Gastrodin protects apoptotic dopaminergic neurons in a toxin-induced Parkinson's disease model

Abstract

Gastrodia elata (GE) Blume is one of the most important traditional plants in Oriental countries and has been used for centuries to improve various conditions. The phenolic glucoside gastrodin is an active constituent of GE. The aim of this study was to investigate the neuroprotective role of gastrodin in 1-methyl-4-phenylpyridinium (MPP(+))/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP) induced human dopaminergic SH-SY5Y cells and mouse model of Parkinson's disease (PD), respectively. Gastrodin significantly and dose dependently protected dopaminergic neurons against neurotoxicity through regulating free radicals, Bax/Bcl-2 mRNA, caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) in SH-SY5Y cells stressed with MPP(+). Gastrodin also showed neuroprotective effects in the subchronic MPTP mouse PD model by ameliorating bradykinesia and motor impairment in the pole and rotarod tests, respectively. Consistent with this finding, gastrodin prevented dopamine depletion and reduced reactive astrogliosis caused by MPTP as assessed by immunohistochemistry and immunoblotting in the substantiae nigrae and striatata of mice. Moreover, gastrodin was also effective in preventing neuronal apoptosis by attenuating antioxidant and antiapoptotic activities in these brain areas. These results strongly suggest that gastrodin has protective effects in experimental PD models and that it may be developed as a clinical candidate to ameliorate PD symptoms.

Figure 1

Effect of gastrodin on MPP⁺-induced neurotoxicity in the dopaminergic neuroblastoma SH-SY5Y cell line. Cells were exposed to gastrodin and 1 mM MPP⁺ for 48 h. Cell viability (A) and morphology of SH-SY5Y cells (B) after treatment with control (a), 1 mM MPP⁺ (b), 25 μM gastrodin (c), 25 μM gastrodin + 1 mM MPP⁺ (d), 5 μM gastrodin + 1 mM MPP⁺ (e), and 1 μM gastrodin + 1 mM MPP⁺ (f). Data are percentages of values in the untreated control cultures and are means ± standard errors of three independent experiments in triplicate. ^### P < 0.001 compared with the control group, **P < 0.01, and ***P < 0.001 compared with the MPP⁺-treated group (one-way analysis of variance followed by Tukey's post hoc test).

Figure 2

Effect of gastrodin on DPPH (a) and alkyl (b) free radical scavenging activities. Gastrodin prevented the ROS generation in 1 mM MPP⁺-treated SH-SY5Y cells as measured by fluorometric analysis using DCFH-DA (c). Gastrodin augmented the MPP⁺- and MPTP-induced perturbation in superoxide dismutase (SOD) activity in SH-SY5Y cells (d), striatum (e), and SNpC (f), respectively. Data are percentages of values in the untreated control cultures and are means ± standard errors of three independent experiments in triplicate. ^### P < 0.001 compared with the control group, **P < 0.01, and ***P < 0.001 compared with the MPP⁺-treated group (one-way analysis of variance followed by Tukey's post hoc test).

Figure 3

Protective effects of gastrodin against MPTP in the mouse substantia nigra pars compacta (SNpC) and striatum. Gastrodin was administered for 15 days at the respective doses, and MPTP was administered for the last 5 days of gastrodin treatment. All groups except the vehicle group received injections of 30 mg/kg/day MPTP for 5 days. Mice were anesthetized for the immunohistochemical study 7 days after MPTP intoxication and after performing the behavioral experiment. Glial fibrillary acidic protein (GFAP) immunofluorescence and immunohistochemistry for tyrosine hydroxylase (TH) were performed in the SNpC and striatum (a). Gastrodin protected against changes in TH and GFAP expression in the SNpC ((b) and (d)) and striatum ((c) and (e)) after MPTP intoxication. TH and GFAP protein levels in the SNpC and striatum were assessed by Western blot analysis. Bar graphs show quantitative data for TH and GFAP signals that are normalized to the β-actin signal (n = 4-5 per group). Values are mean ± standard error (^## P < 0.01, ^### P < 0.001 versus vehicle group) and (*P < 0.05, **P < 0.01, and ***P < 0.001 versus MPTP group).

Figure 4

Effects of gastrodin on Bax and Bcl-2 mRNA expression and caspase-3 activity in SH-SY5Y cells and animal tissue treated with MPTP. Bax and Bcl-2 levels were quantified in SH-SY5Y cells by densitometric analysis (a) and in the substantia nigra pars compacta (SNpC) (c) and striatum (d) with their respective Bax/Bcl-2 ratios. Gastrodin inhibited the MPP⁺- and MPTP-induced increase in caspase-3 activity in SH-SY5Y cells (b), SNpC (e), and striatum (f), respectively. Data are from three independent experiments performed in triplicate. Values are mean ± standard error (^### P < 0.001 versus vehicle group) and (*P < 0.05, **P < 0.01, and ***P < 0.001 versus MPP⁺/MPTP group).

Figure 5

Gastrodin prevents poly(ADP-ribose) polymerase (PARP) cleavage against MPP⁺ in SH-SY5Y cells (a), the substantia nigra pars compacta (SNpC) (b), and striatum (c) after MPTP treatment. PARP protein levels were assessed by Western blot analysis in cells/animal tissues. Bar graphs show quantitative data for PARP signals that are normalized to the β-actin signal (n = 3-4 per group). Values are mean ± standard error (^### P < 0.01 versus vehicle group) and (***P < 0.001 and versus MPP⁺/MPTP group).

Figure 6

Protective effect of gastrodin against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced behavioral dysfunction in a mouse model of Parkinson's disease. Vehicle or different doses (10, 30, and 60 mg/kg) of gastrodin were administered orally once per day for 15 days, and MPTP (30 mg/kg, i.p.) was injected during the last 5 days. Seven days after the last MPTP injection, the time to arrive at the floor (total locomotor activity) (a) and the time to turn completely downward (T-turn) (b) were recorded with a cutoff limit of 60 s for the pole test. Motor deficit was indicated by the latency of fall (c) during the rotarod test. Values are mean ± standard error (^### P < 0.001 versus vehicle group) and (*P < 0.05, **P < 0.01, and ***P < 0.001 versus MPTP group).