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.

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.

白藜芦醇通过MCU抑制mPTP开放减轻H9c2细胞氧化应激损伤

目的观察白藜芦醇(resveratrol)保护心肌细胞减轻氧化应激损伤的过程是否与线粒体通透性转换孔(mPTP)和线粒体钙离子单向转运蛋白(MCU)相关,并探讨可能的机制。方法H9c2心肌细胞常规培养,随机分为对照组、过氧化氢组、白藜芦醇+过氧化氢组、MCU抑制剂(钌红)+白藜芦醇+过氧化氢组。微板法检测细胞外液中乳酸脱氢酶(LDH)的含量;Western印迹法检测内质网应激分子伴侣葡萄糖调节蛋白(GRP)78、GRP94和MCU蛋白表达;激光扫描共聚焦显微镜观察细胞内钙离子(Ca^(2+))、活性氧(ROS)和mPTP变化。结果与对照组相比,过氧化氢显著增加细胞外液中LDH的含量、GRP78、GRP94和MCU蛋白表达、细胞内Ca^(2+)和ROS含量,显著减少mPTP特异性探针TMRE的荧光强度(均P<0.05)。白藜芦醇明显抑制过氧化氢引起的变化,而钌红显著增强白藜芦醇的作用(均P<0.05)。结论白藜芦醇通过MCU减少细胞内Ca^(2+)超载和ROS生成进而抑制mPTP开放,减轻氧化应激引起的内质网应激损伤,保护心肌细胞。

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.

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.

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.

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.

缺氧后线粒体Drp1通过LRRK2-HK2诱导mPTP过度开放的机制研究

目的:探究缺氧后线粒体动力相关蛋白1(dynamin-related protein 1,Drp1)对线粒体膜通透性转换通道(mitochondrial permeability transition pore,mPTP)开放的调控机制。方法:在血管平滑肌细胞中通过免疫荧光方法观察缺氧后mPTP开放情况;通过超速离心法分离线粒体和细胞质成分蛋白;使用Co-IP和Westernblot检测缺氧或者干预Drp1、干预己糖激酶-2(hexokinase-2,HK2)后Drp1的表达分布情况、HK2与电压依赖性阴离子通道蛋白(voltage dependent anion channel,VDAC)结合情况、Drp1与富含亮氨酸重复激酶2(leucine-rich repeat kinase 2,LRRK2)结合情况;使用蛋白分子对接和蛋白芯片方法筛选Drp1的潜在结合蛋白及结合位点;使用Drp1抑制剂和点突变法用于相应机制探究。结果:缺氧后Drp1发生线粒体转位促使mPTP过度开放(P<0.05)。使用Mdivi-1减少线粒体Drp1表达后可抑制mPTP开放,减少细胞色素C(cytochrome C,CytC)释放(P<0.05)。缺氧后HK2-Thr473磷酸化水平减低引起的HK2线粒体分离会导致mPTP结构破坏,通过HK2 T473D点突变恢复HK2活性后,HK2与线粒体结合情况及mPTP开放情况明显改善(P<0.05)。Drp1蛋白芯片结果发现缺氧后Drp1可以与LRRK2结合并封闭其活性位点,通过Drp1 T595A点突变破坏Drp1-LRRK2结合后,HK2活性及HK2线粒体分离情况明显改善(P<0.05),mPTP开放情况明显减少(P<0.05)。结论:缺氧后线粒体Drp1通过封闭激酶LRRK2活性位点导致HK2 Thr473磷酸化水平减低及其线粒体分离,最终诱导mPTP过度开放。

CypD介导mPTP在褪黑素改善七氟醚重复暴露致认知损伤中的作用

目的探讨亲环素D(cyclophilin D,CypD)介导线粒体通透性转化孔(mitochondrial permeability transition pore,mPTP)在褪黑素改善新生动物重复暴露七氟醚后引起认知障碍中的作用。方法新生小鼠随机分为对照组(Con)、七氟醚组(Sevo)、褪黑素预处理+七氟醚组(Sevo+Mel)。检测线粒体膜电位(mitochondrial membrane potential,MMP);Western blot检测CypD、突触素1(Synapsin-1)及突触后致密蛋白95(postsynaptic density protein 95,PSD95)的表达;行为学检测小鼠的认知功能。结果与对照组相比,Sevo组MMP明显下降(P<0.01),CypD表达升高(P<0.05),PSD95及Synapsin-1表达降低(P<0.01),新物体识别指数和空间记忆能力明显下降(P<0.01,P<0.05)。与Sevo组相比,Sevo+Mel组MMP升高(P<0.01),PSD95及Synapsin-1表达增加(P<0.05),CypD表达减少(P<0.01),新物体识别指数和空间记忆能力明显升高(P<0.01)。结论褪黑素通过抑制CypD的表达和mPTP的开放,增加突触蛋白的表达,改善新生动物重复暴露七氟醚后导致的认知损伤。

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.

双下肢缺血后处理保护再灌注心肌时效性及对线粒体通路调控的研究

目的:观察双下肢缺血后处理(即远隔器官缺血后处理,remote ischemic postconditioning,RIpostC)保护缺血再灌注小鼠心肌的时效性及对心肌线粒体依赖性凋亡和坏死通路的调控。方法:成年雄性C57BL/6J野生型小鼠被随机分为假手术(sham)组、心肌缺血再灌注(myocardial ischemia/reperfusion,MI/R)组、缺血后处理组、RIpostC组及RIpostC延迟1、5、10、15、30和60 min组。阻断左冠脉45 min,再灌注24 h,建立MI/R模型;气囊袖带阻断双下肢血流5 min,再灌注5 min,实施RIpostC。再灌注24 h后,Evans blue和TTC染色观察心肌梗死面积与血清心肌钙蛋白I变化。TUNEL和高迁移率族盒蛋白1(high mobility group box protein 1,HMGB1)染色观察心肌凋亡和坏死;线粒体水肿实验观察心肌线粒体膜电位变化;Western blot观察心肌细胞凋亡和线粒体膜通透性转换孔(mitochondrial permeability transition pore,mPTP)相关蛋白表达。结果:与MI/R组比较,RIpostC及RIpostC延迟1、5、10和15 min组心肌梗死面积明显减少,RIpostC延迟30和60 min组则无明显改变。缺血后处理与RIpostC对缺血再灌注心肌有类似保护效应。RIpostC减少缺血再灌注心肌细胞凋亡和坏死发生。RIpostC降低缺血再灌注心肌亲环蛋白D(cyclophilin D,CypD)、Bax和Bak蛋白表达水平。结论:心肌再灌注后15 min内实施RIpostC能够减少心肌梗死面积,其保护作用与缺血后处理类似;RIpostC通过调控线粒体依赖性凋亡和坏死通路减轻MI/R损伤。

MPTP参与硫化氢对大鼠心肌缺血再灌注损伤的延迟性保护作用

目的:探讨硫化氢(H2S)预处理对大鼠心肌缺血再灌注损伤的延迟性保护作用及可能机制。方法:建立大鼠在体心肌缺血再灌注模型,设立假手术组(Sham组)、缺血再灌注组(IR组)、H2S预处理组(HS组)和线粒体通透性转换开孔(MPTP)开放剂苍术苷(ATR)+H2S组(HA组),每组8只。通过经颈动脉插管至左心室监测血流动力学,采用伊文思蓝和TTC染色检测心肌梗死面积。结果:缺血前,各组血流动力学参数差异无统计学意义(P>0.05)。缺血再灌注期间,与IR组比较,HS组各指标(HR、LVESP、±dp/dtmax)均升高,LVEDP降低,差异有统计学意义(P<0.05);与HA组比较,HS组各指标差异均无统计学意义(P>0.05)。与IR组比较,HS组心肌梗死范围明显减少(P<0.05),HA组无明显改变(P>0.05)。结论:硫化氢能改善大鼠心肌缺血再灌注损伤的心功能,这种心肌保护作用可能与MPTP的关闭有关。

防治心肌缺血/再灌注损伤心肌mPTP开放情况的研究进展

本文对最近5-8年来国内外防治心肌缺血/再灌注损伤(MIRI)的机制研究进行了总结和归纳,试图使在防治心肌缺血/再灌注损伤中调控线粒体通透性转换孔(mPTP)开放机制的研究方面有所发现,为防治心肌缺血再灌注损伤提供更多更有力的理论依据。

电针对布比卡因中毒大鼠心肌损伤及线粒体功能的影响

目的:观察电针对布比卡因(bupivacaine,BPV)所致心肌和线粒体功能损伤的影响,探讨电针内关穴抗心肌损伤的作用机制。方法:将30只雄性SD大鼠按照随机数字表法分为对照组[A组,静脉滴注0.9%生理盐水3 mL/(kg·min)]、布比卡因组[B组,先静脉滴注生理盐水30 min,再滴注5%布比卡因2 mg/(kg·min)]、电针组(E组)。经静脉持续泵入BPV建立心肌损伤模型,E组于建模前电针刺激内关穴30 min。磷钼酸比色法检测大鼠心肌组织三磷酸腺苷(adenosine triphosphate,ATP)含量,二氢乙啶(dihydroethidium,DHE)染色法测定大鼠心肌组织活性氧(reactive oxygen species,ROS)相对含量,Fluo-3/AM测定大鼠心肌细胞内Ca^(2+)相对荧光强度,酶联免疫吸附试验检测线粒体膜通道孔(mitochondrial permeability transition pore,MPTP)活性。结果:与A组相比,B组ATP含量、Ca^(2+)荧光强度降低,ROS相对含量升高(P<0.05),提示心肌损伤模型造模成功;与B组相比,E组大鼠发生心脏骤停时间延长,ATP含量、Ca^(2+)荧光强度升高,MPTP升高,ROS相对含量降低(P<0.05)。结论:电针刺激内关穴可有效改善BPV所致心肌损伤,线粒体中ROS、Ca^(2+)是其重要的物质基础,且与MPTP密切相关。

Rho激酶和PKC在环孢素A改善休克血管反应性中的作用及与MPTP的关系

目的观察Rho激酶和PKC在环孢素A(Cs A)改善创伤失血性休克大鼠血管反应性中的作用及与线粒体通透性转换孔(MPTP)开放的关系。方法采用创伤失血性休克大鼠模型和缺氧培养的血管平滑肌细胞(VMSC),观察了Rho激酶和PKC在Cs A调节休克血管反应性中的作用,以及对血管平滑肌细胞线粒体MPTP开放的影响,同时观察Cs A对休克动物炎症因子TNF-α、IL-1β和IL-6水平的影响。结果 Cs A明显改善了休克血管反应性,Rho激酶抑制剂Y27632可显著拮抗Cs A恢复休克血管反应性的作用,但是PKC抑制剂staurosporine对Cs A的作用无明显影响。Cs A和Rho激酶激动剂U46619都可抑制缺氧后线粒体MPTP的开放程度。休克后大鼠血液中TNF-α和IL-1β的浓度均显著增加,但Cs A处理仅使其轻微减少。结论 Cs A可以通过抑制线粒体MPTP开放改善休克后血管的低反应性,发挥对创伤休克的治疗作用。Rho激酶参与了这其中的调节过程。

亲环素D介导的线粒体通透性转换孔开放对骨肉瘤MG63细胞生物学行为的影响

目的:探讨干扰线粒体关键蛋白亲环素D(CypD)对骨肉瘤MG63细胞增殖、迁移、凋亡等生物学行为的影响。方法:构建3种转染不同siRNA的MG63细胞系,分别为CypD-siRNA1、CypD-siRNA2和CypD-siRNA3组,采用qRT-PCR和Western blot检测3组细胞中CypD的表达水平,筛选用于后续实验的CypD-siRNA。将MG63细胞分为阴性对照组(转染siRNA-NC)和敲减组(转染CypD-siRNA2)。采用CCK-8法和平板克隆实验检测细胞的增殖能力,Transwell小室实验检测细胞的迁移能力;采用MitoSOX Red检测线粒体活性氧(mROS)水平,钙黄绿素-钴评价线粒体通透性转换孔的开放状态;采用流式细胞术检测细胞凋亡情况,Western blot法检测细胞迁移和凋亡相关蛋白的表达。结果:与CypD-siRNA1组和CypD-siRNA3组相比,CypD-siRNA2组中CypD mRNA和蛋白表达水平下降(P<0.05)。与阴性对照组相比,敲减组细胞克隆形成数和迁移数减少,细胞增殖能力下降,细胞凋亡率升高,N-cadherin和Vimentin蛋白表达水平降低(P<0.05),Bax/Bcl-2、Cleaved Caspase 3/Caspase 3以及E-cadherin和细胞色素C蛋白表达水平升高(P<0.05)。敲减组细胞mROS水平高于阴性对照组,而线粒体通透性转换孔开放程度低于阴性对照组(P<0.05)。结论:敲减CypD表达可上调MG63细胞氧化应激水平,抑制其增殖、迁移能力,并通过线粒体途径诱导其凋亡。

糖尿病大鼠海马MPTP、Ca^(2+)、Bcl-2/Bax的表达及有氧运动和四叶参的干预作用

目的观察有氧运动和四叶参对Ⅱ型糖尿病大鼠海马区线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)、Ca^(2+)及Bcl-2/Bax的影响,研究有氧运动及中药干预改善糖尿病的可能机制。方法高糖高脂膳食配合链脲佐菌素建立Ⅱ型糖尿病大鼠模型,随机分为糖尿病对照组(DM)、运动组(DMT)、四叶参组(DML)、运动+四叶参组(DMTL),另设正常对照组(C),每组6只。连续运动(15 m/min,5°坡度,60 min,隔日训练)或/和灌胃给药(200 mg/kg) 8 w后,尾静脉取血检测血糖,紫外分光光度计检测海马MPTP的开放情况,双光束紫外分光光度计检测Ca^(2+)转运情况,用免疫印迹法检测Bcl-2/Bax的蛋白表达。结果与C组比较,DM组的线粒体吸光度值显著减小(P﹤0. 01)、Ca^(2+)转运量及Bax蛋白表达显著增加(P﹤0. 01),Bcl-2蛋白表达显著减少(P﹤0. 01),Bcl-2/Bax比值显著减小(P﹤0. 01);与DM组相比,DMT组及DMTL组的血糖值减少(P﹤0. 01),线粒体吸光度值明显增大(P﹤0. 05),Ca^(2+)转运量明显减小(P﹤0. 05),Bax蛋白表达减少(P﹤0. 05,P﹤0. 01),Bcl-2蛋白表达增加(P﹤0. 01),Bcl-2/Bax比值显著增加(P﹤0. 01); 8 w后四叶参干预组Bcl-2/Bax比值显著增加(P﹤0. 05),但两蛋白表达无显著性变化(P﹥0. 05)。结论有氧运动及运动和四叶参共同干预可能通过上调Ⅱ型糖尿病大鼠海马Bcl-2/Bax比值,下调线粒体通透性转换孔的开放及Ca^(2+)转运从而降低血糖。

喜树碱诱导甜菜夜蛾细胞凋亡线粒体途径的MPTP依赖性

【目的】线粒体通透性转换孔(MPTP)的开放可以导致线粒体膜通透性改变,与细胞凋亡关系密切。本研究旨在探索MPTP在喜树碱诱导的昆虫细胞凋亡中的作用,以进一步揭示喜树碱(CPT)诱导昆虫细胞凋亡的机制。【方法】环孢菌素A(CsA)为MPTP开放抑制剂,通过预加入20μmol/L CsA,应用流式细胞仪测定其对CPT和羟基喜树碱(HCPT)诱导的甜菜夜蛾Spodoptera exigua细胞(IOZCAS-SPEX-Ⅱ)凋亡作用的影响,包括细胞内Ca^(2+)浓度变化,线粒体膜电位变化以及活性氧簇(ROS)变化,从而分析MPTP在CPT和HCPT诱导细胞凋亡的作用。【结果】结果显示,10μmol/LCPT和HCPT处理IOZCAS-SPEX-Ⅱ细胞6 h和12 h时,与0.1%DMSO对照组相比,甜菜夜蛾细胞发生凋亡,胞质Ca^(2+)浓度增大,线粒体膜电位降低或丧失,ROS增加,即CPT和HCPT诱导甜菜夜蛾细胞发生凋亡,为线粒体内途径。但经过20μmol/L CsA预处理2 h后再加入CPT和HCPT处理6 h,与0.1%DMSO组相比,细胞凋亡率、胞质Ca^(2+)浓度、线粒体膜电位及ROS产生均无显著差异(P>0.05),即CsA抑制了MPTP的开放,从而抑制了CPT和HCPT诱导的甜菜夜蛾细胞凋亡;而加入CPT和HCPT处理12 h时,CsA对MPTP开放的抑制作用显著降低,与单CPT和HCPT处理组相比,细胞凋亡率、胞质Ca^(2+)浓度、线粒体膜电位及ROS差异不显著,即CPT和HCPT诱导的细胞凋亡如常发生。【结论】本研究证实喜树碱和羟基喜树碱诱导甜菜夜蛾细胞凋亡线粒体途径具有MPTP开放依赖性,且首次明确这种依赖性具有时间性。

Ischemia/reperfusion injury and cardioprotective mechanisms:Role of mitochondria and reactive oxygen species

Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction(AMI).However reperfusion is responsible for additional myocardial damage,which likely involves opening of the mitochondrial permeability transition pore(mPTP).In reperfusion injury,mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability.Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species(ROS).Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning,obtained with brief intermittent ischemia or with pharmacological agents.These pathways converge on a common target,the mitochondria,to preserve their function after ischemia/reperfusion.The present review discusses the role of mitochondria in cardioprotection,especially the involvement of adenosine triphosphate-dependent potassium channels,ROS signaling,and the mPTP.Ischemic postconditioning has emerged as a new way to target the mitochondria,and to drastically reduce lethal reperfusion injury.Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects,and an interesting alternative is pharmacological postconditioning.In fact ischemic postconditioning and the mPTP desensitizer,cyclosporine A,have been shown to induce comparable protection in AMI patients.