Atractylenolide-I Protects Human SH-SY5Y Cells from 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

Abstract

Oxidative stress and apoptosis are the major mechanisms that induce dopaminergic cell death. Our study investigates the protective effects of atractylenolide-I (ATR-I) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity in human dopaminergic SH-SY5Y cells, as well as its underlying mechanism. Our experimental data indicates that ATR-I significantly inhibits the loss of cell viability induced by MPP⁺ in SH-SY5Y cells. To further unravel the mechanism, we examined the effect of ATR-I on MPP⁺-induced apoptotic cell death characterized by an increase in the Bax/Bcl-2 mRNA ratio, the release of cytochrome-c, and the activation of caspase-3 leading to elevated levels of cleaved poly(ADP-ribose) polymerase (PARP) resulting in SH-SY5Y cell death. Our results demonstrated that ATR-I decreases the level of pro-apoptotic proteins induced by MPP⁺ and also restored Bax/Bcl-2 mRNA levels, which are critical for inducing apoptosis. In addition, ATR-I demonstrated a significant increase in the protein expression of heme-oxygenase in MPP⁺-treated SH-SY5Y cells. These results suggest that the pharmacological effect of ATR-I may be, at least in part, caused by the reduction in pro-apoptotic signals and also by induction of anti-oxidant protein.

Keywords: MPP+; Parkinson’s disease; apoptosis; atractylenolide-I; neuroprotection.

Figure 1

(A,B) Effects of atractylenolide-I (ATR-I) on cell viability in SH-SY5Y cells intoxicated with or without 1-methyl-4-phenylpyridinium (MPP⁺). The viability of cells was performed as mentioned in the “Material and Method” section. *** p < 0.001 vs. vehicle group. ^$$$ p < 0.001 vs. vehicle group, and *** p < 0.001, ** p < 0.01 vs. MPP⁺-treated group.

Figure 2

(A) Effects of ATR-I on the Bax/Bcl-2 mRNA ratio in MPP⁺-stimulated SH-SY5Y cells. The levels of (B) HO-1 mRNA and (C) HO-1 protein expression were quantitated by densitometric analysis. Quantification data are shown in the lower panel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as internal controls. ^$$$ p < 0.001 and ^$$ p < 0.01 vs. the vehicle group; *** p < 0.001 and ** p < 0.01 vs. the MPP⁺-treated group.

Figure 3

Effect of ATR-I on the MPP⁺-induced p53 and cytochrome-c release in SH-SY5Y cells. SH-SY5Y cells were subjected to Western blotting with specific antibodies for p53 (A) and cytochrome-c (B). Quantification of the relative expression of p53 and cytochrome-c is provided in the bar graph. β-actin was used as an internal control. ^$$$ p < 0.001 vs. the vehicle group; *** p < 0.001 and * p < 0.05 vs. the MPP⁺-treated group.

Figure 4

Effect of ATR-I on MPP⁺-induced expression of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) in SH-SY5Y cells. SH-SY5Y cells were tagged with specific antibodies for caspase-3 (A) and cleaved PARP (B). Quantification of relative expression of caspase-3 and cleaved PARP is provided in the bar graph. β-actin was used as an internal control. ^$$$ p < 0.001 vs. the vehicle group; *** p < 0.001 and * p < 0.05 vs. the MPP⁺-treated group.

Figure 5

SH-SY5Y cells were pre-incubated with or without zinc protoporphyrin-IX (ZnPP-IX ) (5 µM) for 2 h before treating them with ATR (25 µM) for another 2 h. Cells were further exposed to 2 mM MPP⁺ for 24 h. Cell viability was measured by MTT assay. ^$$$ p < 0.001 vs. the MPP⁺ group, ^### p < 0.001 vs. the MPP⁺ + ATR-I group and *** p < 0.001 vs. the ZnPP-IX + MPP⁺-treated group.

Figure 6

Scheme representing MPP⁺-induced apoptotic signaling