The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity

Abstract

This study aims to investigate the neuroprotective effect of the rhizome of Gastrodia elata (GE) aqueous extract on beta-amyloid(A β )-induced toxicity in vivo and in vitro. Transgenic Drosophila mutants with A β -induced neurodegeneration in pan-neuron and ommatidia were used to determine the efficacy of GE. The antiapoptotic and antioxidative mechanisms of GE were also studied in A β -treated pheochromocytoma (PC12) cells. In vivo studies demonstrated that GE (5 mg/g Drosophila media)-treated Drosophila possessed a longer lifespan, better locomotor function, and less-degenerated ommatidia when compared with the A β -expressing control (all P < 0.05). In vitro studies illustrated that GE increased the cell viability of A β -treated PC12 cells in dose-dependent manner, probably through attenuation of A β -induced oxidative and apoptotic stress. GE also significantly upregulated the enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase, leading to the decrease of reactive oxidation species production and apoptotic marker caspase-3 activity. In conclusion, our current data presented the first evidence that the aqueous extract of GE was capable of reducing the A β -induced neurodegeneration in Drosophila, possibly through inhibition of apoptosis and reduction of oxidative stress. GE aqueous extract could be developed as a promising herbal agent for neuroprotection and novel adjuvant therapies for Alzheimer's disease.

Figure 1

Intake of GE increases the (a) lifespan and (b-i) locomotor activity of Aβ-expressing Drosophila. The lifespan of Aβ42 group (squares in red) is shorter than the control group (circles in blue), while GE (asterisks and triangles in purple) or donepezil (triangles in green) treatments delay the mortality of the Drosophila. (b-ii) is an amplification of the region from days 12 to 23 showing the differences among the Aβ42 group and the treatment groups. The percentage of Drosophila climbing up 8 cm in 10 seconds was increased by GE or donepezil treatments when compared with Aβ42 group. Results are the means ± SEM from five independent crosses. ^### P < 0.001 relative to control; *P < 0.05, **P < 0.01, ***P < 0.001 relative to Aβ42 Drosophila by one-way ANOVA for locomotor activity. Log-Rank analysis and chi-square comparison were applied to the survival data and P < 0.001 was obtained when comparing Aβ42 Drosophila and Donepezil 10 μmol/g or GE 1 mg/g or 5 mg/g treated ones (n = 150).

Figure 2

Rhabdomere count in the pseudopupil assay. Regular array of 7 ommatidia (bright red spots) was observed in OregonR eyes. Degeneration of ommatidia was observed in the Aβ42 group, while the degeneration is improved by GE or donepezil treatments. ^### P < 0.001 relative to OregonR; ***P < 0.001 relative to Aβ42 Drosophila with no treatment by one-way ANOVA. Results are the means ± SEM from 3 independent crosses. One hundred ommatidia were observed from 10 eyes of 5 Drosophila from each group in each trial.

Figure 3

Protective effect of GE on Aβ-induced cytotoxicity in PC12 cells. Effect of 48 h treatment of GE extract on the viability of PC12 cells was determined by MTT assay. Results are the means ± SD from three separate experiments. ^### P < 0.001 relative to control; *P < 0.05, ***P < 0.001 relative to Aβ treatment only by one-way ANOVA.

Figure 4

Antiapoptotic effect of GE on Aβ-induced cytotoxicity in PC12 cells. (a) Representative plots for the flow cytometric analysis. (b) GE extract reduced Aβ-induced apoptosis in flow cytometric analysis. The fluorescence intensity was measured after PC12 cells were exposed to 20 μM Aβ for 48 h, followed by incubation with Annexin V-FITC and PI for 15 min. (c) 48 h treatment of GE extract attenuated Aβ-induced activation of caspase-3. Results are the means ± SD from three separate experiments. ^# P < 0.05 relative to control; *P < 0.05, **P < 0.01, ***P < 0.001 relative to Aβ treatment only by one-way ANOVA.

Figure 5

Antioxidative effect of GE on Aβ-induced cytotoxicity in PC12 cells. (a) GE extract reduced Aβ-induced oxidative stress in flow cytometric analysis of DCF positive cells. The fluorescence intensity of DCF was measured after PC12 cells were exposed to 20 μM Aβ for 48 h, followed by 20 μM H2DCF-DA for 15 min. 48 h treatment of GE extract increased the activities of antioxidative enzymes (b) superoxide dismutase (c) catalase and (d) glutathione peroxidase in 20 μM Aβ-treated cells. Results are the means ± SD from three separate experiments. ^# P < 0.05, ^## P < 0.01 relative to control; *P < 0.05, **P < 0.01, ***P < 0.001 relative to Aβ treatment only by one-way ANOVA.