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Neuroprotective Effects of Pomegranate Juice Against Parkinson's Disease and Presence of Ellagitannins-Derived Metabolite-Urolithin A-In the Brain

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Neuroprotective Effects of Pomegranate Juice Against Parkinson's Disease and Presence of Ellagitannins-Derived Metabolite-Urolithin A-In the Brain

Małgorzata Kujawska et al. Int J Mol Sci.

Abstract

Pomegranate juice is a rich source of ellagitannins (ETs) believed to contribute to a wide range of pomegranate's health benefits. While a lot of experimental studies have been devoted to Alzheimer disease and hypoxic-ischemic brain injury, our knowledge of pomegranate's effects against Parkinson's disease (PD) is very limited. It is suggested that its neuroprotective effects are mediated by ETs-derived metabolites-urolithins. In this study, we examined the capability of pomegranate juice for protection against PD in a rat model of parkinsonism induced by rotenone. To evaluate its efficiency, assessment of postural instability, visualization of neurodegeneration, determination of oxidative damage to lipids and α-synuclein level, as well as markers of antioxidant defense status, inflammation, and apoptosis, were performed in the midbrain. We also check the presence of plausible active pomegranate ETs-derived metabolite, urolithin A, in the plasma and brain. Our results indicated that pomegranate juice treatment provided neuroprotection as evidenced by the postural stability improvement, enhancement of neuronal survival, its protection against oxidative damage and α-synuclein aggregation, the increase in mitochondrial aldehyde dehydrogenase activity, and maintenance of antiapoptotic Bcl-xL protein at the control level. In addition, we have provided evidence for the distribution of urolithin A to the brain.

Keywords: antioxidant enzymes; apoptosis; ellagic acid; ellagitannins; mitochondrial aldehyde dehydrogenase; neurodegeneration; pomegranate juice; rotenone; urolithin A.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of pomegranate juice treatment (PJ) on body weight gain of rats injected with rotenone (ROT). Data are presented as mean values of eight rats per group and analyzed using one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. * p < 0.05 vs. Control. # p < 0.05 vs. ROT.
Figure 2
Figure 2
Effect of pomegranate juice treatment (PJ) on postural instability in rotenone (ROT)-injected rats. Data are presented as mean values ±SEM of eight rats per group and analyzed using one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons. * p < 0.05 vs. Control. # p < 0.05 vs. ROT.
Figure 3
Figure 3
Representative photomicrographs of hematoxylin and eosin (H&E) stained substantia nigra (SN) sections of rats. Control and pomegranate juice alone treated (PJ) rats show normal neurons (blue arrows). Rotenone (ROT) administration caused prominent degeneration of neurons (white arrows). A rat treated with pomegranate juice and rotenone shows normal neurons (blue arrows) and a few cells with signs of degeneration (white arrows). Original magnification ×400; Scale bar—20 μm.
Figure 4
Figure 4
Representative photomicrographs of immunofluorescent staining of TH-positive cells in adjacent microtome sections of substantia nigra (SN) neurons. Rotenone (ROT) administration caused the substantial loss of TH+ neurons, as compared to a control rat (control). Administration of pomegranate juice attenuated this loss (PJ + ROT). The pomegranate juice application (PJ) itself did not cause any effect on TH+ cells survival when compared to control rats. Scale bar—10 μm.
Figure 5
Figure 5
Effect of pomegranate juice treatment (PJ) on: (a) Malondialdehyde (MDA) concentration; (b) reduced glutathione (GSH) concentration; (c) catalase (CAT) activity; (d) glutathione peroxidase (GPx) activity; (e) glutathione S-transferase (GST) activity; (f) mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, in the midbrain homogenate of rotenone (ROT) injected rats. Data are presented as mean values ±SEM of eight rats per group and analyzed using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05 vs. Control. # p < 0.05 vs. ROT.
Figure 5
Figure 5
Effect of pomegranate juice treatment (PJ) on: (a) Malondialdehyde (MDA) concentration; (b) reduced glutathione (GSH) concentration; (c) catalase (CAT) activity; (d) glutathione peroxidase (GPx) activity; (e) glutathione S-transferase (GST) activity; (f) mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, in the midbrain homogenate of rotenone (ROT) injected rats. Data are presented as mean values ±SEM of eight rats per group and analyzed using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05 vs. Control. # p < 0.05 vs. ROT.
Figure 6
Figure 6
Effect of pomegranate juice treatment (PJ) on: (a) Tumor necrosis factor (TNF-α) expression; (b) total nitrite concentration, in the midbrain of rotenone (ROT) injected rats. Data are presented as mean values ±SEM of eight rats per group and analyzed using one-way ANOVA followed by Sidak’s multiple comparisons test.
Figure 7
Figure 7
Effect of pomegranate juice treatment (PJ) on: B-cell lymphoma-extra-large (Bcl-xL) expression shown as representative immunoblots (a), % of control value ±SEM of eight rats per group (b), apoptosis regulator Bax expression shown as representative immunoblots (c), % of control value ±SEM of eight rats per group (d) in the midbrain of rotenone (ROT) injected rats. The results are presented as relative levels normalized to β-Actin, which was used as an internal control. Data analyzed using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05 vs. control.
Figure 8
Figure 8
Effect of pomegranate juice treatment (PJ) on levels of the 17 kDa isoform of α-synuclein (monomers) and 50 kDa isoform of α-synuclein (oligomers) shown as representative immunoblots (a), on the level of α-synuclein oligomers (b) and on the ratio of α-synuclein oligomers/monomers (c) shown as % of control value ±SEM of eight rats per group in the midbrain of rotenone (ROT) injected rats. The results are presented as relative levels normalized to β-Actin, which was used as an internal control. Data analyzed using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05 vs. Control; # p < 0.05 vs. ROT group.
Figure 9
Figure 9
Representative UPLC-UV-QTOF chromatograms (a) total ion chromatogram for a brain sample of a rat treated with PJ; (b) selected ion chromatogram of m/z 227.0354 for a brain sample of a rat treated with PJ; (c) selected ion chromatogram of m/z 227.0354 for pure UA standard.
Figure 10
Figure 10
Schematic representation of the experimental design used in the study.

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