Neuroprotective effects of selegiline on rat neural stem cells treated with hydrogen peroxide

Abstract

Oxidative stress and reactive oxygen species generation have been implicated in the pathogenesis of several neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis. In the present study, the neuroprotective effects of selegiline against hydrogen peroxide-induced oxidative stress in hippocampus-derived neural stem cells (NSCs) were evaluated. NSCs isolated from neonatal Wistar rats were pretreated with different doses of selegiline for 48 h and then exposed to 125 µM H2O2 for 30 min. Using MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, acridine orange/ethidium bromide staining and reverse transcription-quantitative polymerase chain reaction, the effects of selegiline on cell survival, apoptosis and the expression of B-cell lymphoma 2 (Bcl-2) and heat shock protein 4 (Hspa4) in pretreated stem cells were assessed compared with a control group lacking pretreatment. The results indicated that the viability of cells pretreated with 20 µM selegiline was significantly increased compared with the control group (P<0.05). Additionally, 20 µM selegiline increased the mRNA expression of Bcl-2 and Hspa4 (P<0.05 vs. control) and suppressed oxidative stress-induced cell death (apoptosis and necrosis; P<0.05 vs. control and 10 µM groups). From these findings, it was concluded that selegiline may be a therapeutic candidate for the treatment of neurological diseases mediated by oxidative stress.

Keywords: B-cell lymphoma 2; heat shock protein 4; neural stem cells; oxidative stress; selegiline.

Figure 1.

Representative photomicrographs of hippocampus-derived NSCs. (A) Floating neurospheres derived from rat hippocampus after culture for 7 days. (B) Hippocampus-derived NSCs at passage 3. (C) Phase contrast micrograph and (D) immunostaining of nestin (green) in the same field. Nuclei were counterstained with ethidium bromide (red). Magnification, ×200. NSC, neural stem cell.

Figure 2.

Effect of selegiline on cell viability, apoptosis and necrosis. (A) Mean percentages of viable, apoptotic and necrotic cells (relative to untreated cells) determined by MTT assay, TUNEL and acridine orange/ethidium bromide staining, respectively, following 125 µM H₂O₂ exposure with selegiline pretreatment. (B-E) Representative TUNEL and acridine orange staining images of NSCs at (B and D) 0 and (C and E) 20 µM selegiline followed by 125 µM H₂O₂ exposure. Red arrows indicate DNA fragmentation. Late necrotic NSCs exhibited orange/red staining in non-condensed, non-fragmented nuclei (white arrows). Counterstaining with hematoxylin is shown in blue. Magnification, ×200. The bars indicate the mean ± standard error of the mean. *P<0.05 vs. 20 µM group. NSC, neural stem cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 3.

Fold changes in Bcl-2 and Hspa4 mRNA expression in H₂O₂-induced NSCs treated with 20 µM selegiline. The results of polymerase chain reaction were presented as relative expression normalized to β2-microglobulin mRNA amplification. The bars indicate the mean ± standard error of the mean. *P<0.05 vs. 20 µM group. Bcl-2, B-cell lymphoma 2; Hspa4, heat shock protein 4; NSC, neural stem cell.