BACKGROUND: Formation of cholesterol oxidation products is a suggested mechanism of neurodegenerative disorders. Neuronal cell death is mediated by an increased release of excitotoxic glutamate from the presynaptic nerve endings. Tyrosine-specific protein kinases modulate neurotransmitter release at the nerve terminals. Tyrphostin AG126 has anti-inflammatory and cytoprotective effects. However, it remains uncertain whether tyrphostin AG126 has a preventive effect on the alteration of nerve terminal function induced by cholesterol oxidation products. METHODS: The present study was performed to assess the effect of cholesterol oxidation products against nerve terminal function using synaptosomes isolated from rat cerebrum. We determined the preventive effect of tyrphostin AG126 against oxysterol toxicity by measuring the effects on the glutamate release, depolarization of the membrane potential, changes in Ca2+ levels, and Na+/K+-ATPase activity. RESULTS: Synaptosomes treated with 7-ketocholesterol or 25-hydroxycholesterol exhibited a sustained release of glutamate, depolarization of membrane potential, early rapid increase in cellular Ca2+ levels and decrease in Na+/K+-ATPase activity. Those responses were concentration-dependent. Treatment of tyrphostin AG126 interfered with alteration of synaptosomal functions and decrease in Na+/K+-ATPase activity induced by 7-ketocholesterol or 25-hydroxycholesterol. CONCLUSIONS: The results show that 7-ketocholesterol and 25-hydroxycholesterol seem to cause the release of glutamate by inducing depolarization of the membrane potential and early rapid increase in cellular Ca2+ levels and by inactivating Na+/K+-ATPase in the cerebral synaptosomes. Treatment of tyrphostin AG126 may prevent the oxysterol-induced nerve terminal dysfunction.
Animals ; Brain ; Cell Death ; Cerebrum ; Cholesterol ; Glutamic Acid ; Hydroxycholesterols ; Ketocholesterols ; Membrane Potentials ; Neurodegenerative Diseases ; Neurons ; Neurotransmitter Agents ; Presynaptic Terminals ; Protein-Tyrosine Kinases ; Rats ; Synaptosomes ; Tyrphostins

OBJECTIVE: Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxycholesterol (7β-OHC). Thus, it is imperative to identify agents that can prevent 7β-OHC-induced side-effects. METHODS: We evaluated the potential protective effects of Carpobrotus edulis ethanol-water extract (EWe) on murine oligodendrocytes (158N) cultured in the absence or presence of 7β-OHC (20 μg/mL, 24 h). The cells were incubated with EWe (20-200 µg/mL) 2 h before 7β-OHC treatment. Mitochondrial activity and cell growth were evaluated with the MTT assay. Photometric methods were used to analyze antioxidant enzyme [catalase (CAT) and glutathione peroxidase (GPx)] activities and the generation of lipid and protein oxidation products [malondialdehyde (MDA), conjugated diene (CD), and carbonylated proteins (CPs)]. RESULTS: Treatment with 7β-OHC induced cell death and oxidative stress (reflected by alteration in CAT and SOD activities). Overproduction of lipid peroxidation products (MDA and CDs) and CPs was also reported. The cytotoxic effects associated with 7β-OHC were attenuated by 160 μg/mL of EWe of C. edulis. Cell death induced by 7β-OHC treatment was ameliorated, GPx and CAT activities were restored to normal, and MDA, CD, and CP levels were reduced following C. edulis extract treatment. CONCLUSION These data demonstrate the protective activities of C. edulis EWe against 7β-OHC-induced disequilibrium in the redox status of 158N cells, indicative of the potential role of this plant extract in the prevention of neurodegenerative diseases.
Aizoaceae ; Animals ; Cell Line ; Drug Evaluation, Preclinical ; Hydroxycholesterols ; Mice ; Neurodegenerative Diseases ; prevention & control ; Neuroprotection ; Oligodendroglia ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use

Cholesterol is an essential component for neuronal physiology not only during development stage but also in the adult life. Cholesterol metabolism in brain is independent from that in peripheral tissues due to blood-brain barrier. The content of cholesterol in brain must be accurately maintained in order to keep brain function well. Defects in brain cholesterol metabolism has been shown to be implicated in neurodegenerative diseases, such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), and some cognitive deficits typical of the old age. The brain contains large amount of cholesterol, but the cholesterol metabolism and its complex homeostasis regulation are currently poorly understood. This review will seek to integrate current knowledge about the brain cholesterol metabolism with molecular mechanisms.
ATP-Binding Cassette Transporters ; genetics ; metabolism ; Alzheimer Disease ; genetics ; metabolism ; pathology ; Blood-Brain Barrier ; Brain ; metabolism ; pathology ; Cholesterol ; metabolism ; Gene Expression Regulation ; Homeostasis ; Humans ; Huntington Disease ; genetics ; metabolism ; pathology ; Hydroxycholesterols ; metabolism ; Lipid Metabolism ; genetics ; Neurons ; metabolism ; pathology ; Parkinson Disease ; genetics ; metabolism ; pathology ; Receptors, Lipoprotein ; genetics ; metabolism

OBJECTIVEThis study was designed to examine the in vitro effects of fenvalerate on steroid production and steroidogenic enzymes mRNA expression level in rat granulosa cells.

METHODSUsing primary cultured rat granulosa cells (rGCs) as model, fenvalerate of various concentrations (0, 1, 5, 25, 125, 625 micromol/L) was added to the medium for 24 h. In some cases, optimal concentrations of 22(R)-hydroxycholesterol (25 micromol/L), Follicle stimulating hormone (FSH, 2 mg/L), or 8-Bromo-cAMP (1 mmol/L) were provided. Concentrations of 17 beta-estradiol(E2) and progesterone (P4) in the medium from the same culture wells were measured by RIA and the steroidogenic enzyme mRNA level was quantified by semi-quantitative RT-PCR.

RESULTSFenvalerate decreased both P4 and E2 production in a dose-dependent manner while it could significantly stimulate rGCs proliferation. This inhibition was stronger in the presence of FSH. Furthermore, it could not be reversed by 22(R)-hydroxycholesterol or 8-Bromo-cAMP. RT-PCR revealed that fenvalerate had no significant effect on 3 beta-HSD, but could increase the P450scc mRNA level. In addition, 17 beta-HSD mRNA level was dramatically reduced with the increase of fenvalerate dose after 24 h treatment.

CONCLUSIONFenvalerate inhibits both P4 and E2 production in rGCs. These results support the view that fenvalerate is considered as a kind of endocrine-disrupting chemicals. The mechanism of its disruption may involve the effects on steroidogenesis signaling cascades and/or steroidogenic enzyme's activity.

3-Hydroxysteroid Dehydrogenases ; analysis ; metabolism ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Animals ; Base Sequence ; Cells, Cultured ; Dose-Response Relationship, Drug ; Estradiol ; analysis ; metabolism ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Hydroxycholesterols ; pharmacology ; Nitriles ; pharmacology ; Progesterone ; analysis ; metabolism ; Pyrethrins ; pharmacology ; RNA, Messenger ; analysis ; metabolism ; Rats ; Steroids ; metabolism

BACKGROUNDABCA7 is a member of the ABCA subfamily that shows a high degree of homology to ABCA1 and, like ABCA1, mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 has been shown to be downregulated by increased cellular cholesterol while ABCA1 was upregulated.

METHODSThe underlying mechanism for this effect was examined in ABCA1 or ABCA7-transfected HEC293. Lipid content in the medium and cells was determined by enzymatic assays. Gene expression was quantitated by real time PCR, and protein content was determined by Western blotting.

RESULTSWhile ABCA7 mRNA was decreased by 25-hydroxycholesterol treatment, ABCA1 was apparently increased. Treatment with the synthetic LXR agonist T0901317 (T09) upregulated ABCA1 expression and apoAI-mediated cellular lipid release in ABCA1-transfected HEC293 cells, but ABCA7 expression and cellular lipid release in ABCA7-transfected HEC293 cells showed no obvious changes.

CONCLUSIONThe ABCA7 gene is regulated by sterol in a direction opposite to that of ABCA1.

ATP Binding Cassette Transporter 1 ; analysis ; genetics ; physiology ; ATP-Binding Cassette Transporters ; analysis ; genetics ; physiology ; Amino Acid Sequence ; Apolipoprotein A-I ; physiology ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Hydrocarbons, Fluorinated ; pharmacology ; Hydroxycholesterols ; pharmacology ; Lipid Metabolism ; Liver X Receptors ; Molecular Sequence Data ; Orphan Nuclear Receptors ; agonists ; Sulfonamides ; pharmacology

OBJECTIVETo study the activation of sterol regulatory element binding protein (SREBP) and its critical role in endothelial cell migration.

METHODSBovine aortic endothelial cells (ECs) were cultured. The expression of SREBP and Cdc42 were determined by Western blot and quantitative real-time PCR. Moreover, outward growth migration model and transwell chamber assay were used to detect ECs migration.

RESULTS(1) SREBP was activated during ECs migration. Western blot analysis demonstrated increased active form SREBP in migrating as compared to non-migrating ECs population. SREBP activation decreased as ECs migration slowed;(2) Coincidental with SREBP activation, mRNA expression of its target genes such as low density lipoprotein receptor, HMG-CoA reductase, and fatty acid synthase also increased in migrating ECs population as detected by real-time PCR; (3) Migration induced SREBP activation in ECs was inhibited by SREBP-acting protein RNAi and pharmacologically by 25-hydroxycholesterol; (4) Inhibition of SREBP led to decreased ECs migration in various models; (5) Cells genetically deficient in SREBP-acting protein, S1P, or S2P, phenotypically exhibited impaired migration; (6) SREBP inhibition in ECs suppressed the activity of small GTPase Cdc42, a key molecule for ECs motility.

CONCLUSIONSSREBP is activated during and plays a critical role in ECs migration. Targeting SREBP could become a novel approach in fighting diseases involving abnormal ECs migration.

Animals ; Aorta ; cytology ; CHO Cells ; Cattle ; Cell Movement ; Cells, Cultured ; Cricetinae ; Cricetulus ; Endothelial Cells ; Fatty Acid Synthases ; genetics ; metabolism ; Hydroxycholesterols ; pharmacology ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; Receptors, LDL ; genetics ; metabolism ; Sterol Regulatory Element Binding Proteins ; metabolism ; physiology