埃他卡林对脑神经血管单元的保护作用及其分子机制
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摘要
脑血管疾病是威胁人类健康的重大疾病之一,具有高死亡率，高发病率和高并发症发生率的特点。调查显示全世界每年大约有795，000名的人群受到其危害，其相关治疗费用更是高达68.9亿美元。其中约70%为缺血性脑血管疾病。我国是脑血管疾病的高发国之一,并且近20年来其发病率一直呈上升趋势，脑血管疾病已成为我国人口的第二大死亡原因。目前针对其临床治疗的药物疗效有限，亟需研发具有新作用靶点和作用途径的缺血性脑血管疾病治疗药物。
缺氧脑损伤的主要病理改变是脑缺氧导致神经元死亡,从而影响脑功能，在此过程中神经元、胶质细胞以及脑微血管内皮细胞自身的损伤、坏死和凋亡以及相互的影响是脑组织损伤过程的细胞变化基础。随着近年来医学界对中枢神经系统多种细胞形态和功能的再认识，神经血管单元（neurovascular unit，NVU）概念逐步得到医学界的认同。所谓NVU，是指由神经元、星形胶质细胞、脑微血管内皮细胞、小胶质细胞及维持脑组织完整性的细胞外基质和他们之间的相互作用和相互联系构成的一个整体概念。NVU扩充了血脑屏障（Blood brain barrier，BBB）的内涵与外延，具有多种细胞组成的动态微环境结构特征。NVU与缺血性脑血管疾病的发生发展具有密切的关系,其作为一个功能模块，已日益成为治疗脑缺氧损伤研究的重要靶点之一。
ATP敏感性钾离子通道（K_(ATP)通道）是细胞膜上可以偶联细胞兴奋性和能量代谢的受细胞内ATP浓度调控的、具有弱内向整流作用的一类重要分子。文献报道，K_(ATP)通道在大脑各区几乎均有表达，在神经血管单元的三种主要细胞神经元、胶质细胞和内皮细胞上也有分布表达。研究显示，该通道在生理条件下往往处于关闭状态，而在缺血/缺氧状态下开放，通过减少兴奋性递质的释放以及启动下游多条细胞信号通路，可在多种组织中发挥抗缺血/缺氧损伤的内源性保护作用。
埃他卡林是我国具有自主知识产权的一类新药，具有脂肪胺结构的新结构类型的K_(ATP)通道开放剂，在国际上被列为第九类钾通道开放剂。他是小分子化合物能够透过血脑屏障。前期研究发现在沙土鼠双侧颈动脉结扎全脑缺血模型上发现埃他卡林对缺血所致动物行为异常具有明显的对抗作用，能够减少兴奋性递质的释放；能够剂量依赖性地减少全脑缺血所致海马CA1区锥体神经元的丢失。在脑卒中易感型自发性高血压大鼠盐负荷模型上发现，埃他卡林剂量依赖性地减少脑卒中的发生率和致死率，延缓卒中发生并延长存活时间；在减压缺氧大鼠脑损伤模型上发现，埃他卡林对高原脑水肿具有防治作用，能够维持血脑屏障的通透性，维持脑内离子平衡。但是埃他卡林作为ATP敏感性钾通道开放剂在缺氧脑损伤的模型上是否可以靶向神经血管单元发挥保护作用以及埃他卡林是如何针对缺氧脑损伤靶向于缺氧脑损伤进程中神经血管单元的功能的动态变化及其相互调节来发挥作用的，即埃他卡林实现神经血管保护作用的分子作用机制尚未阐明。进一步明确埃他卡林对缺氧性脑损伤的保护作用及其机制将为埃他卡林靶向神经血管单元发展成为针对神经损伤多元复杂病理机制诸多关键环节和高度激活的复杂信号转导级联反应的多潜能神经保护剂提供进一步的实验研究基础，也将为缺氧脑损伤的预防和治疗带来新的治疗策略。
本研究从不同的层次来研究埃他卡林对缺氧损伤神经血管单元的多环节多途径保护作用。在整体动物水平上，采用10%氧浓度常压缺氧舱，并结扎大鼠单侧颈总动脉造成缺氧脑损伤模型，证明埃他卡林对大鼠缺氧脑损伤的神经保护作用。在细胞水平上，建立连二亚硫酸钠无糖环境诱导细胞糖氧剥夺缺氧损伤模型，通过神经血管单元(NVU)成员细胞神经元、星形胶质细胞和微血管内皮细胞实验，证实埃他卡林具有抗缺氧损伤NVU细胞的保护作用。此外，本研究进一步探究埃他卡林激活K_(ATP)通道靶向神经血管单元抗缺氧脑损伤涉及的分子机制。
研究得到了如下的结果：
第一部分、埃他卡林对脑神经血管单元缺氧损伤的保护作用
一、埃他卡林对神经血管单元主要细胞类型的保护作用
1、神经血管单元细胞表达K_(ATP)亚型的差异
RT-PCR结果显示，在NVU三种细胞中Kir6.1的m RNA表达不同，微血管内皮细胞﹤神经元﹤星形胶质细胞；SUR2B的m RNA表达也有差异，神经元﹤星形胶质细胞﹤微血管内皮细胞；在微血管内皮细胞中Kir6.1和Sur2B的表达比较也存在显著差异，Kir6.1﹤SUR2B。
2、埃他卡林对神经血管单元缺氧损伤细胞存活率的影响
在体外细胞缺氧损伤模型下，给予0.01,0.1,到1mol/l的埃他卡林可以明显地提高NVU细胞包括神经元、星形胶质细胞、脑微血管内皮细胞缺氧损伤后的存活率。
3、埃他卡林对神经血管单元缺氧损伤细胞LDH的释放的影响
0.01,0.1,和1mol/l的埃他卡林后可以显著的降低NVU成员细胞在低氧损伤下LDH的释放量。埃他卡林的这种作用可以被K_(ATP)的阻断剂格列苯脲所阻断。
二、埃他卡林对大鼠缺血缺氧脑损伤的保护作用
1、埃他卡林对大鼠缺血缺氧脑损伤病理改变的影响
利用大鼠单侧颈总动脉结扎并处于10%常压缺氧环境造成，大鼠缺氧脑损伤模型，用HE染色的方法观察大鼠脑组织的病理变化，结果发现损伤组大鼠脑皮层神经细胞间隙明显增大，血管周围间隙增大，神经元数量减少，神经胶质细胞肿胀增大，毛细血管扩张充血。不同剂量的埃他卡林给药组（2,4,8mg/kg，每天灌胃给予）仍可见少量的胶质细胞和血管周围空晕，随着剂量增大，脑组织损伤程度逐渐减轻。
2、埃他卡林对大鼠缺血缺氧脑损伤血脑屏障通透性变化的影响
通过透射电镜观察大鼠缺血缺氧脑损伤后血脑屏障的结构变化发现，正常情况下，可见血管外周间隙致密；缺血缺氧组大鼠血管和组织外周间隙增大，可见明显的间隙和空泡；不同剂量埃他卡林（2,4,8kg/mg，每天灌胃给予）组作用之后可见血管外周间歇有不同程度的减小。
第二部分、埃他卡林对脑神经血管单元缺氧损伤保护作用分子机制的研究
一、埃他卡林对神经血管单元缺氧损伤凋亡相关蛋白表达的影响
1、埃他卡林对神经血管单元缺氧损伤细胞凋亡形态学的影响
Hoechest33258染色发现，神经血管单元细胞在缺氧损伤的环境中，体现细胞核染色质密度增加，浓缩集聚等典型的凋亡特征，而给予埃他卡林后，凋亡细胞数量下降，染色质浓缩程度减轻。
2、埃他卡林对神经血管单元缺氧损伤细胞凋亡率的影响
流式细胞仪检测神经元、星形胶质细胞和内皮细胞的凋亡率，在缺氧条件下，较之正常组，凋亡率显著增高（P﹤0.05），三种细胞皆有类似的结果。给予0.01,0.1和1mol/l埃他卡林后，凋亡率显著降低。
3、埃他卡林对缺氧损伤神经血管单元细胞抗凋亡作用强弱比较
埃他卡林对神经元的抗凋亡作用与它对内皮细胞的抗凋亡作用存在显著差异，（P﹤0.025）。埃他卡林对抗缺氧损伤引起星形胶质细胞的凋亡作用与它对内皮的抗凋亡作用也明显差异，（P﹤0.017）。
4、埃他卡林对神经血管单元缺氧损伤细胞凋亡相关通路的影响
缺氧损伤之后，神经元、星形胶质细胞以及内皮细胞中caspase-3mRNA和蛋白在模型组较之正常组均明显增高，0.01到1mol/l埃他卡林给药组中caspase-3mRNA和蛋白表达较之模型组都发生显著下调。0.01到1mol/l的埃他卡林处理细胞之后，NVU三种成员细胞中bax的mRNA表达较之模型组显著下降，而bcl-2的mRNA表达则明显升高。模型组细胞中caspase-9的蛋白表达明显升高，与正常组有显著差异，P﹤0.05。而埃他卡林作用组中caspase-9的蛋白表达显著下降。NVU细胞缺氧损伤后，Western blot的方法检查细胞胞浆中的cytC的蛋白表达较之正常对照明显增加，而1mol/l的埃他卡林可以逆转cytC的表达上调。此外，Western blot的方法还证实星形胶质细胞和微血管内皮细胞中pAkt/tAkt的蛋白表达在缺氧损伤后显著下调，而埃他卡林可以明显的上调pAkt/tAkt的蛋白表达。
5、埃他卡林调节大鼠缺氧脑损伤脑组织凋亡相关蛋白表达情况
大鼠缺氧脑损伤后，利用IHC的方法发现脑组织中Bcl-2的表达减少，同时Bax的表达增加，致Bcl-2/Bax的比值减小。当灌胃给予8mg/kg埃他卡林后，可以抑制脑缺血再灌注损伤所致的脑细胞凋亡，表明它具有神经保护作用。这一作用与其逆转Bcl-2表达下调，逆转Bax的表达上调以及对抗Caspase-3的表达上调有关。免疫组化的实验结果证实，在正常大鼠脑皮层中作为维持血脑屏障的成员之一的MMP-9呈现致密分布。大鼠发生缺氧脑损伤后，脑皮层组织中MMP-9的表达下降，血脑屏障通透性增加；8mg/kg的埃他卡林组大鼠脑皮层MMP-9的表达显著上升。正常大鼠脑组织中有一定量的pCREB表达，大鼠缺氧缺血脑损伤后pCREB的表达明显少于正常组，而给予埃他卡林的大鼠，脑组织中可见pCREB的表达较之模型组显著上升。pCREB可促进下游促神经营养因子的释放和细胞存活。
二、埃他卡林对大鼠缺氧脑损伤线粒体功能的影响
1、埃他卡林对大鼠缺氧脑损伤脑线粒体形态的影响
缺氧缺氧损伤组大鼠脑线粒体，单位面积内数量下降，HE染色结果可见大部分线粒体膜出现破损的状态，线粒体嵴间隙变大，出现空泡。缺氧损伤后给予埃他卡林2mg/kg，线粒体数量有所增加，线粒体膜破损减轻，嵴间隙变小。
2、埃他卡林对大鼠缺氧脑损伤脑线粒体功能的影响
大鼠缺氧脑损伤后，利用氧电极检查脑线粒体的各项指标发现发现，缺氧损伤组的ATP合成速率下降至（117.32±1.72），较之正常对照组（128.82±9.68），下降明显，P﹤0.05。而埃他卡林能增加大鼠缺氧脑损伤脑线粒体ATP合成速率达到（128.43±1.12）；埃他卡林能增加大鼠缺氧脑损伤脑线粒体磷/氧比即P/O比值（2.11±0.05）较之缺氧损伤组（2.08±0.04），有显著差异；埃他卡林能增强大鼠缺氧脑损伤脑线粒体的呼吸率（respiratory control rate，RCR）为（3.11±0.24），较之缺氧损伤组（2.91±0.23）有统计学差异。可见埃他卡林可以维持缺氧损伤线粒体后的形态和功能。
3、埃他卡林对缺氧损伤神经血管单元细胞线粒体膜电位的影响
经过缺氧损伤后NVU细胞经罗丹明123染色，发现罗丹明123的荧光强度都明显下降，说明线粒体膜电位明显下降。而埃他卡林预处理过的细胞经缺氧损伤之后，线粒体膜电位的下降得到一定程度的逆转。
4、埃他卡林对维持神经血管单元细胞膜电位作用差异比较
埃他卡林对神经元线粒体膜电位的维持作用和对内皮细胞的作用存在显著区别。
5、埃他卡林对缺氧损伤神经血管单元ROS生成的影响
采用DCFH-DA探针测定细胞内活性氧水平，发现在神经血管单元三种细胞正常状态下，几乎没有ROS的产生，低氧诱导损伤细胞之后，三种细胞内ROS的水平显著增加，较之正常组有明显差别，埃他卡林作用低氧损伤的细胞之后，细胞内的ROS水平较之损伤组显著下降，P﹤0.05。
三、埃他卡林对缺氧损伤神经血管单元释放神经营养因子的影响
1、埃他卡林对缺氧损伤神经血管单元细胞BDNF,GDNF,CNTF,NGF释放的影响
缺氧处理后的神经元分泌BDNF量与正常组比较，下降了78%(P <0.05)，而微血管内皮细胞在埃他卡林处理之后，可以增加显著增加神经元的存活率，星形胶质细胞在缺氧损伤时，BDNF的分泌也出现显著下降的情况，与正常组比较BDNF的分泌下降到37%(P <0.05)。微血管内皮细胞经缺氧损伤后，内皮细胞分泌BDNF下降。经1mol/l的埃他卡林作用后，可以显著性增加细胞分泌BDNF。
缺氧损伤可降低星形胶质细胞分泌GDNF，与正常组相比，GDNF的分泌下降到23%,(P <0.05)。经0.01-1mol/l的埃他卡林预处理缺氧损伤的细胞，其GDNF的分泌均有所增加。NVU细胞分泌CNTF和NGF的分泌也受到缺氧损伤而明显下降，而埃他卡林能不同程度的增加其分泌的减少，但是钾通道的拮抗剂格列苯脲并不能阻断埃他卡林的这种作用，提示埃他卡林虽然可能增加缺氧损伤后CNTF,NGF的分泌，ATP敏感性钾通道的激活并非直接作用，而只是参与了其中的某些调节环节，具体机制有待进一步研究。
2、埃他卡林对大鼠缺氧脑损伤后大鼠皮层BDNF, TrkB蛋白表达的影响
免疫组化的结果证实，在正常的大鼠脑皮层组织，BDNF主要分布于皮层的边缘区域。缺氧脑损伤之后，皮层部分BDNF的表达明显减少，但经埃他卡林2，4，8mg/kg处理后，大鼠皮层组织中BDNF的表达呈现浓度依赖性的增加。TrkB的变化与BDNF的表达呈现出一致的趋势。TrkB的蛋白表达在缺氧缺血之后，较之正常组显著下降，P <0.05。分别给予埃他卡林2，4，8mg/kg治疗，随着药物剂量的增加，可以逆转TrkB蛋白表达的下降，呈浓度依赖性的上调TrkB蛋白表达。
3、埃他卡林处理后ECM,ACM对化学缺氧损伤神经血管单元细胞相互作用的影响
经过缺氧损伤处理的神经元作为对照组不加任何药物干预，内皮细胞经过缺氧处理收集培养液（ECM），按相同的处理过程，从星形胶质细胞培养液中得到（ACM），实验证实，ECM和ACM分别与对照组的神经元共培养，均能增加神经元存活率，(E-CM:54.1%vs38.9%, P=0.04;A-CM:51.4%vs33.2%，P=0.03)。0.01μmol/L的埃他卡林处理ECM和ACM后，再与对照组神经元共培养，神经元的存活率进一步提高，分别为(E-CM vs Ipt+E-CM=54.1%vs78.2%, P=0.025;A-CM vs Ipt+A-CM=51.4%vs55.2%, P=0.045)。
综上所述，本研究结论如下：
1、ATP敏感性钾通道的开放剂埃他卡林能对神经血管单元低氧损伤发挥保护作用，在缺氧损伤的情况下，埃他卡林激活ATP敏感性钾通道可以保护神经元，星形胶质细胞和微血管内皮细胞，表现在缺氧损伤后它能增加细胞存活率，降低细胞释放LDH。
2、ATP敏感性钾通道的开放剂埃他卡林能够对抗缺氧脑损伤，他可以维持血脑屏障的结构，降低MMP-9表达的病理性上调，从而降低脑水肿，维持血脑屏障的通透性而发挥神经保护作用。
2、埃他卡林对神经血管单元缺氧损伤的保护作用与其抑制缺氧损伤诱导的NVU细胞凋亡，维持脑线粒体的功能，阻止线粒体膜电位的丢失有关，降低ROS生成的增加。其抗凋亡作用涉及细胞凋亡线粒体途径的多个信号分子以及pAkt/tAkt的表达水平变化。可能与激活线粒体ATP敏感性钾通道有关。此外，它可以上调后抗凋亡的bcl-2m RNA表达，下调促凋亡bax，caspase-3mRNA的表达；从蛋白水平，埃他卡林同时能下调Caspase-3蛋白表达。埃他卡林还能抑制CytC的蛋白释放，上调pAkt/tAkt的表达水平，下调Caspase-9的蛋白表达，从而多方面的抑制缺氧损伤引起的神经血管单元细胞凋亡的发生。
4、埃他卡林保护神经血管单元的作用与其促进NVU细胞分泌释放神经营养因子BDNF，GDNF,NGF及CNTF相关，神经营养因子分泌的增加强化了神经血管单元细胞-细胞间的相互作用，维持NVU的动态平衡。
5、在缺氧损伤的情况下，埃他卡林激活ATP敏感性钾通道可以靶向保护神经血管单元，这一效应与上游分子pCREB的表达增强有关，他的活化可以同时调节神经营养因子的释放并抑制细胞凋亡过程的发生，从而发挥细胞内源性神经保护作用。
6、埃他卡林对NVU成员细胞的作用具有细胞选择性，对微血管内皮细胞的作用最强，这与内皮细胞上K_(ATP)的靶点Kir6.1，SUR2B表达丰度有关。
Brain is an important organ with active metabolism. Interruption and decrease incerebral blood fluid leads to ischemia and or hypoxia to brain then the consequence isneuronal dysfunction and cerebral diseases. Cerebral ischemia/hypoxia injury is astrengthening problem to health with high mobility and mortality. Once brainischemia and hypoxia happened, it caused increase of oxygen radicals, induction ofreactive oxygen species,overproduction of peroxides, loss of mitochondria membranepotential and so on. Cerebral ischemia and hypoxia injury leads to the self-damage inneurons, astrocytes and microvascular endothelials, then necrosis and apoptosis, astogether became the cellular basic for brain impairment.
Apoptosis is one regarded way for cell death. Apoptosis is a kind of energy-consumption, programmed process. As energy factory, mitochondria play crucial rolein apoptosis. It is known that ischemia and hypoxia can cause cell mitochondriadysfunction then to activate pre-apoptotic proteinases to induce apoptosis.
The study on neuroprotection once focused on the protection of only neuron but itfailed to work. Recently, the neuroprotection means something new, which containsnot only protection for neurons but protections for astrocytes and endothelial cellsalso. Neurovascular unit as a functional module, become the core of neuroprotection.The definition of neurovascular unit is the neurons, astrocytes, endothelial cells andcell matrix even the interation and signal transduciton among them. It is a conceptionemphasize the dynamic homostasis among all elements.The neuroprotecion ofiptakalim, a K_(ATP)opener, on ischemia and hypoxia were confirmed in differentmodels in vivo and in vitro.How about effects of iptakalim on neurovascular unitsunder hypoxia and its property? The meanings of study the effects of iptakalim onneurovascular units under hypoxia are to confirm its pharmacological characteristics,to witness the development of neuroprotectant and to find effective intervention forbrain ischemia and hypoxia injury.
Our study will apply with in vivo brain injury model contains ratMCAO/reperfusion model and unilaterally occluding the carotid artery feeding in aatmospheric chamber with oxygen concentration at10%for12h simulated high altitude equivalent to5000m leading to ischemic brain injury. In vitro, cell ischemiccondition induced by sodium dithionite to verify the neuroprotection of iptakalim onNVU cells including neurons, astrocytes and microvascular endothelial cells, andfurther explore the different elements and multiple ways involved with theneuroprotection of iptakalim and its molecular mechanism.
Results were as follows,
Part1Protection of iptakalim on neurovascular unit against ischemia injury
1. Protection of iptakalim on main component cells of NVU against ischmic injury
1.1Difference in expression of K_(ATP)subtypes among NVU cells
To mRNA expression of Kir6.1: ECs 1.2Ipt restrains cell death under ishemic conditions
Treatment with0.01,0.1, or1μmol/l Ipt inhibited neurons cell death underischemic conditions. Similar results were obtained for astrocytes and endothelialcells under the same conditions, and treatment with Ipt from0.01μmol/lsignificantly inhibited cell death under ischemic conditions in these cells.
1.3Ipt suppresses LDH release from cells under ischemic condition
The level of LDH released into the culture medium for the three cell types underischemic conditions was strikingly higher compared with control groups.Pretreatment with0.01,0.1, or1μmol/l Ipt significantly decreased the level ofLDH released for all three cell types compared with cells under ischemicconditions. Glibenclamide, a K_(ATP)channel blocker, at a concentration of1mol/lreversed the protective effects of Ipt on the three types of cells. This suggeststhat Ipt protects cells from death via activating K_(ATP)channels.
2. Protection of iptakalim on rat ischemic brain injury
1.1Iptakalim Attenuates pathological process of brain tissue
2,4,8mg/kg iptakalim attenuated the brain edema, improved the neuron survivaland maintained the function of brain against ischemia/hypoxia brain injurycompared with ischemia/hypoxia injury groups.
1.2Iptakalim maintains BBB integrity under ischemic brain injury
Treatment with Ipt at doses of2,4, or8mg/kg/day can suppress the perivascularspace widening and endothelial cell swelling compared with the untreatedischemic group by transmission electron microscope.That means ipt maintainedBBB intergrity.
Part2The molecular mechanism of protection of iptakalim on NVU against ischemicinjury
1.1Iptakalim Attenuates Neuronal Apoptosis in vitro
1.1.1Ipt regulated the mRNA expression of bcl-2/bax and down-regulate thecaspase-3protein expression under ischemic condition.The mRNA expressionratio of bcl-2/bax was reduced in ischemic group compared to the normal one. Iptcan significantly increase its ratio. About caspase-3, Ipt can down-regulate itsprotein expression after hypoxia in NVU cells compared to model ones.
1.1.2Ipt inhibits cellular apoptosis in morphology under ischemic conditions
Hoechst33258was applied to neurons, astrocytes, and endothelial cells. Controlgroups for each cell type showed integral nuclei. Thus, cells treated with ischemicsolution exhibited characteristics of apoptotic cells such as nuclei condensationand fragmentation. In contrast, pretreatment with1μmol/l Ipt reduced theapoptosis induced by the ischemic solution.
1.1.3Ipt inhibits cellular apoptosis under ischemic conditions
Certain concentrations of Ipt can reduce the rate of apoptosis in NVU cell.
1.2Iptakalim Attenuates Neuronal Apoptosis in vivo
Caspase-3activity in the cortex was increased in ischemic/hypoxia(H/I) groupcompared with the sham group. In the ipt-treatment group, there was a significantdecrease in caspase-3activity in the2,4,8mg/kg groups compared with that in H/Igroup. Bcl-2and Bax activity were also measured, because activation of Bcl-2incells is a marker for an endogenous protective pathway in anti-apoptosis, whereasthe appearance of Bax indicates that more cells have initiated programmed celldeath. Thus, the ratio of the expression of these two proteins demonstrates thebalance between cellular protection and cell death. The ratio of Bcl-2/Bax wasreduced in ischemic group compared to the sham operation group. Treatment with2,4,8mg/kg iptakalim significantly increased this ratio to different extent. Iptakalimcould arrest apoptosis in rats after cerebral H/I injury.
2.Iptakalim affects brain mitochondria function
2.1Iptakalim maintained the morphology of brain mitochondria under hypoxiainjury
2.2Iptakalim improves the function of brain mitochondria
Ipt can improve the function of brain mitochondria under hypoxia injury byincreasing the ATP synthesis rate, raising the P/O ratio and elevating the RCR.
2.3Ipt prevents loss of the MMP under ischemic conditions
Application of0.01,0.1, and1μmol/l Ipt to the three different cell typesprevented the loss of MMP under ischemic conditions.
2.4Ipt reduced the production of ROS in NVU cells
3Iptakalim affects the interation among NVU cells
3.1Ipt enhances BDNF secretion in neurons and astrocytes under ischemiccondition and improves the interaction among NVU cells
Ipt can enhances BDNF secretion in neurons, astrocytes and ECsunder ischemiccondition.BDNF, as a brain derivative neurotrophyic factor,can improves theinteraction among NVU cells.
3.2Ipt can enhance the interaction among NVU cells through the effects on ECMand ACM
ECM and ACM can increase the neuron survival rate under ischemic condition.Iptcan enhance their effects on neurons survival by strengthening the secretion ofneurotrophic factors.
3.3Ipt influences the BDNF,TrkB expression in brain tissue underischemia/hypoxia brain injury.
Given with Ipt at doses of2,4, or8mg/kg/day can suppress down-regulation ofBDNF, TrkB protein expression in cerebral cortex.
To summarize as conclusions,
1. Activation of K_(ATP),iptakalim plays the neuroprotection role not only in cerebralischemia/reperfusion but also in ischemia/hypoxia brain injury.
2. Iptakalim can protect NVU cells such as neurons,astrocytes and endothelial cells,components of neurovascular unit, from ischemic condition induced by sodiumdithionite. Besides, iptakalim can also enhance the secretion of BDNF to strengthenthe interaction of components of NVU.This protection originates its enhancement ofsecrection of BDNF, which initiates the inner protection pathway.
3. The protection of iptakalim on NVU is the bottom of its neuroprotecion againstischemia/hypoxia brain injury. The possible mechanisms may include as follows,
a. its antiapoptosis pathway
b. the effect on remaining the function of brian mitochondria, maybe involved withactivation of mito K_(ATP).
c. Ipt has selective effect on NVU cells particular on ECs, this is possible related todifferent intensity of m RNA expression of the subtypes of K_(ATP)on of NVU cells.
缺氧脑损伤的主要病理改变是脑缺氧导致神经元死亡,从而影响脑功能，在此过程中神经元、胶质细胞以及脑微血管内皮细胞自身的损伤、坏死和凋亡以及相互的影响是脑组织损伤过程的细胞变化基础。随着近年来医学界对中枢神经系统多种细胞形态和功能的再认识，神经血管单元（neurovascular unit，NVU）概念逐步得到医学界的认同。所谓NVU，是指由神经元、星形胶质细胞、脑微血管内皮细胞、小胶质细胞及维持脑组织完整性的细胞外基质和他们之间的相互作用和相互联系构成的一个整体概念。NVU扩充了血脑屏障（Blood brain barrier，BBB）的内涵与外延，具有多种细胞组成的动态微环境结构特征。NVU与缺血性脑血管疾病的发生发展具有密切的关系,其作为一个功能模块，已日益成为治疗脑缺氧损伤研究的重要靶点之一。
ATP敏感性钾离子通道（K_(ATP)通道）是细胞膜上可以偶联细胞兴奋性和能量代谢的受细胞内ATP浓度调控的、具有弱内向整流作用的一类重要分子。文献报道，K_(ATP)通道在大脑各区几乎均有表达，在神经血管单元的三种主要细胞神经元、胶质细胞和内皮细胞上也有分布表达。研究显示，该通道在生理条件下往往处于关闭状态，而在缺血/缺氧状态下开放，通过减少兴奋性递质的释放以及启动下游多条细胞信号通路，可在多种组织中发挥抗缺血/缺氧损伤的内源性保护作用。
埃他卡林是我国具有自主知识产权的一类新药，具有脂肪胺结构的新结构类型的K_(ATP)通道开放剂，在国际上被列为第九类钾通道开放剂。他是小分子化合物能够透过血脑屏障。前期研究发现在沙土鼠双侧颈动脉结扎全脑缺血模型上发现埃他卡林对缺血所致动物行为异常具有明显的对抗作用，能够减少兴奋性递质的释放；能够剂量依赖性地减少全脑缺血所致海马CA1区锥体神经元的丢失。在脑卒中易感型自发性高血压大鼠盐负荷模型上发现，埃他卡林剂量依赖性地减少脑卒中的发生率和致死率，延缓卒中发生并延长存活时间；在减压缺氧大鼠脑损伤模型上发现，埃他卡林对高原脑水肿具有防治作用，能够维持血脑屏障的通透性，维持脑内离子平衡。但是埃他卡林作为ATP敏感性钾通道开放剂在缺氧脑损伤的模型上是否可以靶向神经血管单元发挥保护作用以及埃他卡林是如何针对缺氧脑损伤靶向于缺氧脑损伤进程中神经血管单元的功能的动态变化及其相互调节来发挥作用的，即埃他卡林实现神经血管保护作用的分子作用机制尚未阐明。进一步明确埃他卡林对缺氧性脑损伤的保护作用及其机制将为埃他卡林靶向神经血管单元发展成为针对神经损伤多元复杂病理机制诸多关键环节和高度激活的复杂信号转导级联反应的多潜能神经保护剂提供进一步的实验研究基础，也将为缺氧脑损伤的预防和治疗带来新的治疗策略。
本研究从不同的层次来研究埃他卡林对缺氧损伤神经血管单元的多环节多途径保护作用。在整体动物水平上，采用10%氧浓度常压缺氧舱，并结扎大鼠单侧颈总动脉造成缺氧脑损伤模型，证明埃他卡林对大鼠缺氧脑损伤的神经保护作用。在细胞水平上，建立连二亚硫酸钠无糖环境诱导细胞糖氧剥夺缺氧损伤模型，通过神经血管单元(NVU)成员细胞神经元、星形胶质细胞和微血管内皮细胞实验，证实埃他卡林具有抗缺氧损伤NVU细胞的保护作用。此外，本研究进一步探究埃他卡林激活K_(ATP)通道靶向神经血管单元抗缺氧脑损伤涉及的分子机制。
研究得到了如下的结果：
第一部分、埃他卡林对脑神经血管单元缺氧损伤的保护作用
一、埃他卡林对神经血管单元主要细胞类型的保护作用
1、神经血管单元细胞表达K_(ATP)亚型的差异
RT-PCR结果显示，在NVU三种细胞中Kir6.1的m RNA表达不同，微血管内皮细胞﹤神经元﹤星形胶质细胞；SUR2B的m RNA表达也有差异，神经元﹤星形胶质细胞﹤微血管内皮细胞；在微血管内皮细胞中Kir6.1和Sur2B的表达比较也存在显著差异，Kir6.1﹤SUR2B。
2、埃他卡林对神经血管单元缺氧损伤细胞存活率的影响
在体外细胞缺氧损伤模型下，给予0.01,0.1,到1mol/l的埃他卡林可以明显地提高NVU细胞包括神经元、星形胶质细胞、脑微血管内皮细胞缺氧损伤后的存活率。
3、埃他卡林对神经血管单元缺氧损伤细胞LDH的释放的影响
0.01,0.1,和1mol/l的埃他卡林后可以显著的降低NVU成员细胞在低氧损伤下LDH的释放量。埃他卡林的这种作用可以被K_(ATP)的阻断剂格列苯脲所阻断。
二、埃他卡林对大鼠缺血缺氧脑损伤的保护作用
1、埃他卡林对大鼠缺血缺氧脑损伤病理改变的影响
利用大鼠单侧颈总动脉结扎并处于10%常压缺氧环境造成，大鼠缺氧脑损伤模型，用HE染色的方法观察大鼠脑组织的病理变化，结果发现损伤组大鼠脑皮层神经细胞间隙明显增大，血管周围间隙增大，神经元数量减少，神经胶质细胞肿胀增大，毛细血管扩张充血。不同剂量的埃他卡林给药组（2,4,8mg/kg，每天灌胃给予）仍可见少量的胶质细胞和血管周围空晕，随着剂量增大，脑组织损伤程度逐渐减轻。
2、埃他卡林对大鼠缺血缺氧脑损伤血脑屏障通透性变化的影响
通过透射电镜观察大鼠缺血缺氧脑损伤后血脑屏障的结构变化发现，正常情况下，可见血管外周间隙致密；缺血缺氧组大鼠血管和组织外周间隙增大，可见明显的间隙和空泡；不同剂量埃他卡林（2,4,8kg/mg，每天灌胃给予）组作用之后可见血管外周间歇有不同程度的减小。
第二部分、埃他卡林对脑神经血管单元缺氧损伤保护作用分子机制的研究
一、埃他卡林对神经血管单元缺氧损伤凋亡相关蛋白表达的影响
1、埃他卡林对神经血管单元缺氧损伤细胞凋亡形态学的影响
Hoechest33258染色发现，神经血管单元细胞在缺氧损伤的环境中，体现细胞核染色质密度增加，浓缩集聚等典型的凋亡特征，而给予埃他卡林后，凋亡细胞数量下降，染色质浓缩程度减轻。
2、埃他卡林对神经血管单元缺氧损伤细胞凋亡率的影响
流式细胞仪检测神经元、星形胶质细胞和内皮细胞的凋亡率，在缺氧条件下，较之正常组，凋亡率显著增高（P﹤0.05），三种细胞皆有类似的结果。给予0.01,0.1和1mol/l埃他卡林后，凋亡率显著降低。
3、埃他卡林对缺氧损伤神经血管单元细胞抗凋亡作用强弱比较
埃他卡林对神经元的抗凋亡作用与它对内皮细胞的抗凋亡作用存在显著差异，（P﹤0.025）。埃他卡林对抗缺氧损伤引起星形胶质细胞的凋亡作用与它对内皮的抗凋亡作用也明显差异，（P﹤0.017）。
4、埃他卡林对神经血管单元缺氧损伤细胞凋亡相关通路的影响
缺氧损伤之后，神经元、星形胶质细胞以及内皮细胞中caspase-3mRNA和蛋白在模型组较之正常组均明显增高，0.01到1mol/l埃他卡林给药组中caspase-3mRNA和蛋白表达较之模型组都发生显著下调。0.01到1mol/l的埃他卡林处理细胞之后，NVU三种成员细胞中bax的mRNA表达较之模型组显著下降，而bcl-2的mRNA表达则明显升高。模型组细胞中caspase-9的蛋白表达明显升高，与正常组有显著差异，P﹤0.05。而埃他卡林作用组中caspase-9的蛋白表达显著下降。NVU细胞缺氧损伤后，Western blot的方法检查细胞胞浆中的cytC的蛋白表达较之正常对照明显增加，而1mol/l的埃他卡林可以逆转cytC的表达上调。此外，Western blot的方法还证实星形胶质细胞和微血管内皮细胞中pAkt/tAkt的蛋白表达在缺氧损伤后显著下调，而埃他卡林可以明显的上调pAkt/tAkt的蛋白表达。
5、埃他卡林调节大鼠缺氧脑损伤脑组织凋亡相关蛋白表达情况
大鼠缺氧脑损伤后，利用IHC的方法发现脑组织中Bcl-2的表达减少，同时Bax的表达增加，致Bcl-2/Bax的比值减小。当灌胃给予8mg/kg埃他卡林后，可以抑制脑缺血再灌注损伤所致的脑细胞凋亡，表明它具有神经保护作用。这一作用与其逆转Bcl-2表达下调，逆转Bax的表达上调以及对抗Caspase-3的表达上调有关。免疫组化的实验结果证实，在正常大鼠脑皮层中作为维持血脑屏障的成员之一的MMP-9呈现致密分布。大鼠发生缺氧脑损伤后，脑皮层组织中MMP-9的表达下降，血脑屏障通透性增加；8mg/kg的埃他卡林组大鼠脑皮层MMP-9的表达显著上升。正常大鼠脑组织中有一定量的pCREB表达，大鼠缺氧缺血脑损伤后pCREB的表达明显少于正常组，而给予埃他卡林的大鼠，脑组织中可见pCREB的表达较之模型组显著上升。pCREB可促进下游促神经营养因子的释放和细胞存活。
二、埃他卡林对大鼠缺氧脑损伤线粒体功能的影响
1、埃他卡林对大鼠缺氧脑损伤脑线粒体形态的影响
缺氧缺氧损伤组大鼠脑线粒体，单位面积内数量下降，HE染色结果可见大部分线粒体膜出现破损的状态，线粒体嵴间隙变大，出现空泡。缺氧损伤后给予埃他卡林2mg/kg，线粒体数量有所增加，线粒体膜破损减轻，嵴间隙变小。
2、埃他卡林对大鼠缺氧脑损伤脑线粒体功能的影响
大鼠缺氧脑损伤后，利用氧电极检查脑线粒体的各项指标发现发现，缺氧损伤组的ATP合成速率下降至（117.32±1.72），较之正常对照组（128.82±9.68），下降明显，P﹤0.05。而埃他卡林能增加大鼠缺氧脑损伤脑线粒体ATP合成速率达到（128.43±1.12）；埃他卡林能增加大鼠缺氧脑损伤脑线粒体磷/氧比即P/O比值（2.11±0.05）较之缺氧损伤组（2.08±0.04），有显著差异；埃他卡林能增强大鼠缺氧脑损伤脑线粒体的呼吸率（respiratory control rate，RCR）为（3.11±0.24），较之缺氧损伤组（2.91±0.23）有统计学差异。可见埃他卡林可以维持缺氧损伤线粒体后的形态和功能。
3、埃他卡林对缺氧损伤神经血管单元细胞线粒体膜电位的影响
经过缺氧损伤后NVU细胞经罗丹明123染色，发现罗丹明123的荧光强度都明显下降，说明线粒体膜电位明显下降。而埃他卡林预处理过的细胞经缺氧损伤之后，线粒体膜电位的下降得到一定程度的逆转。
4、埃他卡林对维持神经血管单元细胞膜电位作用差异比较
埃他卡林对神经元线粒体膜电位的维持作用和对内皮细胞的作用存在显著区别。
5、埃他卡林对缺氧损伤神经血管单元ROS生成的影响
采用DCFH-DA探针测定细胞内活性氧水平，发现在神经血管单元三种细胞正常状态下，几乎没有ROS的产生，低氧诱导损伤细胞之后，三种细胞内ROS的水平显著增加，较之正常组有明显差别，埃他卡林作用低氧损伤的细胞之后，细胞内的ROS水平较之损伤组显著下降，P﹤0.05。
三、埃他卡林对缺氧损伤神经血管单元释放神经营养因子的影响
1、埃他卡林对缺氧损伤神经血管单元细胞BDNF,GDNF,CNTF,NGF释放的影响
缺氧处理后的神经元分泌BDNF量与正常组比较，下降了78%(P <0.05)，而微血管内皮细胞在埃他卡林处理之后，可以增加显著增加神经元的存活率，星形胶质细胞在缺氧损伤时，BDNF的分泌也出现显著下降的情况，与正常组比较BDNF的分泌下降到37%(P <0.05)。微血管内皮细胞经缺氧损伤后，内皮细胞分泌BDNF下降。经1mol/l的埃他卡林作用后，可以显著性增加细胞分泌BDNF。
缺氧损伤可降低星形胶质细胞分泌GDNF，与正常组相比，GDNF的分泌下降到23%,(P <0.05)。经0.01-1mol/l的埃他卡林预处理缺氧损伤的细胞，其GDNF的分泌均有所增加。NVU细胞分泌CNTF和NGF的分泌也受到缺氧损伤而明显下降，而埃他卡林能不同程度的增加其分泌的减少，但是钾通道的拮抗剂格列苯脲并不能阻断埃他卡林的这种作用，提示埃他卡林虽然可能增加缺氧损伤后CNTF,NGF的分泌，ATP敏感性钾通道的激活并非直接作用，而只是参与了其中的某些调节环节，具体机制有待进一步研究。
2、埃他卡林对大鼠缺氧脑损伤后大鼠皮层BDNF, TrkB蛋白表达的影响
免疫组化的结果证实，在正常的大鼠脑皮层组织，BDNF主要分布于皮层的边缘区域。缺氧脑损伤之后，皮层部分BDNF的表达明显减少，但经埃他卡林2，4，8mg/kg处理后，大鼠皮层组织中BDNF的表达呈现浓度依赖性的增加。TrkB的变化与BDNF的表达呈现出一致的趋势。TrkB的蛋白表达在缺氧缺血之后，较之正常组显著下降，P <0.05。分别给予埃他卡林2，4，8mg/kg治疗，随着药物剂量的增加，可以逆转TrkB蛋白表达的下降，呈浓度依赖性的上调TrkB蛋白表达。
3、埃他卡林处理后ECM,ACM对化学缺氧损伤神经血管单元细胞相互作用的影响
经过缺氧损伤处理的神经元作为对照组不加任何药物干预，内皮细胞经过缺氧处理收集培养液（ECM），按相同的处理过程，从星形胶质细胞培养液中得到（ACM），实验证实，ECM和ACM分别与对照组的神经元共培养，均能增加神经元存活率，(E-CM:54.1%vs38.9%, P=0.04;A-CM:51.4%vs33.2%，P=0.03)。0.01μmol/L的埃他卡林处理ECM和ACM后，再与对照组神经元共培养，神经元的存活率进一步提高，分别为(E-CM vs Ipt+E-CM=54.1%vs78.2%, P=0.025;A-CM vs Ipt+A-CM=51.4%vs55.2%, P=0.045)。
综上所述，本研究结论如下：
1、ATP敏感性钾通道的开放剂埃他卡林能对神经血管单元低氧损伤发挥保护作用，在缺氧损伤的情况下，埃他卡林激活ATP敏感性钾通道可以保护神经元，星形胶质细胞和微血管内皮细胞，表现在缺氧损伤后它能增加细胞存活率，降低细胞释放LDH。
2、ATP敏感性钾通道的开放剂埃他卡林能够对抗缺氧脑损伤，他可以维持血脑屏障的结构，降低MMP-9表达的病理性上调，从而降低脑水肿，维持血脑屏障的通透性而发挥神经保护作用。
2、埃他卡林对神经血管单元缺氧损伤的保护作用与其抑制缺氧损伤诱导的NVU细胞凋亡，维持脑线粒体的功能，阻止线粒体膜电位的丢失有关，降低ROS生成的增加。其抗凋亡作用涉及细胞凋亡线粒体途径的多个信号分子以及pAkt/tAkt的表达水平变化。可能与激活线粒体ATP敏感性钾通道有关。此外，它可以上调后抗凋亡的bcl-2m RNA表达，下调促凋亡bax，caspase-3mRNA的表达；从蛋白水平，埃他卡林同时能下调Caspase-3蛋白表达。埃他卡林还能抑制CytC的蛋白释放，上调pAkt/tAkt的表达水平，下调Caspase-9的蛋白表达，从而多方面的抑制缺氧损伤引起的神经血管单元细胞凋亡的发生。
4、埃他卡林保护神经血管单元的作用与其促进NVU细胞分泌释放神经营养因子BDNF，GDNF,NGF及CNTF相关，神经营养因子分泌的增加强化了神经血管单元细胞-细胞间的相互作用，维持NVU的动态平衡。
5、在缺氧损伤的情况下，埃他卡林激活ATP敏感性钾通道可以靶向保护神经血管单元，这一效应与上游分子pCREB的表达增强有关，他的活化可以同时调节神经营养因子的释放并抑制细胞凋亡过程的发生，从而发挥细胞内源性神经保护作用。
6、埃他卡林对NVU成员细胞的作用具有细胞选择性，对微血管内皮细胞的作用最强，这与内皮细胞上K_(ATP)的靶点Kir6.1，SUR2B表达丰度有关。
Brain is an important organ with active metabolism. Interruption and decrease incerebral blood fluid leads to ischemia and or hypoxia to brain then the consequence isneuronal dysfunction and cerebral diseases. Cerebral ischemia/hypoxia injury is astrengthening problem to health with high mobility and mortality. Once brainischemia and hypoxia happened, it caused increase of oxygen radicals, induction ofreactive oxygen species,overproduction of peroxides, loss of mitochondria membranepotential and so on. Cerebral ischemia and hypoxia injury leads to the self-damage inneurons, astrocytes and microvascular endothelials, then necrosis and apoptosis, astogether became the cellular basic for brain impairment.
Apoptosis is one regarded way for cell death. Apoptosis is a kind of energy-consumption, programmed process. As energy factory, mitochondria play crucial rolein apoptosis. It is known that ischemia and hypoxia can cause cell mitochondriadysfunction then to activate pre-apoptotic proteinases to induce apoptosis.
The study on neuroprotection once focused on the protection of only neuron but itfailed to work. Recently, the neuroprotection means something new, which containsnot only protection for neurons but protections for astrocytes and endothelial cellsalso. Neurovascular unit as a functional module, become the core of neuroprotection.The definition of neurovascular unit is the neurons, astrocytes, endothelial cells andcell matrix even the interation and signal transduciton among them. It is a conceptionemphasize the dynamic homostasis among all elements.The neuroprotecion ofiptakalim, a K_(ATP)opener, on ischemia and hypoxia were confirmed in differentmodels in vivo and in vitro.How about effects of iptakalim on neurovascular unitsunder hypoxia and its property? The meanings of study the effects of iptakalim onneurovascular units under hypoxia are to confirm its pharmacological characteristics,to witness the development of neuroprotectant and to find effective intervention forbrain ischemia and hypoxia injury.
Our study will apply with in vivo brain injury model contains ratMCAO/reperfusion model and unilaterally occluding the carotid artery feeding in aatmospheric chamber with oxygen concentration at10%for12h simulated high altitude equivalent to5000m leading to ischemic brain injury. In vitro, cell ischemiccondition induced by sodium dithionite to verify the neuroprotection of iptakalim onNVU cells including neurons, astrocytes and microvascular endothelial cells, andfurther explore the different elements and multiple ways involved with theneuroprotection of iptakalim and its molecular mechanism.
Results were as follows,
Part1Protection of iptakalim on neurovascular unit against ischemia injury
1. Protection of iptakalim on main component cells of NVU against ischmic injury
1.1Difference in expression of K_(ATP)subtypes among NVU cells
To mRNA expression of Kir6.1: ECs
Treatment with0.01,0.1, or1μmol/l Ipt inhibited neurons cell death underischemic conditions. Similar results were obtained for astrocytes and endothelialcells under the same conditions, and treatment with Ipt from0.01μmol/lsignificantly inhibited cell death under ischemic conditions in these cells.
1.3Ipt suppresses LDH release from cells under ischemic condition
The level of LDH released into the culture medium for the three cell types underischemic conditions was strikingly higher compared with control groups.Pretreatment with0.01,0.1, or1μmol/l Ipt significantly decreased the level ofLDH released for all three cell types compared with cells under ischemicconditions. Glibenclamide, a K_(ATP)channel blocker, at a concentration of1mol/lreversed the protective effects of Ipt on the three types of cells. This suggeststhat Ipt protects cells from death via activating K_(ATP)channels.
2. Protection of iptakalim on rat ischemic brain injury
1.1Iptakalim Attenuates pathological process of brain tissue
2,4,8mg/kg iptakalim attenuated the brain edema, improved the neuron survivaland maintained the function of brain against ischemia/hypoxia brain injurycompared with ischemia/hypoxia injury groups.
1.2Iptakalim maintains BBB integrity under ischemic brain injury
Treatment with Ipt at doses of2,4, or8mg/kg/day can suppress the perivascularspace widening and endothelial cell swelling compared with the untreatedischemic group by transmission electron microscope.That means ipt maintainedBBB intergrity.
Part2The molecular mechanism of protection of iptakalim on NVU against ischemicinjury
1.1Iptakalim Attenuates Neuronal Apoptosis in vitro
1.1.1Ipt regulated the mRNA expression of bcl-2/bax and down-regulate thecaspase-3protein expression under ischemic condition.The mRNA expressionratio of bcl-2/bax was reduced in ischemic group compared to the normal one. Iptcan significantly increase its ratio. About caspase-3, Ipt can down-regulate itsprotein expression after hypoxia in NVU cells compared to model ones.
1.1.2Ipt inhibits cellular apoptosis in morphology under ischemic conditions
Hoechst33258was applied to neurons, astrocytes, and endothelial cells. Controlgroups for each cell type showed integral nuclei. Thus, cells treated with ischemicsolution exhibited characteristics of apoptotic cells such as nuclei condensationand fragmentation. In contrast, pretreatment with1μmol/l Ipt reduced theapoptosis induced by the ischemic solution.
1.1.3Ipt inhibits cellular apoptosis under ischemic conditions
Certain concentrations of Ipt can reduce the rate of apoptosis in NVU cell.
1.2Iptakalim Attenuates Neuronal Apoptosis in vivo
Caspase-3activity in the cortex was increased in ischemic/hypoxia(H/I) groupcompared with the sham group. In the ipt-treatment group, there was a significantdecrease in caspase-3activity in the2,4,8mg/kg groups compared with that in H/Igroup. Bcl-2and Bax activity were also measured, because activation of Bcl-2incells is a marker for an endogenous protective pathway in anti-apoptosis, whereasthe appearance of Bax indicates that more cells have initiated programmed celldeath. Thus, the ratio of the expression of these two proteins demonstrates thebalance between cellular protection and cell death. The ratio of Bcl-2/Bax wasreduced in ischemic group compared to the sham operation group. Treatment with2,4,8mg/kg iptakalim significantly increased this ratio to different extent. Iptakalimcould arrest apoptosis in rats after cerebral H/I injury.
2.Iptakalim affects brain mitochondria function
2.1Iptakalim maintained the morphology of brain mitochondria under hypoxiainjury
2.2Iptakalim improves the function of brain mitochondria
Ipt can improve the function of brain mitochondria under hypoxia injury byincreasing the ATP synthesis rate, raising the P/O ratio and elevating the RCR.
2.3Ipt prevents loss of the MMP under ischemic conditions
Application of0.01,0.1, and1μmol/l Ipt to the three different cell typesprevented the loss of MMP under ischemic conditions.
2.4Ipt reduced the production of ROS in NVU cells
3Iptakalim affects the interation among NVU cells
3.1Ipt enhances BDNF secretion in neurons and astrocytes under ischemiccondition and improves the interaction among NVU cells
Ipt can enhances BDNF secretion in neurons, astrocytes and ECsunder ischemiccondition.BDNF, as a brain derivative neurotrophyic factor,can improves theinteraction among NVU cells.
3.2Ipt can enhance the interaction among NVU cells through the effects on ECMand ACM
ECM and ACM can increase the neuron survival rate under ischemic condition.Iptcan enhance their effects on neurons survival by strengthening the secretion ofneurotrophic factors.
3.3Ipt influences the BDNF,TrkB expression in brain tissue underischemia/hypoxia brain injury.
Given with Ipt at doses of2,4, or8mg/kg/day can suppress down-regulation ofBDNF, TrkB protein expression in cerebral cortex.
To summarize as conclusions,
1. Activation of K_(ATP),iptakalim plays the neuroprotection role not only in cerebralischemia/reperfusion but also in ischemia/hypoxia brain injury.
2. Iptakalim can protect NVU cells such as neurons,astrocytes and endothelial cells,components of neurovascular unit, from ischemic condition induced by sodiumdithionite. Besides, iptakalim can also enhance the secretion of BDNF to strengthenthe interaction of components of NVU.This protection originates its enhancement ofsecrection of BDNF, which initiates the inner protection pathway.
3. The protection of iptakalim on NVU is the bottom of its neuroprotecion againstischemia/hypoxia brain injury. The possible mechanisms may include as follows,
a. its antiapoptosis pathway
b. the effect on remaining the function of brian mitochondria, maybe involved withactivation of mito K_(ATP).
c. Ipt has selective effect on NVU cells particular on ECs, this is possible related todifferent intensity of m RNA expression of the subtypes of K_(ATP)on of NVU cells.
引文
1Zhou M, Offer A, Yang G, Smith M, Hui G, Whitlock G, et al. Body massindex, blood pressure, and mortality from stroke: a nationally representativeprospective study of212,000Chinese men. Stroke; a journal of cerebralcirculation2008;39(3):753-9.
2Zhang LF, Yang J, Hong Z, Yuan GG, Zhou BF, Zhao LC, et al. Proportion ofdifferent subtypes of stroke in China. Stroke; a journal of cerebral circulation2003;34(9):2091-6.
3Liu HD, Liu, S, Li,H.L, Chen,X.Q, iao2Qian2), Yu, A. C. Activation ofSignal Transduction Pathways after Cerebral Ischemia. Chinese Journal ofBiochemistry and Molecular Biology2007;23(1):4.
4Graham SH, Chen J. Programmed cell death in cerebral ischemia. Journal ofcerebral blood flow and metabolism: official journal of the InternationalSociety of Cerebral Blood Flow and Metabolism2001;21(2):99-109.
5Su DF. Fundamental researches on the pathogenesis, prevention and treatmentof cerebrovascular diseases. Chinese Bulletin of Life Sciences2009;21(4):6.
6Haller M, Mironov SL, Karschin A, Richter DW. Dynamic activation ofK(ATP) channels in rhythmically active neurons. The Journal of physiology2001;537(Pt1):69-81.
7Diarra A, Sheldon C, Brett CL, Baimbridge KG, Church J. Anoxia-evokedintracellular pH and Ca2+concentration changes in cultured postnatal rathippocampal neurons. Neuroscience1999;93(3):1003-16.
8Girn HR, Ahilathirunayagam S, Mavor AI, Homer-Vanniasinkam S.Reperfusion syndrome: cellular mechanisms of microvascular dysfunction andpotential therapeutic strategies. Vascular and endovascular surgery2007;41(4):277-93.
9Choi DW. Cerebral hypoxia: some new approaches and unanswered questions.The Journal of neuroscience: the official journal of the Society forNeuroscience1990;10(8):2493-501.
10Lo EH, Rosenberg GA. The neurovascular unit in health and disease:introduction. Stroke; a journal of cerebral circulation2009;40(3Suppl): S2-3.
11Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in healthand disease. Pharmacological reviews2005;57(2):173-85.
12Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunitiesin stroke. Nature reviews Neuroscience2003;4(5):399-415.
13Janigro D, West GA, Gordon EL, Winn HR. ATP-sensitive K+channels in rataorta and brain microvascular endothelial cells. The American journal ofphysiology1993;265(3Pt1): C812-21.
14Mederos y Schnitzler M, Derst C, Daut J, Preisig-Muller R. ATP-sensitivepotassium channels in capillaries isolated from guinea-pig heart. The Journalof physiology2000;525Pt2:307-17.
15Dunn-Meynell AA, Rawson NE, Levin BE. Distribution and phenotype ofneurons containing the ATP-sensitive K+channel in rat brain. Brain research1998;814(1-2):41-54.
16Yoshida H, Feig JE, Morrissey A, Ghiu IA, Artman M, Coetzee WA. K ATPchannels of primary human coronary artery endothelial cells consist of aheteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits. Journal ofmolecular and cellular cardiology2004;37(4):857-69.
17Wang H, Long C, Duan Z, Shi C, Jia G, Zhang Y. A new ATP-sensitivepotassium channel opener protects endothelial function in cultured aorticendothelial cells. Cardiovascular research2007;73(3):497-503.
18Liss B, Bruns R, Roeper J. Alternative sulfonylurea receptor expressiondefines metabolic sensitivity of K-ATP channels in dopaminergic midbrainneurons. The EMBO journal1999;18(4):833-46.
19Domoki F, Kis B, Nagy K, Farkas E, Busija DW, Bari F. Diazoxide preserveshypercapnia-induced arteriolar vasodilation after global cerebral ischemia inpiglets. American journal of physiology Heart and circulatory physiology2005;
289(1): H368-73.
20Sato T, Sasaki N, O'Rourke B, Marban E. Nicorandil, a potent cardioprotectiveagent, acts by opening mitochondrial ATP-dependent potassium channels.Journal of the American College of Cardiology2000;35(2):514-8.
21Shimizu K, Lacza Z, Rajapakse N, Horiguchi T, Snipes J, Busija DW.MitoK(ATP) opener, diazoxide, reduces neuronal damage after middlecerebral artery occlusion in the rat. American journal of physiology Heart andcirculatory physiology2002;283(3): H1005-11.
22Wang H, Tang Y, Wang L, Long CL, Zhang YL. ATP-sensitive potassiumchannel openers and2,3-dimethyl-2-butylamine derivatives. Current medicinalchemistry2007;14(2):133-55.
23Chen YP, Qiu CR, Wang H. Cardiovascular pharmacological characterizationof novel2,3-dimethyl-2-butylamine derivatives in rats. Life sciences2004;75(17):2131-42.
24Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke witha novel opener of ATP-sensitive potassium channels in the brain. Molecularpharmacology2004;66(5):1160-8.
25Wu J, Hu J, Chen YP, Takeo T, Suga S, Dechon J, et al. Iptakalim modulatesATP-sensitive K(+) channels in dopamine neurons from rat substantia nigrapars compacta. The Journal of pharmacology and experimental therapeutics2006;319(1):155-64.
26Zhu HL, Luo WQ, Wang H. Iptakalim protects against hypoxic brain injurythrough multiple pathways associated with ATP-sensitive potassium channels.Neuroscience2008;157(4):884-94.
27Yang Y, Liu X, Ding JH, Sun J, Long Y, Wang F, et al. Effects of iptakalim onrotenone-induced cytotoxicity and dopamine release from PC12cells.Neuroscience letters2004;366(1):53-7.
28Yang YJ, Wang QM, Hu LF, Sun XL, Ding JH, Hu G. Iptakalim alleviated theincrease of extracellular dopamine and glutamate induced by1-methyl-4-phenylpyridinium ion in rat striatum. Neuroscience letters2006;404(1-2):187-90.
29Yang YL, Meng CH, Ding JH, He HR, Ellsworth K, Wu J, et al. Iptakalimhydrochloride protects cells against neurotoxin-induced glutamate transporterdysfunction in in vitro and in vivo models. Brain research2005;1049(1):80-8.
30Kis B, Kaiya H, Nishi R, Deli MA, Abraham CS, Yanagita T, et al. Cerebralendothelial cells are a major source of adrenomedullin. Journal ofneuroendocrinology2002;14(4):283-93.
31Hamprecht B, Loffler F. Primary glial cultures as a model for studyinghormone action. Methods in enzymology1985;109:341-5.
32Franke H, Galla H, Beuckmann CT. Primary cultures of brain microvesselendothelial cells: a valid and flexible model to study drug transport throughthe blood-brain barrier in vitro. Brain research Brain research protocols2000;5(3):248-56.
33Lok J, Gupta P, Guo S, Kim WJ, Whalen MJ, van Leyen K, et al. Cell-cellsignaling in the neurovascular unit. Neurochemical research2007;32(12):2032-45.
34Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role forprogrammed cell death in focal cerebral ischemia in rats. Stroke; a journal ofcerebral circulation1993;24(12):2002-8; discussion08-9.
35Li Y, Chopp M, Jiang N, Yao F, Zaloga C. Temporal profile of in situ DNAfragmentation after transient middle cerebral artery occlusion in the rat.Journal of cerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism1995;15(3):389-97.
36Chan PH. Role of oxidants in ischemic brain damage. Stroke; a journal ofcerebral circulation1996;27(6):1124-9.
37Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeabilitytransition and oxidative stress. FEBS letters2001;495(1-2):12-5.
38Krause GS, White BC, Aust SD, Nayini NR, Kumar K. Brain cell deathfollowing ischemia and reperfusion: a proposed biochemical sequence. Criticalcare medicine1988;16(7):714-26.
39Green DR, Reed JC. Mitochondria and apoptosis. Science1998;281(5381):1309-12.
40Plateel M, Dehouck MP, Torpier G, Cecchelli R, Teissier E. Hypoxia increasesthe susceptibility to oxidant stress and the permeability of the blood-brainbarrier endothelial cell monolayer. Journal of neurochemistry1995;65(5):2138-45.
41Sun XL, Zeng XN, Zhou F, Dai CP, Ding JH, Hu G. KATP channel openersfacilitate glutamate uptake by GluTs in rat primary cultured astrocytes.Neuropsychopharmacology: official publication of the American College ofNeuropsychopharmacology2008;33(6):1336-42.
42Wind T, Prehn JH, Peruche B, Krieglstein J. Activation of ATP-sensitivepotassium channels decreases neuronal injury caused by chemical hypoxia.Brain research1997;751(2):295-9.
43Tsuchiya D, Hong S, Matsumori Y, Shiina H, Kayama T, Swanson RA, et al.Overexpression of rat heat shock protein70is associated with reduction ofearly mitochondrial cytochrome C release and subsequent DNA fragmentationafter permanent focal ischemia. Journal of cerebral blood flow andmetabolism: official journal of the International Society of Cerebral BloodFlow and Metabolism2003;23(6):718-27.
44Garlid KD, Dos Santos P, Xie ZJ, Costa AD, Paucek P. Mitochondrialpotassium transport: the role of the mitochondrial ATP-sensitive K(+) channelin cardiac function and cardioprotection. Biochimica et biophysica acta2003;1606(1-3):1-21.
45Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, et al.Postconditioning the human heart. Circulation2005;112(14):2143-8.
46Soundarapandian MM, Zhong X, Peng L, Wu D, Lu Y. Role of K(ATP)channels in protection against neuronal excitatory insults. Journal ofneurochemistry2007;103(5):1721-9.
47Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterizationof a novel member of the nerve growth factor/brain-derived neurotrophicfactor family. Nature1990;344(6264):339-41.
48Abdel-Hamid KM, Baimbridge KG. The effects of artificial calcium buffers oncalcium responses and glutamate-mediated excitotoxicity in culturedhippocampal neurons. Neuroscience1997;81(3):673-87.
49Kamada H, Nito C, Endo H, Chan PH. Bad as a converging signalingmolecule between survival PI3-K/Akt and death JNK in neurons after transientfocal cerebral ischemia in rats. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism2007;27(3):521-33.
50Valable S, Montaner J, Bellail A, Berezowski V, Brillault J, Cecchelli R, et al.VEGF-induced BBB permeability is associated with an MMP-9activityincrease in cerebral ischemia: both effects decreased by Ang-1. Journal ofcerebral blood flow and metabolism: official journal of the InternationalSociety of Cerebral Blood Flow and Metabolism2005;25(11):1491-504.
51Liu W, Hendren J, Qin XJ, Shen J, Liu KJ. Normobaric hyperoxia attenuatesearly blood-brain barrier disruption by inhibiting MMP-9-mediated occludindegradation in focal cerebral ischemia. Journal of neurochemistry2009;108(3):811-20.
52Bonoiu A, Mahajan SD, Ye L, Kumar R, Ding H, Yong KT, et al. MMP-9gene silencing by a quantum dot-siRNA nanoplex delivery to maintain theintegrity of the blood brain barrier. Brain research2009;1282:142-55.
53Martin F, Laorden ML, Milanes MV. Morphine withdrawal regulatesphosphorylation of cAMP response element binding protein (CREB) throughPKC in the nucleus tractus solitarius-A2catecholaminergic neurons. Journalof neurochemistry2009;110(5):1422-32.
54Grossmann C, Ruhs S, Seiferth A, Gekle M. Interaction between mineralocor-ticoid receptor and cAMP/CREB signaling. Steroids2010;75(8-9):539-43.
55De Rasmo D, Signorile A, Roca E, Papa S. cAMP response element-bindingprotein (CREB) is imported into mitochondria and promotes protein synthesis.The FEBS journal2009;276(16):4325-33.
56Sakai R, Ukai W, Sohma H, Hashimoto E, Yamamoto M, Ikeda H, et al.Attenuation of brain derived neurotrophic factor (BDNF) by ethanol andcytoprotective effect of exogenous BDNF against ethanol damage in neuronalcells. J Neural Transm2005;112(8):1005-13.
57Kearns CM, Gash DM. GDNF protects nigral dopamine neurons against6-hydroxydopamine in vivo. Brain research1995;672(1-2):104-11.
58Abe K. Therapeutic potential of neurotrophic factors and neural stem cellsagainst ischemic brain injury. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism2000;20(10):1393-408.
59Lingor P, Tonges L, Pieper N, Bermel C, Barski E, Planchamp V, et al. ROCKinhibition and CNTF interact on intrinsic signalling pathways anddifferentially regulate survival and regeneration in retinal ganglion cells.Brain: a journal of neurology2008;131(Pt1):250-63.
60Cagnon L, Braissant O. CNTF protects oligodendrocytes from ammoniatoxicity: intracellular signaling pathways involved. Neurobiology of disease2009;33(1):133-42.
61Peng CH, Chiou SH, Chen SJ, Chou YC, Ku HH, Cheng CK, et al.Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosisin hippocampus-derived neural stem cells mediated by activation of BDNFand the MAPK pathway. European neuropsychopharmacology: the journal ofthe European College of Neuropsychopharmacology2008;18(2):128-40.
62Gershtein ES, Levkina NV, Digayeva MA, Laktionov KP, Tereshkina IV,Kushlinsky NE. Matrix metalloproteinases2,7, and9and tissue inhibitor ofmetalloproteinases-1in tumors and serum of patients with ovarian neoplasms.Bulletin of experimental biology and medicine2010;149(5):628-31.
63De Sanctis P, Elmakky A, Farina A, Caramelli E, Seracchioli R, Mabrouk M,et al. Matrix metalloproteinase-3mRNA: a promising peripheral blood markerfor diagnosis of endometriosis. Gynecologic and obstetric investigation2011;71(2):118-23.
64Aid S, Silva AC, Candelario-Jalil E, Choi SH, Rosenberg GA, Bosetti F.Cyclooxygenase-1and-2differentially modulate lipopolysaccharide-inducedblood-brain barrier disruption through matrix metalloproteinase activity.Journal of cerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism2010;30(2):370-80.
65Kumar B, Koul S, Petersen J, Khandrika L, Hwa JS, Meacham RB, et al. p38mitogen-activated protein kinase-driven MAPKAPK2regulates invasion ofbladder cancer by modulation of MMP-2and MMP-9activity. Cancer research2010;70(2):832-41.
66Chen JR, Wang YJ, Tseng GF. The effects of decompression and exogenousNGF on compressed cerebral cortex. Journal of neurotrauma2004;21(11):1640-51.
67Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, et al.Regulation of neuronal survival by the serine-threonine protein kinase Akt.Science1997;275(5300):661-5.
68Ouyang YB, Tan Y, Comb M, Liu CL, Martone ME, Siesjo BK, et al.Survival-and death-promoting events after transient cerebral ischemia:phosphorylation of Akt, release of cytochrome C and Activation ofcaspase-like proteases. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism1999;19(10):1126-35.
69Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9complexinitiates an apoptotic protease cascade. Cell1997;91(4):479-89.
70Lewen A, Matz P, Chan PH. Free radical pathways in CNS injury. Journal ofneurotrauma2000;17(10):871-90.
71Guo S, Kim WJ, Lok J, Lee SR, Besancon E, Luo BH, et al. Neuroprotectionvia matrix-trophic coupling between cerebral endothelial cells and neurons.Proceedings of the National Academy of Sciences of the United States ofAmerica2008;105(21):7582-7.
72Holownia A, Mroz RM, Wielgat P, Skiepko A, Sitko E, Jakubow P, et al.Propofol protects rat astroglial cells against tert-butyl hydroperoxide-inducedcytotoxicity; the effect on histone and cAMP-response-element-bindingprotein (CREB) signalling. Journal of physiology and pharmacology2009;60(4):63-9.
1Direct costs were calculated by Thomas A. Hodgson, chief economist and actingdirector, Division of Health This picture argues powerfully against the concept ofand Utilization Analysis,OAEHP, NCHS, CDC. Estimates of indirect costs weremade by Thomas J. Thom, statistician in the Division of Epidemiology andClinical Applications, NHLBI.
2Krause GS, Kumar K, White BC, Aust SD, Wiegenstein JG. and clinical trials.Antioxidants and NOS inhibitors can Ischemia, resuscitation, and reperfusion:mechanisms of tissue injury and prospects for protection. Am Heart J1986;111:768–80.
3Brain Resuscitation Clinical Trial I Study Group. Randomized reason to believethat they induce survival signal-transduc-clinical study of thiopental loading incomatose survivors of cardiac arrest. New Engl J Med1986;314:397–401.
4Brain Resuscitation Clinical Trial II Study Group. A randomized clinical studyof a calcium-entry blocker (lidoflazine) in the treatment of comatose survivors ofcardiac arrest. New Engl J Med1991;324:1225–31.
5The RANTTAS Investigators. A randomized trial of trilazad mesylate in patientswith acute stroke. Stroke1996;27:1453–8.
6Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G et al.Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDAantagonist. Stroke2000;31:347–54.
7Krause GS, White BC, Aust SD, Nayini NR, Kumar K. Brain cell hibitors ofcalpain and radical damage; death following ischemia and reperfusion: aproposed biochemical sequence. Crit Care Med1988;16:714–26.
8Hossman KA, Sakaki S, Kimoto K. Cerebral uptake of glucose and oxygen inthecat brain after prolonged ischemia. Stroke1976;7:301–5.
9Siesjo BK, Ljunggren B. Cerebral energy reserves after prolonged hypoxiaandischemia. Arch Neurol1973;29:400–7.
10Negovskii VA. Resuscitation and artificial hypothermia, NewYork: ConsultantsBureau,1962.
11Jenkins LW, Povlishock JT, Lewelt W, Miller JD, Becker DP. The role ofpostischemic recirculation in the development of ischemic neuronal injuryfollowing complete cerebral ischemia. Acta Neuro-pathol1981;5:205–20.
12Kumar K, Goosmann M, Krause GS, Nayini NR, Estrada R, Hoehner TJ et al.Ultrastructural and ionic changes in global ischemic dog brain. ActaNeuropathol (Berlin)1987;73:393–9.
13Sato M, Hashimoto H, Kosaka F. Histological changes of neuronal damage invegetative dogs induced by18minutes of complete global brain ischemia:two-phase damage of Purkinje cells and hippocampal CA1pyramidal cells.Acta Neuropathol (Berlin)11990;80:527–34.
14Sterz F, Leonov Y, Safar P, Radovsky A, Shearman G. No improved outcomeafter prolonged cardiac arrest and treatment with excitatory neurotransmitterreceptor blocker MK-801in dogs. Ann Emerg Med1989;18:456.
15Pellegrini-Giampietro DE, Cherici G, Alesiani M, Carla V, Moroni F.Excitatory amino acid release and free radical formation may cooperate in thegenesis of ischemia-induced neuronal damage. J Neurosci1990;10:1035–41.
16Kumar K, White BC, Krause GS, Indrieri RJ, Evans AT, Hoehner TJ et al. Aquantitative morphological assessment of the effect of lidoflazine anddeferoxamine therapy for global brain ischemia. Neurol Res1988;10:136–40.
17White BC, Krause GS, Nayini NR, Aust SD. Effect of deferoxamine orsuperoxide dismutase therapy on biochemical markers of neuro-nal injury at8hours of reperfusion following a15minute cardiac arrest in dogs. Am J EmergMed1988;6:569–76.
18Cerchiari EL, Hoel TM, Safar P, Sclabassi RJ. Protective effects of combinedsuperoxide dismutase and deferoxamine on recovery of cerebral blood flow andfunction after cardiac arrest in dogs. Stroke1987;18:869–78.
19Rosenthal RE, Chanderbhan R, Marshall G, Fiskum G. Prevention ofpost-ischemic brain lipid conjugated diene production and neurological injuryby hydroxyethyl starch-conjugated deferoxamine. Free Radic Biol Med1992;12:29–33.
20Natale JE, Schott RJ, Hall ED, Braughler JM, D’Alecy LG. Effect of theaminosteroid U74006F after cardiopulmonary arrest in dogs. Stroke1988;19:1371–8.
21Sterz F, Safar P, Johnson DW, Oku KI, Tisherman SA. Effects of U74006F onmultifocal cerebral blood flow and metabolism after cardiac arrest in dogs.Stroke1991;22:889–95.
22Skjaerlund JM, Krause GS, O’Neil BJ, White BC. The effect of EMHP onpost-cardiac arrest survival of rats. Resuscitation1991;22:139–49.
23Pierucci O, Rickert M. Duplication of E. coli during inhibition of net phospho-lipid synthesis. J Bacteriol1985;162:374–82.
24Joseleau-Petit D, Ke′pe`s F, Peutat L, D’Ari R, Ke′pe`s A. DNA replicationinitiation, doubling of rate of phospholipid synthesis, and cell division in E. coli.J Bacteriol1987;169:3701–6.
25Loewy B, Marczynski GT, Dingwall A, Shapiro L. Regulatory interactionsbetween phospholipid synthesis and DNA replication in Caulobacter crescentus.J Bacteriol1990;172:5523–30.
26Contreras IR, Bender RA, Mansour J, Henry S, Shapiro L. Caulobactercresentus mutant defective in membrane phospholipid synthesis. J Bacteriol1979;140:612–9.
27Cooper RA, Ip SHC, Cassileth PA, Kuo AL. Inhibition of sterol andphospholipid synthesis in HL-60promyelocytic leukemia cells by inducers ofmyeloid differentiation. Cancer Res1981;41:1847–52.
28Siperstein MD. Role of cholesterogenesis and isoprenoid synthesis in DNAreplication and cell growth. J Lipid Res1984;25:1462–8.
29Bredesen DE. Keeping neurons alive: the molecular control of apoptosis.Neuroscientist1996;2:211–6.
30Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, CulhaneAC et al. Apoptosis: molecular regulation of cell death. Eur J Biochem1995;236:1–26.
31Arends M J, Morris RG, Wyllie AH. Apoptosis. The role of the endonuclease.Am J Pathol1990;136:593–608.
32Jacobson MD, Burne JF, Raff MC. Programmed cell death and Bcl-2protectionin the absence of a nucleus. EMBO J1994;13:1899–910.
33Ellis RE, Jacobson DM, Horvitz HR. Genes required for the engulfment of cellcorpses during programmed cell death in C.elegans. Genetics1991;129:79–94.
34Nagata S, Golstein P. The Fas death factor. Science1995;267:1449–56.
35Hu WH, Johnson H, Shu HB. Tumor necrosis factor-related apoptosis-inducingligand receptors signal NF-kB and JNK activation and apoptosis throughdistinct pathways. J Biol Chem1999;274:30603–10.
36Ahmad M,Srinivasula SM, WangL, Talanian RV, Litwack G,FernandesAlnemri T et al. CRADD, a novel human apoptotic adaptor molecule forcaspase-2and FasL/tumor necrosis factor receptor-interacting protein RIP.Cancer Res1997;57:615–9.
37Muzio M, Salvesen GS, Dixit VM. FLICE-induced apoptosis in a cell-freesystem. Cleavage of caspase zymogens. J Biol Chem1997;272:2952–6.
38Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. Target proteasespecificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem1997;272:7797–800.
39Bertin J, Armstrong RC, Ottilie S, Martin DA,Wang Y, Banks S et al. Deatheffector domain-containing herpesvirus and poxvirus proteins inhibit both FAS-and TNFR1-induced apoptosis. Proc Natl Acad Sci USA1997;94:1172–6.
40Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Kajstura J, ChengW, Reiss K, Clark WA, Sonnenblick EH,independent contributing variables ininfarct size in rats. Lab Invest1996;74:86–107.
2Zhang LF, Yang J, Hong Z, Yuan GG, Zhou BF, Zhao LC, et al. Proportion ofdifferent subtypes of stroke in China. Stroke; a journal of cerebral circulation2003;34(9):2091-6.
3Liu HD, Liu, S, Li,H.L, Chen,X.Q, iao2Qian2), Yu, A. C. Activation ofSignal Transduction Pathways after Cerebral Ischemia. Chinese Journal ofBiochemistry and Molecular Biology2007;23(1):4.
4Graham SH, Chen J. Programmed cell death in cerebral ischemia. Journal ofcerebral blood flow and metabolism: official journal of the InternationalSociety of Cerebral Blood Flow and Metabolism2001;21(2):99-109.
5Su DF. Fundamental researches on the pathogenesis, prevention and treatmentof cerebrovascular diseases. Chinese Bulletin of Life Sciences2009;21(4):6.
6Haller M, Mironov SL, Karschin A, Richter DW. Dynamic activation ofK(ATP) channels in rhythmically active neurons. The Journal of physiology2001;537(Pt1):69-81.
7Diarra A, Sheldon C, Brett CL, Baimbridge KG, Church J. Anoxia-evokedintracellular pH and Ca2+concentration changes in cultured postnatal rathippocampal neurons. Neuroscience1999;93(3):1003-16.
8Girn HR, Ahilathirunayagam S, Mavor AI, Homer-Vanniasinkam S.Reperfusion syndrome: cellular mechanisms of microvascular dysfunction andpotential therapeutic strategies. Vascular and endovascular surgery2007;41(4):277-93.
9Choi DW. Cerebral hypoxia: some new approaches and unanswered questions.The Journal of neuroscience: the official journal of the Society forNeuroscience1990;10(8):2493-501.
10Lo EH, Rosenberg GA. The neurovascular unit in health and disease:introduction. Stroke; a journal of cerebral circulation2009;40(3Suppl): S2-3.
11Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in healthand disease. Pharmacological reviews2005;57(2):173-85.
12Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunitiesin stroke. Nature reviews Neuroscience2003;4(5):399-415.
13Janigro D, West GA, Gordon EL, Winn HR. ATP-sensitive K+channels in rataorta and brain microvascular endothelial cells. The American journal ofphysiology1993;265(3Pt1): C812-21.
14Mederos y Schnitzler M, Derst C, Daut J, Preisig-Muller R. ATP-sensitivepotassium channels in capillaries isolated from guinea-pig heart. The Journalof physiology2000;525Pt2:307-17.
15Dunn-Meynell AA, Rawson NE, Levin BE. Distribution and phenotype ofneurons containing the ATP-sensitive K+channel in rat brain. Brain research1998;814(1-2):41-54.
16Yoshida H, Feig JE, Morrissey A, Ghiu IA, Artman M, Coetzee WA. K ATPchannels of primary human coronary artery endothelial cells consist of aheteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits. Journal ofmolecular and cellular cardiology2004;37(4):857-69.
17Wang H, Long C, Duan Z, Shi C, Jia G, Zhang Y. A new ATP-sensitivepotassium channel opener protects endothelial function in cultured aorticendothelial cells. Cardiovascular research2007;73(3):497-503.
18Liss B, Bruns R, Roeper J. Alternative sulfonylurea receptor expressiondefines metabolic sensitivity of K-ATP channels in dopaminergic midbrainneurons. The EMBO journal1999;18(4):833-46.
19Domoki F, Kis B, Nagy K, Farkas E, Busija DW, Bari F. Diazoxide preserveshypercapnia-induced arteriolar vasodilation after global cerebral ischemia inpiglets. American journal of physiology Heart and circulatory physiology2005;
289(1): H368-73.
20Sato T, Sasaki N, O'Rourke B, Marban E. Nicorandil, a potent cardioprotectiveagent, acts by opening mitochondrial ATP-dependent potassium channels.Journal of the American College of Cardiology2000;35(2):514-8.
21Shimizu K, Lacza Z, Rajapakse N, Horiguchi T, Snipes J, Busija DW.MitoK(ATP) opener, diazoxide, reduces neuronal damage after middlecerebral artery occlusion in the rat. American journal of physiology Heart andcirculatory physiology2002;283(3): H1005-11.
22Wang H, Tang Y, Wang L, Long CL, Zhang YL. ATP-sensitive potassiumchannel openers and2,3-dimethyl-2-butylamine derivatives. Current medicinalchemistry2007;14(2):133-55.
23Chen YP, Qiu CR, Wang H. Cardiovascular pharmacological characterizationof novel2,3-dimethyl-2-butylamine derivatives in rats. Life sciences2004;75(17):2131-42.
24Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke witha novel opener of ATP-sensitive potassium channels in the brain. Molecularpharmacology2004;66(5):1160-8.
25Wu J, Hu J, Chen YP, Takeo T, Suga S, Dechon J, et al. Iptakalim modulatesATP-sensitive K(+) channels in dopamine neurons from rat substantia nigrapars compacta. The Journal of pharmacology and experimental therapeutics2006;319(1):155-64.
26Zhu HL, Luo WQ, Wang H. Iptakalim protects against hypoxic brain injurythrough multiple pathways associated with ATP-sensitive potassium channels.Neuroscience2008;157(4):884-94.
27Yang Y, Liu X, Ding JH, Sun J, Long Y, Wang F, et al. Effects of iptakalim onrotenone-induced cytotoxicity and dopamine release from PC12cells.Neuroscience letters2004;366(1):53-7.
28Yang YJ, Wang QM, Hu LF, Sun XL, Ding JH, Hu G. Iptakalim alleviated theincrease of extracellular dopamine and glutamate induced by1-methyl-4-phenylpyridinium ion in rat striatum. Neuroscience letters2006;404(1-2):187-90.
29Yang YL, Meng CH, Ding JH, He HR, Ellsworth K, Wu J, et al. Iptakalimhydrochloride protects cells against neurotoxin-induced glutamate transporterdysfunction in in vitro and in vivo models. Brain research2005;1049(1):80-8.
30Kis B, Kaiya H, Nishi R, Deli MA, Abraham CS, Yanagita T, et al. Cerebralendothelial cells are a major source of adrenomedullin. Journal ofneuroendocrinology2002;14(4):283-93.
31Hamprecht B, Loffler F. Primary glial cultures as a model for studyinghormone action. Methods in enzymology1985;109:341-5.
32Franke H, Galla H, Beuckmann CT. Primary cultures of brain microvesselendothelial cells: a valid and flexible model to study drug transport throughthe blood-brain barrier in vitro. Brain research Brain research protocols2000;5(3):248-56.
33Lok J, Gupta P, Guo S, Kim WJ, Whalen MJ, van Leyen K, et al. Cell-cellsignaling in the neurovascular unit. Neurochemical research2007;32(12):2032-45.
34Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role forprogrammed cell death in focal cerebral ischemia in rats. Stroke; a journal ofcerebral circulation1993;24(12):2002-8; discussion08-9.
35Li Y, Chopp M, Jiang N, Yao F, Zaloga C. Temporal profile of in situ DNAfragmentation after transient middle cerebral artery occlusion in the rat.Journal of cerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism1995;15(3):389-97.
36Chan PH. Role of oxidants in ischemic brain damage. Stroke; a journal ofcerebral circulation1996;27(6):1124-9.
37Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeabilitytransition and oxidative stress. FEBS letters2001;495(1-2):12-5.
38Krause GS, White BC, Aust SD, Nayini NR, Kumar K. Brain cell deathfollowing ischemia and reperfusion: a proposed biochemical sequence. Criticalcare medicine1988;16(7):714-26.
39Green DR, Reed JC. Mitochondria and apoptosis. Science1998;281(5381):1309-12.
40Plateel M, Dehouck MP, Torpier G, Cecchelli R, Teissier E. Hypoxia increasesthe susceptibility to oxidant stress and the permeability of the blood-brainbarrier endothelial cell monolayer. Journal of neurochemistry1995;65(5):2138-45.
41Sun XL, Zeng XN, Zhou F, Dai CP, Ding JH, Hu G. KATP channel openersfacilitate glutamate uptake by GluTs in rat primary cultured astrocytes.Neuropsychopharmacology: official publication of the American College ofNeuropsychopharmacology2008;33(6):1336-42.
42Wind T, Prehn JH, Peruche B, Krieglstein J. Activation of ATP-sensitivepotassium channels decreases neuronal injury caused by chemical hypoxia.Brain research1997;751(2):295-9.
43Tsuchiya D, Hong S, Matsumori Y, Shiina H, Kayama T, Swanson RA, et al.Overexpression of rat heat shock protein70is associated with reduction ofearly mitochondrial cytochrome C release and subsequent DNA fragmentationafter permanent focal ischemia. Journal of cerebral blood flow andmetabolism: official journal of the International Society of Cerebral BloodFlow and Metabolism2003;23(6):718-27.
44Garlid KD, Dos Santos P, Xie ZJ, Costa AD, Paucek P. Mitochondrialpotassium transport: the role of the mitochondrial ATP-sensitive K(+) channelin cardiac function and cardioprotection. Biochimica et biophysica acta2003;1606(1-3):1-21.
45Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, et al.Postconditioning the human heart. Circulation2005;112(14):2143-8.
46Soundarapandian MM, Zhong X, Peng L, Wu D, Lu Y. Role of K(ATP)channels in protection against neuronal excitatory insults. Journal ofneurochemistry2007;103(5):1721-9.
47Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterizationof a novel member of the nerve growth factor/brain-derived neurotrophicfactor family. Nature1990;344(6264):339-41.
48Abdel-Hamid KM, Baimbridge KG. The effects of artificial calcium buffers oncalcium responses and glutamate-mediated excitotoxicity in culturedhippocampal neurons. Neuroscience1997;81(3):673-87.
49Kamada H, Nito C, Endo H, Chan PH. Bad as a converging signalingmolecule between survival PI3-K/Akt and death JNK in neurons after transientfocal cerebral ischemia in rats. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism2007;27(3):521-33.
50Valable S, Montaner J, Bellail A, Berezowski V, Brillault J, Cecchelli R, et al.VEGF-induced BBB permeability is associated with an MMP-9activityincrease in cerebral ischemia: both effects decreased by Ang-1. Journal ofcerebral blood flow and metabolism: official journal of the InternationalSociety of Cerebral Blood Flow and Metabolism2005;25(11):1491-504.
51Liu W, Hendren J, Qin XJ, Shen J, Liu KJ. Normobaric hyperoxia attenuatesearly blood-brain barrier disruption by inhibiting MMP-9-mediated occludindegradation in focal cerebral ischemia. Journal of neurochemistry2009;108(3):811-20.
52Bonoiu A, Mahajan SD, Ye L, Kumar R, Ding H, Yong KT, et al. MMP-9gene silencing by a quantum dot-siRNA nanoplex delivery to maintain theintegrity of the blood brain barrier. Brain research2009;1282:142-55.
53Martin F, Laorden ML, Milanes MV. Morphine withdrawal regulatesphosphorylation of cAMP response element binding protein (CREB) throughPKC in the nucleus tractus solitarius-A2catecholaminergic neurons. Journalof neurochemistry2009;110(5):1422-32.
54Grossmann C, Ruhs S, Seiferth A, Gekle M. Interaction between mineralocor-ticoid receptor and cAMP/CREB signaling. Steroids2010;75(8-9):539-43.
55De Rasmo D, Signorile A, Roca E, Papa S. cAMP response element-bindingprotein (CREB) is imported into mitochondria and promotes protein synthesis.The FEBS journal2009;276(16):4325-33.
56Sakai R, Ukai W, Sohma H, Hashimoto E, Yamamoto M, Ikeda H, et al.Attenuation of brain derived neurotrophic factor (BDNF) by ethanol andcytoprotective effect of exogenous BDNF against ethanol damage in neuronalcells. J Neural Transm2005;112(8):1005-13.
57Kearns CM, Gash DM. GDNF protects nigral dopamine neurons against6-hydroxydopamine in vivo. Brain research1995;672(1-2):104-11.
58Abe K. Therapeutic potential of neurotrophic factors and neural stem cellsagainst ischemic brain injury. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism2000;20(10):1393-408.
59Lingor P, Tonges L, Pieper N, Bermel C, Barski E, Planchamp V, et al. ROCKinhibition and CNTF interact on intrinsic signalling pathways anddifferentially regulate survival and regeneration in retinal ganglion cells.Brain: a journal of neurology2008;131(Pt1):250-63.
60Cagnon L, Braissant O. CNTF protects oligodendrocytes from ammoniatoxicity: intracellular signaling pathways involved. Neurobiology of disease2009;33(1):133-42.
61Peng CH, Chiou SH, Chen SJ, Chou YC, Ku HH, Cheng CK, et al.Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosisin hippocampus-derived neural stem cells mediated by activation of BDNFand the MAPK pathway. European neuropsychopharmacology: the journal ofthe European College of Neuropsychopharmacology2008;18(2):128-40.
62Gershtein ES, Levkina NV, Digayeva MA, Laktionov KP, Tereshkina IV,Kushlinsky NE. Matrix metalloproteinases2,7, and9and tissue inhibitor ofmetalloproteinases-1in tumors and serum of patients with ovarian neoplasms.Bulletin of experimental biology and medicine2010;149(5):628-31.
63De Sanctis P, Elmakky A, Farina A, Caramelli E, Seracchioli R, Mabrouk M,et al. Matrix metalloproteinase-3mRNA: a promising peripheral blood markerfor diagnosis of endometriosis. Gynecologic and obstetric investigation2011;71(2):118-23.
64Aid S, Silva AC, Candelario-Jalil E, Choi SH, Rosenberg GA, Bosetti F.Cyclooxygenase-1and-2differentially modulate lipopolysaccharide-inducedblood-brain barrier disruption through matrix metalloproteinase activity.Journal of cerebral blood flow and metabolism: official journal of theInternational Society of Cerebral Blood Flow and Metabolism2010;30(2):370-80.
65Kumar B, Koul S, Petersen J, Khandrika L, Hwa JS, Meacham RB, et al. p38mitogen-activated protein kinase-driven MAPKAPK2regulates invasion ofbladder cancer by modulation of MMP-2and MMP-9activity. Cancer research2010;70(2):832-41.
66Chen JR, Wang YJ, Tseng GF. The effects of decompression and exogenousNGF on compressed cerebral cortex. Journal of neurotrauma2004;21(11):1640-51.
67Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, et al.Regulation of neuronal survival by the serine-threonine protein kinase Akt.Science1997;275(5300):661-5.
68Ouyang YB, Tan Y, Comb M, Liu CL, Martone ME, Siesjo BK, et al.Survival-and death-promoting events after transient cerebral ischemia:phosphorylation of Akt, release of cytochrome C and Activation ofcaspase-like proteases. Journal of cerebral blood flow and metabolism:official journal of the International Society of Cerebral Blood Flow andMetabolism1999;19(10):1126-35.
69Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9complexinitiates an apoptotic protease cascade. Cell1997;91(4):479-89.
70Lewen A, Matz P, Chan PH. Free radical pathways in CNS injury. Journal ofneurotrauma2000;17(10):871-90.
71Guo S, Kim WJ, Lok J, Lee SR, Besancon E, Luo BH, et al. Neuroprotectionvia matrix-trophic coupling between cerebral endothelial cells and neurons.Proceedings of the National Academy of Sciences of the United States ofAmerica2008;105(21):7582-7.
72Holownia A, Mroz RM, Wielgat P, Skiepko A, Sitko E, Jakubow P, et al.Propofol protects rat astroglial cells against tert-butyl hydroperoxide-inducedcytotoxicity; the effect on histone and cAMP-response-element-bindingprotein (CREB) signalling. Journal of physiology and pharmacology2009;60(4):63-9.
1Direct costs were calculated by Thomas A. Hodgson, chief economist and actingdirector, Division of Health This picture argues powerfully against the concept ofand Utilization Analysis,OAEHP, NCHS, CDC. Estimates of indirect costs weremade by Thomas J. Thom, statistician in the Division of Epidemiology andClinical Applications, NHLBI.
2Krause GS, Kumar K, White BC, Aust SD, Wiegenstein JG. and clinical trials.Antioxidants and NOS inhibitors can Ischemia, resuscitation, and reperfusion:mechanisms of tissue injury and prospects for protection. Am Heart J1986;111:768–80.
3Brain Resuscitation Clinical Trial I Study Group. Randomized reason to believethat they induce survival signal-transduc-clinical study of thiopental loading incomatose survivors of cardiac arrest. New Engl J Med1986;314:397–401.
4Brain Resuscitation Clinical Trial II Study Group. A randomized clinical studyof a calcium-entry blocker (lidoflazine) in the treatment of comatose survivors ofcardiac arrest. New Engl J Med1991;324:1225–31.
5The RANTTAS Investigators. A randomized trial of trilazad mesylate in patientswith acute stroke. Stroke1996;27:1453–8.
6Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G et al.Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDAantagonist. Stroke2000;31:347–54.
7Krause GS, White BC, Aust SD, Nayini NR, Kumar K. Brain cell hibitors ofcalpain and radical damage; death following ischemia and reperfusion: aproposed biochemical sequence. Crit Care Med1988;16:714–26.
8Hossman KA, Sakaki S, Kimoto K. Cerebral uptake of glucose and oxygen inthecat brain after prolonged ischemia. Stroke1976;7:301–5.
9Siesjo BK, Ljunggren B. Cerebral energy reserves after prolonged hypoxiaandischemia. Arch Neurol1973;29:400–7.
10Negovskii VA. Resuscitation and artificial hypothermia, NewYork: ConsultantsBureau,1962.
11Jenkins LW, Povlishock JT, Lewelt W, Miller JD, Becker DP. The role ofpostischemic recirculation in the development of ischemic neuronal injuryfollowing complete cerebral ischemia. Acta Neuro-pathol1981;5:205–20.
12Kumar K, Goosmann M, Krause GS, Nayini NR, Estrada R, Hoehner TJ et al.Ultrastructural and ionic changes in global ischemic dog brain. ActaNeuropathol (Berlin)1987;73:393–9.
13Sato M, Hashimoto H, Kosaka F. Histological changes of neuronal damage invegetative dogs induced by18minutes of complete global brain ischemia:two-phase damage of Purkinje cells and hippocampal CA1pyramidal cells.Acta Neuropathol (Berlin)11990;80:527–34.
14Sterz F, Leonov Y, Safar P, Radovsky A, Shearman G. No improved outcomeafter prolonged cardiac arrest and treatment with excitatory neurotransmitterreceptor blocker MK-801in dogs. Ann Emerg Med1989;18:456.
15Pellegrini-Giampietro DE, Cherici G, Alesiani M, Carla V, Moroni F.Excitatory amino acid release and free radical formation may cooperate in thegenesis of ischemia-induced neuronal damage. J Neurosci1990;10:1035–41.
16Kumar K, White BC, Krause GS, Indrieri RJ, Evans AT, Hoehner TJ et al. Aquantitative morphological assessment of the effect of lidoflazine anddeferoxamine therapy for global brain ischemia. Neurol Res1988;10:136–40.
17White BC, Krause GS, Nayini NR, Aust SD. Effect of deferoxamine orsuperoxide dismutase therapy on biochemical markers of neuro-nal injury at8hours of reperfusion following a15minute cardiac arrest in dogs. Am J EmergMed1988;6:569–76.
18Cerchiari EL, Hoel TM, Safar P, Sclabassi RJ. Protective effects of combinedsuperoxide dismutase and deferoxamine on recovery of cerebral blood flow andfunction after cardiac arrest in dogs. Stroke1987;18:869–78.
19Rosenthal RE, Chanderbhan R, Marshall G, Fiskum G. Prevention ofpost-ischemic brain lipid conjugated diene production and neurological injuryby hydroxyethyl starch-conjugated deferoxamine. Free Radic Biol Med1992;12:29–33.
20Natale JE, Schott RJ, Hall ED, Braughler JM, D’Alecy LG. Effect of theaminosteroid U74006F after cardiopulmonary arrest in dogs. Stroke1988;19:1371–8.
21Sterz F, Safar P, Johnson DW, Oku KI, Tisherman SA. Effects of U74006F onmultifocal cerebral blood flow and metabolism after cardiac arrest in dogs.Stroke1991;22:889–95.
22Skjaerlund JM, Krause GS, O’Neil BJ, White BC. The effect of EMHP onpost-cardiac arrest survival of rats. Resuscitation1991;22:139–49.
23Pierucci O, Rickert M. Duplication of E. coli during inhibition of net phospho-lipid synthesis. J Bacteriol1985;162:374–82.
24Joseleau-Petit D, Ke′pe`s F, Peutat L, D’Ari R, Ke′pe`s A. DNA replicationinitiation, doubling of rate of phospholipid synthesis, and cell division in E. coli.J Bacteriol1987;169:3701–6.
25Loewy B, Marczynski GT, Dingwall A, Shapiro L. Regulatory interactionsbetween phospholipid synthesis and DNA replication in Caulobacter crescentus.J Bacteriol1990;172:5523–30.
26Contreras IR, Bender RA, Mansour J, Henry S, Shapiro L. Caulobactercresentus mutant defective in membrane phospholipid synthesis. J Bacteriol1979;140:612–9.
27Cooper RA, Ip SHC, Cassileth PA, Kuo AL. Inhibition of sterol andphospholipid synthesis in HL-60promyelocytic leukemia cells by inducers ofmyeloid differentiation. Cancer Res1981;41:1847–52.
28Siperstein MD. Role of cholesterogenesis and isoprenoid synthesis in DNAreplication and cell growth. J Lipid Res1984;25:1462–8.
29Bredesen DE. Keeping neurons alive: the molecular control of apoptosis.Neuroscientist1996;2:211–6.
30Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, CulhaneAC et al. Apoptosis: molecular regulation of cell death. Eur J Biochem1995;236:1–26.
31Arends M J, Morris RG, Wyllie AH. Apoptosis. The role of the endonuclease.Am J Pathol1990;136:593–608.
32Jacobson MD, Burne JF, Raff MC. Programmed cell death and Bcl-2protectionin the absence of a nucleus. EMBO J1994;13:1899–910.
33Ellis RE, Jacobson DM, Horvitz HR. Genes required for the engulfment of cellcorpses during programmed cell death in C.elegans. Genetics1991;129:79–94.
34Nagata S, Golstein P. The Fas death factor. Science1995;267:1449–56.
35Hu WH, Johnson H, Shu HB. Tumor necrosis factor-related apoptosis-inducingligand receptors signal NF-kB and JNK activation and apoptosis throughdistinct pathways. J Biol Chem1999;274:30603–10.
36Ahmad M,Srinivasula SM, WangL, Talanian RV, Litwack G,FernandesAlnemri T et al. CRADD, a novel human apoptotic adaptor molecule forcaspase-2and FasL/tumor necrosis factor receptor-interacting protein RIP.Cancer Res1997;57:615–9.
37Muzio M, Salvesen GS, Dixit VM. FLICE-induced apoptosis in a cell-freesystem. Cleavage of caspase zymogens. J Biol Chem1997;272:2952–6.
38Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. Target proteasespecificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem1997;272:7797–800.
39Bertin J, Armstrong RC, Ottilie S, Martin DA,Wang Y, Banks S et al. Deatheffector domain-containing herpesvirus and poxvirus proteins inhibit both FAS-and TNFR1-induced apoptosis. Proc Natl Acad Sci USA1997;94:1172–6.
40Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Kajstura J, ChengW, Reiss K, Clark WA, Sonnenblick EH,independent contributing variables ininfarct size in rats. Lab Invest1996;74:86–107.
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