Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

ACEA改善线粒体复合体活性诱导神经保护作用研究

目的:探讨线粒体复合体活性对大麻素CB1受体选择性激动剂ACEA神经保护作用的影响。方法:将原代大鼠皮层神经元分为4组:对照组(Control)、氧糖剥夺组(OGD)、ACEA+OGD组和溶剂(Vehicle)+OGD组,分别检测各组神经元损伤程度和线粒体复合体Ⅰ、Ⅱ和Ⅳ的活性。为进一步证实线粒体复合体活性对ACEA神经保护的影响,将原代大鼠皮层神经元分为5组:对照组(Control)、氧糖剥夺组(OGD)、ACEA+OGD组、线粒体复合体Ⅰ抑制剂(rotenone)+ACEA+OGD组和线粒体复合体Ⅱ抑制剂(TTFA)+ACEA+OGD组,检测和比较各组神经元细胞的损伤情况。结果:在OGD后24小时,ACEA明显增加神经元活性,减少LDH释放,降低神经元凋亡率(P0.05),改善OGD损伤后线粒体复合体Ⅰ和Ⅳ的活性(P0.05),而对复合体Ⅱ的活性没有影响;rotenone可以部分逆转ACEA的神经保护作用(P0.05),但TTFA却没有这一作用。结论:ACEA可以诱导神经保护作用,其机制是与改善线粒体呼吸链复合体活性有关。     

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.     

Oxygen Glucose Deprivation (OGD)/Re-Oxygenation-Induced In Vitro Neuronal Cell Death Involves Mitochondrial Cyclophilin-D/P53 Signaling Axis

Oxidative stress-induced neuronal cell death requires opening of the mitochondrial permeability transition pore. P53 mitochondrial translocation and association with Cyclophilin D (Cyp-D) is required for the pore opening. Here we tested this signaling axis in oxygen glucose deprivation (OGD)/re-oxygenation-induced in vitro neuronal death. Using mitochondrion immunoprecipitation, we found that p53 translocated to mitochondrion and associated with Cyp-D in SH-SY5Y cells exposed to (OGD)/re-oxygenation. Disruption of this complex by Cyp-D inhibitor Cyclosporine A (CsA), or by Cyp-D or p53 deficiency, significantly inhibited OGD/re-oxygenation-induced apoptosis-independent cell death. Conversely, over-expression of Cyp-D in SH-SY5Y cells caused spontaneous cell death, and these cells were more vulnerable to OGD/re-oxygenation. Finally, CsA or Cyp-D RNAi suppressed OGD/re-oxygenation-induced neuronal cell death in primary cultures. Together, our study suggests that OGD/re-oxygenation-induced in vitro cell death involves a mitochondrial Cyp-D/p53 signaling axis.     

Coenzyme Q and the regulation of intracellular steady-state levels of superoxide in HL-60 cells

The present work was set to study how CoQ concentrations affected steady-state levels of superoxide in a cellular model of partial CoQ(10) deficiency in cultured human myeloid leukemia HL-60 cells. Culturing HL-60 cells in the presence of p-aminobenzoate, a competitive inhibitor of polyprenyl-4-hydroxybenzoate transferase (Coq2p), produced a significant decrease of CoQ(10) levels without affecting cell viability. Concomitant decreases in CoQ-dependent electron transport activity and mitochondrial membrane potential were observed under these conditions. Intracellular superoxide was significantly elevated in cells treated with p-aminobenzoate, both under serum-containing and serum-free conditions, and this effect was reversed by exogenous CoQ(10). A slight increase of superoxide was also observed in CoQ(10)-supplemented cells in the absence of serum. Our results support a requirement for CoQ(10) to control superoxide levels in HL-60 cells. The importance of extramitochondrial sources of superoxide in cells with impaired CoQ(10) biosynthesis is discussed.     

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.     

Role of mitochondrial electron transport complex I in coenzyme Q1 reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.     

Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.     

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.     

Nrf2对缺氧/复氧诱导的SH-SY5Y细胞线粒体分裂的影响

目的:通过体外模拟脑缺血再灌注损伤的细胞模型,探究核转录相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)与线粒体分裂是否存在调控关系。方法:通过三气培养箱和无糖培养基模拟氧糖剥夺/复氧(Oxygen and glucose deprivation/reperfusion,OGD/R)的条件,将OGD4 h、6h、8h和10h与R0h、6h、12h、18h、24h多个时间点组合,通过CCK8试剂盒(Cell Counting Kit-8,CCK8)检测细胞的存活率,最终以细胞凋亡明显但仍有半数存活的OGD4 h/恢复R18 h为条件建立细胞OGD/R模型;用Nrf2激动剂叔丁基对苯二酚(Tert-butylhydroquinone,t BHQ)和抑制剂鸦胆子苦醇(Brusatol,Bru)对细胞进行干预处理;蛋白免疫印迹(Western blot,WB)法检测Nrf2、动力相关蛋白1(Dynamin-related protein 1,Drp1)的表达量;制备细胞沉淀的切片,通过电镜观察细胞内线粒体的形态。结果:OGD/R+t BHQ组Nrf2蛋白的表达明显高于OGD/R组,且OGD/R+t BHQ组Drp1蛋白的表达则低于OGD/R组(P0.05),而OGD/R+Bru组Nrf2蛋白的表达低于OGD/R组,且OGD/R+Bru组Drp1蛋白的表达高于OGD/R组(P0.05);电镜观察结果显示OGD/R+t BHQ组线粒体分裂的程度和比例较OGD/R组有所减少,而在OGD/R+Bru组有增多(P0.05)。结论:Nrf2对线粒体分裂有负向调控作用,可能机制是经由Drp1蛋白发挥作用。     

Neuroprotective actions of deferiprone in cultured cortical neurones and SHSY-5Y cells

Alzheimer's disease (AD) is a common neurodegenerative disorder, but the initiating molecular processes contributing to neuronal death are not well understood. AD is associated with elevated soluble and aggregated forms of amyloid beta (Abeta) and with oxidative stress. Furthermore, there is increasing evidence for a detrimental role of iron in the pathogenic process. In this context, iron chelation by compounds such as 3-hydroxypyridin-4-one, deferiprone (Ferriprox) may have potential neuroprotective effects. We have evaluated the possible neuroprotective actions of deferiprone against a range of AD-relevant insults including ferric iron, H(2)O(2) and Abeta in primary mouse cortical neurones. We have investigated the possible neuroprotective actions of deferiprone (1, 3, 10, 30 or 100 microM) in primary neuronal cultures following exposure to ferric iron [ferric nitrilotriacetate (FeNTA); 3 and 10 microM], H(2)O(2) (100 microM) or Abeta1-40 (3, 10 and 20 microM). Cultures were treated with deferiprone or vehicle either immediately or up to 6 h after the insult in a 24-well plate format. In order to elucidate a possible neuroprotective action of deferiprone against Parkinson's disease relevant insults another group of experiments were performed in the human neuroblastoma catecholaminergic SHSY-5Y cell line. SHSY-5Y cells were treated with MPP(+) iodide, the active metabolite of the dopaminergic neurotoxin MPTP and the neuroprotective actions of deferiprone evaluated. Cytotoxicity was assessed at 24 h by lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide turnover (FeNTA and hydrogen peroxide) and morphometric analysis of cell viability by Hoechst 33324/propidium iodide (FeNTA, Abeta and MPP(+)) or 6-carboxyfluorescein diacetate and annexin V-Cy3 (Abeta). The present study demonstrates that deferiprone protects against FeNTA, hydrogen peroxide, MPP(+) and Abeta1-40-induced neuronal cell death in vitro, which is consistent with previous in vitro and in vivo studies that have demonstrated similar protection with other iron chelators.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Coenzyme q10 prevents apoptosis by inhibiting mitochondrial depolarization independently of its free radical scavenging property

The permeability transition pore (PTP) is a mitochondrial channel whose opening causes the mitochondrial membrane potential (deltapsi) collapse that leads to apoptosis. Some ubiquinone analogues have been demonstrated previously to modulate the PTP open-closed transition in isolated mitochondria and thought to act through a common PTP-binding site rather than through oxidation-reduction reactions. We have demonstrated recently both in vitro and in vivo that the ubiquitous free radical scavenger and respiratory chain coenzyme Q10 (CoQ10) prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. On this basis, we hypothesized that the antiapoptotic property of CoQ10 could be independent of its free radical scavenging ability and related to direct inhibition of PTP opening. In this study, we have verified this hypothesis by evaluating the antiapoptotic effects of CoQ10 in response to apoptotic stimuli, serum starvation, antimycin A, and ceramide, which do not generate free radicals, in comparison to control, free radical-generating UVC irradiation. As hypothesized, CoQ10 dramatically reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli. This was accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and caspase 9 activation. Because these events are consequent to mitochondrial PTP opening, we suggest that the antiapoptotic activity of CoQ10 could be related to its ability to prevent this phenomenon.     

Mitochondria are direct targets of the lipoxygenase inhibitor MK886. A strategy for cell killing by combined treatment with MK886 and cyclooxygenase inhibitors

We have investigated the mitochondrial and cellular effects of the lipoxygenase inhibitor MK886. Low concentrations (1 microM) of MK886 selectively sensitized the permeability transition pore (PTP) to opening, whereas higher concentrations of MK886 (10 microM) caused depolarization through combination of an ionophoretic effect with inhibition of respiration. MK886 killed prostate cancer PC3 cells only at the higher, toxic concentration (10 microM), whereas the lower concentration (1 microM) had no major effect on cell survival. However, 1 microM MK886 alone demonstrably induced PTP-dependent mitochondrial dysfunction; and it caused cell death through the mitochondrial pathway when it was used in combination with the cyclooxygenase inhibitor, indomethacin, which had no effects per se. Treatment with 1 microM MK886 plus indomethacin sensitized cells to killing by exogenous arachidonic acid, which induces PTP opening and cytochrome c release (Scorrano, L., Penzo, D., Petronilli, V., Pagano, F., and Bernardi, P. (2001) J. Biol. Chem. 276, 12035-12040). Combination of MK886 and cyclooxygenase inhibitors may represent a viable therapeutic strategy to force cell death through the mitochondrial pathway. This approach should be specifically useful to kill cells possessing a high flux of arachidonic acid and its metabolites like prostate and colon cancer cells.     

Cytoplasmic Ca2+ signals and cellular death by apoptosis in myocardiac H9c2 cells

The incubation of H9c2 cells with 10 microM thapsigargin (TG) was associated with the appearance of a two-component cytoplasmic Ca2+ peak. Experiments performed in a Ca2+-free medium indicated that both components came from intracellular sources. The first component of the signal corresponded to the discharge of the sarco-endoplasmic reticulum (SER) Ca2+ store. The appearance of the second component was prevented by cell preincubation with cyclosporin A (CsA) and gave rise to a clear and permanent depolarization of the mitochondrial inner membrane. These features were indication of a mitochondrial origin. The observed release of mitochondrial Ca2+ was related with opening of the permeability transition pore (PTP). The two-component cytoplasmic Ca2+ peak, i.e., treatment with 10 microM TG, as compared with the first component alone, i.e., treatment with 3 microM TG, was associated with a faster process of cellular death. In both cases, chromatin fragmentation and condensation at the nuclear periphery were observed. Other prominent apoptotic events such as loss of DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) were also dependent on TG concentration and occurred in different time windows. PTP opening induced by 10 microM TG was responsible for the faster apoptotic death.     

Pituitary adenylate cyclase-activating polypeptide inhibits caspase-3 activity but does not protect cerebellar granule neurons against beta-amyloid (25-35)-induced apoptosis

The beta-amyloid (Abeta) peptide Abeta25-35 provokes apoptosis of cerebellar granule cells through activation of caspase-3 while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes granule cell survival by inhibiting caspase-3 activation through the intrinsic apoptotic pathway. The aim of the present study was to determine whether PACAP could prevent Abeta25-35 neurotoxicity by inhibiting caspase-3 activity. A 24-h exposure of cultured cerebellar granule cells to Abeta25-35 induced shrinkage of cell bodies, neurite retraction and alteration of mitochondrial activity. Administration of graded concentrations (10-80 microM) of Abeta25-35 induced a dose-related decrease of the number of living cells, and the neurotoxic effect was highly significant after a 24-h exposure to 80 microM Abeta25-35. Exposure of cerebellar granule cells to Abeta25-35 markedly enhanced caspase-3 but not caspase-9 activity. Co-incubation with 1 microM PACAP significantly reduced Abeta25-35-evoked caspase-3 activation. In contrast, PACAP did not prevent the deleterious effects of Abeta25-35 on mitochondrial potential and granule cell survival. Taken together, these data suggest that caspase-3 activation is not the main pathway activated by Abeta25-35 that leads to granule cell death. The results also demonstrate that PACAP cannot be considered as a potent neuroprotective factor against Abeta25-35-induced apoptosis in cerebellar granule neurons.     

Neuroprotective effects of Ptychopetalum olacoides Bentham (Olacaceae) on oxygen and glucose deprivation induced damage in rat hippocampal slices

Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae) are used in traditional medicine by patients presenting age associated symptoms and those recovering from stroke. The aim of this study is to evaluate the neuroprotective properties of PO ethanol extract (POEE) using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation). Mitochondrial activity, an index of cell viability, was assessed by the MTT assay; in addition, the free radicals content was estimated by the use of dichlorofluorescein diacetate as probe. The OGD ischemic condition significantly impaired cellular viability, and increased free radicals generation. In non-OGD slices, incubation with POEE (0.6 microg/ml) increased (approximately 40%) mitochondrial activity, without affecting free radicals levels. In comparison to OGD controls, slices incubated with POEE (0.6 microg/ml) during and after OGD exposure had significantly increased cellular viability. In addition, at this same concentration, POEE prevented the increase of free radicals content induced by OGD. In view of the fact that respiratory chain inhibition and increased generation of free radicals are major consequences of the ischemic injury, this study suggests that Ptychopetalum olacoides contains useful neuroprotective compounds and, therefore, deserves further scrutiny.     

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.     

Effects of Coenzyme Q10 on TNF-alpha secretion in human and murine monocytic cell lines

Studies in humans and cell culture as well as bioinformatics suggested that Coenzyme Q(10) (CoQ10) functions as an anti-inflammatory molecule. Here we studied the influence of CoQ10 (Kaneka Q10) on secretion of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) by using the human and murine monocytic cell lines THP-1 and RAW264.7 expressing human apolipoprotein E3 (apoE3) or pro-inflammatory apoE4. Incubation of cells with physiological (0.1-10 microM) and supra-physiological (> 10 to < 100 microM) concentrations of CoQ10 led to an intracellular accumulation of its reduced form without any cytotoxic effects. Stimulation of cell models with lipopolysaccharide (LPS) resulted in a substantially release of TNF-alpha. When THP-1 cells were pre-incubated with 10 microM CoQ10, the LPS-induced TNF-alpha release was significantly decreased to 72 +/- 32%. This effect is similar to those obtained by 10 microM N-Acetyl-Cysteine, a well known reference antioxidant. In RAW264.7-apoE3 and -apoE4 cells, significant reductions of LPS-induced TNF-alpha secretion to 73.3 +/- 2.8% and 74.7 +/- 8.9% were found with 2.5 microM and 75 microM CoQ10, respectively. In conclusion, CoQ10 has moderate anti-inflammatory effects in two monocytic cell lines which could be mediated by its antioxidant activity.     

The spirostenol (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate blocks mitochondrial uptake of Abeta in neuronal cells and prevents Abeta-induced impairment of mitochondrial function

Abeta(1-42) has been shown to uncouple the mitochondrial respiratory chain and promote the opening of the membrane permeability transition (MPT) pore, leading to cell death. We have previously reported that the spirostenol derivative (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate (SP-233) protects neuronal cells against Abeta(1-42) toxicity by binding to and inactivating the peptide. Picomolar concentrations of Abeta(1-42) decreased the mitochondrial respiratory coefficient in mitochondria isolated from the rat forebrain, and this decrease was partially reversed by SP-233. SP-233 abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone on isolated mitochondria. These results are consistent with a direct effect of SP-233 on the MPT. Moreover, SP-233 displayed a neuroprotective effect on SK-N-AS human neuroblastoma cells treated with the MPT promoter, phenylarsine oxide. Treatment of SK-N-AS cells with Abeta(1-42) resulted in an accumulation of the peptide in the mitochondrial matrix; SP-233 completely scavenged Abeta(1-42) from the matrix. In addition, SP-233 protected the cells against mitochondrial toxins targeting complexes IV and V of the respiratory chain. These results indicate that Abeta(1-42) and SP-233 exert direct effects on mitochondrial function and SP-233 protects neuronal cells against Abeta-induced toxicity by targeting Abeta directly.