川芎嗪抗大鼠局灶性脑缺血再灌注损伤的机制研究
摘要
研究目的
中药抗脑缺血再灌注损伤是一个重要的课题。随着社会的发展，脑血管病已经成为威胁人类生存和健康的主要疾病之一。缺血再灌注是脑血管病发展过程中的一个重要的病理过程，能导致进一步组织损伤和功能障碍，抑制再灌注损伤已经成为目前治疗缺血性脑血管病的关键环节。目前治疗脑缺血病的药物往往作用机制单一，毒副作用较大，而中药多可以从多个层次和多个环节上起到治疗作用，因此研究和阐明中药治疗脑缺血病的机制具有重大的理论和临床价值。
脑血管病与中医血瘀证紧密相关，中医治疗多采用活血化瘀法，配伍理气药以行气活血。川芎为临床常用活血化瘀药，具有活血行气之效，是临床常用于治疗缺血性脑血管病的中药之一。阐明川芎抗脑缺血再灌注的机理对于研究中药治疗缺血性脑病具有重大意义。
根据现代医学和传统中医关于脑缺血病的理论，我们将进行以下研究：(1)证明川芎的有效成分——盐酸川芎嗪可以透过血脑屏障，为其可以直接对脑产生作用提供证据。(2)对盐酸川芎嗪在抗脑缺血再灌注过程中所发挥的作用进行多层次系统研究，探讨其作用机制。希望通过此研究为临床使用川芎或川芎嗪治疗脑缺血病提供理论依据，同时也为今后研究和筛选治疗脑缺血病的中药提供一个实验方法学平台。
研究方法
我们选用SD大鼠为研究对象，制备了阻断大鼠大脑中动脉局灶性脑缺血(MCAO)再灌注模型，研究盐酸川芎嗪对抗脑缺血再灌注的机制。(1)高效液相(HPLC)检测盐酸川芎嗪能否通过大鼠血脑屏障到达脑组织；(2)称量假手术组、缺血再灌注组和各用药组大鼠脑组织重量，检测脑组织含水量；(3)苏木精伊红(HE)染色，观察各组大鼠脑细胞形态的改变；(4)免疫组化染色观察GFAP表达的改变，进而反映星形胶质细胞的变化情况；(5)生化法检测各组大鼠血清NOS、iNOS含量的变化；(6)生化法检测各组大鼠血清和脑匀浆SOD含量的变化；(7)流式细胞术(FCM)检测各组大鼠脑细胞增殖周期、细胞凋亡的改变；(8)Real-Time PCR技术检测各组大鼠脑组织中缺氧诱导因子-1α表达的改变。
结果
(1)鉴于大鼠脑脊液很少，难以达到高效液相测定所需的量，我们选择测定大鼠组织中盐酸川芎嗪的含量。结果各组大鼠脑组织中均检测到盐酸川芎嗪，提示其可以穿透血脑屏障，到达脑从而对中枢产生作用，为研究盐酸川芎嗪乃至川芎对脑的作用机理提供了前提条件。(2)缺血再灌注组脑组织含水量明显高于假手术组(P＜0.01)，而三个用药组与缺血再灌注组比较，脑组织含水量明显减少(P＜0.05)，其中盐酸川芎嗪高剂量组和尼莫地平组变化最为明显，显示盐酸川芎嗪能明显减少梗塞侧脑组织的肿胀程度，减少患侧脑组织含水量。(3)缺血再灌注组大鼠梗死侧脑组织明显肿胀，外观黯淡。镜下观察可见锥体细胞排列紊乱，细胞间隙增宽，神经元变性明显，部分退变的神经元呈现凋亡特征：细胞皱缩成圆形或卵圆形，胞浆皱缩，嗜酸性增加，亦可见均质红染坏死物质和细胞核固缩、溶解等坏死特征。用药组细胞变性程度较缺血再灌注组明显减轻，其中尤以川芎嗪高剂量组和尼莫地平组最为明显，仅有少数锥体细胞胞浆深染，细胞核固缩。(4)缺血再灌注组GFAP免疫染色呈强阳性，胞体肥大，染色加深，突起增粗、变长，而假手术组仅可见极少量阳性细胞表达，用药组GFAP免疫染色则呈阳性——弱阳性改变，说明AST反应性增生减弱，提示盐酸川芎嗪可抑制脑缺血再灌注时AST的过度表达。(5)缺血再灌注组大鼠血清中NOS和iNOS均比假手术组有显著提高，用药组大鼠血清中NOS和iNOS有所下降，其中以川芎嗪高剂量组和尼莫地平组下降最为显著(P＜0.05)，提示川芎嗪可降低脑缺血再灌注时NOS尤其是iNOS的含量，减少NO的合成，减轻神经毒性反应以及由NO所诱导的神经细胞凋亡。(6)缺血再灌注组的血清和脑组织中SOD活力明显降低(P＜0.01)，三个用药组的血清和脑组织中SOD活力均有提高，其中尼莫地平组血清中SOD活力和川芎嗪高剂量组脑组织中SOD活力提高最为明显(P＜0.05)。说明盐酸川芎嗪可提高脑组织SOD活性，有利于清除自由基，减少脂质过氧化损伤。(7)缺血再灌注后细胞周期发生了明显变化，处于Go／G1期的细胞百分率明显减少，进入S期和G2／M期的细胞明显增加，与假手术组比较有显著差异(P＜0.05)，说明了缺血性脑损伤后细胞异常活跃，细胞可以重新进入细胞周期。在细胞凋亡发生率方面，缺血再灌注组大鼠脑细胞为(21.61±4.4)％，较假手术组显著增加(P＜0.05)，盐酸川芎嗪高剂量组和尼莫地平组细胞凋亡发生率较缺血再灌注组显著降低(P＜0.05)，其中以盐酸川芎嗪高剂量组变化最大。(8)经Real-Time PCR测定，发现HIF-1α在缺血再灌注组中的表达远高于假手术组，在川芎嗪低剂量组中的表达略有降低，而在川芎嗪高剂量组和尼莫地平组中的表达有明显降低，其中尼莫地平组基本接近假手术组。
结论与意义
盐酸川芎嗪抗缺血再灌注的机制已经成为近年研究热点之一，取得了有目共睹的成果，但仍存在一些问题：(1)研究多集中于盐酸川芎嗪治疗缺血性心脏病的作用机制，而对于其抗脑缺血再灌注损伤的作用机制研究较少，这和盐酸川芎嗪在脑血管病治疗中的广泛应用不相符合。(2)大多数研究均着眼于盐酸川芎嗪对功能指标的调控，并没有证据证明其可以透过血脑屏障直接作用于中枢。尽管一些学者提出盐酸川芎嗪可以透过正常的血脑屏障，但是对其在脑缺血再灌注、血脑屏障受损的情况下能否透过血脑屏障，以及透过的水平与正常情况有无区别，并没有报道。(3)研究方法比较单一。多数学者仅从某一个角度研究其抗脑缺血再灌注的机制，而脑缺血再灌注的病理生理变化是多方面、多层次的，因此只研究盐酸川芎嗪对其中一个病理环节的影响显然是不全面的。此外由于不同研究人员采用的缺血再灌注模型不尽相同，造成各种实验结果缺乏横向对比的可行性。这些问题造成现有对盐酸川芎嗪抗脑缺血再灌注的机制研究缺乏系统性，整体性。
本实验研究的创新性体现在：(1)证明了盐酸川芎嗪能够穿透血脑屏障，进入脑组织发挥直接作用，而在缺血再灌注后给药脑组织中盐酸川芎嗪含量增多，说明在此状态下药物可以更多的到达脑组织，为研究其进入脑组织对中枢产生作用提供了先决条件。(2)本研究从整体水平、细胞水平、分子水平和基因水平，针对多种病理变化，较完整系统的阐述了盐酸川芎嗪抗脑缺血再灌注损伤的机制。(3)实验采用了多种检测方法，其中Real-Time PCR具有技术水平上的先进性，与传统PCR技术相比，不仅实现了从定性到定量的飞跃，而且具有特异性更强、有效解决PCR污染问题、自动化程度高等特点。(4)实验证明了盐酸川芎嗪在多层次多方面对脑缺血再灌注损伤起到了保护作用，作用机制与降低脑水肿、调整AST对损伤的反应、抗自由基损伤、降低NO毒性、防止血管痉挛、抗细胞凋亡、减少神经细胞变性坏死以及基因调控等多方面关系密切，具有重要的临床和理论价值。(5)本实验为研究中药通过血脑屏障、抗脑缺血再灌注损伤建立了一整套实验方法学平台，对促进中药现代化具有重要意义。
Purpose of research
The inhibitory effect of Traditional Chinese Medicine on cerebralischemia-reperfusion injury is an important topic. With the development of the society, the incidence of the cerbralvascular diseases keeps increasing and become one of themajor diseases threatening the existing and health of the human kind.Ischemia-reperfusion is one of the important pathologic processes in the developmentprocess of Cerbralvascular Disease. It can induce further tissue injury and dysfunction.To control the ischemia-reperfusion injury has become the key factor in the treatmentof ischemic stroke in the present day. At present, the mechanism of the medicine forcerebral ischemia diseases is usually simple and it often has big side effects. On theother hand, Tradition Chinese Medicine has multi-level and multi-link treatmenteffects. So, research on the mechanism of the TCM treatment of cerebral ischemia isvery important in theory and clinic.
The Cerbralvascular Disease is closely associated with Blood stasis syndrome(TCM). The TCM treatment of blood stasis syndrome always adopts the role ofblood-activating and stasis-resolving and use drags that regulated flow of qi incombination to invigorate qi and promote blood circulation. Chuanxiong is ablood-activating and stasis-resolving medicine which is often used in clinic. It has notonly the function of promoting blood, but also the effect of invigorating qi. It is one ofthe common TCM medicines for the treatment of ischemia cerbralvascular disease inclinic. To explain the mechanism of the inhibitory effects of Chuanxiong onischemia-reperfusion is very important for the research on the TCM treatment ofischemic encephalopathy.
Based on the theory of Traditional Chinese Medicine and Modern Medicine about Cerebral ischemia disease, we will do the following research: (1)Prove that theeffective compound of Chuanxiong- Ligustrazine Hydrochloride can go through thenormal and Ischemia-Reperfusion Blood-Brain Barrier. Provide the evidence that ithas direct effect on brain tissues. (2) Do the multi-level research systemically on thefunctions of Ligustrazine Hydrochloride in the process of anti Ischemia-Reperfusion.Hope this research can provide the theory basis for using Chuangxiong orLigustrazine Hydrochloride in the treatment of cerbralvascular disease in clinic, andprovide a methodology platform of screening the TCM for treating cerebral ischemiadiseases.
Research method
We used SD rats as research objects. We prepared the model of Focal CerebralIschemia-reperfusion of Middle Cerebral Artery in rats, and studied the inhibitorymechanism of Ligustrazine Hydrochloride on ischemia-reperfusion injury.
(1) To test if the Ligustrazine Hydrochloride can go through the Blood-BrainBarrier of the normal rats and the rats with cerebral ischemia-reperfusion injury andreach the brain tissue using High-performance Liquid Chromatography (HPLC); (2)Measure the weight of rat brain tissues of the normal group, ischemia-reperfusiongroup and other treatment groups. And the water contents in brain tissue weremeasured. (3) Stain by HE staining, and observe the shape change of the brain cell ofdifferent groups. (4) Observe the expression of GFAP using the method ofimmunohistochemistry stain. And show the change of the astrocyte; (5) Measure thechange of contents of NOS and iNOS in serum of rats in each group usingbiochemical method. (6) Measure the change of contents of SOD in serum and brainhomogenate of rats in each group using biochemical method; (7) Measure the changeof brain cell proliferation cycle and apoptosis of rats in each group using FCM; (8)Measure the expression change of hypoxia-inducible factor 1 alpha in the brain tissueof rats in each group using Real-Time PCR.
Results
Ligustrazine Hydrochloride can be detected in the brain tissues of rats in eachgroup. That indicates it can go through the Blood-Brain Barrier, reach the brain and has effects on central nervous system. This result provides the premise conditions ofthe research of the effects and mechanisms of Ligustrazine Hydrochloride or evenChuanxiong on brain. (2) Water content of ischemia-reperfusion group is obviouslyhigher than sham-operation group (P＜0.01), and the water content of three medicinegroups is apparently reduced comparing with ischemia-reperfusion group (P＜0.05).The change is more apparent in the Ligustrazine Hydrochloride high-dose group andnimodipine group. It indicates that Ligustrazine Hydrochloride can apparently reducethe swelling dimension of contralateral hemisphere, and reduce the water content inthe lesion hemisphere. (3) The brain tissue of rats in ischemia-reperfusion groupswelled apparently and the appearance is dim. It can be seen under microscope thatthe pyramid cells are arranged disorderly, the cell space got widening and neurondegeneration is apparent. Some degenerated neuron appear apoptosis: The cells shrinkto rounded or ovoid shape. The cytoplastic shrinks and eosinophilic character increase.Homogeneous red-stained necrotic material, nucleus condensation and dissolution, and other necrotic features also can be seen. The degree of degeneration wassignificantly reduced in medicine group than in ischemia-reperfusion group. And it ismore apparent in the Ligustrazine Hydrochloride high-dose group and nimodipinegroup. Only a little of pyramid cells were deeply stained and had nucleuscondensation. (4) ischemia-reperfusion group' s GFAP immunostaining was strongpositive. Cell body is mast, stain is deepening and the neurite is thickening and gettinglonger. But sham-operation group only had very few positive cell expressions. Theimmunostaining of medicine only had positive-feebly positive change. Thisdemonstrates that AST reactive hyperplasia is decreased and suggest that LigustrazineHydrochloride can inhibit the over-expression of AST with ischemia-reperfusion. Thecontents of NOS and iNOS in serum of rats in ischemia-reperfusion group issignificantly increased than sham-operated group. The contents of NOS and iNOS inserum of rats in medicine group decreased. And it is more apparent in the LigustrazineHydrochloride high-dose group and nimodipine group (P＜0.05). It suggested thatLigustrazine can decrease the content of NOS, especially iNOS, whenischemia-reperfusion occurs. Reduce the synthesis of NO. Reduce the nerve toxic reactions and the NO-induced neuronal apoptosis. (6) Activity of SOD in the serumand brain tissue of the ischemia-reperfusion group significantly decreased (P＜0.01), Activity of SOD in the serum and brain tissue of the three medicine group allincreased. And it is more apparent in the Ligustrazine Hydrochloride high-dose groupand nimodipine group (P＜0.05). It suggests that igustrazine can increase the activityof SOD in brain tissue, and help to eliminate free radicals, and reduce lipidperoxidation injury. (7) After ischemia-reperfusion, noticeable changes had takenplace in the cell cycle. The percentage of cells in G0/G1 phase decreased apparently.The cells entered S and G2/M phase increased a lot. It had a significantly differencecomparing with sham-operated group (P＜0.05). It shown that, afterischemia-reperfusion injury, cells were very active, and can reenter the cell cycle. Inthe rate of cell apoptsis, the brain cell of rats in the ischemia-reperfusion group is(21.61±4.4)%, a significantly increase comparing with sham-operated group(P＜0.05). The rate of cell apoptsis in the Ligustrazine Hydrochloride high-dose groupand nimodipine group is significantly lower than ischemia-reperfusion group (P＜0.05).The Ligustrazine Hydrochloride high-dose group had the biggest change. (8) Theexpression of HIF-1αin the ischemia-reperfusion group was much higher thansham-operated group measured by Real-Time PCR. It is lower in the LigustrazineHydrochloride low-dose group. But it significantly decreased in the LigustrazineHydrochloride high-dose group and nimodipine group. The nimodipine group isalmost same as sham-operated group.
Conclusions and significance
The mechanism of the inhibitory effect of Ligustrazine Hydrochloride onischemia-reperfusion is a hot topic in recent years and has got significant results. Butit still has some problems: (1) the researches are mostly focused on the mechanism ofusing Ligustrazine Hydrochloride to treat the ischemic heart disease. But the researchon the mechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion injury is much less. This is inconsistent with the widely using ofLigustrazine Hydrochloride in the treatment of Cerebrovascular Disease. (2) Most ofthe researches are focused the regulation of Ligustrazine Hydrochloride on the functional indices, and didn' t prove it can pass through the BBB and has directeffects on central nervous system. Although some scholars prove that LigustrazineHydrochloride can go through the BBB, there isn' t report about whether it still cango through the BBB, with ischemia-reperfusion and damaged BBB. Also there isn' treport about the whether there is any difference in the going through level comparingwith the normal cases. (3) The research method is relatively simple. Most scholarsonly research on one aspect of the mechanism of the inhibitory effect of LigustrazineHydrochloride on ischemia-reperfusion. But the pathophysiological changes ofcerebral ischemia-reperfusion are in many ways and at different levels. So, it isobviously not comprehensive to study the effect of Ligustrazine Hydrochloride ononly one pathology link. In addition, because different researchers use differentischemia-reperfusion model, the various experimental results don' t have thehorizontal comparison feasibility. These problems induced that the research onmechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion is not systemic and holistic.
The innovation of this experiment proves is (1) Proveing LigustrazineHydrochloride can go through the normal and Ischemia-Reperfusion Blood-BrainBarrier, reach the brain tissue and has direct effect on brain tissues. And theincensement of the content of Ligustrazine Hydrochloride in the ischemia-reperfusionbrain tissue after medical treatment, indices that in this case, more medicine can reachthe brain tissue. This provides the preconditions for the research on how it reaches thebrain tissue and has direct effect on brain tissues. (2) This study descripts themechanism for the treatment of cerebral ischemia-reperfusion injury usingLigustrazine Hydrochloride at the overall level, and the level of cells, molecules andgenes according to a variety of pathological changes. (3)This experiment used a lot ofmeasure methods, among which the Real-time PCR is at the advanced technologicallevel. Comparing with traditional PCR technology, it realized not only the leap from aqualitative level to a quantitative level, but also has the characters of more specificity, effectively solving the pollution problems of PCR, and a higher degree ofautomation. (4)Experiment proved that Ligustrazine Hydrochloride has protection function on cerebral ischemia-reperfusion injury. The mechanism of effect is closelyrelated to decreasing brain edema, adjusting the reaction of AST to injury, anti-freeradical damage, reducing the toxicity of NO, preventing vasospasm, anti-apoptotic, reducing nerve cell degeneration and necrosis, Gene regulation and other aspects.(5)This experiment built a whole set of experimental methodology platform forfurther research on going through the BBB of TCM and anti Ischemia-Reperfusion, and also has great significance to promoting the modernization of traditional Chinesemedicine.
中药抗脑缺血再灌注损伤是一个重要的课题。随着社会的发展，脑血管病已经成为威胁人类生存和健康的主要疾病之一。缺血再灌注是脑血管病发展过程中的一个重要的病理过程，能导致进一步组织损伤和功能障碍，抑制再灌注损伤已经成为目前治疗缺血性脑血管病的关键环节。目前治疗脑缺血病的药物往往作用机制单一，毒副作用较大，而中药多可以从多个层次和多个环节上起到治疗作用，因此研究和阐明中药治疗脑缺血病的机制具有重大的理论和临床价值。
脑血管病与中医血瘀证紧密相关，中医治疗多采用活血化瘀法，配伍理气药以行气活血。川芎为临床常用活血化瘀药，具有活血行气之效，是临床常用于治疗缺血性脑血管病的中药之一。阐明川芎抗脑缺血再灌注的机理对于研究中药治疗缺血性脑病具有重大意义。
根据现代医学和传统中医关于脑缺血病的理论，我们将进行以下研究：(1)证明川芎的有效成分——盐酸川芎嗪可以透过血脑屏障，为其可以直接对脑产生作用提供证据。(2)对盐酸川芎嗪在抗脑缺血再灌注过程中所发挥的作用进行多层次系统研究，探讨其作用机制。希望通过此研究为临床使用川芎或川芎嗪治疗脑缺血病提供理论依据，同时也为今后研究和筛选治疗脑缺血病的中药提供一个实验方法学平台。
研究方法
我们选用SD大鼠为研究对象，制备了阻断大鼠大脑中动脉局灶性脑缺血(MCAO)再灌注模型，研究盐酸川芎嗪对抗脑缺血再灌注的机制。(1)高效液相(HPLC)检测盐酸川芎嗪能否通过大鼠血脑屏障到达脑组织；(2)称量假手术组、缺血再灌注组和各用药组大鼠脑组织重量，检测脑组织含水量；(3)苏木精伊红(HE)染色，观察各组大鼠脑细胞形态的改变；(4)免疫组化染色观察GFAP表达的改变，进而反映星形胶质细胞的变化情况；(5)生化法检测各组大鼠血清NOS、iNOS含量的变化；(6)生化法检测各组大鼠血清和脑匀浆SOD含量的变化；(7)流式细胞术(FCM)检测各组大鼠脑细胞增殖周期、细胞凋亡的改变；(8)Real-Time PCR技术检测各组大鼠脑组织中缺氧诱导因子-1α表达的改变。
结果
(1)鉴于大鼠脑脊液很少，难以达到高效液相测定所需的量，我们选择测定大鼠组织中盐酸川芎嗪的含量。结果各组大鼠脑组织中均检测到盐酸川芎嗪，提示其可以穿透血脑屏障，到达脑从而对中枢产生作用，为研究盐酸川芎嗪乃至川芎对脑的作用机理提供了前提条件。(2)缺血再灌注组脑组织含水量明显高于假手术组(P＜0.01)，而三个用药组与缺血再灌注组比较，脑组织含水量明显减少(P＜0.05)，其中盐酸川芎嗪高剂量组和尼莫地平组变化最为明显，显示盐酸川芎嗪能明显减少梗塞侧脑组织的肿胀程度，减少患侧脑组织含水量。(3)缺血再灌注组大鼠梗死侧脑组织明显肿胀，外观黯淡。镜下观察可见锥体细胞排列紊乱，细胞间隙增宽，神经元变性明显，部分退变的神经元呈现凋亡特征：细胞皱缩成圆形或卵圆形，胞浆皱缩，嗜酸性增加，亦可见均质红染坏死物质和细胞核固缩、溶解等坏死特征。用药组细胞变性程度较缺血再灌注组明显减轻，其中尤以川芎嗪高剂量组和尼莫地平组最为明显，仅有少数锥体细胞胞浆深染，细胞核固缩。(4)缺血再灌注组GFAP免疫染色呈强阳性，胞体肥大，染色加深，突起增粗、变长，而假手术组仅可见极少量阳性细胞表达，用药组GFAP免疫染色则呈阳性——弱阳性改变，说明AST反应性增生减弱，提示盐酸川芎嗪可抑制脑缺血再灌注时AST的过度表达。(5)缺血再灌注组大鼠血清中NOS和iNOS均比假手术组有显著提高，用药组大鼠血清中NOS和iNOS有所下降，其中以川芎嗪高剂量组和尼莫地平组下降最为显著(P＜0.05)，提示川芎嗪可降低脑缺血再灌注时NOS尤其是iNOS的含量，减少NO的合成，减轻神经毒性反应以及由NO所诱导的神经细胞凋亡。(6)缺血再灌注组的血清和脑组织中SOD活力明显降低(P＜0.01)，三个用药组的血清和脑组织中SOD活力均有提高，其中尼莫地平组血清中SOD活力和川芎嗪高剂量组脑组织中SOD活力提高最为明显(P＜0.05)。说明盐酸川芎嗪可提高脑组织SOD活性，有利于清除自由基，减少脂质过氧化损伤。(7)缺血再灌注后细胞周期发生了明显变化，处于Go／G1期的细胞百分率明显减少，进入S期和G2／M期的细胞明显增加，与假手术组比较有显著差异(P＜0.05)，说明了缺血性脑损伤后细胞异常活跃，细胞可以重新进入细胞周期。在细胞凋亡发生率方面，缺血再灌注组大鼠脑细胞为(21.61±4.4)％，较假手术组显著增加(P＜0.05)，盐酸川芎嗪高剂量组和尼莫地平组细胞凋亡发生率较缺血再灌注组显著降低(P＜0.05)，其中以盐酸川芎嗪高剂量组变化最大。(8)经Real-Time PCR测定，发现HIF-1α在缺血再灌注组中的表达远高于假手术组，在川芎嗪低剂量组中的表达略有降低，而在川芎嗪高剂量组和尼莫地平组中的表达有明显降低，其中尼莫地平组基本接近假手术组。
结论与意义
盐酸川芎嗪抗缺血再灌注的机制已经成为近年研究热点之一，取得了有目共睹的成果，但仍存在一些问题：(1)研究多集中于盐酸川芎嗪治疗缺血性心脏病的作用机制，而对于其抗脑缺血再灌注损伤的作用机制研究较少，这和盐酸川芎嗪在脑血管病治疗中的广泛应用不相符合。(2)大多数研究均着眼于盐酸川芎嗪对功能指标的调控，并没有证据证明其可以透过血脑屏障直接作用于中枢。尽管一些学者提出盐酸川芎嗪可以透过正常的血脑屏障，但是对其在脑缺血再灌注、血脑屏障受损的情况下能否透过血脑屏障，以及透过的水平与正常情况有无区别，并没有报道。(3)研究方法比较单一。多数学者仅从某一个角度研究其抗脑缺血再灌注的机制，而脑缺血再灌注的病理生理变化是多方面、多层次的，因此只研究盐酸川芎嗪对其中一个病理环节的影响显然是不全面的。此外由于不同研究人员采用的缺血再灌注模型不尽相同，造成各种实验结果缺乏横向对比的可行性。这些问题造成现有对盐酸川芎嗪抗脑缺血再灌注的机制研究缺乏系统性，整体性。
本实验研究的创新性体现在：(1)证明了盐酸川芎嗪能够穿透血脑屏障，进入脑组织发挥直接作用，而在缺血再灌注后给药脑组织中盐酸川芎嗪含量增多，说明在此状态下药物可以更多的到达脑组织，为研究其进入脑组织对中枢产生作用提供了先决条件。(2)本研究从整体水平、细胞水平、分子水平和基因水平，针对多种病理变化，较完整系统的阐述了盐酸川芎嗪抗脑缺血再灌注损伤的机制。(3)实验采用了多种检测方法，其中Real-Time PCR具有技术水平上的先进性，与传统PCR技术相比，不仅实现了从定性到定量的飞跃，而且具有特异性更强、有效解决PCR污染问题、自动化程度高等特点。(4)实验证明了盐酸川芎嗪在多层次多方面对脑缺血再灌注损伤起到了保护作用，作用机制与降低脑水肿、调整AST对损伤的反应、抗自由基损伤、降低NO毒性、防止血管痉挛、抗细胞凋亡、减少神经细胞变性坏死以及基因调控等多方面关系密切，具有重要的临床和理论价值。(5)本实验为研究中药通过血脑屏障、抗脑缺血再灌注损伤建立了一整套实验方法学平台，对促进中药现代化具有重要意义。
Purpose of research
The inhibitory effect of Traditional Chinese Medicine on cerebralischemia-reperfusion injury is an important topic. With the development of the society, the incidence of the cerbralvascular diseases keeps increasing and become one of themajor diseases threatening the existing and health of the human kind.Ischemia-reperfusion is one of the important pathologic processes in the developmentprocess of Cerbralvascular Disease. It can induce further tissue injury and dysfunction.To control the ischemia-reperfusion injury has become the key factor in the treatmentof ischemic stroke in the present day. At present, the mechanism of the medicine forcerebral ischemia diseases is usually simple and it often has big side effects. On theother hand, Tradition Chinese Medicine has multi-level and multi-link treatmenteffects. So, research on the mechanism of the TCM treatment of cerebral ischemia isvery important in theory and clinic.
The Cerbralvascular Disease is closely associated with Blood stasis syndrome(TCM). The TCM treatment of blood stasis syndrome always adopts the role ofblood-activating and stasis-resolving and use drags that regulated flow of qi incombination to invigorate qi and promote blood circulation. Chuanxiong is ablood-activating and stasis-resolving medicine which is often used in clinic. It has notonly the function of promoting blood, but also the effect of invigorating qi. It is one ofthe common TCM medicines for the treatment of ischemia cerbralvascular disease inclinic. To explain the mechanism of the inhibitory effects of Chuanxiong onischemia-reperfusion is very important for the research on the TCM treatment ofischemic encephalopathy.
Based on the theory of Traditional Chinese Medicine and Modern Medicine about Cerebral ischemia disease, we will do the following research: (1)Prove that theeffective compound of Chuanxiong- Ligustrazine Hydrochloride can go through thenormal and Ischemia-Reperfusion Blood-Brain Barrier. Provide the evidence that ithas direct effect on brain tissues. (2) Do the multi-level research systemically on thefunctions of Ligustrazine Hydrochloride in the process of anti Ischemia-Reperfusion.Hope this research can provide the theory basis for using Chuangxiong orLigustrazine Hydrochloride in the treatment of cerbralvascular disease in clinic, andprovide a methodology platform of screening the TCM for treating cerebral ischemiadiseases.
Research method
We used SD rats as research objects. We prepared the model of Focal CerebralIschemia-reperfusion of Middle Cerebral Artery in rats, and studied the inhibitorymechanism of Ligustrazine Hydrochloride on ischemia-reperfusion injury.
(1) To test if the Ligustrazine Hydrochloride can go through the Blood-BrainBarrier of the normal rats and the rats with cerebral ischemia-reperfusion injury andreach the brain tissue using High-performance Liquid Chromatography (HPLC); (2)Measure the weight of rat brain tissues of the normal group, ischemia-reperfusiongroup and other treatment groups. And the water contents in brain tissue weremeasured. (3) Stain by HE staining, and observe the shape change of the brain cell ofdifferent groups. (4) Observe the expression of GFAP using the method ofimmunohistochemistry stain. And show the change of the astrocyte; (5) Measure thechange of contents of NOS and iNOS in serum of rats in each group usingbiochemical method. (6) Measure the change of contents of SOD in serum and brainhomogenate of rats in each group using biochemical method; (7) Measure the changeof brain cell proliferation cycle and apoptosis of rats in each group using FCM; (8)Measure the expression change of hypoxia-inducible factor 1 alpha in the brain tissueof rats in each group using Real-Time PCR.
Results
Ligustrazine Hydrochloride can be detected in the brain tissues of rats in eachgroup. That indicates it can go through the Blood-Brain Barrier, reach the brain and has effects on central nervous system. This result provides the premise conditions ofthe research of the effects and mechanisms of Ligustrazine Hydrochloride or evenChuanxiong on brain. (2) Water content of ischemia-reperfusion group is obviouslyhigher than sham-operation group (P＜0.01), and the water content of three medicinegroups is apparently reduced comparing with ischemia-reperfusion group (P＜0.05).The change is more apparent in the Ligustrazine Hydrochloride high-dose group andnimodipine group. It indicates that Ligustrazine Hydrochloride can apparently reducethe swelling dimension of contralateral hemisphere, and reduce the water content inthe lesion hemisphere. (3) The brain tissue of rats in ischemia-reperfusion groupswelled apparently and the appearance is dim. It can be seen under microscope thatthe pyramid cells are arranged disorderly, the cell space got widening and neurondegeneration is apparent. Some degenerated neuron appear apoptosis: The cells shrinkto rounded or ovoid shape. The cytoplastic shrinks and eosinophilic character increase.Homogeneous red-stained necrotic material, nucleus condensation and dissolution, and other necrotic features also can be seen. The degree of degeneration wassignificantly reduced in medicine group than in ischemia-reperfusion group. And it ismore apparent in the Ligustrazine Hydrochloride high-dose group and nimodipinegroup. Only a little of pyramid cells were deeply stained and had nucleuscondensation. (4) ischemia-reperfusion group' s GFAP immunostaining was strongpositive. Cell body is mast, stain is deepening and the neurite is thickening and gettinglonger. But sham-operation group only had very few positive cell expressions. Theimmunostaining of medicine only had positive-feebly positive change. Thisdemonstrates that AST reactive hyperplasia is decreased and suggest that LigustrazineHydrochloride can inhibit the over-expression of AST with ischemia-reperfusion. Thecontents of NOS and iNOS in serum of rats in ischemia-reperfusion group issignificantly increased than sham-operated group. The contents of NOS and iNOS inserum of rats in medicine group decreased. And it is more apparent in the LigustrazineHydrochloride high-dose group and nimodipine group (P＜0.05). It suggested thatLigustrazine can decrease the content of NOS, especially iNOS, whenischemia-reperfusion occurs. Reduce the synthesis of NO. Reduce the nerve toxic reactions and the NO-induced neuronal apoptosis. (6) Activity of SOD in the serumand brain tissue of the ischemia-reperfusion group significantly decreased (P＜0.01), Activity of SOD in the serum and brain tissue of the three medicine group allincreased. And it is more apparent in the Ligustrazine Hydrochloride high-dose groupand nimodipine group (P＜0.05). It suggests that igustrazine can increase the activityof SOD in brain tissue, and help to eliminate free radicals, and reduce lipidperoxidation injury. (7) After ischemia-reperfusion, noticeable changes had takenplace in the cell cycle. The percentage of cells in G0/G1 phase decreased apparently.The cells entered S and G2/M phase increased a lot. It had a significantly differencecomparing with sham-operated group (P＜0.05). It shown that, afterischemia-reperfusion injury, cells were very active, and can reenter the cell cycle. Inthe rate of cell apoptsis, the brain cell of rats in the ischemia-reperfusion group is(21.61±4.4)%, a significantly increase comparing with sham-operated group(P＜0.05). The rate of cell apoptsis in the Ligustrazine Hydrochloride high-dose groupand nimodipine group is significantly lower than ischemia-reperfusion group (P＜0.05).The Ligustrazine Hydrochloride high-dose group had the biggest change. (8) Theexpression of HIF-1αin the ischemia-reperfusion group was much higher thansham-operated group measured by Real-Time PCR. It is lower in the LigustrazineHydrochloride low-dose group. But it significantly decreased in the LigustrazineHydrochloride high-dose group and nimodipine group. The nimodipine group isalmost same as sham-operated group.
Conclusions and significance
The mechanism of the inhibitory effect of Ligustrazine Hydrochloride onischemia-reperfusion is a hot topic in recent years and has got significant results. Butit still has some problems: (1) the researches are mostly focused on the mechanism ofusing Ligustrazine Hydrochloride to treat the ischemic heart disease. But the researchon the mechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion injury is much less. This is inconsistent with the widely using ofLigustrazine Hydrochloride in the treatment of Cerebrovascular Disease. (2) Most ofthe researches are focused the regulation of Ligustrazine Hydrochloride on the functional indices, and didn' t prove it can pass through the BBB and has directeffects on central nervous system. Although some scholars prove that LigustrazineHydrochloride can go through the BBB, there isn' t report about whether it still cango through the BBB, with ischemia-reperfusion and damaged BBB. Also there isn' treport about the whether there is any difference in the going through level comparingwith the normal cases. (3) The research method is relatively simple. Most scholarsonly research on one aspect of the mechanism of the inhibitory effect of LigustrazineHydrochloride on ischemia-reperfusion. But the pathophysiological changes ofcerebral ischemia-reperfusion are in many ways and at different levels. So, it isobviously not comprehensive to study the effect of Ligustrazine Hydrochloride ononly one pathology link. In addition, because different researchers use differentischemia-reperfusion model, the various experimental results don' t have thehorizontal comparison feasibility. These problems induced that the research onmechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion is not systemic and holistic.
The innovation of this experiment proves is (1) Proveing LigustrazineHydrochloride can go through the normal and Ischemia-Reperfusion Blood-BrainBarrier, reach the brain tissue and has direct effect on brain tissues. And theincensement of the content of Ligustrazine Hydrochloride in the ischemia-reperfusionbrain tissue after medical treatment, indices that in this case, more medicine can reachthe brain tissue. This provides the preconditions for the research on how it reaches thebrain tissue and has direct effect on brain tissues. (2) This study descripts themechanism for the treatment of cerebral ischemia-reperfusion injury usingLigustrazine Hydrochloride at the overall level, and the level of cells, molecules andgenes according to a variety of pathological changes. (3)This experiment used a lot ofmeasure methods, among which the Real-time PCR is at the advanced technologicallevel. Comparing with traditional PCR technology, it realized not only the leap from aqualitative level to a quantitative level, but also has the characters of more specificity, effectively solving the pollution problems of PCR, and a higher degree ofautomation. (4)Experiment proved that Ligustrazine Hydrochloride has protection function on cerebral ischemia-reperfusion injury. The mechanism of effect is closelyrelated to decreasing brain edema, adjusting the reaction of AST to injury, anti-freeradical damage, reducing the toxicity of NO, preventing vasospasm, anti-apoptotic, reducing nerve cell degeneration and necrosis, Gene regulation and other aspects.(5)This experiment built a whole set of experimental methodology platform forfurther research on going through the BBB of TCM and anti Ischemia-Reperfusion, and also has great significance to promoting the modernization of traditional Chinesemedicine.
引文
[1] 周鸿雁，毛海峰，王一蓉，等．脑缺血损伤的研究进展[J]．湖南文理学院学报，2005，17(2)：72-75
[2] 张国瑾，赵增荣．国外脑血管疾病研究进展[M]．北京：中国医药科技出版社，2000：177-197
[3] Xu Z, Xu RX, Liu BS, et al. Time window characteristics of cultured rat hippocampal rat hippocampal neurons subjected to ischemia and reperfusion[J]. Chin J Traumatol, 2005,8(3):179-182
[4] 高维娟．大鼠脑缺血再灌注后海马组织兴奋性氨基酸含量的动态观察[J]．承德医学院学报，2003，20(3)：185-187
[5] Dohmen C, Kumura E, Rosner G, et al. Extracellular correlates of glutamate toxicity in short-term cerebral ischemia and reperfusion:a direct in vivo comparison between white and gray matter[J]. Brain Res, 2005, 1037(1-2): 43-51
[6] 蔡竖平，桂秋萍．脑缺血再灌注后海马迟发性神经元死亡的实验研究[J]．军医进修学院学报，2002，23(4)：268-270
[7] Oimagl U, adeeola C, Moskonitz MA. Pathobiology of ischaemic stroke:integrated view[J]. Trends Nuerosci, 1999,22(9):391-397
[8] OSUGA H, HAIGTI AM. Relevance of interstital Glutamate to selectice vulnerability in focal Cerebral ischemi a [J].J cereb Blood FlowMetab, 1994,14(2):343
[9] Makrs AR. Intracellular calcium-release channels: regulators of cell life and death[J]. Am J Physiol, 1997,272:597
[10] Zhang C, Shen W, Zhang G. N-methyl-D-aspartate receptor and L-type voltage-gated Ca2+ channel antagonists suppress the release of cytochrome c and the expression of procaspase-3 in rat hippocampus after global brain ischemia[J].Neurosci Lett, 2002,328(3):265-268
[11] Langham J, Goldfrad C, easdale G, et al. Calcium channel blockers for acute traumatic brain injury[J].Cochrane Database Syst Rev 2003, (4):CD000565
[12] Biala G. Calcium channel antagonists suppers nicotine-induced place preference and locomotor sensitization in rodents[J]. Pol J Pharmacol, 2003,53(3):327-335
[13] 杨惠玲主编．高级病理生理学[M]．北京：科学出版社，1998，24-38
[14] 陈敏，李长青．钙与脑缺血再灌注后神经细胞凋亡[J]．脑与神经疾病杂志，2003，11(4)：253-255
[15] 马玉羡．脑缺血再灌注损伤病理生理研究机制进展[J]．河南医学研究．1998，7(4)：375-378
[16] Carolina M, Salvatore C, Dennis PR, et al. On the selectivity of surperoxide dismutase mimetics and its importance in pharmacological studies[J]. Br J Pharmacol, 2003,140(3): 445-460
[17] LEWEN A, MQTZ P, CHAN PH. Free radial Pathways in CNS injury[J].Neurotra uma, 2000,17(10):871-890
[18] Aito H, Aalto KT, Raivio KO. Biphasic ATP depletion caused by transient oxidative exposure is associated with apoptotic cell death in rat embryonal cortical neurons[J]. Pediatr Res, 2002,52(1):40-45
[19] 吴伟，史继新．脑缺血再灌注后神经元损伤机制及治疗研究进展[J]．中国微循环，2003，7(6)：391-394
[20] 刘熠敏，徐仁四．兴奋性氨基酸、氧自由基与脑缺血再灌注损伤关系的实验研究[J]．卒中与神经疾病，2000，7(2)：98-100
[21] Selmen WR, Cramrine RC, Ricci AJ et al. Impairment of metabolic recovery with increasing periods of middle artery occlusion in rats[J]. Stroke, 1990,21(3):467-471
[22] Cao G, Minzmi M, Pei W, et al. Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death[J]. Cere B Blood Flow Metab, 2001,21(4):321-333
[23] Shigeno T, Yamask i Y,Kato G, et al. Reduction ofdelayed neuronal death by inhibition protein synthesis[J].NeurosciLett, 1990,120:1171-l184
[24] Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats[J].Stroke, 1993,24:2002
[25] Lepp in C, Finiels Marlier F. Failure of a protein synthesis inhibitor to modify glutamate receptor mediated neuro toxicity in vivo[J].Brain Res, 1992,581:168
[26] Kure S, Tom inaga T, Yoshimo to T, et al. Glutamate triggers internucleomal DNA cleacage in neuronal cell, Biochem, Biophys Res[J].Commun, 1991,179: 39.
[27] Tominaga T, Kure S, Narsiawa K, et al. Endonuclease action following focal ischemic injury in the ratbrain[J].Brain Res,1993,608:21-34
[28] Heron A, Pollard H, Dessi F, et al. Regional variabiligy in DNA fragmentation after global ischemia evidence by combined h isto logical and gelelectropho rerss observations in the rat brain[J]. Neurochem, 1993, 61:1973-1976
[29] ManusMCP J, Buchan AM, Hill IE, et al. Global ischemia can cause DNA fragmentation actative of apoptosis in rat brain[J]. NeurosciLett, 1993,164: 89
[30] Wyllie AH. Apoptosis[M]. BR J Cancer, 1993,67: 205-214
[31] Nitaton T, Sato IV, W aguri S, Karasawa T, et al. Delayed neuronal death in the CA lpyramidal cell layer of he gerbil hippocampus following transient ischemia is apotosis [J]. N eurosci,1995,13:1001-1009
[32] Li Y, Sharov G, Jiang N, et al. Ultratructral and light microscop is evidence of apoptosis after middle cerebral artery occlusion in the rat[J]. Am J Physio 1, 1995, 146:1045-1052.
[33] Gavrieli Y, Sherman Y, Ben2Sasson SA, Identification of programmed cell death in situvia specific labeling of nuclear DNA fragmentation[J]. Cell Bio 1,1992:119
[34] Esmond J, Sander, Michael A, et al. Ultractural identification of apop to ticnuclear using the TUNEL technique[J]. Histochem, 1996, 28: 275
[35] Ding HL, Zhu HF, Dong JW, et al. Inducible nitric oxide synthase contributes to intermittent hypoxia against ischemia reperfusion injury[J]. Acta Pharmacol Sin, 2005, 26(3):315-322
[36] Niwa M, Inao S, Takayasu M., et al . Time couxse of expretion of the three nitric desynthase isoforms after trailsient middle cerebral artery occlusion in rats[J]. Neurol Med Chir(Tokyo), 2001, 41(2):63-72
[37] Gourmala NG, Buttinii M, Limonta S, et al . DifemnfiM and independent expression of monocyte chemoattractant protein mRNA by astrocytes and macrophages in rat brain and peripheral hpo vdysaecharide admistration[J]. Neuroimmunol, 1997, 74: 35-40
[38] Sairanen TR, lindsberg PJ, Brenner M, et al. Globalforebrainischemia results in diferenfial celular expression of interleukin and its receptor at mRNA and proteinlevel[J]. Cerob Blood FlowMetab, 1997, 17: 1107-1110
[39] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury[J]. Trends Neurosci, 1997, 20: 132-139
[40] 李惠．缺血性脑血管疾病[M]．北京：北京科学技术出版社，2002：47-57
[41] Liu XF. Current status of neuroprotection in cerebrovascular disease[J]. Journal of Medical Postgraduates, 2003, 16(6): 52-455
[1] 张伯臾，董建华，周仲英主编．中医内科学[M]．上海科学技术出版社，1996：208
[2] 王键，刘听．益气活血法治疗缺血性中风的实验研究进展[J]．中医药学刊，2002，20(2)：141
[3] 王永炎．关于提高脑血管疾病疗效难点的思考[J]．中国中西医结合杂志，1997，17(4)：196
[4] 林兴栋．“热毒”在中风发病中的作用与证治探讨[J]．中国中医药信息杂志．2002：9(1)：43
[1] 刘金烘，陈华德．血脑屏障与中医药研究[J]．浙江中医学院学报，1999，23(2)：591
[2] 张伟，林凯．HPLC测定舒胸胶囊中盐酸川芎嗪的含量[J]．中成药，2006，28(6)：920-922
[3] Zea Longa EL, Weinstein PR, Canson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke, 1989, 20(1): 84-91
[1] 汪枫．脑损伤时间推断的分子生物学研究进展[J]．皖南医学院学报，2003，22(3)：232-234
[2] Gornes FG, Paulin D, Moura Neto V. Giial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation[J]. Braz J Med Biol Res 1999;32(6): 619-31
[3] 蒲传强主编．脑血管病学[M]．北京：人民军医出版社，1999，95-97
[4] Dalby NO, Rondouin G, Leaner-Natoli M. Increase in GAP-43 and GFAP immunoreactivity in the rat hippocampus subsequent to perforant path kindling[J].Neurosci Res, 1995,41:613-619
[5] ASTBury SD, Eichhorn AC, Kotzer CM, et al. Reactive astrocytic responses to denervation in the motor cortex of adult rats are sensitive to manipulations of behavioral experience[J].Neuropharmacoloev, 2000,39(5):743-755
[6] Kim Y, Truettner J, Zhao W, et al.The influence of delayed postischemic hyperthermia following transient focal ischemia:alterations of gene. expression[J]. Neurolscience,1998,159(1):1-10
[7] Schmidt KR, Aguirre CC, Saul I, et al. Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats[J].Brain Res, 2005 , 1052 (1):28-39
[8] Bury SD, Eichhom AC, Kotzer CM, et al .Reactive astrocytic rsponses to denervation in the motor codex of adult rats are sensitive to manipulations of behavioral experience[J]. Neuropharmacology,2000,39(5): 743-755
[9] 刘之荣，段德新．星形胶质细胞在缺血性脑损伤中的作用[J]．国外医学·神经病学与神经外科学分册，2000，8(3)：148-150
[10] Hohnin S, Schalling M, Hojeberg B, et al. Delayed cytodine expression in rat brain following experimental contusion [J]. Neurosurg, 1997, 86(3):493-497.
[11] 王晓梅，冯加纯，饶明俐．反应性星形胶质细胞对脑缺血神经元的保护[J]．中风与神经疾病杂志，2001，18(1)：63-64．
[1] 王晓娟，余华峰．一氧化氮与脑缺血再灌注损伤[J]．国外医学(老年医学分册)，2000，21(3)：114-117
[2] Zhou G, Zha B, Hou J, et al. Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction[J]. Biotechnol Tech, 1999,13:507-11
[3] ladecola C. Bright and dark side off nitric oxide in ischemic brain injury[J]. TINS 1997,20:1132-9
[4] Mayer B, Hemmens B. Biosynthesis and action off nitric oxide in mammalian cell[J]. Trends Biochem Sci 1997,22:447-81
[5] 王志浩，张岫美．一氧化氮与缺血性脑血管疾病[J]国际病理科学与临床杂志，2005，25(5)：466-470
[6] Mason RB, Pluta RM,Walbridge S, et al. Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide[J]. J Neurosurg, 2000, 93(1):99-107
[1] Matsuda T, Arakawa N, Takuma K, et al. SEA0400, a novel and selective inhibitor of the Na~+-Ca ~(2+)exchanger, attenuates reperthsion injury in the in vitro and in viva cerebral ischemic mod-els[J]. J Phannacol Exp Ther, 2001,298(1):112-116
[2] Xiong Z G, Zhu X M, Chu X P, et al. Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels[J]. Cell,2004,118(6):687-98
[3] 贾健民，贾健平，李泓，等．脑缺血与自由基[J]．国外医学神经病学神经外科学分册，1995，22(8)：207-209
[4] Qu L, Wang W T, Guo L J. Protective effects of 1-(2,6-dimethyl phenoxy)-2-(3,4-dimethoxy-phenylethylamino) propane hydro chloride(DDPH) on brain ischemia injury in rats[J]. Acta Pharmaceutica Sinica,2003,(10):725
[5] 司徒镇强，吴军正．《细胞培养》[M]．世界图书出版公司，1996，186-187
[1] Love S. Neuronal experssion of cell cycle-related proteins after brain ischaemia in man[J].Neurosci Lett, 2003,363(1):29-32
[2] Osuga B, Osuga S, Wang F, et al. Cyclin-dependent kinases as a therapeutic target fox stroke[J]. Proc Natl Acad Sci USA, 2000,97(18):10254-10259
[3] Charriaut-Marlangue C, Aggoun-Zouzoui D, Represa A, et al. Apoptosis features of selective neuronal death in ischemia, epilepsy and gp 120 toxicity [J]. Trends Neurosci, 1996,19:109-114
[4] 赵士福，蔡文琴，邓晓林，等．大鼠脑缺血过程中神经细胞死亡的研究仁[J]．解剖学报，1997，28(4)：342-345
[5] NITATORI T, SATO N, WAGURI S, et al. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis[J]. J Neurosci, 1995,15:1001-1011
[6] MATSUDA T, ARAKAWA N, TAKUMAK, et al. SEA0400, a novel and selective inhibitor of the Na~+-Ca~(2+) exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models[J]. J Pharmacol Exp Ther,2001,298(1):249-256
[7] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis[J]. Semin Hematol, 2001, 38(2):179-193
[8] Becket EB, Bonni A. Beyond proliferationTcell cycle control of neuronal survival and differentiation in the developing mammalian brain[J].Se min Cell Dev Biol, 2005, 16 (3): 439-448
[9] Zhu X, MeShea A, Harris PL, et al. Elevated expression of a regulator of the G2/M phase of the cell cycle, neuronal CIP-1-as-sociated regulator of cyclin B, in Alzhemefs disease[J]. J Neurosci Res, 2004, 76(6):698-703
[10] Volpe BT, Wessel TC, Mukherjee B, et al. Temporal pattern of internuclosomal DNA fragmentation in the Striatumand hippocampus after transient forebrain ischemia[J]. Neurosci Lett, 1995, 156-157
[11] HakimAM. Ischemic penumbra: the therapeutic window[J]. Neurology, 1998, 51 (3):544-546
[12] 左连富主编．流式细胞术与生物医学[M]．沈阳：辽宁科学技术出版社，1996，68-73
[13] Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats [J]. Stroke, 1993, 24:2002-2009
[1] ZhongH, De Marzo AM, laughner E, et al. Over-expression of hypoxia-induciable factor-lain common human cancers and their metastases[J]. Cancer Res, 1999,59:5830-5835
[2] Palmer LA et al. Am J Physiol[M], 1998, 274-212
[3] Senenza GL. HIF-1a, and the 3 PHDs how animal cells signal hypoxia to the nucleus [J]. Cell, 2001,107:123
[4] Wenger RH et al. Eur J Biochem[M], 1998,253-771
[5]Nagle DG,Zhou YD. Natural product-based inhibitors of hypoxiainducible factor-1(HIF-1)[J]. Curr Drug Targets, 2006, 7(3):355-369
[6] Zelzer E et al. EMBO J[M], 1998,17:5085
[7] Lefer DJ. Induction of HIF-1 alpha and iNOS with siRNA: a novel mechanism for myocardial protection[J]. Circ Res, 2006, 98(1): 10-11
[8] Semenza GL et al. Kidney Int[M], 1997, 51-513
[9] Lee PJ et al. J Biol Chem[M]. 1997, 272:5375
[10] Kallio PJ, Wilson WJ, O'Brien SMakino Y. PoellhgerL Regulation of the hypoxia inducible transcription factor 1 alpha by the ubiquitin proteasane pathway[J]. JBiolChem, 1999, 274:6519-6525
[11] Hu J et al. Biochem Biophys Res Commun[M], 1998,245:894
[12] Cormier-regard S et al. J Biol Chem[M], 1998, 273:17787
[13] Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O_2 tension[J]. Am J Physiol, 1996, 271:C1172-1180
[14] Lok-CN et al. J Biol Chem[M], 1999,274(34):24147
[15] Millhorn DE et al. Kidney Int[M], 1997, 51:527
[16] Ren XQ, Meng JM, Fu KD. Study on relationship between expression of p53 HIF-1 α and angiogengesis of colorectal carcinoma[J]. Zhongguo Yixue Gongcheng, 2004, 12(4):31-35:
[20] Senenza GL. Expression of hypoxia inducible factor-1 α echanims and coinsequences[J]. Biochem Phamacology, 2000, 59:47-53
[17] Feldser-D et al. Cancer Res[M], 1999, 59: 3915
[18] Carmeliet P et al. Nature[M], 1998, 394: 485
[19] Tazuke SI et al. Proc Natl Acad Sci USA[M], 1998, 95: 10188
[21] 宋虎平，惠延年，王丽丽，韩晓霞，王海燕．缺氧诱导因子-1α在早期糖尿病大鼠角膜上皮细胞中的表达[J]．国际眼科杂志，2000，6(2)：352-356
[22] Wiener CM et al. Biochem Biophys Res Commun[M], 1996, 225-485
[1] Min HJ, Tomohiro Kaku, Junichi Hada, el al. Different effects of eNOS and nNOS inhibition on transient forebrain ischemia[J]. Brain Res. 2002, 946:139-142
[2] Vaughan CJ, Delanty N, Basson CT. Statin therapy and stroke prevention[J]. CurrOpin Cardiol,2001,16(4):219-228
[3] Oconnell AW, Fox GB, Kjoiler C. el al . Antiischemic and cognition-enhancing properties of NNC-711. agamma-aminobutyricacid reuptakeinhibitor[J]. Eur J Pharmacol, 2001,424(1):37-43
[4] Eguchik, Kariok, Shimada K Beneficial effect of ARB for ischemic stroke[J]. Nippon Rinsho, 2002, 60(10): 1987-1998
[5] Qureshi A1, Ali Z, Suri MF, el al. Intra. arterial third generation recombinant tissue plasminogen activator(reteplase)for acute ischemic stroke[J]. Neurosurgery, 2001,49(1): 41-45
[6] Zeller JA, Tschoepe D, Kessler C Circulating platelet show increases activation in patients with acute cerebral ischemia[J]. Thromb Haemost, 1999,81(3):373-380
[7] Nakamura T, Uchiyama S, Yamazaki M, el al. Effects ofdipyridamole an daspirin on shear induced platelet aggregation in whole blood and platelet rich plasma[J]. Cerebrovasc Dis,2002, 14(3～4): 234-250
[8] Abciximab in acute ischemic stroke:a randomized, double-blind, placetm-controlled, dose escalation study The Abciximab in lschemic Stroke lnve-stigators[J]. Stroke,2000,31(3):601-609
[9] Bath PW, lddenden R, Bath FJ Low Molecular. Weight heparins and hepadnoids in acute ischemic stroke: A MetaAnalysis ofrandomizeel controlled trials[J], stroke,2000,31 (7): 1770-1774
[10] Eun BL, Liu xH, Barks JD. Pentoxifylline attenuates hypoxic ischemic brain injury in immature rats[J]. Pediatr Res, 2000, 47(1): 73-79
[11] Asai S, Zhao H, Yan A.,el al. Nicergoline enhances glutamate re-uptake and protects against brain damage in rat global brain ischemia[J]. Eur J Pharraacol, 1999, 383(3): 267-275
[12] 刘继生，刘树鹏．刺五加注射液治疗缺血性脑卒中78例临床及血液流变学观察[J]．中西医结合实践杂志，1997，10(4)：322
[13] 寿延红，陈颖，冯加继，等．刺五加对蛛网膜下腔出血患者血浆内皮素含量的影响[J]．中风与神经疾病杂志，2001，18(3)：139
[14] 赖伟坚，王艳华．大剂量血栓通注射液治疗急性脑梗死72例疗效分析[J]．广西医学，2004，26(1)：88-89
[15] 王根发，王文安，周永炜，等．三七皂苷对大鼠脑缺血再灌注损伤的保护作用[J]．中国临床康复，2002，6(9)：1268
[16] 朱卫，张晓彪．银杏叶制剂对脑血管治疗的概况[J]．国外医学脑血管疾病分册，1998，6(4)：236
[17] Zhuang H, Pin S Christen Y, et al. Induction of heine oxygenasel by Ginkgo biioba in neuronal cultures and potential implications in ischemia[J]. CellMolBiol, 2002, 48(6):647-653
[18] 孙莉莎，徐江平，杨雪梅．银杏内酯对大鼠脑缺血再灌注损伤的保护作用[J]．中华现代中西医杂志，2003，1(8)：676
[19] 王东吉，尚改萍，连为民，等．川芎嗪对兔脑缺血再灌注损伤的保护作用[J]．长治医学院学报，2003，17(4)：245
[20] 马仁生，杜爱芹灯．盏花注射液治疗急性脑梗死48例[J]．中国中医药信息杂志，2004，11(12)：1092
[21] 韦登明，林兴明，张朝辉，等．绞股兰总皂苷对大鼠局灶性脑缺血再灌注酯质过氧化脑损伤影响的实验研究[J]．中华实用医学杂志，2003，2(6)：484
[22] 徐立新，何绘敏，曲石霞，等．新生大鼠缺氧缺血性脑病脑组织酯质过氧化改变及丹参保护作用探讨[J]．新生儿科杂志，2000，15(1)：63-65
[23] 董少龙．水蛭注射液对高血压性脑出血颅内血肿吸收的影响[J]．广西中医药，1998，21(3)：4-6
[24] 黄修玲，郑丽．清开灵治疗中风后遗症[J]．光明中医，2003，18(4)：107
[25] 程梦琳，吴基良，罗德生．参附注射液对大鼠脑缺血再灌注大鼠脑组织NO含量和NOS活性的影响[J]．中国中医急诊，2004，13(4)：234
[26] 江莲，戎小平．参麦、乌司他丁对大鼠脑缺血再灌注损伤保护作用的研究[J]．河北医科大学学报，2002，23(3)：144
[27] 刘艳，罗祖明，冀玲，等．华佗再造丸对大鼠局灶性脑缺血损伤的保护作用[J]．华西药学杂志，2004，19(2)：103
[28] 杨赶梅，夏娣文，余江健，等．芪蛭胶囊对中风高粘血症患者血液流变学变化的影响[J]．中国中医药科技，2002，9(4)：231
[1] 高长越，周华东，邓娟，等．川芎嗪对脑缺血再灌注损伤后细胞间粘附作用的影响[J]．中国中西医结合急救杂志，2002，9(3)：135
[2] 钟瑜，张焱，杨国汗．川芎嗪对脑缺血丙灌注损伤后细胞粘附作用的影响[J]．深圳中西医结合杂志，2003，13(3)：141
[3] 张兆辉，卫涛涛，余绍祖，等．川芎嗪的抗氧化机制对缺血性脑损伤的保护作用[J]．中国临床康复，2004，8(34)：7742
[4] 王万铁，陈寿权，徐正妏，等．川芎嗪注射液抗脑缺血再灌流损伤作用机制的实验研究[J]．中华急诊医学杂志，2001，10(3)：182
[5] 汪远金，申国明，王钦茂．川芎嗪对大鼠脑缺血再灌注损伤的保护作用[J]．安徽中医学院学报，2000，19(1)：44
[6] 游言文，田新红．川芎嗪对大鼠脑缺血再灌注后神经元突触超微结构的影响[J]．河南中医学院学报，2004，19(114)：19
[7] 李效敏，孙慧，赵勇，等．川芎嗪对脑梗死患者内皮细胞功能影响的研究[J]．中国急救医学，2004，24(10)：740
[8] 钟平，杨任民．中药川芎嗪治疗急性脑缺血机理的实验研究[J]．实用全科医学，2003，1(1)：19
[9] 曲友直，高国栋，赵振伟，等．川芎嗪、黄芪对脑缺血再灌注后神经细胞凋亡及Fos蛋自表达的影响[J]．卒中与神经疾病，2004，11(2)：111
[10] 俞海国，汤云珍，杨锡强．川芎嗪对新生鼠缺血缺氧脑损伤FOS蛋白表达的影响[J]．重庆医学，2003，32(4)：416
[11] 吕少平，孙锋，王春霞．川芎嗪对脑缺血再灌注时细胞凋亡的影响[J]．河北医学，2000，6(8)：673
[2] 张国瑾，赵增荣．国外脑血管疾病研究进展[M]．北京：中国医药科技出版社，2000：177-197
[3] Xu Z, Xu RX, Liu BS, et al. Time window characteristics of cultured rat hippocampal rat hippocampal neurons subjected to ischemia and reperfusion[J]. Chin J Traumatol, 2005,8(3):179-182
[4] 高维娟．大鼠脑缺血再灌注后海马组织兴奋性氨基酸含量的动态观察[J]．承德医学院学报，2003，20(3)：185-187
[5] Dohmen C, Kumura E, Rosner G, et al. Extracellular correlates of glutamate toxicity in short-term cerebral ischemia and reperfusion:a direct in vivo comparison between white and gray matter[J]. Brain Res, 2005, 1037(1-2): 43-51
[6] 蔡竖平，桂秋萍．脑缺血再灌注后海马迟发性神经元死亡的实验研究[J]．军医进修学院学报，2002，23(4)：268-270
[7] Oimagl U, adeeola C, Moskonitz MA. Pathobiology of ischaemic stroke:integrated view[J]. Trends Nuerosci, 1999,22(9):391-397
[8] OSUGA H, HAIGTI AM. Relevance of interstital Glutamate to selectice vulnerability in focal Cerebral ischemi a [J].J cereb Blood FlowMetab, 1994,14(2):343
[9] Makrs AR. Intracellular calcium-release channels: regulators of cell life and death[J]. Am J Physiol, 1997,272:597
[10] Zhang C, Shen W, Zhang G. N-methyl-D-aspartate receptor and L-type voltage-gated Ca2+ channel antagonists suppress the release of cytochrome c and the expression of procaspase-3 in rat hippocampus after global brain ischemia[J].Neurosci Lett, 2002,328(3):265-268
[11] Langham J, Goldfrad C, easdale G, et al. Calcium channel blockers for acute traumatic brain injury[J].Cochrane Database Syst Rev 2003, (4):CD000565
[12] Biala G. Calcium channel antagonists suppers nicotine-induced place preference and locomotor sensitization in rodents[J]. Pol J Pharmacol, 2003,53(3):327-335
[13] 杨惠玲主编．高级病理生理学[M]．北京：科学出版社，1998，24-38
[14] 陈敏，李长青．钙与脑缺血再灌注后神经细胞凋亡[J]．脑与神经疾病杂志，2003，11(4)：253-255
[15] 马玉羡．脑缺血再灌注损伤病理生理研究机制进展[J]．河南医学研究．1998，7(4)：375-378
[16] Carolina M, Salvatore C, Dennis PR, et al. On the selectivity of surperoxide dismutase mimetics and its importance in pharmacological studies[J]. Br J Pharmacol, 2003,140(3): 445-460
[17] LEWEN A, MQTZ P, CHAN PH. Free radial Pathways in CNS injury[J].Neurotra uma, 2000,17(10):871-890
[18] Aito H, Aalto KT, Raivio KO. Biphasic ATP depletion caused by transient oxidative exposure is associated with apoptotic cell death in rat embryonal cortical neurons[J]. Pediatr Res, 2002,52(1):40-45
[19] 吴伟，史继新．脑缺血再灌注后神经元损伤机制及治疗研究进展[J]．中国微循环，2003，7(6)：391-394
[20] 刘熠敏，徐仁四．兴奋性氨基酸、氧自由基与脑缺血再灌注损伤关系的实验研究[J]．卒中与神经疾病，2000，7(2)：98-100
[21] Selmen WR, Cramrine RC, Ricci AJ et al. Impairment of metabolic recovery with increasing periods of middle artery occlusion in rats[J]. Stroke, 1990,21(3):467-471
[22] Cao G, Minzmi M, Pei W, et al. Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death[J]. Cere B Blood Flow Metab, 2001,21(4):321-333
[23] Shigeno T, Yamask i Y,Kato G, et al. Reduction ofdelayed neuronal death by inhibition protein synthesis[J].NeurosciLett, 1990,120:1171-l184
[24] Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats[J].Stroke, 1993,24:2002
[25] Lepp in C, Finiels Marlier F. Failure of a protein synthesis inhibitor to modify glutamate receptor mediated neuro toxicity in vivo[J].Brain Res, 1992,581:168
[26] Kure S, Tom inaga T, Yoshimo to T, et al. Glutamate triggers internucleomal DNA cleacage in neuronal cell, Biochem, Biophys Res[J].Commun, 1991,179: 39.
[27] Tominaga T, Kure S, Narsiawa K, et al. Endonuclease action following focal ischemic injury in the ratbrain[J].Brain Res,1993,608:21-34
[28] Heron A, Pollard H, Dessi F, et al. Regional variabiligy in DNA fragmentation after global ischemia evidence by combined h isto logical and gelelectropho rerss observations in the rat brain[J]. Neurochem, 1993, 61:1973-1976
[29] ManusMCP J, Buchan AM, Hill IE, et al. Global ischemia can cause DNA fragmentation actative of apoptosis in rat brain[J]. NeurosciLett, 1993,164: 89
[30] Wyllie AH. Apoptosis[M]. BR J Cancer, 1993,67: 205-214
[31] Nitaton T, Sato IV, W aguri S, Karasawa T, et al. Delayed neuronal death in the CA lpyramidal cell layer of he gerbil hippocampus following transient ischemia is apotosis [J]. N eurosci,1995,13:1001-1009
[32] Li Y, Sharov G, Jiang N, et al. Ultratructral and light microscop is evidence of apoptosis after middle cerebral artery occlusion in the rat[J]. Am J Physio 1, 1995, 146:1045-1052.
[33] Gavrieli Y, Sherman Y, Ben2Sasson SA, Identification of programmed cell death in situvia specific labeling of nuclear DNA fragmentation[J]. Cell Bio 1,1992:119
[34] Esmond J, Sander, Michael A, et al. Ultractural identification of apop to ticnuclear using the TUNEL technique[J]. Histochem, 1996, 28: 275
[35] Ding HL, Zhu HF, Dong JW, et al. Inducible nitric oxide synthase contributes to intermittent hypoxia against ischemia reperfusion injury[J]. Acta Pharmacol Sin, 2005, 26(3):315-322
[36] Niwa M, Inao S, Takayasu M., et al . Time couxse of expretion of the three nitric desynthase isoforms after trailsient middle cerebral artery occlusion in rats[J]. Neurol Med Chir(Tokyo), 2001, 41(2):63-72
[37] Gourmala NG, Buttinii M, Limonta S, et al . DifemnfiM and independent expression of monocyte chemoattractant protein mRNA by astrocytes and macrophages in rat brain and peripheral hpo vdysaecharide admistration[J]. Neuroimmunol, 1997, 74: 35-40
[38] Sairanen TR, lindsberg PJ, Brenner M, et al. Globalforebrainischemia results in diferenfial celular expression of interleukin and its receptor at mRNA and proteinlevel[J]. Cerob Blood FlowMetab, 1997, 17: 1107-1110
[39] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury[J]. Trends Neurosci, 1997, 20: 132-139
[40] 李惠．缺血性脑血管疾病[M]．北京：北京科学技术出版社，2002：47-57
[41] Liu XF. Current status of neuroprotection in cerebrovascular disease[J]. Journal of Medical Postgraduates, 2003, 16(6): 52-455
[1] 张伯臾，董建华，周仲英主编．中医内科学[M]．上海科学技术出版社，1996：208
[2] 王键，刘听．益气活血法治疗缺血性中风的实验研究进展[J]．中医药学刊，2002，20(2)：141
[3] 王永炎．关于提高脑血管疾病疗效难点的思考[J]．中国中西医结合杂志，1997，17(4)：196
[4] 林兴栋．“热毒”在中风发病中的作用与证治探讨[J]．中国中医药信息杂志．2002：9(1)：43
[1] 刘金烘，陈华德．血脑屏障与中医药研究[J]．浙江中医学院学报，1999，23(2)：591
[2] 张伟，林凯．HPLC测定舒胸胶囊中盐酸川芎嗪的含量[J]．中成药，2006，28(6)：920-922
[3] Zea Longa EL, Weinstein PR, Canson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke, 1989, 20(1): 84-91
[1] 汪枫．脑损伤时间推断的分子生物学研究进展[J]．皖南医学院学报，2003，22(3)：232-234
[2] Gornes FG, Paulin D, Moura Neto V. Giial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation[J]. Braz J Med Biol Res 1999;32(6): 619-31
[3] 蒲传强主编．脑血管病学[M]．北京：人民军医出版社，1999，95-97
[4] Dalby NO, Rondouin G, Leaner-Natoli M. Increase in GAP-43 and GFAP immunoreactivity in the rat hippocampus subsequent to perforant path kindling[J].Neurosci Res, 1995,41:613-619
[5] ASTBury SD, Eichhorn AC, Kotzer CM, et al. Reactive astrocytic responses to denervation in the motor cortex of adult rats are sensitive to manipulations of behavioral experience[J].Neuropharmacoloev, 2000,39(5):743-755
[6] Kim Y, Truettner J, Zhao W, et al.The influence of delayed postischemic hyperthermia following transient focal ischemia:alterations of gene. expression[J]. Neurolscience,1998,159(1):1-10
[7] Schmidt KR, Aguirre CC, Saul I, et al. Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats[J].Brain Res, 2005 , 1052 (1):28-39
[8] Bury SD, Eichhom AC, Kotzer CM, et al .Reactive astrocytic rsponses to denervation in the motor codex of adult rats are sensitive to manipulations of behavioral experience[J]. Neuropharmacology,2000,39(5): 743-755
[9] 刘之荣，段德新．星形胶质细胞在缺血性脑损伤中的作用[J]．国外医学·神经病学与神经外科学分册，2000，8(3)：148-150
[10] Hohnin S, Schalling M, Hojeberg B, et al. Delayed cytodine expression in rat brain following experimental contusion [J]. Neurosurg, 1997, 86(3):493-497.
[11] 王晓梅，冯加纯，饶明俐．反应性星形胶质细胞对脑缺血神经元的保护[J]．中风与神经疾病杂志，2001，18(1)：63-64．
[1] 王晓娟，余华峰．一氧化氮与脑缺血再灌注损伤[J]．国外医学(老年医学分册)，2000，21(3)：114-117
[2] Zhou G, Zha B, Hou J, et al. Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction[J]. Biotechnol Tech, 1999,13:507-11
[3] ladecola C. Bright and dark side off nitric oxide in ischemic brain injury[J]. TINS 1997,20:1132-9
[4] Mayer B, Hemmens B. Biosynthesis and action off nitric oxide in mammalian cell[J]. Trends Biochem Sci 1997,22:447-81
[5] 王志浩，张岫美．一氧化氮与缺血性脑血管疾病[J]国际病理科学与临床杂志，2005，25(5)：466-470
[6] Mason RB, Pluta RM,Walbridge S, et al. Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide[J]. J Neurosurg, 2000, 93(1):99-107
[1] Matsuda T, Arakawa N, Takuma K, et al. SEA0400, a novel and selective inhibitor of the Na~+-Ca ~(2+)exchanger, attenuates reperthsion injury in the in vitro and in viva cerebral ischemic mod-els[J]. J Phannacol Exp Ther, 2001,298(1):112-116
[2] Xiong Z G, Zhu X M, Chu X P, et al. Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels[J]. Cell,2004,118(6):687-98
[3] 贾健民，贾健平，李泓，等．脑缺血与自由基[J]．国外医学神经病学神经外科学分册，1995，22(8)：207-209
[4] Qu L, Wang W T, Guo L J. Protective effects of 1-(2,6-dimethyl phenoxy)-2-(3,4-dimethoxy-phenylethylamino) propane hydro chloride(DDPH) on brain ischemia injury in rats[J]. Acta Pharmaceutica Sinica,2003,(10):725
[5] 司徒镇强，吴军正．《细胞培养》[M]．世界图书出版公司，1996，186-187
[1] Love S. Neuronal experssion of cell cycle-related proteins after brain ischaemia in man[J].Neurosci Lett, 2003,363(1):29-32
[2] Osuga B, Osuga S, Wang F, et al. Cyclin-dependent kinases as a therapeutic target fox stroke[J]. Proc Natl Acad Sci USA, 2000,97(18):10254-10259
[3] Charriaut-Marlangue C, Aggoun-Zouzoui D, Represa A, et al. Apoptosis features of selective neuronal death in ischemia, epilepsy and gp 120 toxicity [J]. Trends Neurosci, 1996,19:109-114
[4] 赵士福，蔡文琴，邓晓林，等．大鼠脑缺血过程中神经细胞死亡的研究仁[J]．解剖学报，1997，28(4)：342-345
[5] NITATORI T, SATO N, WAGURI S, et al. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis[J]. J Neurosci, 1995,15:1001-1011
[6] MATSUDA T, ARAKAWA N, TAKUMAK, et al. SEA0400, a novel and selective inhibitor of the Na~+-Ca~(2+) exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models[J]. J Pharmacol Exp Ther,2001,298(1):249-256
[7] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis[J]. Semin Hematol, 2001, 38(2):179-193
[8] Becket EB, Bonni A. Beyond proliferationTcell cycle control of neuronal survival and differentiation in the developing mammalian brain[J].Se min Cell Dev Biol, 2005, 16 (3): 439-448
[9] Zhu X, MeShea A, Harris PL, et al. Elevated expression of a regulator of the G2/M phase of the cell cycle, neuronal CIP-1-as-sociated regulator of cyclin B, in Alzhemefs disease[J]. J Neurosci Res, 2004, 76(6):698-703
[10] Volpe BT, Wessel TC, Mukherjee B, et al. Temporal pattern of internuclosomal DNA fragmentation in the Striatumand hippocampus after transient forebrain ischemia[J]. Neurosci Lett, 1995, 156-157
[11] HakimAM. Ischemic penumbra: the therapeutic window[J]. Neurology, 1998, 51 (3):544-546
[12] 左连富主编．流式细胞术与生物医学[M]．沈阳：辽宁科学技术出版社，1996，68-73
[13] Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats [J]. Stroke, 1993, 24:2002-2009
[1] ZhongH, De Marzo AM, laughner E, et al. Over-expression of hypoxia-induciable factor-lain common human cancers and their metastases[J]. Cancer Res, 1999,59:5830-5835
[2] Palmer LA et al. Am J Physiol[M], 1998, 274-212
[3] Senenza GL. HIF-1a, and the 3 PHDs how animal cells signal hypoxia to the nucleus [J]. Cell, 2001,107:123
[4] Wenger RH et al. Eur J Biochem[M], 1998,253-771
[5]Nagle DG,Zhou YD. Natural product-based inhibitors of hypoxiainducible factor-1(HIF-1)[J]. Curr Drug Targets, 2006, 7(3):355-369
[6] Zelzer E et al. EMBO J[M], 1998,17:5085
[7] Lefer DJ. Induction of HIF-1 alpha and iNOS with siRNA: a novel mechanism for myocardial protection[J]. Circ Res, 2006, 98(1): 10-11
[8] Semenza GL et al. Kidney Int[M], 1997, 51-513
[9] Lee PJ et al. J Biol Chem[M]. 1997, 272:5375
[10] Kallio PJ, Wilson WJ, O'Brien SMakino Y. PoellhgerL Regulation of the hypoxia inducible transcription factor 1 alpha by the ubiquitin proteasane pathway[J]. JBiolChem, 1999, 274:6519-6525
[11] Hu J et al. Biochem Biophys Res Commun[M], 1998,245:894
[12] Cormier-regard S et al. J Biol Chem[M], 1998, 273:17787
[13] Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O_2 tension[J]. Am J Physiol, 1996, 271:C1172-1180
[14] Lok-CN et al. J Biol Chem[M], 1999,274(34):24147
[15] Millhorn DE et al. Kidney Int[M], 1997, 51:527
[16] Ren XQ, Meng JM, Fu KD. Study on relationship between expression of p53 HIF-1 α and angiogengesis of colorectal carcinoma[J]. Zhongguo Yixue Gongcheng, 2004, 12(4):31-35:
[20] Senenza GL. Expression of hypoxia inducible factor-1 α echanims and coinsequences[J]. Biochem Phamacology, 2000, 59:47-53
[17] Feldser-D et al. Cancer Res[M], 1999, 59: 3915
[18] Carmeliet P et al. Nature[M], 1998, 394: 485
[19] Tazuke SI et al. Proc Natl Acad Sci USA[M], 1998, 95: 10188
[21] 宋虎平，惠延年，王丽丽，韩晓霞，王海燕．缺氧诱导因子-1α在早期糖尿病大鼠角膜上皮细胞中的表达[J]．国际眼科杂志，2000，6(2)：352-356
[22] Wiener CM et al. Biochem Biophys Res Commun[M], 1996, 225-485
[1] Min HJ, Tomohiro Kaku, Junichi Hada, el al. Different effects of eNOS and nNOS inhibition on transient forebrain ischemia[J]. Brain Res. 2002, 946:139-142
[2] Vaughan CJ, Delanty N, Basson CT. Statin therapy and stroke prevention[J]. CurrOpin Cardiol,2001,16(4):219-228
[3] Oconnell AW, Fox GB, Kjoiler C. el al . Antiischemic and cognition-enhancing properties of NNC-711. agamma-aminobutyricacid reuptakeinhibitor[J]. Eur J Pharmacol, 2001,424(1):37-43
[4] Eguchik, Kariok, Shimada K Beneficial effect of ARB for ischemic stroke[J]. Nippon Rinsho, 2002, 60(10): 1987-1998
[5] Qureshi A1, Ali Z, Suri MF, el al. Intra. arterial third generation recombinant tissue plasminogen activator(reteplase)for acute ischemic stroke[J]. Neurosurgery, 2001,49(1): 41-45
[6] Zeller JA, Tschoepe D, Kessler C Circulating platelet show increases activation in patients with acute cerebral ischemia[J]. Thromb Haemost, 1999,81(3):373-380
[7] Nakamura T, Uchiyama S, Yamazaki M, el al. Effects ofdipyridamole an daspirin on shear induced platelet aggregation in whole blood and platelet rich plasma[J]. Cerebrovasc Dis,2002, 14(3～4): 234-250
[8] Abciximab in acute ischemic stroke:a randomized, double-blind, placetm-controlled, dose escalation study The Abciximab in lschemic Stroke lnve-stigators[J]. Stroke,2000,31(3):601-609
[9] Bath PW, lddenden R, Bath FJ Low Molecular. Weight heparins and hepadnoids in acute ischemic stroke: A MetaAnalysis ofrandomizeel controlled trials[J], stroke,2000,31 (7): 1770-1774
[10] Eun BL, Liu xH, Barks JD. Pentoxifylline attenuates hypoxic ischemic brain injury in immature rats[J]. Pediatr Res, 2000, 47(1): 73-79
[11] Asai S, Zhao H, Yan A.,el al. Nicergoline enhances glutamate re-uptake and protects against brain damage in rat global brain ischemia[J]. Eur J Pharraacol, 1999, 383(3): 267-275
[12] 刘继生，刘树鹏．刺五加注射液治疗缺血性脑卒中78例临床及血液流变学观察[J]．中西医结合实践杂志，1997，10(4)：322
[13] 寿延红，陈颖，冯加继，等．刺五加对蛛网膜下腔出血患者血浆内皮素含量的影响[J]．中风与神经疾病杂志，2001，18(3)：139
[14] 赖伟坚，王艳华．大剂量血栓通注射液治疗急性脑梗死72例疗效分析[J]．广西医学，2004，26(1)：88-89
[15] 王根发，王文安，周永炜，等．三七皂苷对大鼠脑缺血再灌注损伤的保护作用[J]．中国临床康复，2002，6(9)：1268
[16] 朱卫，张晓彪．银杏叶制剂对脑血管治疗的概况[J]．国外医学脑血管疾病分册，1998，6(4)：236
[17] Zhuang H, Pin S Christen Y, et al. Induction of heine oxygenasel by Ginkgo biioba in neuronal cultures and potential implications in ischemia[J]. CellMolBiol, 2002, 48(6):647-653
[18] 孙莉莎，徐江平，杨雪梅．银杏内酯对大鼠脑缺血再灌注损伤的保护作用[J]．中华现代中西医杂志，2003，1(8)：676
[19] 王东吉，尚改萍，连为民，等．川芎嗪对兔脑缺血再灌注损伤的保护作用[J]．长治医学院学报，2003，17(4)：245
[20] 马仁生，杜爱芹灯．盏花注射液治疗急性脑梗死48例[J]．中国中医药信息杂志，2004，11(12)：1092
[21] 韦登明，林兴明，张朝辉，等．绞股兰总皂苷对大鼠局灶性脑缺血再灌注酯质过氧化脑损伤影响的实验研究[J]．中华实用医学杂志，2003，2(6)：484
[22] 徐立新，何绘敏，曲石霞，等．新生大鼠缺氧缺血性脑病脑组织酯质过氧化改变及丹参保护作用探讨[J]．新生儿科杂志，2000，15(1)：63-65
[23] 董少龙．水蛭注射液对高血压性脑出血颅内血肿吸收的影响[J]．广西中医药，1998，21(3)：4-6
[24] 黄修玲，郑丽．清开灵治疗中风后遗症[J]．光明中医，2003，18(4)：107
[25] 程梦琳，吴基良，罗德生．参附注射液对大鼠脑缺血再灌注大鼠脑组织NO含量和NOS活性的影响[J]．中国中医急诊，2004，13(4)：234
[26] 江莲，戎小平．参麦、乌司他丁对大鼠脑缺血再灌注损伤保护作用的研究[J]．河北医科大学学报，2002，23(3)：144
[27] 刘艳，罗祖明，冀玲，等．华佗再造丸对大鼠局灶性脑缺血损伤的保护作用[J]．华西药学杂志，2004，19(2)：103
[28] 杨赶梅，夏娣文，余江健，等．芪蛭胶囊对中风高粘血症患者血液流变学变化的影响[J]．中国中医药科技，2002，9(4)：231
[1] 高长越，周华东，邓娟，等．川芎嗪对脑缺血再灌注损伤后细胞间粘附作用的影响[J]．中国中西医结合急救杂志，2002，9(3)：135
[2] 钟瑜，张焱，杨国汗．川芎嗪对脑缺血丙灌注损伤后细胞粘附作用的影响[J]．深圳中西医结合杂志，2003，13(3)：141
[3] 张兆辉，卫涛涛，余绍祖，等．川芎嗪的抗氧化机制对缺血性脑损伤的保护作用[J]．中国临床康复，2004，8(34)：7742
[4] 王万铁，陈寿权，徐正妏，等．川芎嗪注射液抗脑缺血再灌流损伤作用机制的实验研究[J]．中华急诊医学杂志，2001，10(3)：182
[5] 汪远金，申国明，王钦茂．川芎嗪对大鼠脑缺血再灌注损伤的保护作用[J]．安徽中医学院学报，2000，19(1)：44
[6] 游言文，田新红．川芎嗪对大鼠脑缺血再灌注后神经元突触超微结构的影响[J]．河南中医学院学报，2004，19(114)：19
[7] 李效敏，孙慧，赵勇，等．川芎嗪对脑梗死患者内皮细胞功能影响的研究[J]．中国急救医学，2004，24(10)：740
[8] 钟平，杨任民．中药川芎嗪治疗急性脑缺血机理的实验研究[J]．实用全科医学，2003，1(1)：19
[9] 曲友直，高国栋，赵振伟，等．川芎嗪、黄芪对脑缺血再灌注后神经细胞凋亡及Fos蛋自表达的影响[J]．卒中与神经疾病，2004，11(2)：111
[10] 俞海国，汤云珍，杨锡强．川芎嗪对新生鼠缺血缺氧脑损伤FOS蛋白表达的影响[J]．重庆医学，2003，32(4)：416
[11] 吕少平，孙锋，王春霞．川芎嗪对脑缺血再灌注时细胞凋亡的影响[J]．河北医学，2000，6(8)：673