Natural Food Supplements Reduce Oxidative Stress in Primary Neurons and in the Mouse Brain, Suggesting Applications in the Prevention of Neurodegenerative Diseases

Abstract

Neurodegenerative diseases pose a major health problem for developed countries, and stress has been identified as one of the main risk factors in the development of these disorders. Here, we have examined the protective properties against oxidative stress of several bioactive natural food supplements. We found that MecobalActive^®, Olews^®, and red and white grape seed polyphenol extracts may have a neuroprotective effect in vitro, both in the SH-SY 5Y cell line and in hippocampal neuron cultures, mainly by reducing reactive oxygen species levels and decreasing caspase-3 activity. In vivo, we demonstrated that oral administration of the supplements reduces the expression of genes involved in inflammation and oxidation mechanisms, whereas it increments the expression of genes related to protection against oxidative stress. Furthermore, we found that preventive treatment with these natural extracts increases the activity of antioxidant enzymes and prevents lipid peroxidation in the brain of stressed mice. Thus, our results indicate that some natural bioactive supplements may have important protective properties against oxidative stress processes occurring in the brain.

Keywords: MecobalActive®; Olews®; ROS; neurodegenerative diseases; oxidative stress; red grape polyphenol extract; white grape seed polyphenol extract.

Figure 1

Neuroprotective effects of the extracts on the SH-SY 5Y cell line. Dose-response curve effects of the extracts on the SH-SY 5Y cell line. Cells were incubated with different concentrations of Cardiose^® (A), Olews^® (B), citicoline (C), MecobalActive^® (D), red grape (E), or white grape extracts (F) for 72 h in the absence (A–F) or presence (A′–F′) of 125-µM Al(mal)₃. Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett’s multiple comparison post-hoc test were used for statistical analysis. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus cells or Al(mal)₃ treatment. Cs: control cells, not exposed to Al(mal)₃.

Figure 2

Dose-response curves of the stressor on the SH-SY 5Y cell line. Cells were incubated with different concentrations of Al(mal)₃ for 24, 48, or 72 h. Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett’s multiple comparison post-hoc test were used for statistical analysis; 24 h, p < 0.00001; 48 h, p < 0.0001; and 72 h, p < 0.0001.

Figure 3

Neuroprotective effects of the extracts on hippocampal neuron cultures. Dose-response curves of the extracts on hippocampal neuron cultures. Cells were incubated with different concentrations of Cardiose^® (A), Olews^® (B), citicoline (C), MecobalActive^® (D), red grape (E), or white grape extracts (F) for 72 h in the absence (A–F) or presence (A′–F′) of 125-µM Al(mal)₃. Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett’s multiple comparison post-hoc test were used for statistical analysis. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus cells or Al(mal)₃ treatment. Cs: control cells, not exposed to Al(mal)_3.

Figure 4

Reactive oxygen species (ROS) levels (A) and caspase-3 activity (B) on SH-SY 5Y cells after extract treatment. Cells were treated with red grape, white grape, MecobalActive^®, or Olews^® for 48 h in the absence or presence of 125-µM Al(mal)₃. ROS activity (A) was quantified by measuring the fluorescence at 535/590 nm. Caspase-3 activity (B) was quantified by measuring the absorbance at 405 nm. Values are presented as mean ± SEM from at least three independent experiments; Kruskal-Wallis test followed by Mann Whitney post-hoc test were used for statistical analysis. ### p < 0.001 versus untreated cells and ** p < 0.01 versus Al(mal)3. Abbreviations: Mal-Al: Al(mal)_3.

Figure 5

Immobilization causes oxidative stress in mouse brains. Mice were immobilized for different times: 0 (CN), 2, 4, or 6 h. The mRNA expression of IL-6 (A), NOX-2 (B), and TNF-alpha (C) were quantified by real-time (RT)-PCR. The mRNA expression was normalized with GAPDH. All data were related to that from the control and are expressed as a fold change. Values are presented as mean ± SEM from at least three independent experiments. Kruskal-Wallis test followed by Mann Whitney post-hoc test were used for statistical analysis. * p < 0.05 versus CN. Abbreviations: CN: control.

Figure 6

Natural extracts protect against oxidative stress. Red and white grape seed extracts, MecobalActive^®, and Olews^® were administered orally during 5 consecutive days. Then, mice were immobilized for 6 h. The mRNA expressions of IL-6 (A), TNF-alpha (B), NOX-2 (C), HMOX1 (D), and Nrf2 (E) were quantified in mouse brains by RT-PCR. Gene expression was normalized with GAPDH. All data were normalized to levels found in nonstressed mice (normal) and are expressed as a fold change. Values are presented as mean ± SEM from eight experimental animals. One-way ANOVA and Dunnett’s multiple comparison post-hoc test were used for statistical analysis. * p < 0.05 and ** p < 0.01 versus normal mice, and # p < 0.05 and ## p < 0.01 versus restrained mice (stress).

Figure 7

Natural extracts increase the activity of antioxidant enzymes. Mouse brains were isolated, and the catalase activity (A), SOD activity (B), and TBARS (C) were analyzed. The values are presented as mean ± SEM from eight experimental animals. One-way ANOVA and Dunnett’s multiple comparison post-hoc test were used for statistical analysis. * p < 0.05 versus normal mice, and # p < 0.05 and ## p < 0.01 versus restrained (stress) mice. Abbreviations: SOD: superoxide dismutase; TBARS: thiobarbituric acid reactive substances.