Study of the antioxidant and anti-inflammatory properties of argan oil in the deregulation of peroxisomal metabolism

L’huile d’argan (HA) est une huile comestible extraite à partir des amandons de l’arganier Argania spinosa (L.) Skeels. Les travaux de cette thèse avait pour but en premier lieu l’étude de l’effet antioxydant du HA sur un modèle unicellulaire qu’est le protozoaire Tetrahymena pyriformis, ensuite étudier l’effet hépato et neuroprotecteur de le HA contre la dérégulation de la fonction peroxysomale et l’inflammation induite par le LPS dans un modèle in-vivo de souris C57BL/6J. en dernier lieu et afin d’explorer les composés de le HA responsable de son activité pharmacologique nous avons focaliser nos études sur l’étude de l’effet des deux phytostérols majeurs de le HA le schotténol et le spinastérol sur le stress oxydant et l’inflammation induite par le LPS chez un modèle de cellules microgliale BV-2 wild type et déficiente en Acox1 générée dans notre laboratoire par la technique CRISPR-Cas9.Ce travail a permis de mieux caractériser la composition de le HA en polyphénols totaux et en flavonoïdes grâce au screening phytochimique qui a permis aussi de tester l’activité antioxydante et antiradicalaire de le HA par les tests DPPH, FRAP et ABTS. Nous avons montré que le HA a un effet protecteur contre le stress oxydatif et nitrosatif induit par le H2O2 et le SNP respectivement chez T. pyriformis cet effet a été traduit par le rétablissement des activités des enzymes antioxydantes (CAT, SOD et GPX) perturbé par le traitement avec le H2O2 et le SNP. Il a été montré que le LPS possède un pouvoir d’induction de stress oxydant et de l’inflammation, dans notre étude menée in-vivo nous avons montré pour la première fois l’activité neuro protectrice de le HA contre le stress oxydant et l’inflammation induite par le LPS comparé avec l’activité hépato-protectrice, chez la souris. Le traitement avec le HA atténuait l’activité des enzymes antioxydantes induites par le LPS ainsi que l’expression du cytokines inflammatoire TNF-α. Le HA aussi a réagi au niveau peroxysomal par la régulation de l’Acox1 et la catalase. Il a aussi montré un effet protecteur contre la peroxydation lipidique par la diminution de son marqueur MDA induit par le LPS.En outre nous avons démontré pour la première fois que les deux phytostérols majeurs de le HA SC et SP possèdent une activité antioxydante et anti-inflammatoire. Dans la microglie le traitement avec SC et SP a permis la suppression du stress oxydatif induit par le LPS testé par la sonde H2DCFDA et DHE, en plus les deux PS ont montré une forte inhibition de la production du NO. Au niveau peroxysomale SC et SP ont diminué l’activité et l’expression de la catalase induite par le LPS et ont augmenté l’activité et l’expression de l’Acox1 et l’expression des transporteurs peroxysomaux ABCD1 et ABCD2 réprimé par le traitement avec le LPS. Aussi nous avons montré que SC et SP possèdent un effet modulatoire du métabolisme lipidique par la régulation de l’expression du facteur de transcription PPAR-α ainsi qu’une activité anti-inflammatoire exhibé par la répression de l’expression des cytokines inflammatoires iNOS, TNF-α et IL-1β et par l’induction de la cytokine anti-inflammatoire IL-4. L’ensemble des résultats obtenues montrait que le HA grâce à sa composition unique en acides gras polyinsaturés, et en antioxydants, possède une activité antioxydante et anti-inflammatoires au niveau cérébrale et hépatique, les PS, SC et SP montrait une grande efficacité dans l’atténuation de la neuro-inflammation au niveau de la microglie.Mot clés : Argan, Tetrahymena pyriformis, Acide gras, CAT, SOD, GPX, H2O2, SNP, MDA Inflammation, Microglie, BV-2, Phytostérols, Peroxysome, β-oxydation, Schotténol, Spinastérol, Antydant, LPS, PPARα, ABCD1, ABCD2, ACOX1, IL4, TNFαDue to the possible toxicity of synthetic antioxidants and anti-inflammatories, increasing attention has been directed to natural compounds possessing biological activity. Argan oil (AO) is an edible oil extracted from the kernels of the argan tree Argania spinosa (L.) Skeels. The aim of this thesis work was primarily to study the antioxidant effect of AO on a unicellular model which is the protozoan Tetrahymena pyriformis, then studying the hepato and neuroprotective effect of AO against LPS-induced dysregulation of peroxisomal function and inflammation in an in-vivo C57BL/6J mouse model. Finally, and in order to explore the compounds of AO responsible for its pharmacological activity, we have focused our work on the study of the effect of the two major phytosterols of AO, schottenol and spinasterol, on oxidative stress and LPS-induced inflammation in a wild-type, Acox1-deficient BV-2 microglial cell model obtained in our laboratory by the CRISPR-Cas9 technique.The first part of this work aimed to better characterize the composition of the AO in term of total polyphenols and flavonoids using a phytochemical screening which also permitted to test the antioxidant and antiradical activity of the AO by the DPPH, FRAP and ABTS tests. We showed that AO has a protective effect against oxidative and nitrosative stress induced by H2O2 and SNP respectively in T. pyriformis this effect was translated by the restoration of the activities of antioxidant enzymes (CAT, SOD and GPX) dysregulated by treatment with H2O2 and SNP. It has been shown that LPS induce oxidative stress and inflammation, in our in-vivo study we showed for the first time the neuroprotective activity of AO against oxidative stress and LPS-induced inflammation compared to hepatoprotective activity in mice brain and liver. Treatment with AO attenuated the activity of LPS-induced antioxidant enzymes as well as the expression of the inflammatory cytokines TNF-α. AO also reacted at the peroxisomal level through the regulation of Acox1 and catalase. It also showed a protective effect against lipid peroxidation by decreasing its LPS-induced marker MDA.Furthermore, we demonstrated for the first time that the two major phytosterols of AO SC and SP exhibit antioxidant and anti-inflammatory activity. In the microglia, the treatment with SC and SP suppressed oxidative stress induced by the LPS tested by the H2DCFDA and DHE probe, in addition the two PSs showed a strong inhibition of the production of NO. At the peroxisomal level SC and SP decreased LPS-induced catalase activity and expression and increased Acox1 activity and expression of two peroxisomal transporters ABCD1 and ABCD2 repressed by LPS. Also, we showed that SC and SP conveyed a modulatory effect of the lipid metabolism by upregulating the expression of the transcription factor PPAR-α as well as an anti-inflammatory activity exhibited by the repression of the expression of the inflammatory cytokines iNOS, TNF-α and IL-1β and by induction of the anti-inflammatory cytokine IL-4. All the results obtained showed that AO, with its unique composition of polyunsaturated fatty acids and antioxidants, has antioxidant and anti-inflammatory activity at the cerebral and hepatic level, the PSs, SC, and SP showed great efficacy in attenuation of neuro-inflammation in microglia.Keywords: Argan, Tetrahymena pyriformis, Fatty acid, CAT, SOD, GPX, H2O2, SNP, MDA Inflammation, Microglia, BV-2, Phytosterols, Peroxisome, β-oxidation, Schottenol, Spinasterol, Antioxidant, LPS, PPARα, ABCD1, ABCD2, ACOX1, IL4, TNF

Effects of a Short-Term Lipopolysaccharides Challenge on Mouse Brain and Liver Peroxisomal Antioxidant and β-oxidative Functions: Protective Action of Argan Oil

During sepsis, the imbalance between oxidative insult and body antioxidant response causes the dysfunction of organs, including the brain and liver. Exposing mice to bacterial lipopolysaccharides (LPS) results in a similar pathophysiological outcome. The protection offered by argan oil was studied against LPS-induced oxidative stress, dysregulation of peroxisomal antioxidants, and β-oxidation activities in the brain and liver. In a short-term LPS treatment, lipid peroxidation (malonaldehyde assay) increased in the brain and liver with upregulations of proinflammatory tumor necrosis factor (Tnf)-α and anti-inflammatory interleukin (Il)-10 genes, especially in the liver. Although exposure to olive oil (OO), colza oil (CO), and argan oil (AO) prevented LPS-induced lipid peroxidation in the brain and liver, only AO exposure protected against liver inflammation. Remarkably, only exposure to AO prevented LPS-dependent glutathione (GSH) dysregulation in the brain and liver. Furthermore, exposure to AO increased more efficiently than OO and CO in both organs, peroxisomal antioxidant capacity via induction of catalase (Cat) gene, protein and activity expression levels, and superoxide dismutase (Sod1) mRNA and activity levels. Interestingly, LPS decreased protein levels of the peroxisomal fatty acid-ATP binding cassette (ABC) transporters, ABCD1 and ABCD2, and increased acyl-CoA oxidase 1 (ACOX1) protein expression. Moreover, these LPS effects were attenuated for ABCD1 and ACOX1 in the brain of mice pretreated with AO. Our data collectively highlight the protective effects of AO against early oxidative stress caused by LPS in the brain and liver and their reliance on the preservation of peroxisomal functions, including antioxidant and β-oxidation activities, making AO a promising candidate for the prevention and management of sepsis

Two Argan Oil Phytosterols, Schottenol and Spinasterol, Attenuate Oxidative Stress and Restore LPS-Dysregulated Peroxisomal Functions in Acox1−/− and Wild-Type BV-2 Microglial Cells

International audienceOxidative stress and inflammation are the key players in neuroinflammation, in which microglia dysfunction plays a central role. Previous studies suggest that argan oil attenuates oxidative stress, inflammation, and peroxisome dysfunction in mouse brains. In this study, we explored the effects of two major argan oil (AO) phytosterols, Schottenol (Schot) and Spinasterol (Spina), on oxidative stress, inflammation, and peroxisomal dysfunction in two murine microglial BV-2 cell lines, wild-ype (Wt) and Acyl-CoA oxidase 1 (Acox1)-deficient cells challenged with LPS treatment. Herein, we used an MTT test to reveal no cytotoxicity for both phytosterols with concentrations up to 5 µM. In the LPS-activated microglial cells, cotreatment with each of these phytosterols caused a significant decrease in intracellular ROS production and the NO level released in the culture medium. Additionally, Schot and Spina were able to attenuate the LPS-dependent strong induction of Il-1β and Tnf-α mRNA levels, as well as the iNos gene and protein expression in both Wt and Acox1−/− microglial cells. On the other hand, LPS treatment impacted both the peroxisomal antioxidant capacity and the fatty acid oxidation pathway. However, both Schot and Spina treatments enhanced ACOX1 activity in the Wt BV-2 cells and normalized the catalase activity in both Wt and Acox1−/− microglial cells. These data suggest that Schot and Spina can protect cells from oxidative stress and inflammation and their harmful consequences for peroxisomal functions and the homeostasis of microglial cells. Collectively, our work provides a compelling argument for the protective mechanisms of two major argan oil phytosterols against LPS-induced brain neuroinflammation

Two Argan Oil Phytosterols, Schottenol and Spinasterol, Attenuate Oxidative Stress and Restore LPS-Dysregulated Peroxisomal Functions in <i>Acox1^−/−</i> and Wild-Type BV-2 Microglial Cells

Oxidative stress and inflammation are the key players in neuroinflammation, in which microglia dysfunction plays a central role. Previous studies suggest that argan oil attenuates oxidative stress, inflammation, and peroxisome dysfunction in mouse brains. In this study, we explored the effects of two major argan oil (AO) phytosterols, Schottenol (Schot) and Spinasterol (Spina), on oxidative stress, inflammation, and peroxisomal dysfunction in two murine microglial BV-2 cell lines, wild-ype (Wt) and Acyl-CoA oxidase 1 (Acox1)-deficient cells challenged with LPS treatment. Herein, we used an MTT test to reveal no cytotoxicity for both phytosterols with concentrations up to 5 µM. In the LPS-activated microglial cells, cotreatment with each of these phytosterols caused a significant decrease in intracellular ROS production and the NO level released in the culture medium. Additionally, Schot and Spina were able to attenuate the LPS-dependent strong induction of Il-1β and Tnf-α mRNA levels, as well as the iNos gene and protein expression in both Wt and Acox1−/− microglial cells. On the other hand, LPS treatment impacted both the peroxisomal antioxidant capacity and the fatty acid oxidation pathway. However, both Schot and Spina treatments enhanced ACOX1 activity in the Wt BV-2 cells and normalized the catalase activity in both Wt and Acox1−/− microglial cells. These data suggest that Schot and Spina can protect cells from oxidative stress and inflammation and their harmful consequences for peroxisomal functions and the homeostasis of microglial cells. Collectively, our work provides a compelling argument for the protective mechanisms of two major argan oil phytosterols against LPS-induced brain neuroinflammation

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

International audiencePeroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7β-hydroxycholesterol (7β-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 7β-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7β-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 7β-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 7β-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 β-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal β-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)

Sources of 7-ketocholesterol, metabolism and inactivation strategies: food and biomedical applications

Graphical abstract Abstract 7-Ketocholesterol (or 7-oxocholesterol) is an oxysterol essentially formed by cholesterol autoxidation. It is often found at enhanced levels in the body fluids and/or target tissues of patients with age-related diseases (cardiovascular, neuronal, and ocular diseases) as well as in subjects concerned with civilization diseases (type 2 diabetes, bowel diseases, and metabolic syndrome). The involvement of increased 7-ketocholesterol levels in the pathophysiology of these diseases is widely suspected. Indeed, 7-ketocholesterol at elevated concentrations is a powerful inducer of oxidative stress, inflammation, and cellular degeneration which are common features of all these diseases. It is important to better know the origin of 7-ketocholesterol (diet, incidence of environmental factors, and endogenous formation (autoxidation and enzymatic synthesis)) and its inactivation mechanisms which include esterification, sulfation, oxidation, and reduction. This knowledge will make it possible to act at different levels to regulate 7-ketocholesterol level and counteract its toxicity in order to limit the incidence of diseases associated with this oxysterol. These different points as well as food and biomedical applications are addressed in this review