Neuroprotective effects of vanillyl alcohol in Gastrodia elata Blume through suppression of oxidative stress and anti-apoptotic activity in toxin-induced dopaminergic MN9D cells

Abstract

Gastrodia elata Blume (GE) has long been used in oriental countries as a traditional herbal medicine to relieve symptoms associated with neurological ailments such as vertigo, general paralysis and epilepsy. In this study, we have investigated the effects of GE extracts and its major bioactive components on 1-methyl-4-phenylpyridinium (MPP+)-treated MN9D dopaminergic cells, a classic in vitro model for Parkinson's disease (PD). We found that vanillyl alcohol effectively inhibited the cytotoxicity and improved cell viability in MPP+-induced MN9D dopaminergic cells. The underlying mechanisms of vanillyl alcohol action were also studied. Vanillyl alcohol attenuated the elevation of reactive oxygen species (ROS) levels, decreased in the Bax/Bcl-2 ratio and poly (ADP-ribose) polymerase proteolysis. These results indicate that vanillyl alcohol protected dopaminergic MN9D cells against MPP+-induced apoptosis by relieving oxidative stress and modulating the apoptotic process and is therefore a potential candidate for treatment of neurodegenerative diseases such as Parkinson's disease.

Figure 1

Effect of GE on MPP⁺-induced neuronal cell death in dopaminergic MN9D cells. Cells were exposed to 25 μM MPP⁺ in the absence or presence of GE (10, 100, 200 µg/mL), and cell viability was assessed by MTT assay (A); The mRNA expression of Bax and Bcl-2 was determined by RT-PCR (B); and PARP proteolysis was analyzed by immunoblot analysis (C). Data are expressed as the percentage of values in untreated control cultures and are means ± S.E.M. of three independent experiments in triplicate. ^# P < 0.05, compared with the control group. * P < 0.05, compared with the MPP⁺-treated group (one-way ANOVA followed by Bonferroni post hoc test).

Figure 2

The chemical structure of vanillyl alcohol (A); Effect of vanillyl alcohol on cell viability in MN9D cells exposed to MPP⁺ (B). Cells were exposed to 25 μM MPP⁺ in the absence or presence of vanillyl alcohol (1, 10, 20 µM) and cell viability was assessed by MTT assay. Data are expressed as the percentage of values in untreated control cultures and are means ± S.E.M. of three independent experiments in triplicate. ^# P < 0.05, compared with the control group. ^* P < 0.05, compared with the MPP⁺-treated group (one-way ANOVA followed by Bonferroni post hoc test).

Figure 3

Effect of vanillyl alcohol against MPP⁺-induced neurotoxicity in MN9D cells by flow cytometric DNA analysis. (A). Control cells; (B). the cells exposed to 25 μM MPP+ alone; (C). the cells exposed to 20 μM alone; (D–F). the cells pre-treated with vanillyl alcohol at 1, 10, 20 μM, respectively, in the presence of 25 μM MPP⁺. Bar (├─┤) represents a sub-G0/G1 or hypodiploid DNA fraction.

Figure 4

Effect of vanillyl alcohol on the free radical scavenging activity. (A). Left: relationship between the signal intensity of DPPH radical and the various concentrations of vanillyl alcohol. Right: ESR spectra of DPPH radicals recorded; (B). Left: relationship between the signal intensity of the POBN-alkyl radicals and the various concentrations of vanillyl alcohol. Right: ESR spectra of 2-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)-trapped alkyl radicals recorded.

Figure 5

Effects of VA on MPP⁺-induced reactive oxygen species production. Cells were exposed to 25 µM MPP⁺ with or without different concentration of VA (1, 10, 20 µM) for 48 h. ROS generation was detected by fluorometric analysis using DCFH-DA. Data are means ± S.E.M. of three independent experiments in triplicate. ^# P < 0.05, compared with control group; * P < 0.05, ** P < 0.01 compared with MPP⁺-treated group in one-way ANOVA followed by Bonferroni post hoc test.

Figure 6

Effects of vanillyl alcohol on the expression of Bcl-2 and Bax in MN9D cells. Cells were treated with 25 μM MPP⁺ in the absence or presence of vanillyl alcohol, and total RNA was collected for expression analysis. The levels of Bax and Bcl-2 were quantitated by densitometric analysis (A) and the Bax/Bcl-2 ratio was determined (B). Data are means ± S.E.M. of three independent experiments in triplicate. ^# P < 0.05, compared with control group. * P < 0.05, ** P < 0.01, compared with MPP⁺-treated group (one-way ANOVA followed by Bonferroni post hoc test).

Figure 7

Vanillyl alcohol inhibits MPP+-induced PARP proteolysis. PARP proteolysis was analyzed by immunoblot analysis (A) and the levels of cleaved PARP were quantitated by densitometric analysis (B). Data are means ± S.E.M. of three independent experiments in triplicate. ^# P < 0.05, compared with control group; * P < 0.05, ** P < 0.01 compared with MPP+-treated group in one-way ANOVA followed by Bonferroni post hoc test.