延迟整流钾通道Kv2.1在氧糖剥夺介导细胞损伤中的作用及相关药理学研究
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摘要
缺血性脑卒中(Ishemic Stroke)是位列于心血管疾病、癌症之后,导致中老年人死亡的主要脑血管意外类疾病。缺血性脑卒中的特点是其高死亡率和高致残率,约有1／3的患者在发病后死亡,而幸存者由于偏瘫、失语、痴呆等后遗症丧失工作甚至生活自理能力；严重危害人们的健康,并为家庭和社会带来沉重的负担。
目前用于缺血性脑卒中一线临床治疗的药物多集中于溶栓抗凝类药物,而神经保护剂所占比重较小。本研究拟以缺血性脑卒中病理过程中神经元离子稳态失衡为切入点,围绕钾通道的过度开放及细胞内钾的丢失,探讨慢性失活型延迟整流钾通道Kv2.1在由氧糖剥夺模型所介导的细胞损伤中发挥的作用；已知药物多奈派齐(donepezil)具有钾通道阻断的特性,研究其能否独立于胆碱能系统之外,仅通过对电压依赖性钾通道的调节即可发挥抗脑缺血细胞损伤保护作用；围绕钠钙交换体(NCX)功能,筛选NCX调节剂类化合物,探讨NCX激动剂在缺血性脑卒中发挥的神经元保护作用。为脑缺血及再灌注所造成神经元损伤的治疗、以及抗脑缺血神经保护剂类药物的研发提供线索和理论依据。本论文的主要研究内容如下：
第一部分：延迟整流钾通道Kv2.1在氧糖剥夺介导细胞损伤中的作用
1.延迟整流钾通道阻断剂对脑缺血的保护作用
在脑缺血病理过程中,由于能量供给障碍引发的离子稳态平衡失调是造成神经元损伤的始动因素之一。目前的研究结果表明,钾离子的过度外流和细胞内钾的大量丢失是造成脑缺血细胞损伤和凋亡的主要机制，抑制钾通道的过度开放则可能发挥对脑缺血细胞损伤的保护作用。本课题组的前期研究结果表明：大鼠制备脑缺血模型后,脑缺血半影区域延迟整流钾通道Kv2.1的mRNA表达水平显著升高。所以本研究先应用延迟整流钾通道阻断剂四乙铵(TEA)验证了阻断延迟整流钾通道在脑缺血中的神经元保护作用,实验结果显示：大脑中动脉阻塞模型大鼠侧脑室注射TEA(5ug·kg-1)可以显著降低实验动物的脑梗死体积。
2.野生型HEK293细胞转染Kv2.1钾通道后对氧糖剥夺损伤敏感性增强
Kv2.1钾通道在中枢神经系统广泛表达,并且研究表明Kv2.1电流是神经元外向延迟整流钾电流的主要成分；为了研究Kv2.1钾通道在由脑缺血所造成细胞损伤中的作用同时排除神经元上其他类型离子通道的影响,本研究应用高表达Kv2.1钾通道亚型的转基因细胞—Kv2.1／HEK293细胞。在钾通道电流无明显衰减的前提下,应用全细胞膜片钳技术观察和记录野生型HEK293细胞和转染Kv2.1钾通道的HEK293细胞外向钾通道电流差别。实验结果显示：同野生型HEK293细胞相比,Kv2.1／HEK293细胞的外向电流密度增高超过10倍；并通过电生理学实验验证其属于延迟整流钾电流成分。
应用氧糖剥夺模型模拟脑缺血病理过程中的缺血缺氧状态,MTT实验显示：同野生型HEK293细胞相比,氧糖剥夺使转染Kv2.1钾通道的HEK293细胞存活率显著降低。由Hoechst33342标记的细胞凋亡实验显示：氧糖剥夺使转染Kv2.1钾通道的HEK293细胞凋亡率比其载体细胞显著升高。以上结果说明Kv2.1钾通道同氧糖剥夺引起的细胞损伤密切相关。
3.氧糖剥夺对转染Kv2.1钾通道的HEK293细胞电生理学性质的影响
Kv2.1钾离子通道属于氧敏感型钾通道,文献报道在缺氧条件下,Kv2.1钾通道会受到轻微抑制。本研究发现Kv2.1／HEK293细胞氧糖剥夺处理后,Kv2.1电流降低为有氧状态的约82%,同文献报道结果基本一致；同时本研究观察到氧糖剥夺对Kv2.1钾通道的激活和失活无显著影响。
但在应用膜片钳全细胞电流钳制观察细胞膜电位的实验中发现：氧糖剥夺可以使Kv2.1／HEK293细胞膜电位向去极化方向移动并维持在+2mV左右。由于Kv2.1通道特点是该电流激活的电压和时间依赖性,基本上无自主灭活,除非细胞的复极化；所以由氧糖剥夺引起的细胞膜持续去极化状态可以激活Kv2.1通道,使其持续开放并介导钾离子的过度外流,从而引起继发的细胞损伤。
4.钾离子浓度对提取线粒体膜电位的影响
线粒体膜电位的稳定不但是线粒体能量代谢正常进行的前提,也是减缓线粒体通透转运孔开放,避免凋亡通路激活的重要因素。以线粒体对于荧光染料Rhodamin123的摄取速率和摄取量表示线粒体膜电位的高低,观察细胞内液中钾离子浓度同线粒体膜电位间的关系。结果显示：提取线粒体的膜电位随着孵化液中钾离子浓度的降低(用氯化铯或氯化胆碱代替细胞内液中的钾)而降低。提示线粒体膜电位同细胞内钾离子浓度密切相关,抑制钾离子的过度外流有助于稳定线粒体膜电位,减少线粒体凋亡因子的释放。
5.Kv2.1钾通道对氧糖剥夺损伤介导的线粒体凋亡因子释放的影响
氧糖剥夺损伤可以引起线粒体呼吸和能量代谢障碍,同时造成线粒体膜电位降低,最终导致线粒体通透转运孔(MPTP)开放,膜通透性增加,凋亡相关蛋白释放,进而引发凋亡级联反应。本研究应用Westen blot方法观察了氧糖剥夺对Kv2.1／HEK293线粒体细胞凋亡因子Cyto-C和AIF释放的影响,实验结果显示：同野生型HEK293细胞相比,氧糖剥夺6小时使转染Kv2.1钾通道的HEK293细胞胞浆中的Cyto-C和AIF水平显著升高；提示Kv2.1钾通道同氧糖剥夺损伤介导的线粒体凋亡因子释放密切相关。
第二部分：Donepezil对氧糖剥夺介导Kv2.1／HEK293细胞损伤的保护作用1. Donepezil对Kv2.1钾通道的阻断作用
在Kv2.1钾通道电流无明显衰减的前提下,应用全细胞膜片钳技术观察和记录第二代胆碱酯酶抑制剂donepezil对转染Kv2.1钾通道HEK293细胞Kv2.1电流的抑制作用。实验结果显示：donepezil可以呈剂量依赖性的抑制Kv2.1钾电流；对其量效曲线进行分析得到donepezil抑制Kv2.1钾通道的IC50值为7.59μM；donepezil浓度为30μM时对Kv2.1钾通道的抑制接近最大值,当去极化到+60mV时30μMdonepezil可以将Kv2.1电流抑制到对照组的49.93±6.59%。同时,选择经典延迟整流钾通道阻断剂TEA作为阳性对照药物,30μM donepezil和10mM TEA对Kv2.1钾电流的抑制程度接近。
2. Donepezil对氧糖剥夺Kv2.1／HEK293细胞电生理性质的影响
氧糖剥夺可以使转染Kv2.1钾通道HEK293细胞的Kv2.1电流有所降低,donepezil则可以使氧糖剥夺处理的Kv2.1／HEK293细胞Kv2.1电流进一步降低,并且这种抑制程度要远远高于氧糖剥夺对Kv2.1电流的抑制。Donepezil对氧糖剥夺Kv2.1／HEK293细胞Kv2.1钾通道稳态激活曲线无显著影响,但Donepezil可以使氧糖剥夺Kv2.1／HEK293细胞Kv2.1钾通道失活曲线向超极化方向移动8.73mV；提示donepezil对氧糖剥夺Kv2.1钾通道的抑制作用可能同加速通道的失活密切相关。
Kv2.1／HEK293细胞膜电位在-30mV左右,氧糖剥夺处理后细胞膜电位去极化至2.23±2.06mV,氧糖剥夺和30μM donepezil或10mM TEA共同作用后细胞膜电位仍然向去极化方向移动到-5.36±1.37mV和-3.08±1.55mV；提示donepezil和TEA可以起到稳定氧糖剥夺Kv2.1／HEK293细胞膜电位的作用,进而降低钾通道的开放频率以减少由细胞膜去极化造成的钾离子外流。
但在0mV左右的膜电位下,Kv2.1还是可以被激活开放,所以本研究进一步观察了细胞膜电位是0mV时donepezil对氧糖剥夺Kv2.1／HEK293细胞钾电流的影响。实验结果显示：氧糖剥夺处理后,Kv2.1／HEK293细胞膜电位0mV时的电流密度为89.90±8.26 pA／pF；30μMdonepezil和10mM TEA分别可以使氧糖剥夺Kv2.1／HEK293细胞0mV时的电流密度降低到27.11±5.22 pA／pF和38.71±5.53pA／pF；提示在由氧糖剥夺引起细胞去极化到0mV左右时donepezil可以通过对Kv2.1钾电流的显著抑制作用避免细胞内钾的过度丢失,从而发挥保护作用。
3. Donepezil对氧糖剥夺介导Kv2.1／HEK293细胞损伤的保护作用
应用MTT法观察donepezil对氧糖剥夺Kv2.1／HEK293细胞存活率的影响,结果表明：同有氧对照组相比,氧糖剥夺损伤模型组的细胞生存率显著降低至33.26±1.09%; donepezil可以剂量依赖性的提高氧糖剥夺损伤Kv2.1／HEK293细胞存活率,30μM donepezi和10mM TEA分别可以使细胞存活率升高至47.29±2.59%和44.03±1.95%。
细胞凋亡实验发现：同野生型HEK293细胞相比,氧糖剥夺24小时使Kv2.1／HEK293细胞的凋亡率显著升高至52.82±2.01%;30μM donepezil可以使细胞凋亡率显著降低至23.70±1.75%；10mM TEA可以使细胞凋亡率显著降低至26.35±2.11%。提示在氧糖剥夺介导的Kv2.1／HEK293细胞凋亡中,donepezil通过阻断Kv2.1钾通道可以发挥对抗细胞凋亡作用。
4. Donepezil对氧糖剥夺损伤Kv2.1／HEK293细胞线粒体凋亡因子释放的抑制作用
氧糖剥夺损伤可以使Kv2.1／HEK293细胞胞浆细胞色素C和凋亡诱导因子水平升高,并且在氧糖剥夺6小时后同野生型HEK293细胞相比有显著性差异,实验结果显示：同氧糖剥夺6小时损伤模型相比,30μM donepezil和10mM TEA可以显著降低氧糖剥夺Kv2.1／HEK293细胞胞浆细胞色素C水平；同时30μM donepezil还可以使Kv2.1／HEK293细胞胞浆凋亡诱导因子水平显著降低,而TEA对Kv2.1／HEK293细胞胞浆凋亡诱导因子水平影响不明显。以上结果提示,donepezil可以通过阻断Kv2.1钾离子通道,进而降低由氧糖剥夺介导钾离子大量外流所引起的胞浆细胞色素C和凋亡诱导因子水平的升高,发挥抗凋亡作用。
5. Donepezil对氢糖剥夺损伤Kv2.1／HEK293细胞PKC-delta水平的影响
PKC-delta属于蛋白激酶C家族成员。研究表明PKC-delta参与细胞凋亡的发生,细胞内PKC-delta水平同脑缺血损伤造成的神经元凋亡相关。PKC-delta被活化后才可以诱导细胞凋亡,目前报道有三种方式可以使PKC-delta活化,分别是PKC-delta的膜转位、酪氨酸磷酸化和溶蛋白性裂解；PKC-delta激活的结果是使细胞内完整形式的PKC-delta蛋白水平降低,其激活产物参与其他信号通路的调节。本研究发现,在氧糖剥夺6小时后,Kv2.1／HEK293细胞胞浆内完整形式的PKC-delta水平显著降低,提示PKC-delta的激活；但在给予donepezil和TEA后对胞浆内已经降低PKC-delta水平无明显影响。提示donepezil阻断钾通道并不通过对胞浆内PKC-delta水平的调节发挥其抗凋亡细胞保护作用。
第三部分：基于NCX功能的抗脑缺血药物筛选
钠钙交换体(Na+/Ca2+exchanger, NCX)是一种双向转运蛋白,以3 Na+：1Ca2+的方式转运细胞膜两侧的钠离子和钙离子。在脑缺血的病理过程中,神经元的钠钙稳态失衡是造成细胞损伤的重要因素之一,近期研究表明NCX在脑缺血的病理过程中发挥着神经元保护作用,是一个潜在的脑缺血治疗药物作用靶点。本实验中采用Port-a-patch全细胞膜片钳技术,观察了高通量筛选出的可能影响NCX功能的化合物对转基因细胞上NCX1、NCX2和NCX3三种亚型的作用。结果表明,有4个化合物对NCX呈抑制作用；有9个化合物对NCX的激动作用较明显；对其进一步研究期望可以开发成为具有脑缺血治疗前景的药物。
Cerebral ischaemia is a major cause of disability and death globally and has a profoundly negative impact on the individuals it affects, those that care for them and society as a whole. The most common and familiar manifestation is stroke,85% of which are ischaemic and which is the third leading cause of death after cardiovascular diseases and cancer. Meanwhile, ischemic stroke is the most common cause of complex chronic disability worldwide. Stroke survivors often suffer from long-term neurological disabilities significantly reducing their ability to integrate effectively in society with all the financial and social consequences that this implies.
Among clinical trials for ischemic stroke, those involving thrombolytic, anti-thrombotic, anti-platelet agents are by far more numerous than clinical trials of neuroprotectants and these agents protect the brain do so primarily via hemodynamic rather than metabolic mechanisms. Take the ischemic neuron ionic homeostasis imbalance as a cut-in point; Centering on excessive potassium channel activating and intracellular K+ depletion happened in ischemic stroke pathological process, our study investigated the role of slow-inactivating delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult and whether the drug which is already adopted in neurodegenerative diseases clinical trails—donepezil could protect cells against hypoxia/ischemia apoptotic death by blocking Kv2.1 potassium channel independent of acetylcholine receptor system; centering on the function of Na+/Ca2+ exchanger performed in ischemic stroke pathological process, we screened the NCX activators. Our researching is aim at providing the clues and theoretical foundation for the research and development of anti-ischemic stroke neruoprotective agents.
Part I:Role of delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult
1. Neruoprotective effect of delayed rectifier potassium channel blocker in MCAO rats
Ionic homeostasis imbalance leads by energy failure play a critical role in the ischemic neuron damage during cerebral ischemia pathological process. Since excessively loss of intracellular K+is one of the important factor result in ischemia neuron insult, K+channel blockers could attenuated hypoxia/ischemia cell death in cerebral ischemia. In the former research work of our group, we found that rats after middle cerebral artery occlusion and reperfusion, infarction region mRNA expression of Kv2.1 increased dramatically, so we hypothesized that delayed rectifier potassium channel Kv2.1 might be one target for anti-ischemic stroke drugs. Therefore, the effects of delayed rectifier potassium channel blocker TEA on middle cerebral artery occlusion (MCAO) rats were investigated in the present study:compared with vehicle-control group, intracerebroventricular injection TEA (5ug·kg-1) could significantly reduce the infarct volume.
2. Kv2.1/HEK293 cells were more susceptible to OGD insult than Wt/HEK293 cells
Delayed rectifier-type potassium channel Kv2.1 is expressed at high levels on all neuronal somata and proximal dendrites of neurons in brain and it has been proved to be a major component of neuron outward delayed rectifier potassium current. Furthermore, Kv2.1 has been suggested as a necessary delayed rectifier K+channel subtype in regulating apoptotic signaling cascade in mammalian cortical neurons in culture. In order to identify the role of Kv2.1 played in oxygen-glucose deprivation induced cell insult and rule out the impact of other channels, we selected the Kv2.1 transfected HEK293 cells. Results showed that the delayed rectifier K+current (IK(DR)) density of Kv2.1/HEK23 cells (416.05±13.45 pA/pF**P<0.01) was more than 10 times higher than that of Wt/HEK293 cells (35.18±4.05 pA/pF). To evaluate the cell resistance of Kv2.1/HEK293 cells and Wt/HEK293 cells to OGD, these cells were exposed to OGD for 2hr,6hr and 12hr. Consistent chemical hypoxia affected the cell viability of Kv2.1/HEK293 cells to a much greater degree than it did in Wt/HEK293 cells. To study OGD induced cell apoptosis, chromatin condensation were evaluated after OGD treatment. Results by Hoechst33342 staining assay showed that hypoxia increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. Compared with HEK293 cells, OGD induced a higher cell apoptosis rate in Kv2.1/HEK293 cells.
3. Electrophysiological properties of Kv2.1/HEK293 cells were changed by OGD
Voltage-clamp and current-clamp recording mode was applied to detect the current and membrane potential of Kv2.1/HEK293 cells. Results indicated OGD slightly inhibited Kv2.1 current without influencing the kinetic property of this channel, however, OGD exposure could induced a membrane depolarization to around 2mV within a short period of time. At this membrane potential, Kv2.1 could be activated then lead to massive K+efflux and the subsequent cell damage if the membrane potential maintain at this level persistently due to Kv2.1 channel slow inactivation or almost without auto inactivation property after they were activated.
4. Effect of K+concentration on isolated mitochondrial membrane potential
Mitochondria are intracellular organelles in which high energy phosphate is produced. The role of the mitochondria in apoptotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic death and the stability of mitochondrial membrane potential is important for the decrease of mitochondrial membrane permeability and prevents apoptosis factor release from mitochondrial. Mitochondrial membrane potential (MMP) was measured by mitochondrial uptake of Rhodamin123, a kind of fluorochrome. After the mitochondrial was isolated, they were coincubated in intracellular solution with different K+ concentration. Results showed that:Mitochondrial membrane potential decreased with the reduction of K+concentration in the intracellular solution. This result indicated that the K+concentration is important for the stability of mitochondrial membrane potential.
5. Effect of Kv2.1 potassium channel on OGD induced Kv2.1/HEK293 mitochondrial apoptosis factor release.
Mitochondria are often centrally involved in the development of apoptosis after cerebral ischemia. Mitochondria are the targets for many intracellular anti-apoptotic and pro-apoptotic signals. In response to pro-apoptotic signals, cytochrome C (Cytc) and apoptosis induce factor (AIF) are released from the intermembrane space. In the cytoplasm, Cytc and AIF could produce further downstream events including the activation of caspase-dependent DNase leading to intemucleosomal fragementation of DNA and ect. Therefore, the release of Cytc, AIF and other mitochondrial proteins from the intermembrane space is commonly a critical step in the death of cells by apoptosis. So in our study, the release of Cytc and AIF into the cytoplasm as a consequence of OGD was detected by Western blotting. Compared with HEK293 cells, OGD could markedly increase the release of Cytc from mitochondria after 6h treatment in Kv2.1/HEK293 cells and similar result was observed in AIF release. These results confirmed the effect of Kv2.1 on OGD induced mitochondrial apoptosis and suggested that the antiapoptotic ability of Kv2.1 blocker might be performed partially by suppressing Cytc and AIF release after OGD treatment.
Part II:Donepezil attenuate oxygen-glucose deprivation insult through blocking Kv2.1 potassium channel on transfected HEK293 cells
Donepezil is a widely used drug that improves cognitive and global functions in patients with mild, moderate Alzheimer's disease, as well as vascular dementia. Besides its acetylcholinesterase inhibition ability, neuroprotective effect of donepezil has also been supported by the results of many preclinical studies in hypoxia/ischemia insult models, such as middle cerebral artery occlusion (MCAO) in rats; oxygen-glucose deprivation, glutamate, and Nmethyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)/kainite-induced excitatory amino acids insult, veratridine induce membrane depolarization insult using primary-cultured neurons. These results imply that the neuroprotective effect of donepezil may not be totally dependent on acetylcholinesterase inhibitory activity and the precise neuroprotection mechanism of donepezil is remaining to be clarified. However, interference with the K+-dependent cell damage pathway may be one of them; because among all the above injury models, neurons suffer from sustained membrane depolarization and subsequent excessive K+efflux.
1. Donepezil inhibited Kv2.1 currents in a dose-dependent manner
The Kv2.1 current did not alter significantly during the recording in the time matched control (without donepezil), so the run-down of current could be ruled out. In the presence of 30μM of donepezil, Kv2.1 currents were inhibited to 49.93±6.59%(n=10) of control and the current density of Kv2.1 decreased from 412.42±37.76 pA/pF to 179.63±26.95 pA/pF. Analysis of the concentration-response relationship revealed an IC50 value of 7.59μM. Meanwhile, lOmM TEA decreased Kv2.1 current density from 360.05±53.81 pA/pF to 153.76±25.23 pA/pF.
2. Effect of donepezil on OGD Kv2.1/HEK293 cell Electrophysiological property
OGD induced membrane depolarization is sure to lead to activation of Kv2.1 potassium channel. OGD could decreased Kv2.1 currents amplitude, but this blockade effect of OGD on Kv2.1 currents was not as potent as donepezil did. In the presence of 30μM of donepezil, the current density of OGD Kv2.1 decreased from 354.80±19.25 pA/pF to 123.79±22.69 pA/pF while the depolarizing test pulse was to+60mV.
Membrane potential of Kv2.1/HEK293 cells were moved toward to depolarization direction after OGD treatment. In the presence of 30μM of donepezil and lOmM TEA, OGD induced membrane depolarization was decreased for 5-7mV. Although donepezil and TEA attenuated the membrane depolarization induced by OGD, membrane potential at around 0mV still could result in the activation of Kv2.1 potassium channel.
The steady-state activation curve of Kv2.1 showed that donepezil didn't shift the OGD Kv2.1 half-maximal activation potential (V1/2) significantly. Whereas, donepezil at 30μM significantly shifted the inactivation curve to negative potential by 8.73mV, these results implicated that donepezil blocked Kv2.1 channel mainly due to accelerating the inactivation of this channel and further inhibited the massive K+loss induced by OGD.
3. Donepezil attenuate OGD induced apoptotic damage in Kv2.1/HEK293 cells
MTT staining and Hoechst33342 dye were used to assess the cell viability and apoptois. HEK293 cells become more susceptible to OGD insult after they were transfected with Kv2.1 potassium channel. Donepezil 30μM significantly increase the viability of OGD insulted Kv2.1/HEK293 cells. Since the blockade effects of 30μM donepezil on normal and OGD insulted kv2.1 currents is close to that of 10mM TEA, we observed the antiapoptotic effect of donepezil with TEA at this concentration. In the present study, donepezil and TEA significantly attenuated OGD induced apoptotic morphological alteration, including unhealthy bodies and chromatin fragmentation.
4. Donepezi inhibit OGD induced mitochondrial apoptosis factor release in Kv2.1/HEK293 cells
OGD markedly increased the release of Cytc and AIF from mitochondria into cytoplasm after 6h treatment in Kv2.1/HEK293 cells. In present of 30μM of donepezil, OGD induced the release of Cytc and AIF from mitochondria decreased significantly. These results imply that the anti-OGD-induced apoptotic effect of donepezil maybe contributed to its ability that blocked Kv2.1 potassium channel then stabilizing mitochondria membrane potential, inhibited Cytoc and AIF form mitochondria; and thereby inhibited cell apoptosis, promoted the survival of Kv2.1/HEK293 cells in response to OGD damage.
5. Effect of donepezil on OGD Kv2.1/HEK293 cell cytoplasm PKC-delta level
PKC-delta is a member of the novel PKC family that can be activated in response to numerous cellular stimuli by various mechanisms. The activation pathways include membrane translocation, tyrosine phosphorylation or proteolytic cleavage by an activated enzyme and activated PKC-delta participate in cell apoptosis procedure. By detecting the decrease of complete from PKC-delta, we can observe PKC-delta activation extent. In our research, Kv2.1/HEK293 cell complete from PKC-delta level in cytoplasm decreased signifcantly after 6hr of OGD treatment; but both donepezil and TEA didn't impact the decreased complete from PKC-delta level in cytoplasm. This result imply that anti-OGD-induced apoptotic effect of donepezil may not correlate with PKC-delta activation pathway.
PartⅢ:Anti-ischemic stroke drug screen based on NCX function
The Na+/Ca2+exchanger (NCX) is a bi-directional membrane ion transporter. Under normal physiological conditions, the exchanger transports one calcium ion out of the cell and three sodium ions into the cell. Because dysregulation of sodium and calcium homeostasis is an integral feature of ischemic brain injury, the role of the NCX in neurons following ischemia has been investigated. Studies using in vitro ischemia-related models have produced conflicting results. However, the majority of in vivo studies using the focal cerebral ischemia model and NCX transgenic animals indicate that blocking NCX activity or knocking out NCX gene is neurodamaging while increasing NCX activity is neuroprotective, especially targeting at NCX3. In our research, we studied 9 compounds on NCX1, NCX2 and NCX3 in transgenic cells using Port-a-patch whole cell patch-clamp techniques. Results:4 compounds were found to be the activator of all three NCX isoform,6 compounds could active NCX3 and further study is needed on these compounds in ischemic stoke models to explore promising anti-cerebral ischemia drugs.
目前用于缺血性脑卒中一线临床治疗的药物多集中于溶栓抗凝类药物,而神经保护剂所占比重较小。本研究拟以缺血性脑卒中病理过程中神经元离子稳态失衡为切入点,围绕钾通道的过度开放及细胞内钾的丢失,探讨慢性失活型延迟整流钾通道Kv2.1在由氧糖剥夺模型所介导的细胞损伤中发挥的作用；已知药物多奈派齐(donepezil)具有钾通道阻断的特性,研究其能否独立于胆碱能系统之外,仅通过对电压依赖性钾通道的调节即可发挥抗脑缺血细胞损伤保护作用；围绕钠钙交换体(NCX)功能,筛选NCX调节剂类化合物,探讨NCX激动剂在缺血性脑卒中发挥的神经元保护作用。为脑缺血及再灌注所造成神经元损伤的治疗、以及抗脑缺血神经保护剂类药物的研发提供线索和理论依据。本论文的主要研究内容如下：
第一部分：延迟整流钾通道Kv2.1在氧糖剥夺介导细胞损伤中的作用
1.延迟整流钾通道阻断剂对脑缺血的保护作用
在脑缺血病理过程中,由于能量供给障碍引发的离子稳态平衡失调是造成神经元损伤的始动因素之一。目前的研究结果表明,钾离子的过度外流和细胞内钾的大量丢失是造成脑缺血细胞损伤和凋亡的主要机制，抑制钾通道的过度开放则可能发挥对脑缺血细胞损伤的保护作用。本课题组的前期研究结果表明：大鼠制备脑缺血模型后,脑缺血半影区域延迟整流钾通道Kv2.1的mRNA表达水平显著升高。所以本研究先应用延迟整流钾通道阻断剂四乙铵(TEA)验证了阻断延迟整流钾通道在脑缺血中的神经元保护作用,实验结果显示：大脑中动脉阻塞模型大鼠侧脑室注射TEA(5ug·kg-1)可以显著降低实验动物的脑梗死体积。
2.野生型HEK293细胞转染Kv2.1钾通道后对氧糖剥夺损伤敏感性增强
Kv2.1钾通道在中枢神经系统广泛表达,并且研究表明Kv2.1电流是神经元外向延迟整流钾电流的主要成分；为了研究Kv2.1钾通道在由脑缺血所造成细胞损伤中的作用同时排除神经元上其他类型离子通道的影响,本研究应用高表达Kv2.1钾通道亚型的转基因细胞—Kv2.1／HEK293细胞。在钾通道电流无明显衰减的前提下,应用全细胞膜片钳技术观察和记录野生型HEK293细胞和转染Kv2.1钾通道的HEK293细胞外向钾通道电流差别。实验结果显示：同野生型HEK293细胞相比,Kv2.1／HEK293细胞的外向电流密度增高超过10倍；并通过电生理学实验验证其属于延迟整流钾电流成分。
应用氧糖剥夺模型模拟脑缺血病理过程中的缺血缺氧状态,MTT实验显示：同野生型HEK293细胞相比,氧糖剥夺使转染Kv2.1钾通道的HEK293细胞存活率显著降低。由Hoechst33342标记的细胞凋亡实验显示：氧糖剥夺使转染Kv2.1钾通道的HEK293细胞凋亡率比其载体细胞显著升高。以上结果说明Kv2.1钾通道同氧糖剥夺引起的细胞损伤密切相关。
3.氧糖剥夺对转染Kv2.1钾通道的HEK293细胞电生理学性质的影响
Kv2.1钾离子通道属于氧敏感型钾通道,文献报道在缺氧条件下,Kv2.1钾通道会受到轻微抑制。本研究发现Kv2.1／HEK293细胞氧糖剥夺处理后,Kv2.1电流降低为有氧状态的约82%,同文献报道结果基本一致；同时本研究观察到氧糖剥夺对Kv2.1钾通道的激活和失活无显著影响。
但在应用膜片钳全细胞电流钳制观察细胞膜电位的实验中发现：氧糖剥夺可以使Kv2.1／HEK293细胞膜电位向去极化方向移动并维持在+2mV左右。由于Kv2.1通道特点是该电流激活的电压和时间依赖性,基本上无自主灭活,除非细胞的复极化；所以由氧糖剥夺引起的细胞膜持续去极化状态可以激活Kv2.1通道,使其持续开放并介导钾离子的过度外流,从而引起继发的细胞损伤。
4.钾离子浓度对提取线粒体膜电位的影响
线粒体膜电位的稳定不但是线粒体能量代谢正常进行的前提,也是减缓线粒体通透转运孔开放,避免凋亡通路激活的重要因素。以线粒体对于荧光染料Rhodamin123的摄取速率和摄取量表示线粒体膜电位的高低,观察细胞内液中钾离子浓度同线粒体膜电位间的关系。结果显示：提取线粒体的膜电位随着孵化液中钾离子浓度的降低(用氯化铯或氯化胆碱代替细胞内液中的钾)而降低。提示线粒体膜电位同细胞内钾离子浓度密切相关,抑制钾离子的过度外流有助于稳定线粒体膜电位,减少线粒体凋亡因子的释放。
5.Kv2.1钾通道对氧糖剥夺损伤介导的线粒体凋亡因子释放的影响
氧糖剥夺损伤可以引起线粒体呼吸和能量代谢障碍,同时造成线粒体膜电位降低,最终导致线粒体通透转运孔(MPTP)开放,膜通透性增加,凋亡相关蛋白释放,进而引发凋亡级联反应。本研究应用Westen blot方法观察了氧糖剥夺对Kv2.1／HEK293线粒体细胞凋亡因子Cyto-C和AIF释放的影响,实验结果显示：同野生型HEK293细胞相比,氧糖剥夺6小时使转染Kv2.1钾通道的HEK293细胞胞浆中的Cyto-C和AIF水平显著升高；提示Kv2.1钾通道同氧糖剥夺损伤介导的线粒体凋亡因子释放密切相关。
第二部分：Donepezil对氧糖剥夺介导Kv2.1／HEK293细胞损伤的保护作用1. Donepezil对Kv2.1钾通道的阻断作用
在Kv2.1钾通道电流无明显衰减的前提下,应用全细胞膜片钳技术观察和记录第二代胆碱酯酶抑制剂donepezil对转染Kv2.1钾通道HEK293细胞Kv2.1电流的抑制作用。实验结果显示：donepezil可以呈剂量依赖性的抑制Kv2.1钾电流；对其量效曲线进行分析得到donepezil抑制Kv2.1钾通道的IC50值为7.59μM；donepezil浓度为30μM时对Kv2.1钾通道的抑制接近最大值,当去极化到+60mV时30μMdonepezil可以将Kv2.1电流抑制到对照组的49.93±6.59%。同时,选择经典延迟整流钾通道阻断剂TEA作为阳性对照药物,30μM donepezil和10mM TEA对Kv2.1钾电流的抑制程度接近。
2. Donepezil对氧糖剥夺Kv2.1／HEK293细胞电生理性质的影响
氧糖剥夺可以使转染Kv2.1钾通道HEK293细胞的Kv2.1电流有所降低,donepezil则可以使氧糖剥夺处理的Kv2.1／HEK293细胞Kv2.1电流进一步降低,并且这种抑制程度要远远高于氧糖剥夺对Kv2.1电流的抑制。Donepezil对氧糖剥夺Kv2.1／HEK293细胞Kv2.1钾通道稳态激活曲线无显著影响,但Donepezil可以使氧糖剥夺Kv2.1／HEK293细胞Kv2.1钾通道失活曲线向超极化方向移动8.73mV；提示donepezil对氧糖剥夺Kv2.1钾通道的抑制作用可能同加速通道的失活密切相关。
Kv2.1／HEK293细胞膜电位在-30mV左右,氧糖剥夺处理后细胞膜电位去极化至2.23±2.06mV,氧糖剥夺和30μM donepezil或10mM TEA共同作用后细胞膜电位仍然向去极化方向移动到-5.36±1.37mV和-3.08±1.55mV；提示donepezil和TEA可以起到稳定氧糖剥夺Kv2.1／HEK293细胞膜电位的作用,进而降低钾通道的开放频率以减少由细胞膜去极化造成的钾离子外流。
但在0mV左右的膜电位下,Kv2.1还是可以被激活开放,所以本研究进一步观察了细胞膜电位是0mV时donepezil对氧糖剥夺Kv2.1／HEK293细胞钾电流的影响。实验结果显示：氧糖剥夺处理后,Kv2.1／HEK293细胞膜电位0mV时的电流密度为89.90±8.26 pA／pF；30μMdonepezil和10mM TEA分别可以使氧糖剥夺Kv2.1／HEK293细胞0mV时的电流密度降低到27.11±5.22 pA／pF和38.71±5.53pA／pF；提示在由氧糖剥夺引起细胞去极化到0mV左右时donepezil可以通过对Kv2.1钾电流的显著抑制作用避免细胞内钾的过度丢失,从而发挥保护作用。
3. Donepezil对氧糖剥夺介导Kv2.1／HEK293细胞损伤的保护作用
应用MTT法观察donepezil对氧糖剥夺Kv2.1／HEK293细胞存活率的影响,结果表明：同有氧对照组相比,氧糖剥夺损伤模型组的细胞生存率显著降低至33.26±1.09%; donepezil可以剂量依赖性的提高氧糖剥夺损伤Kv2.1／HEK293细胞存活率,30μM donepezi和10mM TEA分别可以使细胞存活率升高至47.29±2.59%和44.03±1.95%。
细胞凋亡实验发现：同野生型HEK293细胞相比,氧糖剥夺24小时使Kv2.1／HEK293细胞的凋亡率显著升高至52.82±2.01%;30μM donepezil可以使细胞凋亡率显著降低至23.70±1.75%；10mM TEA可以使细胞凋亡率显著降低至26.35±2.11%。提示在氧糖剥夺介导的Kv2.1／HEK293细胞凋亡中,donepezil通过阻断Kv2.1钾通道可以发挥对抗细胞凋亡作用。
4. Donepezil对氧糖剥夺损伤Kv2.1／HEK293细胞线粒体凋亡因子释放的抑制作用
氧糖剥夺损伤可以使Kv2.1／HEK293细胞胞浆细胞色素C和凋亡诱导因子水平升高,并且在氧糖剥夺6小时后同野生型HEK293细胞相比有显著性差异,实验结果显示：同氧糖剥夺6小时损伤模型相比,30μM donepezil和10mM TEA可以显著降低氧糖剥夺Kv2.1／HEK293细胞胞浆细胞色素C水平；同时30μM donepezil还可以使Kv2.1／HEK293细胞胞浆凋亡诱导因子水平显著降低,而TEA对Kv2.1／HEK293细胞胞浆凋亡诱导因子水平影响不明显。以上结果提示,donepezil可以通过阻断Kv2.1钾离子通道,进而降低由氧糖剥夺介导钾离子大量外流所引起的胞浆细胞色素C和凋亡诱导因子水平的升高,发挥抗凋亡作用。
5. Donepezil对氢糖剥夺损伤Kv2.1／HEK293细胞PKC-delta水平的影响
PKC-delta属于蛋白激酶C家族成员。研究表明PKC-delta参与细胞凋亡的发生,细胞内PKC-delta水平同脑缺血损伤造成的神经元凋亡相关。PKC-delta被活化后才可以诱导细胞凋亡,目前报道有三种方式可以使PKC-delta活化,分别是PKC-delta的膜转位、酪氨酸磷酸化和溶蛋白性裂解；PKC-delta激活的结果是使细胞内完整形式的PKC-delta蛋白水平降低,其激活产物参与其他信号通路的调节。本研究发现,在氧糖剥夺6小时后,Kv2.1／HEK293细胞胞浆内完整形式的PKC-delta水平显著降低,提示PKC-delta的激活；但在给予donepezil和TEA后对胞浆内已经降低PKC-delta水平无明显影响。提示donepezil阻断钾通道并不通过对胞浆内PKC-delta水平的调节发挥其抗凋亡细胞保护作用。
第三部分：基于NCX功能的抗脑缺血药物筛选
钠钙交换体(Na+/Ca2+exchanger, NCX)是一种双向转运蛋白,以3 Na+：1Ca2+的方式转运细胞膜两侧的钠离子和钙离子。在脑缺血的病理过程中,神经元的钠钙稳态失衡是造成细胞损伤的重要因素之一,近期研究表明NCX在脑缺血的病理过程中发挥着神经元保护作用,是一个潜在的脑缺血治疗药物作用靶点。本实验中采用Port-a-patch全细胞膜片钳技术,观察了高通量筛选出的可能影响NCX功能的化合物对转基因细胞上NCX1、NCX2和NCX3三种亚型的作用。结果表明,有4个化合物对NCX呈抑制作用；有9个化合物对NCX的激动作用较明显；对其进一步研究期望可以开发成为具有脑缺血治疗前景的药物。
Cerebral ischaemia is a major cause of disability and death globally and has a profoundly negative impact on the individuals it affects, those that care for them and society as a whole. The most common and familiar manifestation is stroke,85% of which are ischaemic and which is the third leading cause of death after cardiovascular diseases and cancer. Meanwhile, ischemic stroke is the most common cause of complex chronic disability worldwide. Stroke survivors often suffer from long-term neurological disabilities significantly reducing their ability to integrate effectively in society with all the financial and social consequences that this implies.
Among clinical trials for ischemic stroke, those involving thrombolytic, anti-thrombotic, anti-platelet agents are by far more numerous than clinical trials of neuroprotectants and these agents protect the brain do so primarily via hemodynamic rather than metabolic mechanisms. Take the ischemic neuron ionic homeostasis imbalance as a cut-in point; Centering on excessive potassium channel activating and intracellular K+ depletion happened in ischemic stroke pathological process, our study investigated the role of slow-inactivating delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult and whether the drug which is already adopted in neurodegenerative diseases clinical trails—donepezil could protect cells against hypoxia/ischemia apoptotic death by blocking Kv2.1 potassium channel independent of acetylcholine receptor system; centering on the function of Na+/Ca2+ exchanger performed in ischemic stroke pathological process, we screened the NCX activators. Our researching is aim at providing the clues and theoretical foundation for the research and development of anti-ischemic stroke neruoprotective agents.
Part I:Role of delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult
1. Neruoprotective effect of delayed rectifier potassium channel blocker in MCAO rats
Ionic homeostasis imbalance leads by energy failure play a critical role in the ischemic neuron damage during cerebral ischemia pathological process. Since excessively loss of intracellular K+is one of the important factor result in ischemia neuron insult, K+channel blockers could attenuated hypoxia/ischemia cell death in cerebral ischemia. In the former research work of our group, we found that rats after middle cerebral artery occlusion and reperfusion, infarction region mRNA expression of Kv2.1 increased dramatically, so we hypothesized that delayed rectifier potassium channel Kv2.1 might be one target for anti-ischemic stroke drugs. Therefore, the effects of delayed rectifier potassium channel blocker TEA on middle cerebral artery occlusion (MCAO) rats were investigated in the present study:compared with vehicle-control group, intracerebroventricular injection TEA (5ug·kg-1) could significantly reduce the infarct volume.
2. Kv2.1/HEK293 cells were more susceptible to OGD insult than Wt/HEK293 cells
Delayed rectifier-type potassium channel Kv2.1 is expressed at high levels on all neuronal somata and proximal dendrites of neurons in brain and it has been proved to be a major component of neuron outward delayed rectifier potassium current. Furthermore, Kv2.1 has been suggested as a necessary delayed rectifier K+channel subtype in regulating apoptotic signaling cascade in mammalian cortical neurons in culture. In order to identify the role of Kv2.1 played in oxygen-glucose deprivation induced cell insult and rule out the impact of other channels, we selected the Kv2.1 transfected HEK293 cells. Results showed that the delayed rectifier K+current (IK(DR)) density of Kv2.1/HEK23 cells (416.05±13.45 pA/pF**P<0.01) was more than 10 times higher than that of Wt/HEK293 cells (35.18±4.05 pA/pF). To evaluate the cell resistance of Kv2.1/HEK293 cells and Wt/HEK293 cells to OGD, these cells were exposed to OGD for 2hr,6hr and 12hr. Consistent chemical hypoxia affected the cell viability of Kv2.1/HEK293 cells to a much greater degree than it did in Wt/HEK293 cells. To study OGD induced cell apoptosis, chromatin condensation were evaluated after OGD treatment. Results by Hoechst33342 staining assay showed that hypoxia increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. Compared with HEK293 cells, OGD induced a higher cell apoptosis rate in Kv2.1/HEK293 cells.
3. Electrophysiological properties of Kv2.1/HEK293 cells were changed by OGD
Voltage-clamp and current-clamp recording mode was applied to detect the current and membrane potential of Kv2.1/HEK293 cells. Results indicated OGD slightly inhibited Kv2.1 current without influencing the kinetic property of this channel, however, OGD exposure could induced a membrane depolarization to around 2mV within a short period of time. At this membrane potential, Kv2.1 could be activated then lead to massive K+efflux and the subsequent cell damage if the membrane potential maintain at this level persistently due to Kv2.1 channel slow inactivation or almost without auto inactivation property after they were activated.
4. Effect of K+concentration on isolated mitochondrial membrane potential
Mitochondria are intracellular organelles in which high energy phosphate is produced. The role of the mitochondria in apoptotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic death and the stability of mitochondrial membrane potential is important for the decrease of mitochondrial membrane permeability and prevents apoptosis factor release from mitochondrial. Mitochondrial membrane potential (MMP) was measured by mitochondrial uptake of Rhodamin123, a kind of fluorochrome. After the mitochondrial was isolated, they were coincubated in intracellular solution with different K+ concentration. Results showed that:Mitochondrial membrane potential decreased with the reduction of K+concentration in the intracellular solution. This result indicated that the K+concentration is important for the stability of mitochondrial membrane potential.
5. Effect of Kv2.1 potassium channel on OGD induced Kv2.1/HEK293 mitochondrial apoptosis factor release.
Mitochondria are often centrally involved in the development of apoptosis after cerebral ischemia. Mitochondria are the targets for many intracellular anti-apoptotic and pro-apoptotic signals. In response to pro-apoptotic signals, cytochrome C (Cytc) and apoptosis induce factor (AIF) are released from the intermembrane space. In the cytoplasm, Cytc and AIF could produce further downstream events including the activation of caspase-dependent DNase leading to intemucleosomal fragementation of DNA and ect. Therefore, the release of Cytc, AIF and other mitochondrial proteins from the intermembrane space is commonly a critical step in the death of cells by apoptosis. So in our study, the release of Cytc and AIF into the cytoplasm as a consequence of OGD was detected by Western blotting. Compared with HEK293 cells, OGD could markedly increase the release of Cytc from mitochondria after 6h treatment in Kv2.1/HEK293 cells and similar result was observed in AIF release. These results confirmed the effect of Kv2.1 on OGD induced mitochondrial apoptosis and suggested that the antiapoptotic ability of Kv2.1 blocker might be performed partially by suppressing Cytc and AIF release after OGD treatment.
Part II:Donepezil attenuate oxygen-glucose deprivation insult through blocking Kv2.1 potassium channel on transfected HEK293 cells
Donepezil is a widely used drug that improves cognitive and global functions in patients with mild, moderate Alzheimer's disease, as well as vascular dementia. Besides its acetylcholinesterase inhibition ability, neuroprotective effect of donepezil has also been supported by the results of many preclinical studies in hypoxia/ischemia insult models, such as middle cerebral artery occlusion (MCAO) in rats; oxygen-glucose deprivation, glutamate, and Nmethyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)/kainite-induced excitatory amino acids insult, veratridine induce membrane depolarization insult using primary-cultured neurons. These results imply that the neuroprotective effect of donepezil may not be totally dependent on acetylcholinesterase inhibitory activity and the precise neuroprotection mechanism of donepezil is remaining to be clarified. However, interference with the K+-dependent cell damage pathway may be one of them; because among all the above injury models, neurons suffer from sustained membrane depolarization and subsequent excessive K+efflux.
1. Donepezil inhibited Kv2.1 currents in a dose-dependent manner
The Kv2.1 current did not alter significantly during the recording in the time matched control (without donepezil), so the run-down of current could be ruled out. In the presence of 30μM of donepezil, Kv2.1 currents were inhibited to 49.93±6.59%(n=10) of control and the current density of Kv2.1 decreased from 412.42±37.76 pA/pF to 179.63±26.95 pA/pF. Analysis of the concentration-response relationship revealed an IC50 value of 7.59μM. Meanwhile, lOmM TEA decreased Kv2.1 current density from 360.05±53.81 pA/pF to 153.76±25.23 pA/pF.
2. Effect of donepezil on OGD Kv2.1/HEK293 cell Electrophysiological property
OGD induced membrane depolarization is sure to lead to activation of Kv2.1 potassium channel. OGD could decreased Kv2.1 currents amplitude, but this blockade effect of OGD on Kv2.1 currents was not as potent as donepezil did. In the presence of 30μM of donepezil, the current density of OGD Kv2.1 decreased from 354.80±19.25 pA/pF to 123.79±22.69 pA/pF while the depolarizing test pulse was to+60mV.
Membrane potential of Kv2.1/HEK293 cells were moved toward to depolarization direction after OGD treatment. In the presence of 30μM of donepezil and lOmM TEA, OGD induced membrane depolarization was decreased for 5-7mV. Although donepezil and TEA attenuated the membrane depolarization induced by OGD, membrane potential at around 0mV still could result in the activation of Kv2.1 potassium channel.
The steady-state activation curve of Kv2.1 showed that donepezil didn't shift the OGD Kv2.1 half-maximal activation potential (V1/2) significantly. Whereas, donepezil at 30μM significantly shifted the inactivation curve to negative potential by 8.73mV, these results implicated that donepezil blocked Kv2.1 channel mainly due to accelerating the inactivation of this channel and further inhibited the massive K+loss induced by OGD.
3. Donepezil attenuate OGD induced apoptotic damage in Kv2.1/HEK293 cells
MTT staining and Hoechst33342 dye were used to assess the cell viability and apoptois. HEK293 cells become more susceptible to OGD insult after they were transfected with Kv2.1 potassium channel. Donepezil 30μM significantly increase the viability of OGD insulted Kv2.1/HEK293 cells. Since the blockade effects of 30μM donepezil on normal and OGD insulted kv2.1 currents is close to that of 10mM TEA, we observed the antiapoptotic effect of donepezil with TEA at this concentration. In the present study, donepezil and TEA significantly attenuated OGD induced apoptotic morphological alteration, including unhealthy bodies and chromatin fragmentation.
4. Donepezi inhibit OGD induced mitochondrial apoptosis factor release in Kv2.1/HEK293 cells
OGD markedly increased the release of Cytc and AIF from mitochondria into cytoplasm after 6h treatment in Kv2.1/HEK293 cells. In present of 30μM of donepezil, OGD induced the release of Cytc and AIF from mitochondria decreased significantly. These results imply that the anti-OGD-induced apoptotic effect of donepezil maybe contributed to its ability that blocked Kv2.1 potassium channel then stabilizing mitochondria membrane potential, inhibited Cytoc and AIF form mitochondria; and thereby inhibited cell apoptosis, promoted the survival of Kv2.1/HEK293 cells in response to OGD damage.
5. Effect of donepezil on OGD Kv2.1/HEK293 cell cytoplasm PKC-delta level
PKC-delta is a member of the novel PKC family that can be activated in response to numerous cellular stimuli by various mechanisms. The activation pathways include membrane translocation, tyrosine phosphorylation or proteolytic cleavage by an activated enzyme and activated PKC-delta participate in cell apoptosis procedure. By detecting the decrease of complete from PKC-delta, we can observe PKC-delta activation extent. In our research, Kv2.1/HEK293 cell complete from PKC-delta level in cytoplasm decreased signifcantly after 6hr of OGD treatment; but both donepezil and TEA didn't impact the decreased complete from PKC-delta level in cytoplasm. This result imply that anti-OGD-induced apoptotic effect of donepezil may not correlate with PKC-delta activation pathway.
PartⅢ:Anti-ischemic stroke drug screen based on NCX function
The Na+/Ca2+exchanger (NCX) is a bi-directional membrane ion transporter. Under normal physiological conditions, the exchanger transports one calcium ion out of the cell and three sodium ions into the cell. Because dysregulation of sodium and calcium homeostasis is an integral feature of ischemic brain injury, the role of the NCX in neurons following ischemia has been investigated. Studies using in vitro ischemia-related models have produced conflicting results. However, the majority of in vivo studies using the focal cerebral ischemia model and NCX transgenic animals indicate that blocking NCX activity or knocking out NCX gene is neurodamaging while increasing NCX activity is neuroprotective, especially targeting at NCX3. In our research, we studied 9 compounds on NCX1, NCX2 and NCX3 in transgenic cells using Port-a-patch whole cell patch-clamp techniques. Results:4 compounds were found to be the activator of all three NCX isoform,6 compounds could active NCX3 and further study is needed on these compounds in ischemic stoke models to explore promising anti-cerebral ischemia drugs.
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