Prevention of 7-Ketocholesterol-Induced Overproduction of Reactive Oxygen Species, Mitochondrial Dysfunction and Cell Death with Major Nutrients (Polyphenols, ω3 and ω9 Unsaturated Fatty Acids) of the Mediterranean Diet on N2a Neuronal Cells

Abstract

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV; quercetin, QCT; and apigenin, API) as well as ω3 and ω9 unsaturated fatty acids (α-linolenic acid, ALA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 µM)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: ≤6.25 µM; fatty acids: ≤25 µM) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of α-tocopherol (400 µM) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (ΔΨm) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as ω3 and ω9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.

Prevention by Dietary Polyphenols (Resveratrol, Quercetin, Apigenin) Against 7-Ketocholesterol-Induced Oxiapoptophagy in Neuronal N2a Cells: Potential Interest for the Treatment of Neurodegenerative and Age-Related Diseases

“…These two tests were performed because they give complementary information and can be carried out in microplates, allowing easy determination of the 50% inhibiting concentration (IC50) values. Compared to untreated cells, and in agreement with previous data [ 16 , 70 ], significant cytotoxic effects were observed with RSV ( Figure 1 A,E) and QCT ( Figure 1 B,F) from 12.5 to 100 µM, whereas slight cytotoxic effects were observed with API at 50 and 100 µM ( Figure 1 C,G). With 7KC (1.5625 to 100 µM), the IC50 was around 50 µM, both with the FDA and the SR101 assay ( Figure 1 D–H).…”

“…Consequently, before simultaneously treating N2a cells in combination with 7KC and RSV, QCT or API for 48 h, we determined the cytotoxicity of these polyphenols. To this end, two assays were choosen: the fluorescein diacetate (FDA) assay, which is a widely used assay of cytotoxicity based on the measurement of esterase activity [ 16 , 17 , 70 ], and the sulforhodamine 101 (SR101) assay which permits the quantification of adherent cells to evaluate cell growth [ 16 ]. These two tests were performed because they give complementary information and can be carried out in microplates, allowing easy determination of the 50% inhibiting concentration (IC50) values.…”

“…The viability of N2a, SK-N-BE and SH-SY5Y cells was measured with fluorescein diacetate (FDA) (Sigma-Aldrich) as previously described [ 16 , 17 , 70 ]. The FDA assay evaluates the ability of living cells to convert the non-fluorescent FDA into the green fluorescent metabolite fluorescein after cleavage by cellular esterases.…”

“…As previously reported [ 70 ], sulforhodamine 101 (SR101) assay was used to assess the cytotoxic effect of 7KC and polyphenols on N2a cells at different concentrations ranging from 0.5 to 100 µM. SR101 is an anionic dye that electrostatically binds to cellular proteins [ 16 ].…”

“…PI enters in the cells with damaged, permeabilized plasma membranes which are considered as dead cells [ 68 ]. Flow cytometric evaluation of cellular permeability and cell death with PI was carried out as previously described [ 70 ]. Ten thousand cells were acquired for each sample, and the data were analyzed with FlowJo (Tree Star Inc., Ashland, OR, USA) software.…”