Vincamine Alleviates Amyloid-β 25-35 Peptides-induced Cytotoxicity in PC12 Cells

Abstract

Objective: Vincamine is a plant alkaloid used clinically as a peripheral vasodilator that increases cerebral blood flow and oxygen and glucose utilization by neural tissue to combat the effect of aging. The main purpose of the present study is to investigate the influence of vincamine on amyloid-β 25-35 (Aβ25-35) induced cytotoxicity, to gain a better understanding of the neuroprotective effects of this clinically used anti-Alzheimer's disease drug.

Materials and methods: Oxidative stress was assessed by measuring malondialdehyde, glutathione, and superoxide dismutase (SOD) levels. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis detection was performed using an Annexin-V-FITC Apoptosis Detection Kit. The production of reactive oxygen species (ROS) was determined using an ROS Assay Kit. Western blot detection was carried out to detect the protein expression.

Results: Our studies showed that pretreatment with vincamine could reduce Aβ25-35 induced oxidative stress. Vincamine markedly inhibited cell apoptosis dose-dependently. More importantly, vincamine increased the phosphatidylinositol-3 kinase (PI3K)/Akt and Bcl-2 family protein ratios on preincubation with cells for 2 h.

Conclusion: Above observation led us to assume that one possible mechanism of vincamine protects Aβ25-35-induced cell death could be through upregulation of SOD and activation of the PI3K/Akt pathway.

Summary: Vincamine ameliorates amyloid-β 25-35 (Aβ25-35) peptides induced cytotoxicity in PC12 cellsVincamine reduces Aβ 25-35 peptides induced apoptosis of PC12 cellsVincamine activates the phosphatidylinositol-3 kinase/Akt pathwayVincamine up-regulates the superoxide dismutase. Abbreviation used: Aβ25-35: Amyloid-β 25-35; AD: Alzheimer's disease; BCA: Bicinchoninic acid; GSH: glutathione; PBS: Phosphate buffered solution; SDS: Sodium dodecylsulphate; SOD: Superoxide dismutase.

Figure 1

Chemical structure of vincamine

Figure 2

Effect of amyloid-β 25–35 on cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in PC12 cells. (a) Cells treated with amyloid-β 25–35 (0–80 μM) for 24 h. (b) Cells treated with 30 μM amyloid-β 25–35 for 0, 12, 24, 36, 48, 60 and 72 h. All results are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the control group

Figure 3

Effect of vincamine on cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in amyloid-β 25–35-treated PC12 cells. The results are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01 as compared with the amyloid-β 25–35-treated group

Figure 4

Intracellular reactive oxygen species produced after amyloid-β 25–35 induced oxidative stress in PC12 cells with and without vincamine. Student's t-test was performed to evaluate the significance of the results. *P < 0.05, **P < 0.01, vincamine-treated cells compared with respective control. Results are mean ± standard deviation (n = 3)

Figure 5

Vincamine attenuated amyloid-β 25–35 induced neurotoxicity in PC12 cells. (A) Apoptosis analysis of PC12 cells treated with amyloid-β 25–35, vincamine, or a combination of them. (a) Untreated cells; (b) 30 μM amyloid-β 25–35-treated cells; (c) 30 μM amyloid-β 25–35 + 20 μM vincamine-treated cells; (d) 30 μM amyloid-β 25–35 + 40 μM vincamine-treated cells; (e) 30 μM amyloid-β 25–35 + 80 μM vincamine-treated cells. Cells were exposed for 48 h. Double staining was used to distinguish between viable (lower left quadrant, annexin V-negative, propidium iodide-negative), early apoptosis (lower right quadrant, annexin V-positive, propidium iodide-negative), late apoptosis and necrotic (upper right quadrant, annexin V-positive, propidium iodide-positive) and cell debris (upper left quadrant). Statistical analysis is shown in (B). *P < 0.05, **P < 0.01, amyloid-β 25–35, vincamine or both treated cells compared with untreated control cells. #P < 0.05, ##P < 0.01, vincamine-treated cells compared with 30 μM amyloid-β 25–35-treated cells. Results are mean ± standard deviation (n = 3)

Figure 6

Phosphatidylinositol-3 kinase/Akt pathway was involved in the anti-apoptotic effects of vincamine. (a) PC12 cells were treated with vincamine for 2 h. Akt and its phosphorylation were detected by Western blotting. β-actin served as a control for loading. (b) Protein expression of Bcl-2, Bax in PC12 cells after indicated treatments was also measured by Western blots. GAPDH served as a control for loading