Cytoprotective activities of representative nutrients from the Mediterranean diet and of Mediterranean oils against 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cytotoxicity: Application to age-related diseases and civilization diseases.

7-ketocholesterol and 7ß-hydroxycholesterol are two oxysterols mainly formed by the autoxidation of cholesterol. These two molecules are interconvertible via specific enzymes. These two oxysterols are often observed at increased amounts in biological fluids as well as tissues and organs affected during age-related diseases and in diseases of civilization such as cardiovascular, neurodegenerative, and ocular diseases as well as type 2 diabetes and metabolic syndrome. Noteworthy, 7-ketocholesterol and 7ß-hydroxycholesterol induce oxidative stress and inflammation, which are frequently observed in patients with age-related and civilization diseases. For this reason, the involvement of these two oxysterols in the pathophysiology of these diseases is widely suspected. In addition, the toxicity of these oxysterols can lead to death by oxiapoptophagy characterized by oxidative stress, apoptosis induction and autophagy criteria. To prevent, or even treat, certain age-related or civilization diseases associated with increased levels of 7-ketocholesterol and 7ß-hydroxycholesterol, the identification of molecules or mixtures of molecules attenuating or inhibiting the toxic effects of these oxysterols allows to consider new treatments. In this context, many nutrients present in significant amounts in the Mediterranean diet, especially tocopherols, fatty acids, and polyphenols, have shown cytoprotective activities as well as several Mediterranean oils (argan and olive oils, milk thistle seed oil, and pistacia lentiscus seed oil). Consequently, a nutraceutical approach, rich in nutrients present in the Mediterranean diet, could thus make it possible to counteract certain age-related and civilization diseases associated with increased levels of 7-ketocholesterol and 7ß-hydroxycholesterol.

Profiles of Fatty Acids, Polyphenols, Sterols, and Tocopherols and Scavenging Property of Mediterranean Oils: New Sources of Dietary Nutrients for the Prevention of Age-related Diseases.

The present study provides the fatty acid, tocopherol, phytosterol, and polyphenol profiles of some Mediterranean oils extracted from pumpkin, melon, and black cumin seed oils and those of dietary argan seed oil. Gas chromatography analysis revealed that oleic and linoleic acids were the most abundant fatty acids. Argan and melon seed oils exhibited the highest levels of oleic acid (47.32±0.02%) and linoleic acid (58.35±0.26%), respectively. In terms of tocopherols, melon seed oil showed the highest amount (652.1±3.26 mg/kg) with a predominance of γ-tocopherol (633.1±18.81 mg/kg). The phytosterol content varied between 2237.00±37.55 µg/g for argan oil to 6995.55±224.01 µg/g for melon seed oil. High Performance Liquid Chromatography analysis also revealed the presence of several polyphenols: vanillin (0.59 mg equivalents Quercetin/100 g) for melon seed oil, and p-hydroxycinnamic acid (0.04 mg equivalents Quercetin/100 g), coumarine (0.05 mg equivalents Quercetin/100 g), and thymoquinone (1.2 mg equivalents Quercetin/100 g) for black cumin seed oil. The "Kit Radicaux Libres" (KRL) assay used to evaluate the scavenging properties of the oils showed that black cumin seed oil was the most efficient. On the light of the richness of all Mediterranean oil samples in bioactive compounds, the seed oils studied can be considered as important sources of nutrients endowed with cytoprotective properties which benefits in preventing age-related diseases which are characterized by an enhanced oxidative stress.

Cytotoxic and Antioxidant Activities of Imine Analogs of Trans-Resveratrol towards Murine Neuronal N2a Cells.

Trans-resveratrol is a natural polyphenol showing numerous biological properties, especially anti-tumoral and antioxidant activity. Among numerous resveratrol derivatives, aza-stilbenes, which bear an imine bound, show interesting biological activities. In the present study, we synthesized a series of imine analogs of trans-resveratrol (seven aza-stilbenes) following an easy and low-cost procedure of green chemistry. The toxicity of synthesized aza-stilbenes, which is currently unknown, was evaluated on murine neuronal N2a cells, comparatively to trans-resveratrol, by considering: cell density evaluated by staining with sulforhodamine 101; esterase activity, which is a criteria of cell viability, by staining with fluorescein diacetate; and transmembrane mitochondrial potential, which is known to decrease during cell death, by staining with DiOC6(3) using flow cytometry. In addition, the antioxidant activity was quantified with the KRL (Kit Radicaux Libres) assay, the DPPH (2,2'-diphenyl-1-picrylhydrazyl radical) assay and the FRAP (ferric reducing antioxidant power) assay. The PAOT (Pouvoir Antioxidant Total) score was also used. The aza-stilbenes provide different cytotoxic and antioxidant activities, which are either higher or lower than those of trans-resveratrol. Based on their cytotoxic and antioxidant characteristics, all synthesized aza-stilbenes are distinguished from trans-resveratrol.

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).

Saffron (Crocus sativus L.): A Source of Nutrients for Health and for the Treatment of Neuropsychiatric and Age-Related Diseases.

Saffron (Crocus sativus L.) is a medicinal plant, originally cultivated in the East and Middle East, and later in some Mediterranean countries. Saffron is obtained from the stigmas of the plant. Currently, the use of saffron is undergoing a revival. The medicinal virtues of saffron, its culinary use and its high added value have led to the clarification of its phytochemical profile and its biological and therapeutic characteristics. Saffron is rich in carotenoids and terpenes. The major products of saffron are crocins and crocetin (carotenoids) deriving from zeaxanthin, pirocrocin and safranal, which give it its taste and aroma, respectively. Saffron and its major compounds have powerful antioxidant and anti-inflammatory properties in vitro and in vivo. Anti-tumor properties have also been described. The goal of this review is to present the beneficial effects of saffron and its main constituent molecules on neuropsychiatric diseases (depression, anxiety and schizophrenia) as well as on the most frequent age-related diseases (cardiovascular, ocular and neurodegenerative diseases, as well as sarcopenia). Overall, the phytochemical profile of saffron confers many beneficial virtues on human health and, in particular, on the prevention of age-related diseases, which is a major asset reinforcing the interest for this medicinal plant.

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.

7-Ketocholesterol: Effects on viral infections and hypothetical contribution in COVID-19.

7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

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.

The Mediterranean diet is associated with health benefits due to bioactive compounds such as polyphenols. The biological activities of three polyphenols (quercetin (QCT), resveratrol (RSV), apigenin (API)) were evaluated in mouse neuronal N2a cells in the presence of 7-ketocholesterol (7KC), a major cholesterol oxidation product increased in patients with age-related diseases, including neurodegenerative disorders. In N2a cells, 7KC (50 µM; 48 h) induces cytotoxic effects characterized by an induction of cell death. When associated with RSV, QCT and API (3.125; 6.25 µM), 7KC-induced toxicity was reduced. The ability of QCT, RSV and API to prevent 7KC-induced oxidative stress was characterized by a decrease in reactive oxygen species (ROS) production in whole cells and at the mitochondrial level; by an attenuation of the increase in the level and activity of catalase; by attenuating the decrease in the expression, level and activity of glutathione peroxidase 1 (GPx1); by normalizing the expression, level and activity of superoxide dismutases 1 and 2 (SOD1, SOD2); and by reducing the decrease in the expression of nuclear erythroid 2-like factor 2 (Nrf2) which regulates antioxidant genes. QCT, RSV and API also prevented mitochondrial dysfunction in 7KC-treated cells by counteracting the loss of mitochondrial membrane potential (ΨΔm) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase α (AMPKα), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) implicated in mitochondrial biogenesis. At the peroxisomal level, QCT, RSV and API prevented the impact of 7KC by counteracting the decrease in ATP binding cassette subfamily D member (ABCD)3 (a peroxisomal mass marker) at the protein and mRNA levels, as well as the decreased expresssion of genes associated with peroxisomal biogenesis (Pex13, Pex14) and peroxisomal ß-oxidation (Abcd1, Acox1, Mfp2, Thiolase A). The 7KC-induced decrease in ABCD1 and multifunctional enzyme type 2 (MFP2), two proteins involved in peroxisomal ß-oxidation, was also attenuated by RSV, QCT and API. 7KC-induced cell death, which has characteristics of apoptosis (cells with fragmented and/or condensed nuclei; cleaved caspase-3; Poly(ADP-ribose) polymerase (PARP) fragmentation) and autophagy (cells with monodansyl cadaverine positive vacuoles; activation of microtubule associated protein 1 light chain 3-I (LC3-I) to LC3-II, was also strongly attenuated by RSV, QCT and API. Thus, in N2a cells, 7KC induces a mode of cell death by oxiapoptophagy, including criteria of OXIdative stress, APOPTOsis and autoPHAGY, associated with mitochondrial and peroxisomal dysfunction, which is counteracted by RSV, QCT, and API reinforcing the interest for these polyphenols in prevention of diseases associated with increased 7KC levels.

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.

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.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Octadecaneuropeptide (ODN) Induces N2a Cells Differentiation through a PKA/PLC/PKC/MEK/ERK-Dependent Pathway: Incidence on Peroxisome, Mitochondria, and Lipid Profiles.

Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10-14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.

Profile of Fatty Acids, Tocopherols, Phytosterols and Polyphenols in Mediterranean Oils (Argan Oils, Olive Oils, Milk Thistle Seed Oils and Nigella Seed Oil) and Evaluation of their Antioxidant and Cytoprotective Activities.

BACKGROUND: The effects of vegetable oils on human health depend on their components. Therefore, their profiles of lipid nutrients and polyphenols were determined. OBJECTIVE: To establish and compare the fatty acid, tocopherol, phytosterol and polyphenol profiles of Mediterranean oils: cosmetic and dietary argan oils (AO; Morocco: Agadir, Berkane); olive oils (OO; Morocco, Spain, Tunisia); milk thistle seed oils (MTSO; Tunisia: Bizerte, Sousse, Zaghouane); nigella seed oil (NSO). METHODS: The biochemical profiles were determined by gas chromatography-flame ionization, high performance liquid chromatography and gas chromatography, coupled with mass spectrometry as required. The antioxidant and cytoprotective activities were evaluated with the KRL (Kit Radicaux Libres) and the fluorescein diacetate tests on nerve cells treated with 7-ketocholesterol (7KC). RESULTS: The fatty acid profile revealed high linoleic acid (C18:2 n-6) content in AO, OO, MTSO and NSO. The highest levels of oleic acid (C18:1 n-9) were found in AO and OO. The tocopherol profile showed that Agadir AO contained the highest amount of α-tocopherol, also present at high level in MTSO and Tunisian OO; Berkane AO was rich in γ-tocopherol. The phytosterol profile indicated that ß-sitosterol was predominant in the oils, except AO; spinasterol was only present in AO. Polyphenol profiles underlined that OO was the richest in polyphenols; hydroxytyrosol was only found in OO; few polyphenols were detected in AO. The oils studied have antioxidant activities, and all of them, except NSO, prevented 7KC-induced cell death. The antioxidant characteristics of AO were positively correlated with procatechic acid and compestanol levels. CONCLUSION: Based on their biochemical profiles, antioxidant and cytoprotective characteristics, AO, OO, and MTSO are potentially beneficial to human health.

Elaboration of Trans-Resveratrol Derivative-Loaded Superparamagnetic Iron Oxide Nanoparticles for Glioma Treatment.

In this work, new nanohybrids based on superparamagnetic iron oxide nanoparticles (SPIONs) were elaborated and discussed for the first time as nanovectors of a derivative molecule of trans-resveratrol (RSV), a natural antioxidant molecule, which can be useful for brain disease treatment. The derivative molecule was chemically synthesized (4'-hydroxy-4-(3-aminopropoxy) trans-stilbene: HAPtS) and then grafted onto SPIONs surface using an organosilane coupling agent, which is 3-chloropropyltriethoxysilane (CPTES) and based on nucleophilic substitution reactions. The amount of HAPtS loaded onto SPIONs surface was estimated by thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) analyses at 116 µmol·g-1 SPIONs. The synthesized HAPtS molecule, as well as the associated nanohybrids, were fully characterized by transmission electron microscopy (TEM), XPS, TGA, infrared (IR) and UV-visible spectroscopies, dynamic light scattering (DLS), and zeta potential measurements. The in vitro biological assessment of the synthesized nanohybrid's efficiency was carried out on C6 glioma cells and showed that the nanovector SPIONs-CPTES-HAPtS do not affect the mitochondrial metabolism (MTT test), but damage the plasma membrane (FDA test), which could contribute to limiting the proliferation of cancerous cells (clonogenic test) at a HAPtS concentration of 50 µM. These nanoparticles have a potential cytotoxic effect that could be used to eliminate cancer cells.

Polydopamine Modified Superparamagnetic Iron Oxide Nanoparticles as Multifunctional Nanocarrier for Targeted Prostate Cancer Treatment.

Polydopamine (pDA)-modified iron oxide core-shell nanoparticles (IONPs) are developed and designed as nanovectors of drugs. Reactive quinone of pDA enhances the binding efficiency of various biomolecules for targeted delivery. Glutathione disulfide (GSSG), an abundant thiol species in the cytoplasm, was immobilized on the pDA-IONP surface. It serves as a cellular trigger to release the drug from the nanoparticles providing an efficient platform for the drug delivery system. Additionally, GSSG on the surface was further modified to form S-nitrosoglutathione that can act as nitric oxide (NO) donors. These NPs were fully characterized using a transmission electronic microscopy (TEM), thermogravimetric analysis (TGA), dynamic light scattering (DLS), zeta potential, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and UV-vis spectroscopies. Doxorubicin (DOX) and docetaxel (DTX) are two anticancer drugs, which were loaded onto nanoparticles with respective loading efficiencies of 243 and 223 µmol/g of IONPs, calculated using TGA measurements. DOX release study, using UV-vis spectroscopy, showed a pH responsive behavior, making the elaborated nanocarrier a potential drug delivery system. (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium (MTS) and apoptosis assays were performed on PC3 cell lines to evaluate the efficiency of the developed nanocarriers. These nanoparticles thus can prove their worth in cancer treatment on account of their easy access to the site and release of drug in response to changes to internal parameters such as pH, chemicals, etc.

Prevention of 7-ketocholesterol-induced side effects by natural compounds.

Cholesterol oxidation products, also named oxysterols, can be formed either by cholesterol auto-oxidation, enzymatically or both. Among these oxysterols, 7-ketocholesterol (7KC) is mainly formed during radical attacks that take place on the carbon 7 of cholesterol. As increased levels of 7KC have been found in the tissues, plasma and/or cerebrospinal fluid of patients with major diseases, especially age-related diseases (cardiovascular diseases, eye diseases, neurodegenerative diseases), some cancers, and chronic inflammatory diseases, it is suspected that 7KC, could contribute to their development. Since 7KC, provided by the diet or endogenously formed, is not or little efficiently metabolized, except in hepatic cells, its cellular accumulation can trigger numerous side effects including oxidative stress, inflammation and cell death. To counteract 7KC-induced side effects, it is necessary to characterize the metabolic pathways activated by this oxysterol to identify potential targets for cytoprotection and geroprotection. Currently, several natural compounds (tocopherols, fatty acids, polyphenols, etc) or mixtures of compounds (oils) used in traditional medicine are able to inhibit the deleterious effects of 7KC. The different molecules identified could be valued in different ways (functional foods, recombinant molecules, theranostic) to prevent or treat diseases associated with 7KC.

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.

Cytoprotective Activities of Milk Thistle Seed Oil Used in Traditional Tunisian Medicine on 7-Ketocholesterol and 24S-Hydroxycholesterol-Induced Toxicity on 158N Murine Oligodendrocytes.

The Asteraceae family is economically very important, because many of these plants are grown mainly for their food value, such as lettuce (Lactuca), chicory (Cichorium), and sunflower (Heliantus aminus). One of the typical properties of this family, which includes milk thistle (Sylibum marianum), is the richness of the oil in various compounds (flavonoids, alkaloids, tocopherols, and unsaturated fatty acids). Currently, and for the coming decades, age-related diseases, including neurodegenerative diseases, are a major public health problem. Preventing their appearance or opposing their evolution is a major objective. In this context, the cytoprotective activities of milk thistle seed oil produced in Tunisia were studied on the 158N model using 7-ketocholesterol (7KC) and 24S-hydroxycholesterol (24S) as cytotoxic agents. 7KC and 24S were used because they can be increased in the brain and body fluids of patients with major age-related neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. In order to evaluate the cytoprotective properties of milk thistle seed oil, complementary techniques of microscopy, flow cytometry, and biochemistry were used. The chemical composition of milk thistle seed oil has also been determined by various chromatography techniques. Milk thistle seed oils from different area of Tunisia are rich in tocopherols and are strongly antioxidant according to various biochemical tests (KRL (Kit Radicaux Libres), FRAP (Ferric Reducing Antioxidant Power), and DPPH (2,2-diphenyl-1-picrylhydrazyl)). The main fatty acids are linoleic acid (C18:2 n-6) and oleic acid (C18:1 n-9). The main polyphenols identified are homovanillic acid, p-coumaric acid, quercetin, and apigenin, with a predominance of vanillic acid. On 158N cells, milk thistle seed oil attenuates the cytotoxicity of 7KC and 24S including: loss of cell adhesion, increased plasma membrane permeability, mitochondrial dysfunction, overproduction of reactive oxygen species, induction of apoptosis, and autophagy. The attenuation of the cytotoxicity of 7KC and 24S observed with the milk thistle seed oil is in the order of that observed with α-tocopherol used as a positive control. In the presence of nigella seed oil, considered potentially cytotoxic, no cytoprotective effects were observed. Given the chemical characteristics, antioxidant properties, and cytoprotective activities of milk thistle seed oil, our results highlight the potential benefit of this oil for human health.

Induction of Neuronal Differentiation of Murine N2a Cells by Two Polyphenols Present in the Mediterranean Diet Mimicking Neurotrophins Activities: Resveratrol and Apigenin.

In the prevention of neurodegeneration associated with aging and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), neuronal differentiation is of interest. In this context, neurotrophic factors are a family of peptides capable of promoting the growth, survival, and/or differentiation of both developing and immature neurons. In contrast to these peptidyl compounds, polyphenols are not degraded in the intestinal tract and are able to cross the blood⁻brain barrier. Consequently, they could potentially be used as therapeutic agents in neurodegenerative pathologies associated with neuronal loss, thus requiring the stimulation of neurogenesis. We therefore studied the ability to induce neuronal differentiation of two major polyphenols present in the Mediterranean diet: resveratrol (RSV), a major compound found in grapes and red wine, and apigenin (API), present in parsley, rosemary, olive oil, and honey. The effects of these compounds (RSV and API: 6.25⁻50 µM) were studied on murine neuro-2a (N2a) cells after 48 h of treatment without or with 10% fetal bovine serum (FBS). Retinoic acid (RA: 6.25⁻50 µM) was used as positive control. Neuronal differentiation was morphologically evaluated through the presence of dendrites and axons. Cell growth was determined by cell counting and cell viability by staining with fluorescein diacetate (FDA). Neuronal differentiation was more efficient in the absence of serum than with 10% FBS or 10% delipidized FBS. At concentrations inducing neuronal differentiation, no or slight cytotoxicity was observed with RSV and API, whereas RA was cytotoxic. Without FBS, RSV and API, as well as RA, trigger the neuronal differentiation of N2a cells via signaling pathways simultaneously involving protein kinase A (PKA)/phospholipase C (PLC)/protein kinase C (PKC) and MEK/ERK. With 10% FBS, RSV and RA induce neuronal differentiation via PLC/PKC and PKA/PLC/PKC, respectively. With 10% FBS, PKA and PLC/PKC as well as MEK/ERK signaling pathways were not activated in API-induced neuronal differentiation. In addition, the differentiating effects of RSV and API were not inhibited by cyclo[DLeu5] OP, an antagonist of octadecaneuropeptide (ODN) which is a neurotrophic factor. Moreover, RSV and API do not stimulate the expression of the diazepam-binding inhibitor (DBI), the precursor of ODN. Thus, RSV and API are able to induce neuronal differentiation, ODN and its receptor are not involved in this process, and the activation of the (PLC/PKC) signaling pathway is required, except with apigenin in the presence of 10% FBS. These data show that RSV and API are able to induce neuronal differentiation and therefore mimic neurotrophin activity. Thus, RSV and API could be of interest in regenerative medicine to favor neurogenesis.

Biomarkers of Amyotrophic Lateral Sclerosis: Current Status and Interest of Oxysterols and Phytosterols.

Amyotrophic lateral sclerosis (ALS) is a non-demyelinating neurodegenerative disease in adults with motor disorders. Two forms exist: a sporadic form (90% of cases) and a family form due to mutations in more than 20 genes including the Superoxide dismutase 1, TAR DNA Binding Protein, Fused in Sarcoma, chromosome 9 open reading frame 72 and VAPB genes. The mechanisms associated with this pathology are beginning to be known: oxidative stress, glutamate excitotoxicity, protein aggregation, reticulum endoplasmic stress, neuroinflammation, alteration of RNA metabolism. In various neurodegenerative diseases, such as Alzheimer's disease or multiple sclerosis, the involvement of lipids is increasingly suggested based on lipid metabolism modifications. With regard to ALS, research has also focused on the possible involvement of lipids. Lipid involvement was suggested for clinical arguments where changes in cholesterol and LDL/HDL levels were reported with, however, differences in positivity between studies. Since lipids are involved in the membrane structure and certain signaling pathways, it may be considered to look for oxysterols, mainly 25-hydroxycholesterol and its metabolites involved in immune response, or phytosterols to find suitable biomarkers for this pathology.

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.