Gastrodin protects dopaminergic neurons via insulin-like pathway in a Parkinson's disease model

Abstract

Background: Recently, the use of traditional Chinese medicine (TCM) has become more generally accepted, including by the Food and Drug Administration. To expand the use of TCM worldwide, it is important to study the molecular mechanisms by which TCM and its active ingredients produce effects. Gastrodin is an active ingredient from Gastrodia elata Blume. It is reported that gastrodin has neuroprotective function in Parkinson's disease. But its mechanisms of neuroprotection remain not clear in PD. Here, we build two C. elegans PD model using 6-OHDA and transgenic animal to observe the changes of PD worms treated with or without gastrodin to confirm the function of gastrodin, then utilize mutant worms to investigate DAF-2/DAF-16 signaling pathway, and finally verify the mechanism of gastrodin in PD.

Results: Gastrodin attenuates the accumulation of α-synuclein and the injury of dopaminergic neurons, improves chemotaxis behavior in Parkinson's disease models, then recovers chemotaxis behavior by insulin-like pathway. DAF-2/DAF-16 is required for neuroprotective effect of dopamine neuron in PD.

Conclusions: Our study demonstrated that gastrodin rescued dopaminergic neurons and reduced accumulation of α-synuclein protein, and the activity of gastrodin against Parkinson's disease depended on the insulin-like DAF-2/DAF-16 signaling pathway. Our findings revealed that this insulin-like pathway mediates neuroprotection of gastrodin in a Parkinson's disease model.

Keywords: DAF-2/DAF-16 pathway; Dopamine neurons; Gastrodin; Parkinson’s disease; α-Synuclein.

Fig. 1

Determination of the appropriate assay concentration of gastrodin. a The schematic diagram of the timeline about building PD model and gastrodin treatment. b The food clearance rates of worms for different concentrations of gastrodin were determined. The statistical differences were analyzed using two-way RM ANOVA following by Bonferroni post-tests to compare replicate means by row (c) Gastrodin at 200 μM obviously reduced the pumping rates of worms. The statistical differences were analyzed using one-way ANOVA. ns P ≥ 0.05, *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Gastrodin can alleviate the injury of dopamine neurons and accumulated α-synuclein in two PD models. a Both 50 and 100 μM gastrodin restored the loss of GFP of dopaminergic neurons with 6-OHDA treatment. b Quantification of dopaminergic neurons in C. elegans using Image J software by one-way ANOVA. (The scale bar and magnification of left panel is 50 μm, 200; The scale bar and magnification of right panel is 25 μm, 400, respectively). c Gastrodin markedly lessened GFP of α-synuclein in the PD model with overexpressed α-synuclein. d The fluorescence intensity of α-synuclein was also analyzed by Image J. (The scale bar and magnification of left panel is 200 μm, 40; The scale bar and magnification of right panel is 100 μm, 100). The used statistical differences were t-test. ns P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Gastrodin rescues chemotaxis behavior by DAF-2/DAF-16. a In the two PD models, the chemotaxis behavior of worms was increased after treatment with gastrodin. b Gastrodin obviously increased chemotaxis behavior in two PD models. c The mutant daf-16(mu86) shows decreased chemotaxis compared to wild type, and treatment of the mutant with gastrodin did not rescue the behavior. d Gastrodin increased the nuclear accumulation of DAF-16::GFP. The scale bar and magnification of left panel is 50 μm, 200. e Quantification of DAF-16::GFP nuclear accumulation in no gastrodin and gastrodin conditions. f daf-2 mutation exhibited an increased chemotaxis index, and the double-mutants daf-2;daf-16 restored the level to that of wild type animals. The statistical differences were analyzed using t-test method, ns P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

DAF-2 is involved in the protection role of dopamine neurons in PD. a The expression of BZ555 egIs1 [dat-1p::GFP] was similar as the expression in daf-2(e1370) mutants. The scale bar and magnification of left panel is 50 μm, 200; The scale bar and magnification of right panel is 25 μm, 400, respectively. b Quantification of dat-1p::GFP was no changed in wild type and daf-2(e1370) mutant animals. c daf-2 mutants were more resistant to the injury of 6-OHDA treatment. The scale bar and magnification of left panel is 50 μm, 200; The scale bar and magnification of right panel is 25 μm, 400, respectively. d Quantification of dat-1p::GFP animals was induced than that of daf-2(e1370) mutants treated with 6-OHDA. e DAF-2 is not required for the accumulation of α-synuclein. (The scale bar and magnification of left panel is 200 μm, 40; The scale bar and magnification of right panel is 100 μm, 100). f Quantification of α-synuclein in wild type was consistent with that in daf-2(e1370) mutant animals. The statistical differences were analyzed using t-test, ns P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001