Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells

Abstract

Amyloid beta (Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer's disease. Cyanidin, a natural flavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreatment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment significantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial membrane potential via upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity.

Keywords: amyloid-beta; apoptosis; cyanidin; nerve regeneration; neural regeneration; oxidative damage; reactive oxide species.

Figure 1

Cyanidin (Cya) suppressed amyloid-beta (Aβ)-induced cytotoxicity in PC12 cells. (A–C) Effect of Cya on PC12 cell viability (MTT assay). PC12 cells seeded in a 96-well plate were treated with Cya (5–80 μM) for 48 hours (A), Aβ (5–80 μM) for 24 hours (B), or both (Cya 5–80 µM plus Aβ 10 μM; C). Cya protected cells against Aβ-induced cytotoxicity. Cell viability was quantified by MTT assay. Aβ treatment alone (5–80 μM) dose-dependently decreased cell viability (P < 0.05), while Cya dose-dependently increased cell viability at 20–80 μM (P < 0.05). Multiple comparisons were performed using one-way analysis of variance. Data are presented as the mean ± SD of four independent experiments. (D) Morphological changes in PC12 cells with Cya and Aβ. Arrows, apoptotic bodies.

Figure 2

Cyanidin (Cya) prevented amyloid-beta Aβ)-induced apoptosis in PC12 cells. (A) TUNEL/DAPI double staining. Cells were exposed to 80 µM Cya for 24 hours and/or 10 µM Aβ for 24 hours, and apoptosis was detected by TUNEL/DAPI double staining. Arrows indicate TUNEL-positive cells. (B) Cya blocked Aβ-induced activation of caspase-3, measured by fluorometric assay. *P < 0.05, vs. control; #P < 0.05, vs. Aβ treatment. Multiple comparisons were performed using one-way analysis of variance. (C) Caspase-3 expression (western blot). Full length caspase-3 (35 kDa) and the cleavage fraction (CF) of caspase-3 (19 kDa) were examined. Protein expression was measured using Quantity-One software, and all measurements were normalized against β-actin. All experiments were performed three times.

Figure 3

Cyanidin (Cya) inhibited amyloid-beta (Aβ)-induced loss of mitochondrial membrane potential (Δψm) by modulating Bcl-2 family proteins. (A) Cya suppresses Aβ-mediated depletion of Δψm. JC-1 probe was used to detect Δψm. Red, mitochondrial fluorescence; green, cytoplasmic fluorescence. The shift of fluorescence from red to green indicates change in Δψm. (B) Cya prevented the Aβ-induced decrease in Bcl-2 family protein expression (western blot assay). All experiments were carried out three times.

Figure 4

Cyanidin (Cya) prevented amyloid-beta (Aβ)-induced DNA damage by suppressing reactive oxygen species (ROS) accumulation. (A) Cya prevented Aβ-induced accumulation of ROS in PC12 cells. Cells were pretreated with Cya (20–80 µM) for 1 hour and then incubated in the presence or absence of 10 µM Aβ for 2 hours. ROS accumulation was monitored using DCFH-DA dye. Treatment with Cya at 20–80 µM dose-dependently decreased Aβ-induced elevation of ROS (P < 0.05). Multiple comparisons were performed using one-way analysis of variance. (B) Cya inhibited Aβ-induced superoxide production in PC12 cells. The superoxide was observed using the mitochondria-targeted dye, MitoSOX. DAPI was used to stain the nucleus. All images shown here are representative of three independent experiments. Red fluorescence, mitochondrial superoxide. (C) Protection of Cya against Aβ-induced DNA damage (western blot assay). Experiments were conducted three times.

Figure 5

Scheme illustrating potential signaling pathways. Amyloid-beta (Aβ) directly caused reactive oxygen species (ROS) accumulation, triggered DNA damage and induced PC12 cell apoptosis. However, cyanidin inhibited ROS accumulation, attenuated DNA damage, and eventually reversed Aβ-induced apoptosis in PC12 cells by modulating Bcl-2 family protein expression and restoring the Δψm.