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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Chrysophanol localizes in mitochondria to promote cell death through upregulation of mitochondrial cyclophilin D in HepG2 cells

Objective:Chrysophanol(Chry) displays potent anticancer activity in human cancer cells and animal models,but the cellular targets of Chry have not been fully defined.Herein,we speculated whether mitochondria were a target involved in Chry-induced cytotoxicity.Methods:Human liver cancer cell line HepG2 was incubated.The cytotoxicity was evaluated by MTT assay.Mitochondria localization was evaluated by a confocal microscopy.Mitochondrial membrane potential ΔΨm was detected by TMRE staining and determined by the flow cytometer.The levels of ATP,mitochondrial superoxide anions,and GSH/GSSG were determined according to the assay kits.The apoptosis were evaluated through Hoechst33342/PI and Annexin V/PI staining,respectively.The expression of cyclophilin D(CyPD) was determined by immunoblot method,and the interaction between CyPD and Chry was analyzed by molecule docking procedure.Results:Chry itself mainly localized in mitocho ndria to cause mitochondrial dysfunction and cell death in HepG2 cells.As regard to the mechanism,cyclosporin A as the inhibitor for the formation of mitochondrial permeability transition pore(mPTP) moderately suppressed cell death,indicating mPTP involved in the process of cell death.Further,Chry enhanced the protein expression of Cyclophilin D(CyPD) which is a molecular componentry and a modulator of mPTP,while antioxidant N-acetyl-L-cysteine inhibited the expression of CyPD.Molecule docking procedure disclosed two hydrogen-bonds existed in CyPD-Chry complex with-11.94 kal/mol of the binding affinity value.Besides,the mtDNA-deficient HepG_2-ρ0 cells were much resistant to Chry-induced cell death,indicating mtDNA at least partly participated in cell death.A combination of Chry and VP-16 produced the synergism effect toward cell viability andΔΨm,while Chry combined with Cis-Pt elicited the antagonism effect.Conclusion:Taken together,enrichment in mitochondria and actions on mPTP,CyPD and mtDNA provides an insight into the anticancer mechanism of Chry.The combination therapy for Chry with clinical drugs may deserve to further explore.

Reducing the oxidative stress mediates the cardioprotection of bicyclol against ischemia-reperfusion injury in rats

Objective:To investigate the beneficial effect of bicyclol on rat hearts subjected to ischemia-reperfusion(IR) injuries and its possible mechanism.Methods:Male Sprague-Dawley rats were intragastrically administered with bicyclol(25,50 or 100 mg/(kg·d)) for 3 d.Myocardial IR was produced by occlusion of the coronary artery for 1 h and reperfusion for 3 h.Left ventricular hemodynamics was continuously monitored.At the end of reperfusion,myocardial infarct was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining,and serum lactate dehydrogenase(LDH) level and myocardial superoxide dismutase(SOD) activity were determined by spectrophotometry.Isolated ventricular myocytes from adult rats were exposed to 60 min anoxia and 30 min reoxygenation to simulate IR injuries.After reperfusion,cell viability was determined with trypan blue;reactive oxygen species(ROS) and mitochondrial membrane potential of the cardiomyocytes were measured with the fluorescent probe.The mitochondrial permeability transition pore(mPTP) opening induced by Ca2+(200 μmol/L) was measured with the absorbance at 520 nm in the isolated myocardial mitochondria.Results:Low dose of bicyclol(25 mg/(kg·d)) had no significant improving effect on all cardiac parameters,whereas pretreatment with high bicyclol markedly reduced the myocardial infarct and improved the left ventricular contractility in the myocardium exposed to IR(P<0.05).Medium dose of bicyclol(50 mg/(kg·d)) markedly improved the myocardial contractility,left ventricular myocyte viability,and SOD activity,as well decreased infarct size,serum LDH level,ROS production,and mitochondrial membrane potential in rat myocardium exposed to IR.The reduction of ventricular myocyte viability in IR group was inhibited by pretreatment with 50 and 100 mg/(kg·d) bicyclol(P<0.05 vs.IR),but not by 25 mg/(kg·d) bicyclol.The opening of mPTP evoked by Ca2+ was significantly inhibited by medium bicyclol.Conclusions:Bicyclol exerts cardioprotection against IR injury,at least,via reducing oxidative stress and its subsequent mPTP opening.

Effects of Panax Quinquefolium Saponin on Phosphatidylinositol 3-Kinase/Serine Threonine Kinase Pathway of Neonatal Rat Myocardial Cells Subjected to Hypoxia

Objective： To explore the effects of Panax Quinquefolium Saponin （PQS） on phosphatidylinositol 3-kinase/serine threonine kinase （P13/Akt） pathway of neonatal rat myocardial cells subjected to hypoxia. Methods： Neonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups： the normal group, the model group, the positive control group （Ciclosporin A, 2 p, mol/L）, the low-dose PQS group （PQSL, 25mg/L）, and the high-dose PQS group （PQSH, 50 mg/L）. Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase （LDH） leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase （GSK）-3β and phospho-Akt as well as cytochrome C were determined by Western blot. Results： LDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group （P〈0.05）. Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group （P〈0.05, P〈0.01）. Compared with the model group （16.41 ± 1.74; 35.28 ± 6.30）, both the integrated optical density of mitochondrial permeability transition pore （MPTP） and the mitochondrial transmembrane potential significantly increased in the PQSH group （42.74± 2.12; 71.36 ± 6.54） and the PQSL group （39.58± 1.49; 66.99± 5.45; P〈0.05, P〈0.01）. However, the protein of cytochrome C outside the mitochonddon decreased in the PQSH group （273.66 ± 14.61） and the PQSL group （259.62 ± 17.31） compared with the model group （502.41 ± 17.76; P〈0.05）. Conclusion： Through activation of the P13K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

Synthesis of hydroxycinnamic acid derivatives as mitochondria-targeted antioxidants and cytotoxic agents

In order to develop agents with superior chemopreventive and chemotherapeutic properties against hepatocellular carcinomas, mitochondria-targeted hydroxycinnamic acids(Mito HCAs) were synthesized by conjugation with a triphenylphosphonium cation. These synthetic compounds were evaluated for their antioxidant activities in hepatic mitochondria, including against OH? àand ROO? àinduced lipid peroxidation. H_2O_2 production was decreased significantly by increasing glutathione peroxidase and catalase activities. In addition, cell proliferation data from three cell lines(HepG2, L02 and WI38) indicated that the Mito HCAs were selective for cancer cells. Interestingly, the Mito HCAs both with or without Ca^(2+)triggered mitochondrial dysfunction by inducing mitochondrial swelling, collapsing the mitochondrial membrane potential and causing cytochrome c release. In particular, an inhibitor of the mitochondrial permeability transition pore(m PTP), cyclosporin A, attenuated mitochondrial damage and cell apoptosis, indicating that m PTP may be involved in the antiproliferative activity of Mito HCAs.Further studies focused on structural optimization of these compounds are onging.