Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside.

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 µmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Bioavailability and Biological Effects of 2- O-ß-d-Glucopyranosyl-carboxyatractyligenin from Green Coffee in Caenorhabditis elegans.

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.

Atractyligenin, a terpenoid isolated from coffee silverskin, inhibits cutaneous photoaging.

Ultraviolet (UV) light exposure-induced photoaging of the skin is a multifactorial process involving both extrinsic and intrinsic cellular mechanisms. Several naturally occurring products are known to confer protection against UV light-induced skin damage. Our preliminary studies confirmed that the ethyl acetate fraction of coffee silverskin exhibits inhibitory effects on matrix metalloproteases (MMPs). Furthermore, we previously isolated and identified atractyligenin, which has MMP-inhibitory activity, from the silverskin ethyl acetate fraction. The aim of this study was to elucidate the anti-photoaging effects of atractyligenin on human dermal fibroblasts and the underlying mechanism. Human dermal fibroblasts were exposed to 8 J/cm2 UVA radiation, and cell viability was analyzed by MTT assay. The fluorescent dye 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) was used to measure the intracellular reactive oxygen species (ROS) levels. Our study showed that atractyligenin significantly suppressed the expression of UVA-induced MMPs by inhibiting intracellular ROS production. Atractyligenin treatment reduced c-Jun phosphorylation and c-Fos expression by inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway activated by UVA irradiation. Additionally, treatment with atractyligenin contributed to the homeostasis of collagen by restoring the loss of collagen absorption-related receptor Endo180 and altered fibroblast morphology induced by UVA irradiation. These results indicate that atractyligenin isolated from coffee silverskin inhibits multiple pathways in the human skin photoaging process and is thus a potential candidate for treatment or prevention of photoaging.

Nutritional Preconditioning of Apigenin Alleviates Myocardial Ischemia/Reperfusion Injury via the Mitochondrial Pathway Mediated by Notch1/Hes1.

Apigenin (Api), a natural flavone found in high amounts in several herbs, has shown potent cardioprotective effects in clinical studies, although the underlying mechanisms are not clear. We hypothesized that Api protects the myocardium from simulated ischemia/reperfusion (SI/R) injury via nutritional preconditioning (NPC). Rats fed with Api-containing food showed improvement in cardiac functions; lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities; infarct size; apoptosis rates; malondialdehyde (MDA) levels; caspase-3, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities; and ferric reducing antioxidant power (FRAP) compared to those fed standard chow following SI/R injury. In addition, Api pretreatment significantly improved the viability, decreased the LDH activity and intracellular reactive oxygen species (ROS) generation, alleviated the loss of mitochondrial membrane potential (MMP), prevented the opening of the mitochondrial permeability transition pore (mPTP), and decreased the caspase-3 activity, cytochrome c (Cyt C) release, and apoptosis induced by SI/R in primary cardiomyocytes. Mechanistically, Api upregulated Hes1 expression and was functionally neutralized by the Notch1 γ-secretase inhibitor GSI, as well as the mPTP opener atractyloside (Atr). Taken together, Api protected the myocardium against SI/R injury via the mitochondrial pathway mediated by the Notch1/Hes1 signaling pathway.

Sasanquasaponin-induced cardioprotection involves inhibition of mPTP opening via attenuating intracellular chloride accumulation.

Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.

Sevoflurane pre-conditioning increases phosphorylation of Erk1/2 and HO-1 expression via inhibition of mPTP in primary rat cortical neurons exposed to OGD/R.

BACKGROUND: As an indispensable clinical inhalation anesthetic, sevoflurane is widely used for peri-operative sedation. The neuroprotective effect of sevoflurane pre-conditioning against cerebral ischemia/reperfusion has been gradually realized, but the underlying mechanism during the early reperfusion period has not been established. METHOD: Primary cultured cortical neurons were treated with 2% sevoflurane pre-conditioning for 30min, exposed to oxygen-glucose deprivation for 90min, and followed by 60min of reperfusion (OGD/R). Additionally, neuronal cells were treated with an inhibitor of extracellular signal-related kinases 1 and 2 (Erk1/2) phosphorylation (PD98059), a mPTP opener (atractyloside), or a mPTP opening inhibitor (cyclosporine A) before sevoflurane pre-conditioning. RESULT: Sevoflurane pre-conditioning decreased neuronal apoptosis (assessed by TUNEL), oxidative stress (assessed by malondialdehyde [MDA], superoxide dismutase [SOD], and heme oxygenase [HO]-1), and opening of mitochondrial permeability transition pores [mPTPs] (assessed by calcein-cobalt), but increased neuronal viability (assessed by MTT) and mitochondrial membrane potential (assessed by JC-1) after OGD/R exposure compared with OGD/R treatment alone. Pre-treatment with the mPTP opener and inhibitor of Erk1/2 phosphorylation abolished the protective effect induced by sevoflurane pre-conditioning. Pre-treatment with the mPTP opener attenuated the phosphorylation of Erk1/2 in mitochondria of neuronal cultures exposed to OGD/R induced by sevoflurane pre-conditioning. The mPTP opening inhibitor, like sevoflurane pre-conditioning, increased phosphorylation of Erk1/2 after OGD/R exposure, while PD98059 failed to reverse inhibition of mPTP opening in cultures exposed to OGD/R induced by sevoflurane pre-conditioning. CONCLUSION: The neuroprotective mechanism of sevoflurane pre-conditioning might be associated with increased Erk1/2 phosphorylation in mitochondria via inhibition of mPTP opening in the early reperfusion period.

Raw coffee based dietary supplements contain carboxyatractyligenin derivatives inhibiting mitochondrial adenine-nucleotide-translocase.

Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-ß-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside, and 2-O-ß-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6µmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement.

Acute starvation in C57BL/6J mice increases myocardial UCP2 and UCP3 protein expression levels and decreases mitochondrial bio-energetic function.

Associations between uncoupling protein (UCP) expression and functional changes in myocardial mitochondrial bio-energetics have not been well studied during periods of starvation stress. Our aim was to study the effects of acute starvation, for 24 or 48 h, on combined cardiac mitochondrial function and UCP expression in mice. Isolated heart mitochondria from female mice starved for 48 h compared to that from mice fed revealed a significantly (p < 0.05) decreased adenosine diphosphate-to-oxygen ratio, a significantly increased proton leak and an increased GTP inhibition on palmitic acid-induced state 4 oxygen consumption (p < 0.05). These bio-energetic functional changes were associated with increases in mitochondrial UCP2 and UCP3 protein expression. In conclusion, our findings suggest that increased UCP2 and UCP3 levels may contribute to decreased myocardial mitochondrial bio-energetic function due to starvation.

Antioxidant action of a Chrysanthemum morifolium extract protects rat brain against ischemia and reperfusion injury.

The present study evaluated the potential neuroprotective effect and underlying mechanism of the total flavones extracted from Chrysanthemum morifolium (TFCM) against ischemia/reperfusion (I/R) injury. An animal model of cerebral ischemia was established by occluding the right middle cerebral artery for 90 minutes followed by reperfusion for 22 hours. The neurobehavioral scores, infarct area, and hemispheric edema were evaluated. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) level in brain were also measured. The results showed that pretreatment with TFCM significantly decreased the neurological deficit scores, percentage of infarction, and brain edema and attenuated the decrease in SOD activity, the elevation of MDA content, and the generation of ROS. In isolated brain mitochondria, Ca(2+)-induced swelling was attenuated by pretreatment with TFCM, and this effect was antagonized by atractyloside. These results showed that pretreatment with TFCM provides significant protection against cerebral I/R injury in rats by, at least in part, its antioxidant action and consequent inhibition of mitochondrial swelling.

Plasmodium falciparum: functional mitochondrial ADP/ATP transporter in Escherichia coli plasmic membrane as a tool for selective drug screening.

Plasmodium falciparum mitochondrial ADP/ATP transporter or adenylate translocase (PfAdT) was previously characterised at the molecular level and intracellularly located by immuno-electromicroscopy. Inhibition of this transporter blocks parasite development in erythrocytes. In this study, PfAdT was expressed in C43 (DE3) Escherichia coli strain under isopropyl beta-d-thiogalacto-pyranoside (IPTG) induction to screen inhibitory molecules. PfAdT was integrated directly into the bacterial cytoplasmic membrane. Whereas IPTG-induced bacterial cells imported radioactively labelled ATP, non-induced cells did not. The transporter bound specifically ADP and ATP, but not AMP. IPTG-induced cells preloaded with labelled ATP exported ATP after exogenous addition of unlabelled ADP or ATP, indicating a counter exchange transport mechanism. Bongrekic acid and atractyloside, two well-known specific inhibitors of mitochondrial ADP/ATP transporter, were tested. This experimental model was evaluated using three Malagasy crude plants extracts which have shown antiplasmodial activity on in vitro parasite cultures.

Cytotoxic activity of some natural and synthetic ent-kauranes.

Atractyligenin (1) and several synthetic derivatives were tested and found to be active against tumor cell replication. Compound 1 was readily converted to the 2,15-diketo (3) or 15-keto (4) derivatives, which contain an alpha,beta-unsaturated ketone. Compounds 3 and 4 showed significant cytotoxic activity against all six tested cancer cell lines and were most potent against 1A9 ovarian cancer cells with EC50 values of 0.2 and 0.3 microM, respectively. These two 1-analogues are promising lead compounds for further investigation.

Antioxidant MCI-186 inhibits mitochondrial permeability transition pore and upregulates Bcl-2 expression.

Reperfusion after a period of ischemia is associated with the formation of reactive oxygen species (ROS) and Ca2+ overload resulting in the opening of a nonspecific pore in the inner membrane of the mitochondria, called the mitochondrial permeability transition pore (PTP), leading to cell damage. Although endogenous antioxidants are activated because of oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury. Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia. Here we demonstrated the effects of the antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in preventing reperfusion injury of the heart by inhibition of PTP opening. Ischemia (30 min) by left coronary artery (LCA) occlusion and reperfusion (120 min) in Wistar rats after pretreatment with MCI-186 (10 mg/kg iv) infusion starting from 30 min before LCA occlusion resulted in 1) less area of myocardial infarction (19.2% vs. 61.6%), 2) well-maintained myocardial ATP content (P < 0.03 vs. control), 3) decreased mitochondrial swelling and reduced cytochrome c release, 4) increased expression of BCl-2, 5) lower prevalence of apoptotic cells (14.3% vs. 2.9%), and 6) reduced DNA fragmentation in the MCI-186-treated group. These cytoprotective effects of MCI-186 were inhibited on opening PTP before MCI-186 treatment with the PTP activators lonidamine (10 mg/kg iv) or atractyloside (5 mg/kg iv) but failed to inhibit the protective effects exerted by another antioxidant, allopurinol, suggesting that the PTP inhibiting property is specific for MCI-186. These results demonstrate that the radical scavenger MCI-186, by inhibiting the opening of the PTP, prevents necrosis and cytochrome c release and hence pathological apoptosis.

Atractyligenine chemistry, Part VI: Synthesis and biological activities of atractyligenine derivatives.

Derivatives of atractyligenine, the aglycone of atractyloside, were tested in vitro for their antimicrobial and antiproliferative activity against K-562 (human chronic myelogeneous leukemia), HL-60 (human leukemia) and MCF-7 (human breast adenocarcinoma) cell lines. The most active compound showed IC50 values in the range 0.8-6.9 microM.

Fatty acid-mediated uncoupling of potato tuber mitochondria.

The present work examined whether the ATP/ADP carrier, other than the plant uncoupling mitochondrial protein, participates in free fatty acid-mediated uncoupling of potato tuber mitochondria. The basal respiration rate of succinate-energized mitochondria was stimulated by a low concentration of palmitate (20 microM). This uncoupling was reversed by 10 microM carboxyatractyloside and by the subsequent addition of 0.1% bovine serum albumin. The decrease in membrane potential caused by palmitate was suppressed by carboxyatractyloside (1 microM) and, to a lesser degree, by bongkrekate (20 microM). GTP could also reversed this decrease via a carboxyatractyloside-independent mechanism. These results indicate that the ATP/ADP carrier, along with the plant uncoupling mitochondrial protein, participates in the protonophoric action of palmitate in potato tuber mitochondria.

Effect of carboxyatractylate on transmembrane electrical potential of plant mitochondria in different metabolic states.

The effects of carboxyatractylate (CAtr) on delta psi in sunflower hypocotyl and pea stem mitochondria were compared. In sunflower mitochondria, (1) CAtr at higher concentration increased delta psi in the presence of palmitate and delta psi in metabolic state 3; (2) ]1 microM CAtr did not prevent delta psi decrease, induced by ADP addition (in contrast to pea mitochondria); (3) The ATP-generated delta psi was small and was insensitive to 40 microM CAtr. Under the same conditions, in pea mitochondria generation of delta psi by ATP was inhibited by 1 microM CAtr.

ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate.

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially the delta psi.

ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate.

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially delta psi.

Metabolic action of N-methyl-D-aspartate in newborn rat brain ex vivo: 31p magnetic resonance spectroscopy.

N-methyl-D-aspartate (NMDA) is an agonist used to identify neuronal receptive sites for dicarboxylic amino acid neurotransmitters; NMDA receptors are implicated in neuronal damage of ischemic or hypoglycemic origin in newborns although involved mechanisms remain to be identified. In the present study, 31P magnetic resonance spectroscopy with fast (6/min) data acquisition was used in newborn rat brain slices to measure changes of intracellular phosphocreatine and nucleotide triphosphate levels following extracellular NMDA applications. The rapid exhaustion of phosphocreatine stores in 50% of the total population of brain cells was induced in all cases by application of NMDA (30-45 s, 25-100 mM). It was not reproduced by other excitatory agents: potassium ions (24.6 mM, 4 min), isobutylxanthine (1mM), muscarine (10 mM), serotonin (0.1 mM) or substance P (10 microM). Such an effect of NMDA was not modified after tetrodotoxin (1 microM) and was reduced by extracellular 2-amino-5-phosphonovalerate (50 microM) or magnesium ions (2.2 mM). However it did develop during NMDA-induce neuronal excitations and was reversible within 10-30 min. This action of NMDA was followed by an irreversible decrease of phosphorus metabolites if mitochondrial creatine kinase and adenosine triphosphatase were decoupled by atractyloside (50 microM). Experiments revealed a link between selective NMDA action at neuronal plasma membranes, neurotoxicity and energy production by mitochondria.

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3.

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.