Ellagic Acid Prevents Dopamine Neuron Degeneration from Oxidative Stress and Neuroinflammation in MPTP Model of Parkinson's Disease

Abstract

Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by progressive dopaminergic neurodegeneration in the substantia nigra pars compacta area. In the present study, treatment of EA for 1 week at a dose of 10 mg/kg body weight prior to MPTP (25 mg/kg body weight) was carried out. MPTP administration caused oxidative stress, as evidenced by decreased activities of superoxide dismutase and catalase, and the depletion of reduced glutathione with a concomitant rise in the lipid peroxidation product, malondialdehyde. It also significantly increased the pro-inflammatory cytokines and elevated the inflammatory mediators like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the striatum. Immunohistochemical analysis revealed a loss of dopamine neurons in the SNc area and a decrease in dopamine transporter in the striatum following MPTP administration. However, treatment with EA prior to MPTP injection significantly rescued the dopaminergic neurons and dopamine transporter. EA treatment further restored antioxidant enzymes, prevented the depletion of glutathione and inhibited lipid peroxidation, in addition to the attenuation of pro-inflammatory cytokines. EA also reduced the levels of COX-2 and iNOS. The findings of the present study demonstrate that EA protects against MPTP-induced PD and the observed neuroprotective effects can be attributed to its potent antioxidant and anti-inflammatory properties.

Keywords: Parkinson’s disease; ellagic acid; neurodegeneration; neurotoxicity.

Figure 1

Immunostaining of tyrosine hydroxylase immune-positive (TH⁺) neurons to quantify the number of dopaminergic (DA) neurons in the substantia nigra (SNc) and dopamine transporter (DAT) in the striatum. (A): Representative images showing the TH⁺ neurons in the SNc area. (B): The number of DA neurons in the SNc was counted in each animal using unbiased Stereo Investigator system as described in Section 2. The number of DA neurons was significantly higher in the SNc of the control group (V + Sal) when compared to the MPTP-injected group (V + 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP)). The ellagic acid (EA) treatment significantly prevented the DA neurons from the MPTP-induced neurodegeneration (n = 3–6 animals). (C): Representative images showing the immunoreactivity of dopamine transporter (DAT) in the striatum. The intensity of dopamine nerve terminals was significantly reduced in the striatum of MPTP-injected mice when compared with that in the control (V + Sal) group. Treatment with EA prior to MPTP injection shows significant attenuation of dopamine nerve terminals intensity. (D): DAT intensity was measured using NIH image J software and is presented in the graph. Values are expressed as a percentage of mean ± SEM relative to 100% control (n = 3–5 animals). The scale bar is 400 µm. Significance is denoted as * p < 0.05 and *** p < 0.001.

Figure 2

Estimation of malondialdehyde (MDA), glutathione (GSH), catalase, and superoxide dismutase (SOD) in the striatum tissue of different experimental groups. The MPTP injection increased the lipid peroxidation product, malondialdehyde (MDA) (A) and decreased the total glutathione (GSH) level (B) in the striatum tissue of mice relative to the control (Vehicle + Saline) group. MPTP injections also significantly decreased the activity of catalase (C) and SOD (D). Ellagic acid (EA) treatment prior to MPTP significantly increased the activities of the antioxidant enzymes SOD and catalase. It significantly reduced the level of MDA and increased the level of total GSH. Values are expressed as mean ± SEM (n = 4–5). Significance is denoted as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Immunofluorescence staining to detect the expression of activated glial fibrillary acidic protein (GFAP)-positive astrocytes (green) and ionized calcium-binding adaptor molecule-1 (Iba-1)-positive microglia (green) in the striatum of different groups of mice. Significantly higher levels of activated GFAP-positive astrocytes (A) and Iba-1-positive microglia (C) were found in the MPTP-treated mice relative to control mice (Vehicle + Saline). EA administration prior to MPTP injection in mice led to significantly lower numbers of activated GFAP and Iba-1 than MPTP-challenged mice. Quantitative analysis of activated astrocytes (B) and microglia (D) revealed a significant increase in the number of activated astrocytes and microglia in the mice injected with MPTP compared with the control mice. EA administration significantly reduced the number of activated astrocytes and microglia in the EA + MPTP group mice relative to the Vehicle + MPTP mice. Values are expressed as percentage of mean ± SEM (n = 3–6). The scale bar is 75 µm. Significance is denoted as * p < 0.05 and *** p < 0.001.

Figure 4

Determination of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) in the striatum tissue of different experimental groups animals using enzyme-linked immunosorbent assay (ELISA). MPTP treatment significantly increased the levels of IL-1β, IL-6, and TNF-α. EA treatment prior to MPTP injections significantly reduced the level of IL-1β (A), IL-6 (B), and TNF-α (C) when compared with MPTP-treated animals. Values are expressed as mean ± SEM (n = 4–5). Significance is denoted as ** p < 0.01 and *** p < 0.001.

Figure 5

Expression of striatal inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as measured by Western blot analysis (A,C). A significant increase in iNOS was observed in the MPTP group relative to the control (Vehicle + Saline) group. Ellagic acid (EA) treatment prior to MPTP injection significantly decreased the expression of iNOS relative to the MPTP group (A,B). Similarly, COX-2 expression was increased significantly in MPTP group relative to the control (Vehicle + Saline) group. EA treatment decreased the COX-2 expression relative to the MPTP group (C,D). Values are expressed as percentage of mean ± SEM relative to control group (Vehicle + Saline). Significance is denoted as * p < 0.05, ** p < 0.01 and *** p < 0.001.