Protective effects of milk thistle (Sylibum marianum) seed oil and α-tocopherol against 7ß-hydroxycholesterol-induced peroxisomal alterations in murine C2C12 myoblasts: Nutritional insights associated with the concept of pexotherapy.

Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7ß-hydroxycholesterol (7ß-OHC; 50 µM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 µg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7ß-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7ß-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7ß-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7ß-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal ß-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal ß-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy).

Antioxidant Properties and Cytoprotective Effect of Pistacia lentiscus L. Seed Oil against 7ß-Hydroxycholesterol-Induced Toxicity in C2C12 Myoblasts: Reduction in Oxidative Stress, Mitochondrial and Peroxisomal Dysfunctions and Attenuation of Cell Death.

Aging is characterized by a progressive increase in oxidative stress, which favors lipid peroxidation and the formation of cholesterol oxide derivatives, including 7ß-hydroxycholesterol (7ß-OHC). This oxysterol, which is known to trigger oxidative stress, inflammation, and cell death, could contribute to the aging process and age-related diseases, such as sarcopenia. Identifying molecules or mixtures of molecules preventing the toxicity of 7ß-OHC is therefore an important issue. This study consists of determining the chemical composition of Tunisian Pistacia lentiscus L. seed oil (PLSO) used in the Tunisian diet and evaluating its ability to counteract the cytotoxic effects induced by 7ß-OHC in murine C2C12 myoblasts. The effects of 7ß-OHC (50 µM; 24 h), associated or not with PLSO, were studied on cell viability, oxidative stress, and on mitochondrial and peroxisomal damages induction. α-Tocopherol (400 µM) was used as the positive control for cytoprotection. Our data show that PLSO is rich in bioactive compounds; it contains polyunsaturated fatty acids, and several nutrients with antioxidant properties: phytosterols, α-tocopherol, carotenoids, flavonoids, and phenolic compounds. When associated with PLSO (100 µg/mL), the 7ß-OHC-induced cytotoxic effects were strongly attenuated. The cytoprotection was in the range of those observed with α-tocopherol. This cytoprotective effect was characterized by prevention of cell death and organelle dysfunction (restoration of cell adhesion, cell viability, and plasma membrane integrity; prevention of mitochondrial and peroxisomal damage) and attenuation of oxidative stress (reduction in reactive oxygen species overproduction in whole cells and at the mitochondrial level; decrease in lipid and protein oxidation products formation; and normalization of antioxidant enzyme activities: glutathione peroxidase (GPx) and superoxide dismutase (SOD)). These results provide evidence that PLSO has similar antioxidant properties than α-tocopherol used at high concentration and contains a mixture of molecules capable to attenuate 7ß-OHC-induced cytotoxic effects in C2C12 myoblasts. These data reinforce the interest in edible oils associated with the Mediterranean diet, such as PLSO, in the prevention of age-related diseases, such as sarcopenia.

Nigella and Milk Thistle Seed Oils: Potential Cytoprotective Effects against 7ß-Hydroxycholesterol-Induced Toxicity on SH-SY5Y Cells.

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7ß-hydroxycholesterol (7ß-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7ß-OHC-induced side effects. The impact of 7ß-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7ß-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7ß-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7ß-OHC.

Association Between Oxidative Stress and Altered Cholesterol Metabolism in Alzheimer's Disease Patients.

BACKGROUND: Oxidative stress is the main feature of several diseases including Alzheimer's disease (AD). The involvement of oxysterols derivates has been recently reported. OBJECTIVE: The aim of this study was to evaluate the implication of oxidative stress in cholesterol impairment in AD patients. METHODS: A case-control study was conducted on 56 AD patients and 97 controls. Levels of oxidative biomarkers, including lipid peroxidation products and antioxidant enzyme activities were measured with spectrophotometric methods on red blood cells (RBCs) and plasma. Cholesterol precursors and oxysterols (7-Ketocholeterol (7KC), 7α-hydroxycholesterol (7α-OHC), 7ß-hydroxycholesterol (7ß-OHC), 24Shydroxycholesterol (24S-OH), 25-hyroxycholesterol (25-OHC), and 27-hydroxycholesterol (27-OHC), in plasma were quantified by gas chromatography coupled with mass spectrometry. RESULTS: In RBCs and plasma of AD patients, a significant decrease of glutathione peroxidase (GPx) activity was detected associated with raised levels of malondialdehyde (MDA). A decreased level of lanosterol and an accumulation of 7ß-OHC, 24S-OHC, 27-OHC, and 25-OHC that were higher in plasma of AD patients, compared to controls, were also observed in AD patients. Mini-Mental State Examination (MMSE) score was correlated with MDA and conjugated dienes (CD) levels in plasma. Besides, the MDA level in RBCs was correlated with 7ß-OHC. Binary logistic regression revealed an association between GPx activity and AD (OR=0.895, 95%CI: 0.848-0.945. P<0.001). CONCLUSION: Our data consolidate the relationship between the rupture of redox homeostasis and lipid and cholesterol oxidation in AD.

Evaluation of pro-inflammatory cytokines in frail Tunisian older adults.

The present study was undertaken to evaluate serum levels of pro-inflammatory cytokines in Tunisian older adults and to examine the relationships between inflammatory marker levels, geriatric, and biochemical parameters. A cross-sectional study was conducted in a population of Tunisian older adults (N = 141, aged 65 and over). Patients were recruited from the Department of Internal Medicine, Fattouma Bourguiba University Hospital (Monastir, Tunisia) and from a nursing home (Sousse, Tunisia). Comprehensive geriatric assessment, history taking and examination including functional and nutritional assessment were done for each participant. Enzyme-linked immunosorbent assay (ELISA) test was used to measure serum cytokine (TNF-α, IL-8, IL-6) levels. The modified Short Emergency Geriatric Assessment score (SEGAm) were used to classify patients as 51 very-frail, 40 frail, and 50 non-frail. The age of the participants (80 men, 61 women) ranged from 65 to 97 years. Serum levels of TNF-α, IL-8 and C-reactive protein (CRP) were significantly higher in very-frail participants compared to frail and non-frail ones. However, no significant differences in IL-6 levels were detected among frailty groups. After adjustment for age, CRP and IL-8 levels remained significantly associated with frailty. Analysis of the receiver operating characteristic (ROC) curve corresponding to IL-8 showed an area under the curve of 0.7 (p = 0.003; 95% CI [0.58-0.81]) and a predictive threshold of 5.27 pg/ml. Positive correlations were found between frailty score, IL-6, and IL-8 levels. In addition, a significant positive correlation was observed between IL-8 levels and Timed Up and Go test results. However, a negative correlation was observed between Mini Nutritional Assessment Short-Form score, IL-6 and CRP levels, as well as between Activities of Daily Living score and serum levels of TNF-α, IL-6, and CRP. In conclusion, the key findings of this study collectively support a role of pro-inflammatory cytokines, TNF-α, CRP, and especially IL-8 in the development of frailty in older adults.

Genetic variants in FADS1 and ELOVL2 increase level of arachidonic acid and the risk of Alzheimer's disease in the Tunisian population.

Polyunsaturated fatty acids (PUFAs) are closely related to various physiological conditions. In several age-related diseases including Alzheimer's disease (AD) altered PUFAs metabolism has been reported. However, the mechanism behind PUFAs impairment and AD developpement remains unclear. In humans, PUFAs biosynthesis requires delta-5 desaturase (D5D), delta-6 desaturase (D6D) and elongase 2 activities; which are encoded by fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), and elongation of very-long-chain fatty acids-like 2 (ELOVL2) genes, respectively. In the present work, we aim to assess whether genetic variants in FADS1, FADS2 and ELOVL2 genes influence plasma and erythrocyte PUFA composition and AD risk. A case-control study was carried out in 113 AD patients and 161 healthy controls.Rs174556, rs174617, and rs3756963 of FADS1, FADS2, and ELOVL2 genes, respectively were genotyped using PCR-RFLP. PUFA levels were quantified using Gas Chromatography. Genotype distributions of rs174556 (FADS1) and rs3756963 (ELOVL2) were different between case and control groups. The genotype TT of rs174556 and rs3756963 single nucleotide polymorphism (SNP) increases significantly the risk of AD in our population. PUFA analysis showed higher plasma and erythrocyte arachidonic acid (AA) level in patients with AD, whereas only plasma docosahexaenoic acid (DHA) was significantly decreased in AD patients. The indexes AA/Dihomo-gamma-linolenic acid (DGLA) and C24:4n-6/Adrenic acid (AdA) were both higher in the AD group. Interestingly, patients with TT genotype of rs174556 presented higher AA level and AA/DGLA index in both plasma and erythrocyte. In addition, higher AA and AA/DGLA index were observed in erythrocyte of TT genotype ofrs3756963 carrier's patients. Along with, positive correlation between AA/DGLA index, age or Gamma-linolenic acid (GLA)/ Linoleic acid (LA) index was seen in erythrocyte and /or plasma of AD patients. After adjustment for confounding factors, the genotype TT of rs174556, erythrocyte AA and AA/DGLA index were found to be predictive risk factors for AD while plasma DHA was found associated with lower AD risk. Both rs174556 and rs3756963 influence AD risk in the Tunisian population and they are likely associated with high AA level. The combination of the two variants increases further the susceptibility to AD. We suggest that FADS1 and ELOVL2 variants could likely regulate the efficiency of AA biosynthesis which could be at the origin of inflammatory derivate.

Association Between Genetic Variants in FADS1-FADS2 and ELOVL2 and Obesity, Lipid Traits, and Fatty Acids in Tunisian Population.

The aim of this study was to determine whether genetic variants in FADS1/FADS2 and ELOVL2 are associated with overweight-obesity and body mass index (BMI) and to assess the association between these genetic variants and lipid profile and fatty acid levels. A total of 259 overweight-obese patients were compared to 369 healthy controls. FADS1, FADS2, and ELOVL2 genes were associated with BMI and overweight-obesity (P ≤ .001). In an additive model, the C allele in each of these variants was associated with a lower BMI: -1.18, -0.90, and -1.23 units, respectively. Higher amounts of total cholesterol, low-density lipoprotein cholesterol, total saturated fatty acids (lauric [12:0], myristic [C14:0], palmitic [C16:0], stearic [C18:0], arachidic [20:0], lignoceric [24:0]), monounsaturated fatty acids (myristoleic [C14:1], erucic [C22:1 n-9]), and polyunsaturated fatty acids (α-linolenic [ALA, 18:3 n-3], docosahexaenoic [DHA, C22:6 n-3], eicosapentaenoic acid [EPA, C20:5n-3], arachidonic acid [AA, 20:4n-6], and conjugated linolenic acids [CLA1 and CLA2]) were shown in patients. A significant increase in D6D activities presented by 20:4n-6/18:2n-6 and 18:3n-6/18:2n-6, Δ9 desaturase (D9D) activity, estimated by the ratio 18:1n-9/18:0 and elongase activities (AE), and estimated by the ratio of docosatetraenoic/AA and DPA/EPA in patients. The C minor allele of FADS1 had significantly lower DHA. A significant decrease in stearic acid, EPA, and AE activity (docosatetraenoic/AA) was revealed in patients with the minor allele carriers of FADS2. The C minor allele of ELOVL2 had significantly lower ALA, EPA, DPA, and D6D activity (C20:4 n-6/C18:2n-6). These data suggest that variations in FADS1, FADS2, and ELOVL2 affect the risk of overweight-obesity and the level of circulating fatty acids and could point to a key molecular pathway of metabolic syndrome and its related comorbidities.