Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro

Abstract

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson's disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-3. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (SOD) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (30 mg/kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-3. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.

Fig. 1

Chemical structure of AST.

Fig. 2

Effect of AST on the MPP+-induced decrease in SH-SY5Y cell viability. Cell viability (A and B) or apoptotic death (C and D) was assessed by MTT assay, flow cytometric assay, or immunoblotting assay as described in the Materials and Methods section. (A) Cells were treated with various concentrations (0.1–2 mM) of MPP+ for 24 h. (B) Cells were pretreated with various concentrations (5–50 μM) of AST for 2 h and then treated with 1 mM MPP+ for 24 h in the presence of AST. (C) Cells were treated with either 0.1% DMSO (control) or 1 mM MPP+ in the absence or presence of 50 μM AST for 24 h as described in B. After treatment, cells were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI). Apoptosis was detected by the flow cytometric assay. In A, B, and C, error bars represent the mean ± SE from three separate experiments. *p < 0.05, compared with control (untreated group); **p < 0.01, compared with the group treated by MPP+ alone. (D) Cells were treated as described C and then harvested. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and immunoblotted with anti-PARP-1 antibody. Actin was shown as an internal standard.

Fig. 3

Effect of AST on MPP+-mediated ROS generation in SH-SY5Y cells. (A) and (B) Cells were treated with 1 mM MPP+ in the absence or presence of 50 μM AST for 24 h as described in Fig. 2B. After treatment, ROS generation was determined with 5 μM CMH₂DCFDA. Morphological features were analyzed with a phase-contrast microscope and fluorescent signals were detected with fluorescence microscopy (A). DCF fluorescence intensity was determined and plotted (B). (C) and (D) Cells were treated with 1 mM MPP+ in the absence or presence of 50 μM AST for 24 h as described in Fig. 2B. After treatment, SOD activity (C) and catalase activity (D) were measured. (E) Cells were treated with 1 mM MPP+ in the presence/absence of 50 μM AST, 20 μg/ml SOD, or 20 μ/ml catalase for 24 h. Data are expressed as percentage of values in untreated control cultures, and are means ± S.E.M. (n = 4). *p < 0.05, compared with control (untreated group); **p < 0.01, compared with the group treated by MPP+ alone.

Fig. 4

Effect of AST on the expression of Bcl-2 and Bax in SH-SY5Y cells. Cells were treated with MPP+ (1 mM) and/or ATS (50 μM) for 24 h, and then cell lysates were subjected to Western blot analysis (upper panel of A). The levels of Bax and Bcl-2 were quantified by densitometric analysis (lower panel of A) and the Bax/Bcl-2 ratio was determined (B). Data are means ± S.E.M. (n = 3). #p < 0.05, compared with control, *p < 0.05, compared with MPP+ alone;

Fig. 5

Effect of AST on MPP+-induced dysfunction of mitochondrial and caspase-3 activity in SH-SY5Ycells. Cells were treated with MPP+ (1 mM) for 24 h in the presence or absence of 50 μM AST. Enzymatic activity of caspase-3 (A), cleaved caspase-3 (active form of caspase-3)-positive cells (B), and mitochondrial membrane potential (C) were determined as described in the Materials and Methods section. Data are from representative experiments repeated at least three times. Data are means ± S.E.M. (n = 3). *p < 0.05, compared with control (untreated cells); **p < 0.05, compared with the group treated by MPP+ alone. (D) Cytochrome c (15 kDa) release into cytosol was determined by immunoblotting for cytochrome c in the cytosolic fraction. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and immunoblotted with anti-cytochrome c antibody. β-tubulin was shown as an internal standard.

Fig. 6

Effects of AST on nigra TH expression and dopaminergic neurons in the mouse MPTP model. (A) Immunohistochemical staining of dopaminergic neurons with an anti-TH antibody in the substantia nigra (SN). Photomicrographs were taken at a magnification of 200×. a, control group. b, MPTP-treated group (30 mg/kg, 1×/day for 28 days). c, AST-treated group (30 mg/kg), d, MPTP + AST-treated group. (B) The immunoreactive cell counts in the SN pars compacta of control group, MPTP-treated group, AST-treated group, and AST + MPTP group. Values represent means ± S.E.M of 4 mice per group. *p <0.05 compared with the control group; and **p <0.01 compared with the MPTP-treated group. (C) and (D) TH and α-synuclein expression was determined in the SN (C) and stria terminalis (ST) (D). Lysates containing equal amounts of protein (20 μg) from SN or ST tissue samples were separated by SDS-PAGE and immunoblotted with anti-α-synuclein or anti-TH antibody. Actin was shown as an internal standard.