Protective effects of resveratrol and quercetin against MPP+ -induced oxidative stress act by modulating markers of apoptotic death in dopaminergic neurons

Abstract

Reactive oxygen species produced by oxidative stress may participate in the apoptotic death of dopamine neurons distinctive of Parkinson's disease. Resveratrol, a red wine extract, and quercetin, found mainly in green tea, are two natural polyphenols, presenting antioxidant properties in a variety of cellular paradigms. The aim of this study was to evaluate the effect of resveratrol and quercetin on the apoptotic cascade induced by the administration of 1-methyl-4-phenylpyridinium ion (MPP(+)), a Parkinsonian toxin, provoking the selective degeneration of dopaminergic neurons. Our results show that a pre-treatment for 3 h with resveratrol or quercetin before MPP(+) administration could greatly reduce apoptotic neuronal PC12 death induced by MPP(+). We also demonstrated that resveratrol or quercetin modulates mRNA levels and protein expression of Bax, a pro-apoptotic gene, and Bcl-2, an anti-apoptotic gene. We then evaluated the release of cytochrome c and the nuclear translocation of the apoptosis-inducing factor (AIF). Altogether, our results indicate that resveratrol and quercetin diminish apoptotic neuronal cell death by acting on the expression of pro- and anti-apoptotic genes. These findings support the role of these natural polyphenols in preventive and/or complementary therapies for several human neurodegenerative diseases caused by oxidative stress and apoptosis.

Fig. 1

Histograms showing the effect of 500 μM MPP⁺ administration in neuronal PC12 cells, as revealed by measuring LDH activity in supernatants after 24 h of treatment. Resveratol (Resv) or Quercetin (Querc) could reduce MPP⁺ -induced toxicity. **P < 0.01 compared with control (CTRL), P < 0.05 compared with MPP⁺ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test

Fig. 2

a Immunofluorescence detection of apoptotic neuronal PC12 cells. Blue Total neuronal PC12 nuclei were counterstained with Hoechst 33342. Red Anti-cleaved caspase-3 antibody. Green TUNEL. Merged: Cells are considered apoptotic when positive for both TUNEL (green) and anti-cleaved caspase-3 antibody (red). They appear as light blue cells because of the third superposition of dark blue Hoechst 33342. Immunofluorescence clearly illustrates that there are less apoptotic PC12 cells (light blue, indicated by white arrows) when cells are treated with resveratrol (MPP⁺RESV) or quercetin (MPP⁺QUER) prior to MPP⁺ addition. Cells were also treated with medium alone (Ctrl), resveratrol alone, and quercetin alone (data not shown). b Histogram showing the number of apoptotic cells counted on triple-stained slides (merged) on a total of 100 nuclei, as described in Materials and Methods. Pre-treatment with resveratrol (RESV) or quercetin (QUER) strongly reduced the number of apoptotic neuronal PC12 cells. c Histogram of DNA fragmentation in apoptotic cells detected with a monoclonal antibody to a single-stranded DNA (ssDNA). Pre-treatment of MPP⁺-treated cells with resveratrol (RESV) or quercetin (QUER) shows a significant decrease in DNA fragmentation. ***P < 0.001 compared with control (CTRL), P < 0.01 and P < 0.001 compared with MPP⁺ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test

Fig. 3

a Bax mRNA levels, as revealed by RT-PCR, are increased with MPP⁺ treatment. Treatment with resveratrol (RESV) or quercetin (QUER) prior to MPP⁺ administration detects a significant decrease of Bax mRNA expression. Bottom: RT-PCR bands of Bax mRNA and 18S as control. b Bcl-2 mRNA levels, as revealed by RT-PCR. Treatment with resveratrol (RESV) or quercetin (QUER) prior to MPP⁺ administration detects a significant increase of Bcl-2 mRNA expression. Bottom: RT-PCR bands of Bcl-2 mRNA and 18S as control. ***P < 0.001 compared with control (CTRL), P < 0.05 and P < 0.001 compared with MPP⁺ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test

Fig. 4

Effect of resveratrol (RESV) or quercetin (QUER) on the Bax/Bcl-2 ratio in PC12 cells. The levels of Bax and Bcl-2 protein expression were analyzed, and the Bax/Bcl-2 ratio was determined for each treatments. Resveratrol (RESV) or quercetin (QUER) alone did not modulate Bax/Bcl-2 ratio. MPP⁺ increased Bax/Bcl-2 ratio, and the addition of resveratrol or quercetin strongly prevented this increase. Bottom: bands od bax and Bcl-2 as revealed by Western blots. **P < 0.01 compared with control (CTRL), P < 0.01 and P < 0.001 compared with MPP+ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test

Fig. 5

a Histogram of kinetic studies showing AIF translocation from the cytoplasm to the nucleus during 24 h. AIF protein increases in the cytosolic and nuclear fraction at 22 h of treatment with MPP⁺. Black line indicates nuclear/cytoplasm ratio of AIF protein. P < 0.001 compared with CTRL (nuclear/cytoplasm ratio) as determined by ANOVA, followed by Dunnett’s multiple-comparisons test. b Histogram revealing total amounts of AIF protein levels in each cellular fraction after resveratrol (RESV), quercetin (QUER), and MPP⁺ treatments. Bands show total amounts of AIF protein levels in each cellular fraction. In each fraction, pretreatment with resveratrol (RESV) or quercetin (QUER) leads to a significant reduction of AIF protein expression. An anti-TH antibody was used as control for cytosolic fraction and an anti-HDAC antibody was used to control for nuclear fraction. ***P < 0.001 and **P < 0.01 compared with control (CTRL), P < 0.05, P < 0.01 and P < 0.001 compared with MPP+ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test

Fig. 6

Western blots analysis of cytosolic cytochrome c after each treatment. Resveratrol and quercetin did not modulate cytochrome c expression when used alone in neuronal PC12 cells. When resveratrol and quercetin were administered 3 h prior to MPP+ treatment, a decrease of cytosolic cytochrome c protein expression was apparent. An anti-TH antibody was used as control for cytosolic fraction. ***P < 0.001 and *P < 0.05 compared with control (CTRL), P < 0.01 and P < 0.001 compared with MPP+ as determined by ANOVA, followed by Dunnett’s multiple-comparisons test