Effect of Ergothioneine on 7-Ketocholesterol-Induced Endothelial Injury.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Anti-inflammatory and Cytoprotective Effect of Clinacanthus nutans Leaf But Not Stem Extracts on 7-Ketocholesterol Induced Brain Endothelial Cell Injury.

Clinacanthus nutans (Lindau) (C. nutans) has diverse uses in traditional herbal medicine for treating skin rashes, insect and snake bites, lesions caused by herpes simplex virus, diabetes mellitus and gout in Singapore, Malaysia, Indonesia, Thailand and China. We previously showed that C. nutans has the ability to modulate the induction of cytosolic phospholipase A2 (cPLA2) expression in SH-SY5Y cells through the inhibition of histone deacetylases (HDACs). In the current study, we elucidated the effect of C. nutans on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induced a dose-dependent loss of hCMEC/D3 cell viability, and such damage was significantly inhibited by C. nutans leaf extracts but not stem extracts. 7KC also induced a marked increase in mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX-2) in brain endothelial cells, and these increases were significantly inhibited by C. nutans leaf but not stem extracts. HPLC analyses showed that leaf extracts have a markedly different chemical profile compared to stem extracts, which might explain their different effects in counteracting 7KC-induced inflammation. Further study is necessary to identify the putative phytochemicals in C. nutans leaves that have anti-inflammatory properties.

Mutagenicity of 7-ketocholesterol in CHO cells: The role of lipid peroxidation.

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 µM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

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.

Inflammatory and cell death mechanisms induced by 7-ketocholesterol in the retina. Implications for age-related macular degeneration.

This review will focus on the inflammatory and toxic mechanism of action of 7-ketocholesterol (7KCh) and the potential implications of its accumulation, especially in the retina. 7KCh is a pro-inflammatory oxysterol usually associated with oxidized lipoprotein deposits present in aged retinas. High amounts of 7KCh can be generated in situ in these lipoprotein deposits possibly through a free radical-mediated mechanism catalyzed by iron. 7KCh seems to activate several kinase signaling pathways that work via multiple transcription factors to induce cytokines and intracellular effectors causing cell death. There seems to be a controversy in the literature in relation to the mechanisms of death induced by 7KCh. Some of the discrepancies arise from the way the oxysterol is delivered because different signaling pathways are activated in different experimental setups. The elucidation of the inflammatory and toxic mechanisms is crucial for the discovery and design of new therapies. Importantly, there is little evidence of 7KCh detoxifying mechanisms in the retina, although some potential enzymes have been described. Thus, continuous formation throughout life and potential toxicity of 7KCh points it out as an "age-related" risk factor in pathologies such as age-related macular degeneration.

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.

7keto-stigmasterol and 7keto-cholesterol induce differential proteome changes to intestinal epitelial (Caco-2) cells.

Recent studies have expanded the appreciation of the roles of oxysterols triggering inflammatory, immune cytotoxic and apoptotic processes, but have not been considered for proteome analysis. A comparative proteomic study in intestinal epithelial cell cultures incubated (60 µM/24 h) with 7keto-cholesterol or 7keto-stigmasterol was performed. The influence of both compounds was studied following the nLC-TripleTOF analysis. Findings were compared to results for control cultures. In the principal component analysis (PCA) of proteome patterns, two components were extracted accounting for 99.8% of the variance in the protein expression. PCA analysis clearly discriminated between the perturbations in the proteome of cell cultures incubated with 7keto-cholesterol and 7keto-stigmasterol. These proteins participate in mitochondrial function, lipid homeostasis, inflammation and immunity and cell proliferation. Remarkable differences between proteome patterns in cell cultures exposed to 7keto-cholesterol and 7keto-stigmasterol affect macrophage migration inhibitory factor, apolipoprotein E, Bcl-2-associated transcription factor and cellular retinoic acid-binding protein. Besides, exposure to 7keto-stigmasterol increased the concentration of ubiquitin-conjugating enzyme E2 and the mitochondrial superoxide dismutase protein. Such findings raise new questions about safety studies and the regulatory potential of oxysterols in the differentiation and function of intestinal and associated immune cells, their response to environmental stimuli and impairment of absorption processes.

Quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside attenuates cholesterol oxidation product-induced apoptosis by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

Cholesterol oxidation products are suggested to be involved in neuronal cell degeneration. We examined the preventive effect of quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside (QGR), a quercetin derivative, on the cholesterol oxidation product-induced neuronal cell death using differentiated PC12 cells in relation to nuclear factor (NF)-κB-mediated apoptotic process. 7-Ketocholesterol and 25-hydroxycholesterol induced a decrease in the levels of BH3 interacting-domain death agonist (Bid) and B cell lymphoma 2 (Bcl-2), increase in the levels of Bcl-2-associated X protein (Bax) and p53, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases, and cleavage of poly(ADP-ribose) polymerase 1 (PARP-1). 7-Ketocholesterol induced increase in cytosolic and nuclear NF-κB p65, nuclear phospho-NF-κB p65, cytosolic NF-κB p50, and cytosolic phospho-IκB-α levels. The addition of QGR, N-acetyl cysteine, or Bay 11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of NF-κB, formation of reactive oxygen species, depletion of glutathione (GSH), nuclear damage, and cell death. The results show that QGR may attenuate the cholesterol oxidation product-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that is mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

Toxicity of 7-ketocholesterol on lethality, growth, reproduction, and germline apoptosis in the nematode Caenorhabditis elegans.

7-Ketocholesterol is one of the most abundant cholesterol oxides, and is known to be cytotoxic to various types of cultured mammalian cells; however, little is known regarding its effects in vivo. With the use of the nematode Caenorhabditis elegans as model organism, in vivo toxicity of 7-ketocholesterol was investigated. The aim of the study was to examine the effects on life span, as well as short-term effects on reproduction, thermotolerance, germline apoptosis, and reactive oxygen species (ROS) generation resulting from C. elegans exposure to 7-ketocholesterol at concentrations ranging from 0 to 200 µg/ml. Results indicated that 7-ketocholesterol reduced reproductive capacity, shortened the life span in a concentration-dependent manner, and impaired thermotolerance of the adult nematode. 7-Ketocholesterol also induced germline apoptotic cell death and increased ROS generation in adult worms. Thus, the model organism C. elegans is recommended for assessment of the safety and bioactivity of cholesterol oxides.

Differential effects of the estrogen receptor agonist estradiol on toxicity induced by enzymatically-derived or autoxidation-derived oxysterols in human ARPE-19 cells.

PURPOSE/AIM OF THE STUDY: Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER. MATERIALS AND METHODS: ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels. RESULTS: Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC. CONCLUSIONS: Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.

Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53.

Egr-1 and p53 are involved in pathology of both atherosclerosis and cancer. However, it is unknown whether p53 and Egr1 are interactively involved in apoptosis in atherosclerosis. We found that in human carotid plaques, the expression of p53 was inversely correlated with Egr1. In U937 cells, 7ß-hydroxycholesterol and 7-ketocholesterol induced production of reactive oxygen species (ROS), transient up-regulation of Egr1 followed by late induction of p53 and apoptosis. Cells with nuclear fragmentation induced by 7-oxysterol or p53 showed increased levels of p53, but decreased levels of Egr1. In conclusion, ROS induced by 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7-oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression.

Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase.

7-Ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 µM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cytotoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications.

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3ß-hydroxysterol-Δ(7)-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4ß-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4ß-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d(7)-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25µmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols.

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7ß-hydroxycholesterol (7ßOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24h, which was preceded by early lipid accumulation (6h) and lysosomal membrane permeabilization (6-12h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7ßOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7ßOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.

Proinflammatory effect of cholesterol and its oxidation products on CaCo-2 human enterocyte-like cells: effective protection by epigallocatechin-3-gallate.

Cholesterol and its oxidation products, namely oxysterols, have very recently been shown to potentially interfere with homeostasis of the human digestive tract, by promoting and sustaining irreversible damage of the colonic epithelial layer. This report concerns the strong proinflammatory action that a dietary oxysterol mixture and, to a lesser extent, an identical concentration of unoxidized cholesterol exert on CaCo-2 colonic epithelial cells by up-regulating both expression and synthesis of interleukin 8. The oxysterol mixture and its most effective component, 7ß-hydroxycholesterol, are also shown to markedly enhance the expression of key inflammatory and chemotactic cytokines in colonic epithelial cells, more efficiently than unoxidized cholesterol. The sterols' proinflammatory effect seems to be mediated by enhanced activation of NOX1, because it is prevented by pretreatment of the cells with DPI, a selective inhibitor of this oxidase. Importantly, NOX1 hyperactivation by the oxysterol mixture or cholesterol was fully prevented by CaCo-2 cell preincubation with epigallocatechin-3-gallate. Consistently, supplementation with this compound fully protected colonic epithelial cells against overexpression of inflammatory and chemotactic genes induced by the sterols investigated.

7-Ketocholesterol and cholesterol-5alpha,6alpha-epoxide induce smooth muscle cell migration and proliferation through the epidermal growth factor receptor/phosphoinositide 3-kinase/Akt signaling pathways.

Oxysterols, the major components of oxidized low-density lipoproteins (ox-LDLs), are present in atherosclerotic plaque and are suggested to play an active role in plaque development. The formation of an atherosclerotic lesion occurs through activation of cellular events that include vascular smooth muscle cell (SMC) migration and proliferation. Therefore, we investigated the roles of two common oxysterols, 7-ketocholesterol (7-keto) and cholesterol-5alpha,6alpha-epoxide (alpha-epoxide) on SMCs. Our results showed that 7-keto and alpha-epoxide promoted SMC migration by a chemotactic assay, and induced mitogenic effects by MTT assay and BrdU assay. Specific inhibitors confirmed that MMPs, EGFR and PI3K are involved in oxysterol-induced SMC migration, while EGFR, ERK, Akt, and sphingomyelin/ceramide pathways might play a role in SMC proliferation. More, the co-immunoprecipitation study indicated that 7-keto and alpha-epoxide caused EGFR phosphorylation and there was an interaction between EGFR and PI3K. At protein expression level, Akt and ERK were activated, at messenger RNA level, MMP-2/9 mRNA was transcribed, at enzyme activity level, the MMP-2/9 enzyme activity were increased in SMCs treated with 7-keto and alpha-epoxide according to Western bolt, RT-PCR and a fluorogenic substrate. Taken together, we concluded that 7-keto and alpha-epoxide may be an atherogenic factor by stimulating SMC migration and proliferation.

7-ketocholesterol induces cell apoptosis by activation of nuclear factor kappa B in mouse macrophages.

We investigated the molecular mechanisms responsible for the induction of apoptosis in mouse monocytic macrophage cell line J774A.1 stimulated by 7-ketocholesterol (7-KC). Cell apoptosis was detected by Annexin V-propidium iodide (PI) staining. The DNA-binding activity of nuclear factor kappa B (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that 7-KC-stimulation in J774A.1 cells activated NF-kappaB, which is involved in cell apoptosis, in a time- and dose-dependent manners. 7-KC was also found to increase the binding activity of NF-kappaB to specific DNA binding sites, a possible mechanism for the induction of the cell apoptosis. Moreover, these effects were partially inhibited by pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor. Taken together, 7-KC may be an important factor in atherosclerosis due to the ability of 7-KC to induce cell apoptosis, which is at least partially mediated through the activation of NF-kappaB.

Tyrosine kinase inhibitor AG126 reduces 7-ketocholesterol-induced cell death by suppressing mitochondria-mediated apoptotic process.

The preventive effect of tyrosine kinase inhibitor AG126 against the 7-ketocholesterol toxicity was investigated in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, the mitochondrial membrane permeability changes, the formation of reactive oxygen species and the depletion of GSH, which leads to cell death in differentiated PC12 cells. Tyrphostin AG126 significantly attenuated the 7-ketocholesterol-induced decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic pro-apoptotic Bax levels, mitochondrial membrane potential loss, cytochrome c release and subsequent caspase-3 activation. The inhibitory effect of tyrphostin AG126 may be supported by the inhibitory effect on another oxysterol 25-hydroxycholesterol-induced cell death. The results show that tyrphostin AG126 may prevent the 7-ketocholesterol toxicity by suppressing the mitochondrial membrane permeability change that leads to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and the depletion of GSH.

Methionine sulfoxide reductase B2 is highly expressed in the retina and protects retinal pigmented epithelium cells from oxidative damage.

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial enzyme that converts methionine sulfoxide (R) enantiomer back to methionine. This enzyme is suspected of functioning to protect mitochondrial proteins from oxidative damage. In this study we report that the retina is one of the human tissues with highest levels of MSRB2 mRNA expression. Other tissues with high expression were heart, kidney and skeletal muscle. Overexpression of an MSRB2-GFP fusion protein increased the MSR enzymatic activity three-fold in stably transfected cultured RPE cells. This overexpression augmented the resistance of these cells to the toxicity induced by 7-ketocholesterol, tert-butyl hydroperoxide and all-trans retinoic acid. By contrast, knockdown of MSRB2 by a miRNA in stably transfected cells did not convey increased sensitivity to the oxidative stress. In the monkey retina MSRB2 localized to the ganglion cell layer (GLC), the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE). MSRB2 expression is most pronounced in the OPL of the macula and foveal regions suggesting an association with the cone synaptic mitochondria. Our data suggests that MSRB2 plays an important function in protecting cones from multiple type of oxidative stress and may be critical in preserving central vision.

Glycyrrhizin prevents 7-ketocholesterol toxicity against differentiated PC12 cells by suppressing mitochondrial membrane permeability change.

Defects in mitochondrial function participate in the induction of neuronal cell injury. In neurodegenerative conditions, oxidative products of cholesterol are elevated and oxysterols seem to be implicated in neuronal cell death. The present work was designed to study the inhibitory effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against the toxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, loss of the mitochondrial transmembrane potential, increase in the cytosolic Bax and cytochrome c levels, caspase-3 activation and cell death in differentiated PC12 cells. Glycyrrhizin and 18beta-glycyrrhetinic acid prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death. The results obtained show that glycyrrhizin and 18beta-glycyrrhetinic acid may prevent the 7-ketocholesterol-induced neuronal cell damage by suppressing changes in the mitochondrial membrane permeability.