hours) incubation was used. HYPX-induced intracellular Ca²⁺ responses to TRPM2 activation were increased in the SH-SY5Y cells but not in the HEK293 cells from coming H₂O₂ and ADPR. RSV treatment improved intracellular Ca²⁺ responses, mitochondrial function, suppressed the generation of cytokine (IL-1β and TNF-α), cytosolic and mitochondrial ROS in the SH-SY5Y cells. Intracellular free Zn²⁺, apoptosis, cell death, PARP-1, TRPM2 expression, caspase -3 and -9 levels are increased through activating TRPM2 in the SH-SY5Y cells exposed to the HYPX. However, the values were decreased in the cells by RSV and TRPM2 blockers (ACA and 2-APB). In SH-SY5Y neuronal cells exposed to HYPX conditions, the neuroprotective effects of RSV were shown to be exerted via modulation of oxidative stress, inflammation, apoptosis and death through modulation of TRPM2 channel. RSV could be used as an effective agent in the treatment of neurodegeneration exposure to HYPX.

Figure 1

Effects of resveratrol (RSV and 50 μM) on the hypoxia (HYPX)-induced increase of [Ca²⁺]_i concentration in the SH-SY5Y cells (mean ± SD). The cells in the four groups were stained with Fluo-8 (1 μM for 30 min) and then the stained cells in the TRPM2 experiments were stimulated by H₂O₂ (1 mM for 10 min), but they were inhibited by 2-APB (100 μM for 5–10 min). Representative images of the effect of RSV and HYPX on the [Ca²⁺]_i levels through TRPM2 in the confocal microscope analyses were shown Fig. 1a. Changes of intensity of the [Ca²⁺]_i levels were shown by columns (Fig. 1b). The scale bar = 5 µm. Objective: 40x oil. One example of each figure was taken from 6 independent experiments, with each experiment examining 20 cells for each condition (^ap ≤ 0.001 versus control (Ctr). ^bp ≤ 0.001 versus HYPX group. ^*p ≤ 0.001 versus - H₂O₂ group).

Figure 2

There was no changes on the [Ca²⁺]_i concentration in the HEK293 cells (in the absence of TRPM2) by hydrogen peroxide (H₂O₂). (mean ± SD). The cells were stained with Fluo-8 calcium dye and mean ± SD of fluorescence in 15 mm² of the cells as arbitrary unit are presented; n = 20 independent experiments. The HEK293 cells were stimulated by H₂O₂ (1 mM for 10 min), but they were inhibited by 2-APB (100 μM for 10 min). The samples were analyzed by the LSC microscopy fitted with a 40× oil objective. The scale bar was 5 µm. Representative images (a) and column (b) of fluorescence intensities of the H₂O₂ and 2-APB on the TRPM2 activation in the LSC microscope analyses are shown in Figs a and b, respectively.

Figure 3

Hypoxia (HYPX)-induced the TRPM2 current densities (pA/pF) in the SH-SY5Y cells but not in the HEK293 cells were reduced by the resveratrol (RSV) treatment. (mean ± SD and n = 4–6). The TRPM2 currents in the SH-SY5Y cells were induced by intracellular ADPR (1 mM in the patch-pipette), but they were blocked by extracellular ACA (25 μM) in the patch-chamber. W.C. is abbreviation of whole cell record configuration. (a) Ctr (without ADPR stimulation): Original recordings from Ctr neuron. (b) ADPR group of control neuron (with ADPR stimulation). (c) HYPOX group. (d) RSV group. (e) RSV + HYPX group. The Fig. (f) was currents densities of a, b, c, d and e patch clamp records. (g) ADPR group of control HEK293 (with ADPR stimulation). (h) HYPOX group of HEK293 cells. The Fig. (i) was currents densities of g and h patch clamp records. (^ap ≤ 0.001 versus control (Ctr). ^bp ≤ 0.001 versus Ctr+ADPR groups. ^cp ≤ 0.001 versus Ctr+ADPR + ACA group. ^dp ≤ 0.001 versus HYPX + ADPR group. ^ep ≤ 0.001 versus HYPX + ADPR + ACA group).

Figure 4

Resveratrol (RSV and 50 μM) and TRPM2 channel blocker (ACA and 25 μM) modulated hypoxia (HYPX)-induced cell viability (MTT), apoptosis, caspase -3, caspase -9, mitochondrial membrane depolarisation (JC-1) and cytosolic ROS generation (DCFH-DA) levels in the SH-SY5Y neuronal cells (mean ± SD and n = 3). The cell viability and apoptosis levels were analyzed in the spectrophotometer by using the MTT and commercial kit, respectively. However, caspase -3, caspase -9, mitochondrial membrane depolarization and cytosolic ROS generation in the SH-SY5Y neuronal cells were assayed in the microplate reader by using caspase -3 substrate (AC-DEVD-AMC), caspase -9 substrate (ACDEVD-AMC), JC-1 and DCFH-DA stains, respectively. (^*p ≤ 0.001 versus control (Ctr) and RSV groups. ^**p ≤ 0.001 versus HYPX group).

Figure 5

Effect of resveratrol (RSV and 50 μM) and 2-APB (100 μM) on the mitochondrial membrane depolarization (JC-1), cytosolic (DHR123 and DCFH-DA) and mitochondria (MitoROS) ROS generation levels in the hypoxia (HYPX)-induced SH-SY5Y cells. Mean ± SD of fluorescence in 15 mm² of the cells as arbitrary unit are presented; n = 20 independent experiments. The SH-SY5Y neuronal cells were stimulated by H₂O₂ (1 mM for 10 min), but they were inhibited by 2-APB (100 μM for 10 min). The samples were analyzed by the LSC microscopy fitted with a 40× oil objective. The scale bar was 5 µm. Representative images and column of fluorescence intensities of the JC-1 + DHR123 (Fig. 5a,b) and DCFH-DA + MitoROS (Fig. 5c,d) on the TRPM2 activation in the LSC microscope analyses are shown in Fig. 5a–d, respectively. (^ap ≤ 0.001 versus control (Ctr) and RSV groups. ^bp ≤ 0.001 versus HYPX group. ^cp ≤ 0.001 versus RSV + HYPX group).

Figure 6

Resveratrol (RSV and 50 μM) and 2-APB (100 μM) protected hypoxia (CoCl₂ and 50 μM)-induced cell death and cell number changes in the SH-SY5Y cells. (mean ± SD). (a) Each panel consists of PI (red) and Hoechst (blue)-staining, and bride field (black-white) images are showing dead and live cells. The scale bar is 50–20 μm. (b) Summary of the mean percentage of PI and Hoechst-positive cells under the indicated conditions from 6 independent experiments, with each experiment examining 20–25 cells for each condition. (c) Mean numbers of the cell count (n = 6). (^ap ≤ 0.001 versus control (Ctr) and RSV groups. ^bp ≤ 0.001 versus HYPX group. ^cp ≤ 0.001 versus RSV + HYPX group).

Figure 7

Resveratrol (RSV and 50 μM) and 2-APB (100 μM) protected hypoxia (CoCl₂ and 50 μM)-induced increase of intracellular free Zn²⁺ concentration and PARP-1 expression level in the SH-SY5Y cells. (mean ± SD). (a) Each panel consists of FluoZn3 and TPEN-staining images are showing presence and absence of intracellular free Zn²⁺, respectively. The scale bar is 5 μm. (b) Summary of the mean percentage of FluoZn3 and TPEN-positive cells under the indicated conditions from 6 independent experiments, with each experiment examining 20–25 cells for each condition. (c) PARP-1 expression level. (^ap ≤ 0.001 versus control (Ctr) and RSV groups. ^bp ≤ 0.001 versus HYPX group. ^cp ≤ 0.001 versus RSV + HYPX group).