Postconditioning of sevoflurane and propofol is associated with mitochondrial permeability transition pore

Background: Sevoflurane and propofol are effective cardioprotective anaesthetic agents, though the cardioprotection of propofol has not been shown in humans. Their roles and underlying mechanisms in anesthetic postconditioning are unclear. Mitochondrial permeability transition pore (MPTP) opening is a major cause of ischemia-reperfusion injury. Here we investigated sevoflurane- and propofol-induced postconditioning and their relationship with MPTP. Methods: Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. During the first 15 min of reperfusion, hearts were treated with either control buffer (CTRL group) or buffer containing 20 μmol/L atractyloside (ATR group), 3% (v/v) sevoflurane (SPC group), 50 μmol/L propofol (PPC group), or the combination of atractyloside with respective anesthetics (SPC+ATR and PPC+ATR groups). Infarct size was determined by dividing the total necrotic area of the left ventricle by the total left ventricular slice area (percent necrotic area). Results: Hearts treated with sevoflurane or propofol showed significantly better recovery of coronary flow, end-diastolic pressures, left ventricular developed pressure and derivatives compared with controls. Sevoflurane resulted in more protective alteration of hemodynamics at most time point of reperfusion than propofol. These improvements were paralleled with the reduction of lactate dehydrogenase release and the decrease of infarct size (SPC vs CTRL: (17.48±2.70)% vs (48.47±6.03)%, P<0.05; PPC vs CTRL: (35.60±2.10)% vs (48.47±6.03)%, P<0.05). SPC group had less infarct size than PPC group (SPC vs PPC: (17.48±2.70)% vs (35.60±2.10)%, P<0.05). Atractyloside coadministration attenuated or completely blocked the cardioprotective effect of postconditioning of sevoflurane and propofol. Conclusion: Postconditioning of sevoflurane and propofol has cardio-protective effect against ischemia-reperfusion injury of heart, which is associated with inhibition of MPTP opening. Compared to propofol, sevoflurane provides superior protection of functional recovery and infarct size.

Ethanol induced mitochondria injury and permeability transition pore opening: Role of mitochondria in alcoholic liver disease

AIM: To observe changes of mitochondria and investigate the effect of ethanol on mitochondrial perme- ability transition pore (PTP), mitochondrial membrane potential (MMP, ΔΨm) and intracellular calcium concentration in hepatocytes by establishing an animal model of alcoholic liver disease (ALD). METHODS: Fourty adult male Wistar rats were randomly divided into two groups, the model group (20) was administered alcohol intragastrically plus an Oliver oil diet to establish an ALD model, and the control group (20) was given an equal amount of normal saline. The ultramicrostructural changes of mitochondria were observed under electron microscopy. Mitochondria of liver was extracted, and patency of PTP, mitochondrial membrane potential (ΔΨm), mitochondrial mass and intracellular calcium concentration of isolated hepacytes were detected by flow cytometry using rhodamine123 (Rh123), Nonyl-Acridine Orange and calcium fluorescent probe Fluo-3/AM, respectively. RESULTS: Membrane and cristae were broken or disappeared in mitochondria in different shapes under electron microscopy. Some mitochondria showed U shape or megamitochondrion. In the model group, liver mitochondria PTP was broken, and mitochondria swelled, the absorbance at 450 nm, A540 decreased (0.0136 ± 0.0025 vs 0.0321 ± 0.0013, model vs control, P < 0.01); mitochondria transmembrane potential (239.4638 ± 12.7263 vs 377.5850 ± 16.8119, P < 0.01) was lowered; mitochondrial mass (17.4350 ± 1.9880 vs 31.6738 ± 3.4930, P < 0.01); and [Ca2+]i was increased in liver cells (7.0020 ± 0.5008 vs 10.2050 ± 0.4701, P < 0.01).CONCLUSION: Chronic alcohol intake might lead to broken mitochondria PTP, decreased mitochondria membrane potential and injury, and elevated intracellular Ca2+ production. Ethanol-induced chondriosome injury may be an important mechanism of alcoholic diseases.

The calcium uniporter regulates the permeability transition pore in isolated cortical mitochondria

To investigate the influence of the mitochondrial calcium uniporter on the mitochondrial permeability transition pore, the present study observed mitochondrial morphology in cortical neurons isolated from adult rats using transmission electron microscopy, and confirmed the morphology and activity of isolated mitochondria by detecting succinic dehydrogenase and monoamine oxidase, two mitochondrial enzymes. Isolated mitochondria were treated with either ruthenium red, an inhibitor of the uniporter, spermine, an activator of the uniporter, or in combination with cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. Results showed that ruthenium red inhibited CaCl2-induced mitochondrial permeability transition pore opening, spermine enhanced opening, and cyclosporin A attenuated the effects of spermine. Results demonstrated that the mitochondrial calcium uniporter plays a role in regulating the mitochondrial permeability transition pore in mitochondria isolated from the rat brain cortex.

Targeting the mitochondrial permeability transition pore in traumatic central nervous system injury

The mitochondrion serves many functions in the central nervous system （CNS） and other organs beyond the well-recognized role of adenosine triphosphate （ATP） production. This includes calcium-dependent cell signaling, regulation of gene expression, synthesis and release of cytotoxic reactive oxygen species, and the release of cytochrome c and other apoptotic cell death factors. Traumatic injury to the CNS results in a rapid and, in some cases, sustained loss of mitochondrial function. One consequence of compromised mitochondrial function is induction of the mitochondrial permeability transition （mPT） state due to formation of the cyclosporine A sensitive permeability transition pore （mPTP）. In this mini-review, we summarize evidence supporting the involvement of the mPTP as a mediator of mitochondrial and cellular demise following CNS traumatic injury and discuss the beneficial effects and limitations of the current ex- perimental strategies targeting the mPTP.

Shexiang Tongxin Dropping Pill(麝香通心滴丸)Reduces Coronary Microembolization in Rats via Regulation of Mitochondrial Permeability Transition Pore Opening and AKT-GSK3βPhosphorylation

Objective To investigate the protective effects of Shexiang Tongxin Dropping Pill(麝香通心滴丸,STDP)following sodium laurate-induced coronary microembolization(CME)in rats.Methods Forty rats were divided into 4 groups:the control(sham)group,CME group,low-dose STDP pretreatment group(20 mg·kg^(−1)·d^(−1)),and high-dose STDP pretreatment group(40 mg·kg^(−1)·d^(−1)).The rats were intragastric administrated with STDP 2 weeks before operation.Moreover,the histopathological alterations were observed using optical microscopy and transmission electron microscopy.Antioxidant biomarkers were analyzed by enzyme-linked immunosorbent assay.Mitochondrial functions including the mitochondrial permeability transition pore(mPTP)mtDNA copy number were determined and proteins of AKT/GSK3βwere analyzed by Western blot.Results The rats in the CME group showed a significant increase in the fibrinogen-like protein 2 expression level and mitochondrial dysfunction and a decrease in the expression level of antioxidant biomarkers(superoxide dismutase and catalase,P<0.01 for all).In contrast,the rats in the low-and high-dose STDP pretreatment groups showed a significant decrease in coronary microthrombi(P<0.05);moreover,STDP restored the antioxidant-related protein activities and mitochondrial function,inhibited mPTP opening,decreased AKT-Ser473 phosphorylation,and increased GSK3β-Ser9 phosphorylation(P<0.05 or P<0.01).Conclusion STDP may be useful for treatment of CME,possibly via regulation of mPTP opening and AKT/GSK3βphosphorylation.

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.

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 demon-strated that it can inhibit sperm apoplosis via afecting 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-mediated serum was prepared from rats following oral adminis-tration 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 afer drugs in-cubation.Cell viability was asessed using CCK-8.Apoptosis was detected using flow cytometry and TUNEL method.The opening of mPTP and mitochondrial membrane potential(MMP)were dected by Calcein AM and JC-1 fuorescent probe respectively.The mRNA and protein levels of voltage-dependent anion channel I(VDACI),cyelophilin D(CypD),adenine nucleide translocator(ANT),cytochrome C(Cyt C),caspase 3,9 were dected by RT-PCR(real time quantity PCR)and Western blotting respectively.The results demonstrated that mPTP of GC-2 cells was opened alpter 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 associ ated with increased MMP and decreased apoptosis.Morcover,the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDACI and CypD,Caspase-3,9 and CylC,as well as a increased ra-tio of BclBax.However,ANT was not significantly ffected.Therefore,these findings indicated that WZYZW inhibited mitochondri-al mediated apoptosis by atenuating the opening of mPTP in GC-2 cells.WZYZW-medicated serum inhibited the expressions of VDACI and CypD and increased the expression of Bcl-2,which afected the opening of mPTP and exerted protective and anti-apop-totic ffects on GC-2 cell induced by ATR.

Opening of liver mitochondrial permeability transition pore in streptozotocin-induced diabetic rats and its inhibition by methanol fraction of Ficus mucoso(Welw) root bark

Objective: Several pathologies arise from the inappropriate opening of the mitochondrial permeability transition(mPT) pore. In this regard, inhibition of mPT pore represents a cytoprotective approach to preserve mitochondrial function for treatment of diseases characterized by excessive tissue wastage such as diabetes mellitus. The aim of this study, therefore, was to study the effects of fractions of Ficus mucoso, a medicinal plant used in the traditional treatment of diabetes, on mPT pore in normal and streptozotocin(STZ)-induced diabetic rat liver.Methods: Different solvent fractions of the crude methanol extract of F. mucoso were obtained by vacuum liquid chromatography and were tested on the mPT pore. Of all the fractions tested, methanol fraction of F. mucoso(MFFM) was the most potent and was used for in vivo studies. Diabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg STZ, while treatment lasted for 14 d. In vivo, 30 male Wistar rats were divided into five groups: A, normo-glycemic control(distilled water);B, STZ(65 mg/kg;diabetic control);C, STZ + MFFM(25 mg/kg);D, STZ + MFFM(50 mg/kg);E, STZ + glibenclamide(5 mg/kg). The mPT, mitochondrial ATPase activity, lipid peroxidation and cytochrome c release were assessed spectrophotometrically while blood glucose levels were monitored using glucometer.Results: In vitro, the solvent fractions of F. mucoso, at all concentrations tested, had no effect on the mPT pore, in the absence of calcium, with no significant release of cytochrome c. Interestingly, calciumdependent pore opening was inhibited by all solvent fractions of F. mucoso, with the MFFM having the highest inhibitory effect of 83% at 3 mg/mL. Induction of opening of the mPT pore, significant(P < 0.001) enhancement of mitochondrial ATPase activity and elevated malondialdehyde(MDA) levels in STZ-induced diabetes were significantly(P < 0.001) reversed by MFFM and were comparable with the effects of glibenclamide, a standard antidiabetic drug. Also, treatment with MFFM at different doses significantly(P < 0.001) reduced high serum blood glucose compared to the diabetic control.Conclusion: F. mucoso could be useful in therapeutic management of diabetes mellitus given its ability to prevent excessive tissue wastage via inhibition of pore opening, and reduction in levels of MDA and serum blood glucose.

Mitochondrial Ca^(2+) transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.

Icariin Ameliorates D-galactose-induced Cell Injury in Neuron-like PC12 Cells by Inhibiting MPTP Opening

Objective Icariin(ICA)has a good neuroprotective effect and can upregulate neuronal basal autophagy in naturally aging rats.Mitochondrial dysfunction is associated with brain aging-related neurodegenerative diseases.Abnormal opening of the mitochondrial permeability transition pore(mPTP)is a crucial factor in mitochondrial dysfunction and is associated with excessive autophagy.This study aimed to explore that ICA protects against neuronal injury by blocking the mPTP opening and down-regulating autophagy levels in a D-galactose(D-gal)-induced cell injury model.Methods A cell model of neuronal injury was established in rat pheochromocytoma cells(PC12 cells)treated with 200 mmol/L D-gal for 48 h.In this cell model,PC12 cells were pre-treated with different concentrations of ICA for 24 h.MTT was used to detect cell viability.Senescence associatedβ-galactosidase(SA-β-Gal)staining was used to observe cell senescence.Western blot analysis was performed to detect the expression levels of a senescence-related protein(p21),autophagy markers(LC3B,p62,Atg7,Atg5 and Beclin 1),mitochondrial fission and fusion-related proteins(Drp1,Mfn2 and Opa1),and mitophagy markers(Pink1 and Parkin).The changes of autophagic flow were detected by using mRFP-GFP-LC3 adenovirus.The intracellular ultrastructure was observed by transmission electron microscopy.Immunofluorescence was used to detect mPTP,mitochondrial membrane potential(MMP),mitochondrial reactive oxygen species(mtROS)and ROS levels.ROS and apoptosis levels were detected by flow cytometry.Results D-gal treatment significantly decreased the viability of PC12 cells,and markedly increased the SA-β-Gal positive cells as compared to the control group.With the D-gal stimulation,the expression of p21 was significantly up-regulated.Furthermore,D-gal stimulation resulted in an elevated LC3B II/I ratio and decreased p62 expression.Meanwhile,autophagosomes and autolysosomes were significantly increased,indicating abnormal activation of autophagy levels.In addition,in this D-gal-induced model of cell injury,the mPTP was abnormally open,the ROS generation was continuously increased,the MMP was gradually decreased,and the apoptosis was increased.ICA effectively improved mitochondrial dysfunction to protect against D-gal-induced cell injury and apoptosis.It strongly inhibited excessive autophagy by blocking the opening of the mPTP.Cotreatment with ICA and an mPTP inhibitor(cyclosporin A)did not ameliorate mitochondrial dysfunction.However,the protective effects were attenuated by cotreatment with ICA and an mPTP activator(lonidamine).Conclusion ICA inhibits the activation of excessive autophagy and thus improves mitochondrial dysfunction by blocking the mPTP opening.

Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

The mitochondrial permeability transition pore is a nonspecific transmembrane channel.Inhibition of mitochondrial permeability transition pore opening has been shown to alleviate mitochondrial swelling,calcium overload,and axonal degeneration.Cyclophilin D is an important component of the mitochondrial permeability transition pore.Whether cyclophilin D participates in mitochondrial impairment and axonal injury after intracerebral hemorrhage is not clear.In this study,we established mouse models of intracerebral hemorrhage in vivo by injection of autologous blood and oxyhemoglobin into the striatum in Thy1-YFP mice,in which pyramidal neurons and axons express yellow fluorescent protein.We also simulated intracerebral hemorrhage in vitro in PC12 cells using oxyhemoglobin.We found that axonal degeneration in the early stage of intracerebral hemorrhage depended on mitochondrial swelling induced by cyclophilin D activation and mitochondrial permeability transition pore opening.We further investigated the mechanism underlying the role of cyclophilin D in mouse models and PC12 cell models of intracerebral hemorrhage.We found that both cyclosporin A inhibition and short hairpin RNA interference of cyclophilin D reduced mitochondrial permeability transition pore opening and mitochondrial injury.In addition,inhibition of cyclophilin D and mitochondrial permeability transition pore opening protected corticospinal tract integrity and alleviated motor dysfunction caused by intracerebral hemorrhage.Our findings suggest that cyclophilin D is used as a key mediator of axonal degeneration after intracerebral hemorrhage;inhibition of cyclophilin D expression can protect mitochondrial structure and function and further alleviate corticospinal tract injury and motor dysfunction after intracerebral hemorrhage.Our findings provide a therapeutic target for preventing axonal degeneration of white matter injury and subsequent functional impairment in central nervous diseases.

Deficit of mitochondria-derived ATP during oxidative stress impairs mouse MII oocyte spindles

Although the role of oxidative stress in maternal aging and infertility has been suggested, the underlying mechanisms are not fully understood. The present study is designed to determine the relationship between mitochondrial function and spindle stability in metaphase II (MII) oocytes under oxidative stress. MII mouse oocytes were treated with H2O2 in the presence or absence of permeability transition pores (PTPs) blockers cyclosporin A (CsA). In addition, antioxidant N-acetylcysteine (NAC), F0/F1 synthase inhibitor oligomycin A, the mitochondria uncoupler carbonyl cyanide 4-trifluoro- methoxyphenylhydrazone (FCCP) or thapsigargin plus 2.5 mM Ca^2+ (Th+2.5 mM Ca^2+) were used in mechanistic studies. Morphologic analyses of oocyte spindles and chromosomes were performed and mitochondrial membrane potential (AWm), cytoplasmic free calcium concentration ([Ca^2+]c) and cytoplasmic ATP content within oocytes were also assayed. In a time- and H202 dose-dependent manner, disruption of meiotic spindles was found after oocytes were treated with H202, which was prevented by pre-treatment with NAC. Administration of H2O2 led to a dissipation of AWm, an increase in [Ca^2+]c and a decrease in cytoplasmic ATP levels. These detrimental responses of oocytes to H2O2 treatment could be blocked by pre-incubation with CsA. Similar to H2O2, both oligomycin A and FCCP dissipated AWm, decreased cytoplasmic ATP contents and disassembled MII oocyte spindles, while high [Ca^2+]c alone had no effects on spindle morphology. In conclusion, the decrease in mitochondria-derived ATP during oxidative stress may cause a disassembly of mouse MII oocyte spindles, presumably due to the opening of the mitochondrial PTPs.

C-reactive protein aggravates myocardial ischemia/reperfusion injury through activation of extracellular-signal-regulated kinase 1/2

Background Ischemia/reperfusion injury （IRI） is an inflammatory response that occurs when tissue is reperfused following a prolonged period of ischemia. Several studies have indicated that C-reactive protein （CRP） might play an important role in inducing IRI. However, the effects of CRP on myocardial IRI and the underlying mechanisms have not been fully elucidated. This study aimed to investigate the association between CRP and myocardial IRI and the underlying mechanisms. Methods We simulated ischemia/reperfusion using oxygen-glucose deprivation/ reoxygenation （OGD/R） in neonatal Sprague-Dawley rat cardiomyocytes; reperfusion injury was induced by three hours of hypoxia with glucose and serum deprivation followed by one hour of reperfusion. Cell viability was tested with MTS assays, and cardiomyocyte damage was evaluated by lactate dehydrogenase （LDH） leakage. Mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester （TMRE） and mitochondrial permeability transition pore （mPTP） opening was measured using calcein/AM; both TMRE and caocein/AM were visualized with laser scanning confocal microscopy. In addition, we studied the signaling pathways underlying CRP-mediated ischemia/reperfusion injury via Western blot analysis. Results Compared with the simple OGD/R group, after intervention with 10 pg/mL CRP, cell viability decreased markedly （82.36 % ± 6.18% vs. 64.84% ± 4.06%, P = 0.0007）, and the LDH leakage significantly increased （145.3 U/L ± 16.06 U/L vs. 208.2 U/L ± 19.23 U/L, P = 0.0122）. CRP also activated mPTP opening and reduced mitochondrial membrane potential during myocardial ischemia/reperfusion. Pretreatment with 1 pM atorvastatin （Ator） before CRP intervention protected cardiomyocytes from IRI. Mitochondrial KATP channel opener diazoxide and mPTP inhibitor cyclosporin A also offset the effects of CRP in this process. The level of phosphorylated extracellular-signal-regulated kinase （ERK） 1/2 was significantly higher after pre-treatment with CRP compared with the OGD/R group （170.4% ± 3.00% v.v. 93.53% ± 1.94%, P 〈 0.0001）. Western blot analysis revealed that Akt expression was markedly activated （184.2% ± 6.96% vs. 122.7% ± 5.30%, P = 0.0003） and ERK 1/2 phosphorylation significantly reduced after co-treatment with Ator and CRP compared with the level after CRP pretreatment alone. Conclusions Our results suggested that CRP directly aggravates myocardial IRI in myocardial cells and that this effect is primarily mediated by inhibiting mitochondrial ATP- sensitive potassium （mitoKATp） channels and promoting mPTP opening. Ator counteracts these effects and can reduce CRP-induced IRI. One of the mechanisms of CRP-induced IRI may be related to the sustained activation of the ERK signaling pathway.

Regulatory role of calpain in neuronal death

Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly （ADP-ribose）polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases.

Role of MGST1 in reactive intermediate-induced injury

Microsomal glutathione transferase (MGST1, EC 2.5.1.18) is a membrane bound glutathione transferase extensively studied for its ability to detoxify reactive intermediates, including metabolic electrophile intermediates and lipophilic hydroperoxides through its glutathione dependent transferase and peroxidase activities. It is expressed in high amounts in the liver, located both in the endoplasmic reticulum and the inner and outer mitochondrial membranes. This enzyme is activated by oxidative stress. Binding of GSH and modification of cysteine 49 (the oxidative stress sensor) has been shown to increase activation and induce conformational changes in the enzyme. These changes have either been shown to enhance the protective effect ascribed to this enzyme or have been shown to contribute to cell death through mitochondrial permeability transition pore formation. The purpose of this review is to elucidate how one enzyme found in two places in the cell subjected to the same conditions of oxidative stress could both help protect against and contribute to reactive oxygen species-induced liver injury.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction

AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

AF-2364 is a prospective spermicide candidate

Inhibition of sperm motility has recently become a promising target for male contraceptive development. AF- 2364, an analogue of Lonidamine （LND）, had a contraceptive effect when orally administered to adult SpragueDawley rats. LND can also target mitochondria to inhibit oxygen consumption and block energy metabolism in tumour cells. However, there are no reports of the effects of AF-2364 on human sperm function. Herein we describe the action of AF-2364 on human sperm in vitro, as well as the mechanisms involved. AF-2364 specifically blocked human sperm motility in vitro. Further experiments revealed that AF-2364 can target sperm mitochondrial permeability transition （MPT） pores to induce the loss of sperm mitochondrial membrane potential （AqUm） and decrease ATP generation; however, no significant changes in the cytoskeletal network or the human sperm proteome were detected after exposure to AF-2364. Incubation of AF-2364 with other human or mouse cell lines indicated that the spermicidal effect at the lower concentration was specific. In summary, the spermicidal effect olAF-2364 involves direct action on sperm MPT pores, and this compound should be further investigated as a new spermicide candidate.

Study on the structure and composition of aortic valve calcific deposits:Etiological aspects

The structures and chemical compositions of valve calcific deposits were investigated. The deposits was chosen arbitrarily and subjected to chemical analysis, observation with scanning microscope, semi-quantitative determination of Ca, Mg, Na, K, P and C elements by energy dispersive X-ray, X-ray diffraction and Fourier transform infrared spectroscopy carried out. These deposits were found to have non-uniform internal structures composed of layers of a structureless aspidinic inorganic material, substantial amounts of voluminous organic material and in a few samples small spheres were also present. Two groups of deposits with distinctly different chemical compositions were identified: one group with a low Ca/P molar ratio (1.59) and the other group with a high (1.82) Ca/P molar ratio. The deposits belonging to the group with a low Ca/P molar ratio contain higher concentration of magnesium and consist of increased amount of amorphous calcium phosphate. The deposits with a high Ca/P molar ratio contain low concentration of magnesium and consist predominantly of carbonated hydroxyapatite. The inorganic material was identified as a poorly crystalline carbonate hydroxyapatite containing molecular water of the average formula Ca9.1Mg0.4(Na,K)(PO4)5.8(CO3)0.3(OH)2. The actual chemical composition of the apatitic solid phase varies not only from deposit to deposit but also within the same deposit. The non-uniform internal structure of the deposits, the occasional presence of spherical particles and the variable point composition of the individual deposits indicate that their formation did not proceed under more or less constant conditions.

Realgar induces differentiation through ROS-dependent mitochondrial pathway in HL-60 cells

Realgar （As 4 S 4 ）, as a mineral drug in traditional Chinese medicine, is currently used as the remedy for acute promyelocytic leukemia and has been proven to have relatively milder side effects as compared to the arsenolite （As 2 O 3 ）-based drugs. We have previously demonstrated that realgar induces differentiation in HL-60 cells, and the differentiation is associated with serine/threonine protein phosphatases, MAPK signaling pathways, and mitochondrial transmembrane potential decrease. In this study, we further explore the roles of mitochondrial permeability transition pore and reactive oxygen species （ROS） in realgar-induced differentiation in HL-60 cells. The differentiation was preceded by marked changes in the cellular level of ROS, and could be enhanced by SB202190, a p38 MAPK inhibitor. In addition, the efficacy of realgar was suppressed by closing the MPTP with an inhibitor. Taken together, these findings indicate that the opening of MPTP and the alteration of ROS generation were involved in realgar-induced differentiation.

Protective role of mitochondrial K-ATP channel and mitochondrial membrane transport pore in rat kidney ischemic postconditioning

Background Previous studies suggested that mechanical intervention during early reperfusion, or ischemia postconditioning （Ipo）, could protect kidneys against renal ischemia reperfusion injury （RIRI）. However, the mechanisms responsible for this protection remain unclear. This study therefore investigated the protection afforded by Ipo in rat kidneys in vivo, and the roles of mitochondrial KATP channels （mitOKATP） and mitochondrial permeability transition pores （MPTPs）, by inhibiting mitOKATP with 5-hydroxydecanoate （5-HD）, and by directly detecting open MPTPs using calcein-AM and CoCl2.Methods Thirty-five male Sprague-Dawley rats were randomly assigned to sham-operation （S）, ischemia-reperfusion （I/R）,Ipo, ischemia reperfusion with 5-HD （I/R＋5-HD）, or Ipo with 5-HD （Ipo ＋5-HD） groups. Rats in each group were sacrificed after 6 hours of reperfusion by heart exsanguination or cervical dislocation under anesthesia. RIRI was assessed by determination of creatinine and blood urea nitrogen （BUN）, and by examination of histologic sections. The roles of mitoKATP and MPTP were investigated by analyzing fluorescence intensities of mitochondria, mitochondrial membrane potential,intracellular reactive oxygen species （ROS） and intracellular calcium, using appropriate fluorescent markers. The relationship between apoptosis and RIRI was assessed by determining the apoptotic index （Al） of kidney tubular epithelial cells.Results The RIRI model was shown to be successful. Significantly higher levels of creatinine and BUN, and abnormal pathology of histologic sections, were observed in group I/R, compared with group S. 5-HD eliminated the renoprotective effects of Ipo. Mitochondrial and mitochondrial membrane potential fluorescence intensities increased, and intracellular calcium, ROS fluorescence intensities and AI decreased in group Ipo, compared with group I/R. However, mitochondrial and mitochondrial membrane potential fluorescence intensities decreased, and intracellular calcium and ROS fluorescence intensities and AI increased in group Ipo＋5-HD, compared with group Ipo.Conclusions mitoKATP and MPTPs participated in Ipo-induced renoprotective mechanisms in rat kidneys subjected to RIRI, possibly through decreased renal tubular epithelial cell apoptosis.