Evaluation of Antioxidant, Anti-Inflammatory and Cytoprotective Properties of Ethanolic Mint Extracts from Algeria on 7-Ketocholesterol-Treated Murine RAW 264.7 Macrophages.

The present study consisted in evaluating the antioxidant, anti-inflammatory and cytoprotective properties of ethanolic extracts from three mint species (Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L.) Huds (MR)) with biochemical methods on murine RAW 264.7 macrophages (a transformed macrophage cell line isolated from ascites of BALB/c mice infected by the Abelson leukemia virus). The total phenolic, flavonoid and carotenoid contents were determined with spectrophotometric methods. The antioxidant activities were quantified with the Kit Radicaux Libres (KRLTM), the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. The MS extract showed the highest total phenolic content, and the highest antioxidant capacity, while the MR extract showed the lowest total phenolic content and the lowest antioxidant capacity. The cytoprotective and anti-inflammatory activities of the extracts were quantified on murine RAW 264.7 macrophages treated with 7-ketocholesterol (7KC; 20 µg/mL: 50 µM) associated or not for 24 h and 48 h with ethanolic mint extracts used at different concentrations (25, 50, 100, 200 and 400 µg/mL). Under treatment with 7KC, an important inhibition of cell growth was revealed with the crystal violet test. This side effect was strongly attenuated in a dose dependent manner with the different ethanolic mint extracts, mainly at 48 h. The most important cytoprotective effect was observed with the MS extract. In addition, the effects of ethanolic mint extracts on cytokine secretion (Interleukin (IL)-6, IL-10, Monocyte Chemoattractant Protein (MCP)-1, Interferon (IFN)-ϒ, Tumor necrosis factor (TNF)-α) were determined at 24 h on lipopolysaccharide (LPS, 0.2 µg/mL)-, 7KC (20 µg/mL)- and (7KC + LPS)-treated RAW 264.7 cells. Complex effects of mint extracts were observed on cytokine secretion. However, comparatively to LPS-treated cells, all the extracts strongly reduce IL-6 secretion and two of them (MP and MR) also decrease MCP-1 and TNF-α secretion. However, no anti-inflammatory effects were observed on 7KC- and (7KC + LPS)-treated cells. Altogether, these data bring new evidences on the potential benefits (especially antioxidant and cytoprotective properties) of Algerian mint on human health.

Modulation of Kv3.1b potassium channel level and intracellular potassium concentration in 158N murine oligodendrocytes and BV-2 murine microglial cells treated with 7-ketocholesterol, 24S-hydroxycholesterol or tetracosanoic acid (C24:0).

Little is known about K+ regulation playing major roles in the propagation of nerve impulses, as well as in apoptosis and inflammasome activation involved in neurodegeneration. As increased levels of 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC) and tetracosanoic acid (C24:0) have been observed in patients with neurodegenerative diseases, we studied the effect of 24 and/or 48 h of treatment with 7KC, 24S-OHC and C24:0 on Kv3.1b potassium channel level, intracellular K+ concentration, oxidative stress, mitochondrial dysfunction, and plasma membrane permeability in 158N oligodendrocytes and BV-2 microglial cells. In 158N cells, whereas increased level of Kv3.1b was only observed with 7KC and 24S-OHC but not with C24:0 at 24 h, an intracellular accumulation of K+ was always detected. In BV-2 cells treated with 7KC, 24S-OHC and C24:0, Kv3.1b level was only increased at 48 h; intracellular K+ accumulation was found at 24 h with 7KC, 24S-OHC and C24:0, and only with C24:0 at 48 h. Positive correlations between Kv3.1b level and intracellular K+ concentration were observed in 158N cells in the presence of 7KC and 24S-OHC, and in 7KC-treated BV-2 cells at 48 h. Positive correlations were also found between Kv3.1b or the intracellular K+ concentration, overproduction of reactive oxygen species, loss of transmembrane mitochondrial potential and increased plasma membrane permeability in 158N and BV-2 cells. Our data support that the lipid environment affects Kv3.1b channel expression and/or functionality, and that the subsequent rupture of K+ homeostasis is relied with oligodendrocytes and microglial cells damages.

Lipid Biomarkers in Alzheimer's Disease.

BACKGROUND: There are now significant evidences that lipid metabolism is affected in numerous neurodegenerative diseases including Alzheimer's disease. These dysfunctions lead to abnormal levels of certain lipids in the brain, cerebrospinal fluid and plasma. It is consequently of interest to establish lipid profiles in neurodegenerative diseases. This approach, which can contribute to identify lipid biomarkers of Alzheimers' disease, can also permit to identify new therapeutic targets. It was therefore of interest to focus on central and peripheral biomarkers in Alzheimer's disease. METHODS: A review of the literature on 148 papers was conducted. Based on this literature, the involvement of lipids (cholesterol and oxysterols, fatty acids, phospholipids) in Alzheimer's disease has been proposed. RESULTS: Of the 148 references cited for lipid biomarkers for Alzheimer's disease, 65 refer to cholesterol and oxysterols, 35 to fatty acids and 40 to phospholipids. Among these lipids, some of them such as 24S-hydroxyckolesterol, open up new therapeutic perspectives in gene therapy, in particular. The results on the very long-chain fatty acids suggest the potential of peroxisomal dysfunctions in Alzheimer's disease. As for the phospholipids, they could constitute interesting biomarkers for detecting the disease at the prodromal stage. CONCLUSION: There are now several lines of evidence that lipids play fundamental roles in the pathogenesis of AD and that some of them have a prognostic and diagnosis value. This may pave the way for the identification of new therapeutic targets, new effective drugs and / or new treatments.

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

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, ß-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.

Flow Cytometric Analysis of the Expression Pattern of Peroxisomal Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells.

Microglial cells play important roles in neurodegenerative diseases including peroxisomal leukodystrophies. The BV-2 murine immortalized cells are widely used in the context of neurodegenerative researches. It is therefore important to establish the expression pattern of peroxisomal proteins by flow cytometry in these cells. So, the expression pattern of various peroxisomal transporters (Abcd1, Abcd2, Abcd3) contributing to peroxisomal ß-oxidation was evaluated on BV-2 cells by flow cytometry and complementary methods (fluorescence microscopy, and RT-qPCR). By flow cytometry a strong expression of peroxisomal proteins (Abcd1, Abcd2, Abcd3) was observed. These data were in agreement with those obtained by fluorescence microscopy (presence of numerous fluorescent dots in the cytoplasm characteristic of a peroxisomal staining pattern) and RT-qPCR (high levels of Abcd1, Abcd2, and Abcd3 mRNAs). Thus, the peroxisomal proteins (Abcd1, Abcd2, Abcd3) are expressed in BV-2 cells, and can be analyzed by flow cytometry.

Comparison of the effects of major fatty acids present in the Mediterranean diet (oleic acid, docosahexaenoic acid) and in hydrogenated oils (elaidic acid) on 7-ketocholesterol-induced oxiapoptophagy in microglial BV-2 cells.

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation+APOPTOsis+autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Masson's trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases.

Evidence of K+ homeostasis disruption in cellular dysfunction triggered by 7-ketocholesterol, 24S-hydroxycholesterol, and tetracosanoic acid (C24:0) in 158N murine oligodendrocytes.

Imbalance in the homeostasis of K+ ions has been reported to contribute to the pathogenesis of neurodegenerative diseases. 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC), and tetracosanoic acid (C24:0), often found at increased levels in patients with Alzheimer's disease, Multiple Sclerosis and X-ALD, are able to trigger numerous nerve cell dysfunctions. We therefore studied the impact of 7KC, 24S-OHC, and C24:0 on 158N murine oligodendrocytes, and determined their impact on K+ homeostasis. The effects of 7KC, 24S-OHC and C24:0 on lipid membrane organization and membrane potential were examined with merocyanine 540 (MC540) and bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3)), respectively. The intracellular concentration of K+ ([K+]i) was measured by flame photometry and the ratiometric approach using the PBFI-AM fluorescence indicator. To determine the relationships between [K+]i and lipotoxicity, 158N cells were pre-treated with a universal Kv channels blocker, 4-aminopyridine (4-AP), without or with 7KC, 24S-OHC or C24:0. Cell adhesion, cell growth, mitochondrial depolarization, cytoplasmic membrane integrity, the presence of SubG1 and the morphological aspect of the nuclei were determined with various microscopy, flow cytometry and biochemistry methods. 7KC, 24S-OHC and C24:0 induced changes in lipid content and polarization of the cytoplasmic membrane. These events were associated with increased [K+]i. Blocking Kv channels with 4-AP exacerbated 7KC-, 24S-OHC- and C24:0-induced cell dysfunction. 4-AP exacerbated loss of cell adhesion and cell growth inhibition, amplified mitochondrial depolarization and cytoplasmic membrane damage, and increased the percentage of SubG1 cells. The positive correlation between [K+]i and cell death supports the potential involvement of K+ in 7KC-, 24S-OHC-, and C24:0-induced cytotoxicity.

Attenuation of 7-ketocholesterol-induced overproduction of reactive oxygen species, apoptosis, and autophagy by dimethyl fumarate on 158N murine oligodendrocytes.

Mitochondrial dysfunctions and oxidative stress are involved in several non demyelinating or demyelinating neurodegenerative diseases. Some of them, including multiple sclerosis (MS), are associated with lipid peroxidation processes leading to increased levels of 7-ketocholesterol (7KC). So, the eventual protective effect of dimethylfumarate (DMF), which is used for the treatment of MS, was evaluated on 7KC-treated oligodendrocytes, which are myelin synthesizing cells. To this end, murine oligodendrocytes 158N were exposed to 7KC (25, 50µM) for 24h without or with DMF (1, 25, 50µM). The biological activities of DMF associated or not with 7KC were evaluated by phase contrast microscopy, crystal violet and MTT tests. The impact on transmembrane mitochondrial potential (ΔYm), O2- and H2O2 production, apoptosis and autophagy was measured by microscopical and flow cytometric methods by staining with DiOC6(3), dihydroethidine and dihydrorhodamine 123, Hoechst 33342, and by Western blotting with the use of specific antibodies raised against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II. DMF attenuates the different effects of 7KC, namely: cell growth inhibition and/or loss of cell adhesion, decrease of ΔΨm, O2- and H2O2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. The ability of DMF to attenuate 7KC-induced reactive oxygen species overproduction, apoptosis, and autophagy on oligodendrocytes reinforces the interest for this molecule for the treatment of MS or other demyelinating diseases.

Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with α-tocopherol: Impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors.

In multiple sclerosis (MS) a process of white matter degradation leading to demyelination is observed. Oxidative stress, inflammation, apoptosis, necrosis and/or autophagy result together into a progressive loss of oligodendrocytes. 7-ketocholesterol (7KC), found increased in the cerebrospinal fluid of MS patients, triggers a rupture of RedOx homeostasis associated with mitochondrial dysfunctions, aptoptosis and autophagy (oxiapoptophagy) in cultured murine oligodendrocytes (158N). α-tocopherol is able to mild the alterations induced by 7KC partially restoring the cellular homeostasis. In presence of 7KC, the amount of adherent 158N cells was decreased and oxidative stress was enhanced. An increase of caspase-3 and PARP degradation (evidences of apoptosis), and an increased LC3-II/LC3-I ratio (criterion of autophagy), were detected. These events were associated with a decrease of the mitochondrial membrane potential (ΔΨm) and by a decrease of oxidative phosphorylation revealed by reduced NAD+ and ATP. The cellular lactate was higher while pyruvate, citrate, fumarate, succinate (tricarboxylic acid (TCA) cycle intermediates) were significantly reduced in exposed cells, suggesting that an impairment of mitochondrial respiratory functions could lead to an increase of lactate production and to a reduced amount of ATP and acetyl-CoA available for the anabolic pathways. The concentration of sterol precursors lathosterol, lanosterol and desmosterol were significantly reduced together with satured and unsatured long chain fatty acids (C16:0 - C18:0, structural elements of membrane phospholipids). Such reductions were milder with α-tocopherol. It is likely that the cell death induced by 7KC is associated with mitochondrial dysfunctions, including alterations of oxidative phosphorylation, which could result from lipid anabolism dysfunctions, especially on TCA cycle intermediates. A better knowledge of mitochondrial associated dysfunctions triggered by 7KC will contribute to bring new information on the demyelination processes which are linked with oxidative stress and lipid peroxidation, especially in MS.

7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder induced by a mutation in the ABCD1 gene, which causes the accumulation of very long-chain fatty acids in tissue and plasma. Oxidative stress may be a hallmark of X-ALD. In the plasma of X-ALD patients with different forms of the disease, characterized by high levels of C24:0 and C26:0, we observed the presence of oxidative stress revealed by decreased levels of GSH, α-tocopherol, and docosahexaenoic acid (DHA). We showed that oxidative stress caused the oxidation of cholesterol and linoleic acid, leading to the formation of cholesterol oxide derivatives oxidized at C7 (7-ketocholesterol (7KC), 7ß-hydroxycholesterol (7ß-OHC), and 7α-hydroxycholesrol (7α-OHC)) and of 9- and 13-hydroxyoctadecadienoic acids (9-HODE, 13-HODE), respectively. High levels of 7KC, 7ß-OHC, 7α-OHC, 9-HODE and 13-HODE were found. As 7KC induces oxidative stress, inflammation and cell death, which could play key roles in the development of X-ALD, the impact of 7KC on the peroxisomal status was determined in microglial BV-2 cells. Indeed, environmental stress factors such as 7KC could exacerbate peroxisomal dysfunctions in microglial cells and thus determine the progression of the disease. 7KC induces oxiapoptophagy in BV-2 cells: overproduction of H2O2 and O2-, presence of cleaved caspase-3 and PARP, nuclear condensation and/or fragmentation; elevated [LC3-II/LC3-I] ratio, increased p62 levels. 7KC also induces several peroxisomal modifications: decreased Abcd1, Abcd2, Abcd3, Acox1 and/or Mfp2 mRNA and protein levels, increased catalase activity and decreased Acox1-activity. However, the Pex14 level was unchanged. It is suggested that high levels of 7KC in X-ALD patients could foster generalized peroxisomal dysfunction in microglial cells, which could in turn intensify brain damage.

Protective Effects of α-Tocopherol, γ-Tocopherol and Oleic Acid, Three Compounds of Olive Oils, and No Effect of Trolox, on 7-Ketocholesterol-Induced Mitochondrial and Peroxisomal Dysfunction in Microglial BV-2 Cells.

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal ß-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.

Fatty acid profiles in demented patients: identification of hexacosanoic acid (C26:0) as a blood lipid biomarker of dementia.

BACKGROUND: Several lipid metabolism alterations have been described in the brain and plasma of Alzheimer's disease (AD) patients, suggesting a relation between lipid metabolism alteration and dementia. OBJECTIVE: We attempted to identify blood fatty acids as biomarkers of dementia. METHODS: Fatty acid profiles were established using gas chromatography with or without mass spectrometry on matched plasma and red blood cells (RBCs) of demented patients diagnosed with AD, vascular dementia, or other dementia, and compared with a control group of elderly individuals. The severity of dementia was evaluated with the Mini-Mental State Examination test. RESULTS: Fatty acid analysis showed significant variations of fatty acid levels in demented patients including AD patients. The highest plasma and RBC accumulation was found with hexacosanoic acid (C26:0). Our data also support that alterations of desaturase and elongase activities may contribute to cognitive dysfunction. CONCLUSION: The variations of fatty acid levels and the accumulation of C26:0 in the plasma and RBCs highlight an alteration of fatty acid metabolism in demented patients and point toward possible peroxisomal dysfunction. It is suggested that C26:0 may constitute a convenient blood biomarker of dementia that could be useful in routine medical practice.