体外模拟缺血再灌注诱导神经细胞线粒体功能改变的研究

目的观察线粒体功能失调在缺血再灌注后神经细胞凋亡中的作用.方法(1)采用体外培养神经母细胞瘤细胞株N2a,模拟缺血再灌注(先缺氧缺营养90 min,然后正常培养不同时间);(2)采用琼脂糖凝胶电泳检测细胞凋亡情况;(3)通过MTT法、细胞色素C释放和跨膜压的改变判断线粒体的功能;(4)Cas-pase 3活性测定采用水解其可见光底物.结果(1)N2a细胞缺血再灌注12 h即出现明显DNA片段化,24h更明显;(2)线粒体琥珀酸脱氢酶活性缺血再灌注24 h明显降低;跨膜电位在缺血再灌注1 h先短暂下降,3 h明显升高,6 h后降低,以后再没回升.缺血再灌注3 h细胞色素C开始释放,6 h达到高峰,并持续到24 h;(3)Caspase 3活性在缺血再灌注10 h升高,24 h达到高峰.线粒体通透性转换孔抑制剂cyclosporine A只能抑制部分caspase 3的活性和DNA片段化改变,而caspase 8抑制剂虽不能完全抑制caspase 3的活性,但能完全抑制DNA的片段化.结论缺血再灌注诱导神经细胞凋亡存在caspase 3依赖性和非依赖性两条途径,线粒体功能失调在缺血再灌注引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用.     

线粒体自噬在褪黑素抗缺血再灌注损伤中的作用

目的初步探讨线粒体自体吞噬在褪黑素抗缺血再灌注损伤中的作用。方法体外培养N2a细胞,模拟缺血再灌注,加入褪黑素(melatonin,Mel),用DNA琼脂糖凝胶电泳和Caspase3活性测定分析细胞凋亡情况,并采用激光共聚焦分析线粒体自噬现象。结果(1)Mel能抑制缺血再灌注介导的N2a细胞caspase3的活性,并减轻N2a细胞DNA的片段化;(2)N2a细胞缺血90min再灌注12h,未观察到线粒体自噬现象,而加入Mel的N2a有大量的线粒体自噬现象。结论线粒体自噬可能是褪黑素抗缺血再灌注损伤的机制之一。     

siRNA细胞色素c基因静默对大鼠脑皮质神经元体外模拟缺血再灌注损伤的影响

目的探讨体外模拟缺血再灌注（ischemia／reperfusion，IS／RE）后大鼠脑皮质神经元内质网应激凋亡的机制及抑制细胞色素c（Cyt c）表达对内质网应激的影响。方法体外培养SD乳鼠脑皮质神经元，用免疫组织化学、免疫荧光染色鉴定神经元纯度；用流式细胞术AnnexinV、PI双标检测调亡率及活性Caspase-3，-7，-9表达水平；用Westernblot免疫印迹方法检测Caspase-12、GRP78、Bcl-2、Cytc蛋白表达水平。结果SD乳鼠皮质神经元可纯化体外培养；空转染组神经元模拟缺血6h再灌注24h及48h（IS6h／RE24h、48h）细胞凋亡率分别是17．95％、22．62％；CytC基冈静默后凋亡率分别降至10．26％、9．37％，空转染组与转染组比较有统计学意义（P＜0．05）；空转染组神经元模拟缺血再灌注后GRP78、Cytc、活性Caspase-3、caspase-9、caspasel2表达均增加．Bcl2表达减少；siRNA Cyt c基因静默后神经元Bcl-2表达增加，GRP78、活性caspase-12、caspase-3、caspase-9表达明显降低，2组比较有统计学意义（P＜0．05）；空转染组和转染组均未检测到caspase-7表达。结论体外模拟缺血再灌注后脑皮质神经元发生凋亡，线粒体、内质网应激参与了神经元凋亡；抑制细胞色素c释放对内质网应激凋亡有抑制作用。     

抑制beclin-1信号通路对缺血再灌注后神经母细胞瘤N2a细胞自噬的影响

目的观察抑制beclin-1信号通路对缺血再灌注后神经母细胞瘤N2a细胞自噬的影响。方法体外培养神经母细胞瘤系N2a细胞,脂质体转染beclin-1的miRNA干扰质粒,以缺氧、缺营养方式模拟缺血再灌注,甲基噻唑基四唑法检测各组N2a细胞活性,免疫印迹法测定各组N2a细胞beclin-1、微管相关蛋白1轻链3(LC3)、天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)的表达,电镜观察该细胞自噬活性。结果在缺血90min、再灌注24h干扰组较相应假干扰组细胞活性明显升高(P<0.05);干扰组beclin-1和Caspase3表达水平明显低于相应的假干扰组(P<0.05),但两组之间LC3表达无明显差异(P>0.05);而在缺血30min再灌注24h细胞活性,beclin-1、Caspase3和LC3的表达真假干扰组间无明显差异(P>0.05)。电镜下在正常组没有观察到细胞自噬现象;在缺血30min和90min再灌注24h,真假干扰组均观察到明显的细胞自噬现象,且两组间无明显差异。结论缺血90min,再灌注24h时,干扰beclin-1的表达虽不能明显改变N2a细胞自噬水平,却减少了凋亡,有利细胞存活。     

褪黑素和6-羟褪黑素保护神经细胞抗缺血再灌注损伤比较研究

目的研究褪黑素(Mel)和6-羟褪黑素(6-OHMel)神经保护作用及作用机理。方法体外培养N2a细胞,模拟缺血再灌注(OGSD),加入Mel和6-OHMel,检测以下指标:①细胞生存能力:MTT法、乳酸脱氢酶释放;②细胞凋亡分析:DNA片断化,细胞色素C,Caspase3活性;③活性氧(ROS)和线粒体跨膜电位。结果①Mel和6-OHMel都能减轻OGSD诱导的N2a细胞损伤,Mel的作用强于6-OHMel。②Mel和6-OHMel均能抑制细胞色素C释放,但6-OHMel强于Mel。③Mel和6-OHMel都能稳定线粒体跨膜电位,但Mel作用时间比6-OHMel长。④Mel和6-OHMel能清除ROS,6-OHMel表现为直接作用,Mel表现为间接作用。⑤Mel和6-OHMel均能抑制caspase3的活性,但是作用时间不同。6-OHMel表现在OGSD后12h,Mel在OGSD后24h。结论Mel和6-OHMel的神经保护作用与其抗氧化、稳定线粒体功能相关,Mel的作用机制更复杂。     

6-羟基褪黑素保护神经细胞抗缺血再灌注损伤的作用机制

目的探讨6-羟基褪黑素的神经保护作用及作用机理。方法体外培养神经母细胞瘤N2a细胞，缺糖、缺血清和缺氧（OGSD）90min，再正常培养不同时间，同时加入6-羟基褪黑素，观察细胞生存能力（MTT法）、线粒体跨膜电位变化（荧光标记）、线粒体细胞色素C释放（western blot）、caspase3活性（荧光底物）以及细胞内活性氧（荧光标记）的产生。结果6-羟基褪黑素抑制细胞色素C释放，抑制caspase3激活，清除细胞内活性氧，稳定线粒体跨膜电位。结论6-羟基褪黑素通过直接抗氧化作用和抑制线粒体凋亡途径促进神经细胞生存。     

Pinacidil抑制线粒体和死亡受体通路减少大鼠脑缺血再灌注后神经元凋亡(英文)

目的探讨ATP敏感性钾通道开放剂pinacidil对大鼠脑缺血再灌注后神经元凋亡的保护作用及信号转导机制。方法 100 只Wistar 雄性大鼠随机分为四组:A 组(假手术组)、B组 (缺血组)、C 组 (KATP开放剂处理组)及D组 (KATP开放剂和阻断剂处理组)。用线栓法制备大鼠大脑中动脉缺血(middle cerebral artery occlusion,MCAO)模型,用DNA断端末端标记法(terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling,TUNEL)检测神经元凋亡,用原位杂交方法检测caspase-3、caspase-8及caspase-9 mRNA的表达。结果 (1) C组12 h、24 h、48 h、72 h 时间点的凋亡细胞数较 B、D 组显著减少(P<0.05 或 P<0.01) ;B 组和 D组之间无显著性差异(P>0.05)。(2) C 组 caspase-3 mRNA 和 caspase-8 mRNA 在各时间点及 caspase-9 mRNA 在 12 h、24 h、48 h、72h 时间点的表达显著少于B组和D组(P<0.01或P<0.05),B组和D组之间无显著性差异(P>0.05)。结论 KATP通道开放剂能显著减少大鼠脑缺血再灌注后的细胞凋亡及caspase-3、caspase-8及caspase-9 mRNA的表达。KATP通道开放剂可能通过抑制线粒体通路和死亡受体通路降低神经元凋亡,保护缺血再灌注损伤后的脑组织。     

褪黑素在缺血再灌注引起神经元凋亡的保护作用

目的探讨模拟缺血再灌注引起神经元凋亡的途径和褪黑素(m elaton in,MT)抗凋亡的作用机理。方法建立原代培养大鼠小脑颗粒细胞的体外模拟缺血(Oxygen G lucose Deprivation,OGD)再灌注模型,测定培养液LDH活性和细胞DNA琼脂糖凝胶电泳;利用Rhodam ine123和激光共聚焦显微镜观察线粒体膜电位的变化;ELISA检测细胞浆中细胞色素C水平;用同样指标观察MT对其损伤的保护作用。结果在体外模拟缺血再灌注模型中培养液LDH活性增加(P<0.05),琼脂糖凝胶电泳DNA出现梯状条带,线粒体膜电位明显降低,细胞浆中细胞色素C含量增加(P<0.05),MT对上述现象有明显的抑制作用。结论(1)缺血再灌注引起的神经元凋亡机理之一是通过线粒体凋亡途径;(2)MT可通过阻止线粒体凋亡途径而保护模拟缺血再灌注诱导小脑颗粒细胞的凋亡。     

P~(38)激酶在PC12细胞缺氧/再给氧损伤中的作用

目的:探讨PC12细胞缺氧/再给氧损伤的信号转导机理。方法:培养的PC12细胞先缺氧(95％N2/5％CO2)6h,然后重新给氧,观测不同时间点细胞的存活率和caspase-3的活性;用MTT法测存活率,caspase-3检测试剂盒测caspase-3活性。用p38拮抗剂SB203580孵育细胞2h,之后缺氧/再给氧,观察SB203580对细胞存活率和caspase-3活性的影响。结果:PC12细胞缺氧/再给氧后caspase-3活性明显增加并使细胞存活率下降,SB203580明显降低缺氧/复氧后caspase-3的活性并使细胞死亡减少。结论:PC12细胞缺氧/再给氧后至少可以通过激活p38、caspase-3信号分子诱导PC12细胞死亡。     

缺血后处理对大鼠脑缺血—再灌注中细胞凋亡的作用及其机制研究

目的观察缺血后处理(IP)对大鼠脑缺血-再灌注(I/R)中细胞凋亡的影响,并探寻其机制。方法开颅永久性阻断大脑中动脉+临时夹闭双侧颈总动脉法制作模型。将大鼠随机分为空白对照组(Control组)12只、假手术组(Sham组)、I/R组、IP组各48只、I/R+caspase-3抑制剂组(I/R+Z-DEVD-FMK组)、I/R+溶剂组(I/R+DMSO组)各12只。通过免疫荧光及Western blot法检测细胞凋亡数、细胞色素c(cyt-c)释放及caspase-3活性。结果 IP组较I/R组TUNEL阳性细胞数减少(P<0.05)。Sham组、IP组和I/R组均有细胞呈cyt-c/TUNEL双阳性,但cyt-c阳性不全伴有TUNEL阳性。I/R组与IP组cyt-c呈双峰样释放(再灌注3 h和48 h),在48 h时IP组较I/R组降低(P<0.05)。I/R组caspase-3活性在再灌注3 h时开始升高,12 h和24 h时最高。各相应时点IP组较I/R组的caspase-3活性降低(P<0.01和P<0.05)。I/R+Z-DEVD-FMK组cyt-c后期释放量小于I/R+DMSO组(P<0.01),但完整细胞数多于I/R+DMSO组(P<0.01)。结论 IP可以抑制凋亡;cyt-c参与凋亡,并与caspase-3形成相互作用的反馈回路。IP对该反馈回路具有调节作用。     

Cytochrome c release and caspase activation after traumatic brain injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage resulting in significant neurological dysfunction. The identification of cell death pathways that mediate this secondary traumatic injury have not been elucidated, however recent studies have implicated a role for apoptosis in the neuropathology of traumatic brain injury. The present study utilized a controlled cortical impact model of brain injury to assess the involvement of apoptotic pathways: release of cytochrome c from mitochondria and the activation of caspase-1- and caspase-3-like proteases in the injured cortex at 6, 12 and 24 h post-injury. Collectively, these results demonstrate cytochrome c release from mitochondria and its redistribution into the cytosol occurs in a time-dependent manner following TBI. The release of cytochrome c is accompanied by a time-dependent increase in caspase-3-like protease activity with no apparent increase in caspase-1-like activity. However, pretreatment with a general caspase inhibitor had no significant effect on the amount of cortical damage observed at 7 days post-injury. Our data suggest that several pro-apoptotic events occur following TBI, however the translocation of cytochrome c itself and/or other events upstream of caspase activation/inhibition may be sufficient to induce neuronal cell death.     

大鼠局灶性脑缺血再灌流后Caspase-3激活与神经元凋亡的关系

目的探讨Caspase-3与局灶性脑缺血后神经元凋亡的关系。方法局灶性脑缺血再灌流大鼠模型,按再灌流时间不同随机分为5组。用半定量RT-PCR观察了脑缺血后不同时程Caspase-3mRNA表达及四肽荧光底物法检测Caspase-3蛋白酶活性;用TUNEL方法检测不同时程细胞凋亡。结果脑缺血2h再灌流2h组Caspase-3mRNA水干即已升高(P＜0.05)),蛋白酶活性轻度升高,再灌流6h后两者均明显升高(P＜0.05);脑缺血再灌流后不同时程细胞凋亡具有与Caspase-3相似的变化趋势。结论提示脑缺血后细胞凋亡的发生与Caspase-3的激活有关。     

Cytochrome c release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells

The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.     

Manganese Superoxide Dismutase Affects Cytochrome c Release and Caspase-9 Activation After Transient Focal Cerebral Ischemia in Mice.

Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 -/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 -/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5;-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 -/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.     

Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.     

Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and Activation of caspase-like proteases.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.     

Induction of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion.

Deoxyribonucleic acid fragmentation at nucleosomal junctions is a hallmark of neuronal apoptosis in ischemic brain injury, for which the mechanism is not fully understood. Using the in vitro cell-free apoptosis assay, the authors found that caspase-3-dependent deoxyribonuclease activity caused internucleosomal DNA fragmentation in brain-cell extracts in a rat model of transient focal ischemia. This in vitro deoxyribonuclease activity was completely inhibited by purified inhibitor of caspase-activated deoxyribonuclease protein, the specific endogenous inhibitor of caspase-activated deoxyribonuclease, or by caspase-activated deoxyribonuclease immunodepletion. The induction of the deoxyribonuclease activity was correlated with caspase-3 activation and caspase-3-mediated degradation of inhibitor of caspase-activated deoxyribonuclease. Furthermore, inhibiting caspase-3-like protease activity prevented the endogenous induction of internucleosomal DNA fragmentation in the ischemic brain. These results suggest that caspase-3-dependent caspase-activated deoxyribonuclease activity plays an important role in mediating DNA fragmentation after focal ischemia.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.