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. 2017 Oct 23;18(10):2220.
doi: 10.3390/ijms18102220.

Argan Oil-Mediated Attenuation of Organelle Dysfunction, Oxidative Stress and Cell Death Induced by 7-Ketocholesterol in Murine Oligodendrocytes 158N

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Argan Oil-Mediated Attenuation of Organelle Dysfunction, Oxidative Stress and Cell Death Induced by 7-Ketocholesterol in Murine Oligodendrocytes 158N

Asmaa Badreddine et al. Int J Mol Sci. .
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Abstract

Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25-50 µM; 24 h) without and with argan oil (0.1% v/v) or α-tocopherol (400 µM, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, β-amyrin and citrostadienol. α- and γ-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood-brain barrier.

Keywords: 158N murine oligodendrocytes; 7-ketocholesterol; argan oil; extra virgin olive oil; lysosome; mitochondria; oxiapoptophagy; peroxisome; α-tocopherol.

Conflict of interest statement

The authors declare no conflicts of interests.

Figures

Figure 1
Figure 1
Evaluation of the effect of argan oil on 7-ketocholesterol (7KC)-induced cell growth inhibition in 158N murine oligodendrocytes with the crystal violet and MTT tests. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The cytoprotective effect of argan oils on 7KC-induced inhibition of cell growth was evaluated with the crystal violet test (measurement of adherent cells) (A) or the MTT test (measurement of the activity of the succinate deshydrogenase, a mitochondrial enzyme belonging to the Krebs cycle) (B). The experiments were carried out three times in triplicate. Data are mean ± standard deviation (SD) of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference was found between control and vehicle-treated cells (EtOH 0.95% v/v). The EtOH value of 0.95% corresponds to the highest EtOH concentration obtained when the cells were simultaneously treated with 7KC used at 25 µM (EtOH, 0.05%) and with argan oil (EtOH, 0.9% v/v).
Figure 2
Figure 2
Evaluation of the effect of argan oil on 7-ketocholesterol-induced overproduction of reactive oxygen species in 158N murine oligodendrocytes by flow cytometry after staining with dihydroethidine. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The cytoprotective effect of argan oils on 7KC-induced overproduction of reactive oxygen species (ROS), mainly superoxide anions, was evaluated by flow cytometry after staining with dihydroethidine (DHE). ROS overproduction was determined by the percentage of DHE-positive cells. The experiments were carried out twice in triplicate. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference between control and vehicle-treated cells (EtOH 0.95% v/v).
Figure 3
Figure 3
Evaluation of the effect of argan oil on 7-ketocholesterol-induced plasma membrane permeability in 158N murine oligodendrocytes by flow cytometry after staining with propidium iodide. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The cytoprotective effect of argan oils on 7KC-induced plasma membrane permeability was evaluated by flow cytometry after staining with propidium iodide (PI). Plasma membrane permeability was determined by the percentage of PI positive cells. The experiments were carried out twice in triplicate. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05.The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference was observed between control and vehicle-treated cells (EtOH 0.95% v/v).
Figure 4
Figure 4
Evaluation of the effect of argan oil on 7-ketocholesterol-induced acidic vesicle formation in 158N murine oligodendrocytes by flow cytometry after staining with acridine orange. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The cytoprotective effect of argan oils on 7KC-induced acidic vesicle formation was evaluated by flow cytometry after staining with acridine orange (AO). Acidic vesicle formation was determined by the percentage of AO-positive cells. The experiments were carried out twice in triplicate. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference was detected between control and vehicle-treated cells (EtOH 0.95% v/v).
Figure 5
Figure 5
Effects of argan oil on 7-ketocholesterol-induced decreased transcription of Abcd1, Acox1, Mfp2, and Abcd3. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The mRNAs of the peroxisomal transporters (Abcd1) (A), and of the peroxisomal enzymes (Acox1, and Mfp2) (C–D) as well as of Abcd3 (a marker of the peroxisomal mass); (B) were quantified by RT-qPCR. To this end, 36B4 was used as the reference gene. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference was found between control and vehicle-treated cells (EtOH 0.95% v/v).
Figure 5
Figure 5
Effects of argan oil on 7-ketocholesterol-induced decreased transcription of Abcd1, Acox1, Mfp2, and Abcd3. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The mRNAs of the peroxisomal transporters (Abcd1) (A), and of the peroxisomal enzymes (Acox1, and Mfp2) (C–D) as well as of Abcd3 (a marker of the peroxisomal mass); (B) were quantified by RT-qPCR. To this end, 36B4 was used as the reference gene. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No significant difference was found between control and vehicle-treated cells (EtOH 0.95% v/v).
Figure 6
Figure 6
Effects of argan oil on 7-ketocholesterol-induced decreased transcription of PPARα. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. The mRNAs of PPARα was quantified by RT-qPCR. 36B4 was used as the reference gene. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No difference was observed between control and vehicle (EtOH 0.95%)-treated cells.
Figure 7
Figure 7
Effects of argan oil on 7-ketocholesterol-induced apoptosis evaluated by condensation and/or fragmentation of the nuclei. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25–50 µM) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. Apoptosis was evaluated by the percentage of apoptotic cells characterized by condensed and/or fragmented nuclei whereas control cells (untreated cells) have round and regular nuclei. Data are mean ± SD of two independent experiments carried out in triplicate. The significance of the relationship between vehicle and cells treated with 7KC, argan oils or α-tocopherol was calculated by the Anova test (Sidak’s multiple comparisons); * p ≤ 0.05. The significance of the relationship between cells treated with 7KC alone, 7KC and argan oils cotreatment, or 7KC and α-tocopherol cotreatment was calculated by the Anova test (Sidak’s multiple comparisons); # p ≤ 0.05. No difference was observed between control and vehicle (EtOH 0.95%)-treated cells.
Figure 8
Figure 8
Effects of argan oil on 7-ketocholesterol-induced cleaved caspase-3 and activation of LC3-I into LC3-II. After 24 h of culture, 158N murine oligodendrocytes were cultured for an additional 24 h without or with 7KC (25 µM) (A) or with 7KC (50 µM) (B) in the absence or presence of argan oils (Agadir or Berkane; Morocco; 0.1% v/v) or of α-tocopherol (400 µM) used as the positive control. Argan oils and α-tocopherol were added to the culture medium 2 h before 7KC. Apoptosis was evaluated by caspase-3 activation (cleaved caspase-3) and autophagy by conversion of LC3-I to LC3-II (increased LC3-II/LC3-I ratio). The EtOH values (0.05%, 0.1% and 0.9% v/v) correspond to the final EtOH concentration in the culture medium with 7KC (25 µM), 7KC (50 µM) and argan oils, respectively. No difference was observed between control and vehicle (EtOH)-treated cells. Data shown are representative of three independent experiments.

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References

    1. German J.B., Dillard C.J. Composition, structure and absorption of milk lipids: A source of energy, fat-soluble nutrients and bioactive molecules. Crit. Rev. Food Sci. Nutr. 2006;46:57–92. doi: 10.1080/10408690590957098. - DOI - PubMed
    1. Giuffrida F., Cruz-Hernandez C., Flück B., Tavazzi I., Thakkar S.K., Destaillats F., Braun M. Quantification of phospholipids classes in human milk. Lipids. 2013;48:1051–1058. doi: 10.1007/s11745-013-3825-z. - DOI - PMC - PubMed
    1. Kamelska A.M., Pietrzak-Fiećko R., Bryl K. Variation of the cholesterol content in breast milk during 10 days collection at early stages of lactation. Acta Biochim. Pol. 2012;59:243–247. - PubMed
    1. Delplanque B., Gibson R., Koletzko B., Lapillonne A., Strandvik B. Lipid Quality in Infant Nutrition: Current Knowledge and Future Opportunities. J. Pediatr. Gastroenterol. Nutr. 2015;61:8–17. doi: 10.1097/MPG.0000000000000818. - DOI - PMC - PubMed
    1. Gow R.V., Hibbeln J.R. Omega-3 fatty acid and nutrient deficits in adverse neurodevelopment and childhood behaviors. Child Adolesc. Psychiatr. Clin. N. Am. 2014;23:555–590. doi: 10.1016/j.chc.2014.02.002. - DOI - PMC - PubMed
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