摘要
缺血性脑卒中是引起人类非自然死亡和致残的主要疾病之一。激肽释放酶—激肽系统(kallikrein—kinin system，KKS)作为公认的炎症调节系统在缺血性脑血管病的病理过程中发挥重要作用。近年来研究提示，这一系统可能在脑缺血后的不同时期发挥截然不同的作用，并且可通过其终末效应物质缓激肽，作用于缓激肽B1和B2受体，对脑缺血后的细胞凋亡产生影响。本研究观察KKS各组分在脑缺血后的动态表达变化及缓激肽受体在缺血组织中的细胞分布，进一步应用缓激肽受体的特异性抑制剂对脑缺血动物进行干预，探讨KKS在脑缺血后不同时期的作用及其深层机制。为排除复杂内环境的影响，本研究采用缺糖、缺氧(oxygen-glucose deprivation，OGD)后复糖、复氧的神经元模型，应用缓激肽受体的特异性激动剂和抑制剂对其进行干预，以验证缓激肽受体在神经元凋亡中的作用。我们拟通过本研究明确KKS在脑缺血后发挥不同作用的病理基础，以期为缺血性脑卒中提供新的治疗靶点。
第一部分：激肽释放酶—激肽系统在脑缺血后的动态表达
研究缺血后KKS各组分的动态表达变化，是探索KKS在脑缺血后不同时期作用的理论前提。本部分制作大鼠左侧大脑中动脉梗塞模型，缺血90min后恢复再灌流。观察实验性脑缺血再灌注损伤后不同时期组织型激肽释放酶(tissuekallikrein，TK)活性、缓激肽(bradykinin，BK)含量、缓激肽B1受体(B1 receptor，B1R)和B2受体(B2 receptor，B2R)的动态表达及细胞分布。
取左侧前囟前、后1mm(相当于缺血核心区)脑组织，通过生物酶学方法测定脑组织TK活性、运用ELISA技术测定脑组织BK浓度，测定时间点为：缺血前和再灌流后2h、4h、6h、12h、24h和48h；采用RT—PCR及western-blot技术，测定缓激肽B1R和B2R的mRNA及蛋白表达水平，测定时间点为：缺血再灌流后3h、6h、12h、24h、48h、72h、4d及7d；根据Western—blot结果，分别在缓激肽B1R和B2R的表达高峰时间点，即再灌流后12h和72h，采用免疫荧光双标技术，观察缺血后缓激肽B1R和B2R在神经元和星型胶质细胞的分布情况。
结果：1、缺血后脑组织TK活性明显增高，于再灌流后2h到达高峰；缺血后脑组织BK浓度显著增高，也在再灌流后2h达到高峰；两者变化趋势一致。2、缺血后脑组织缓激肽B1R的mRNA和蛋白水平迅速上调，于再灌流后12h到达高峰，而后迅速下降，再灌流后第4d回到基线水平；而缓激肽B2R的mRNA和蛋白水平在脑缺血后上调缓慢，于再灌流48～72h到达高峰，之后缓慢下调，再灌流后第7d仍高于基线水平。3、缺血后缓激肽B1R主要分布于反应增生的星形胶质细胞；而缓激肽B2R主要分布于缺血周边区的皮层神经元。
研究提示：缓激肽B1R和B2R在缺血再灌流后的动态表达和细胞分布存在很大差异，这可能是导致KKS在脑缺血不同时期发挥不同作用的病理基础。
第二部分：缓激肽受体在脑缺血后不同时期的作用研究
本研究观察到：缺血后脑组织内缓激肽B1R和B2R在时间动态变化和细胞分布方面存在很大差异。为进一步探讨KKS在脑缺血中的作用，我们分别应用缓激肽B1R的特异性抑制剂Lys-(des-Arg~9-Leu~8)-BK和B2R的特异性抑制剂bradyzide，对脑缺血动物进行干预，以深入了解这两个受体对缺血后组织损伤的影响。仍采用上述大鼠左侧大脑中动脉梗塞模型，缺血90min后恢复再灌流。
脑缺血模型分为即刻干预和24h后干预两大组。即刻干预指在缺血再灌流后即刻尾静脉给药，再灌流24h处死动物；而24h后干预是指在缺血再灌流后24h方开始尾静脉给药，每日一次，7d后处死动物；每大组动物根据干预方式不同，又随机分为四组：假手术组、生理盐水干预组(生理盐水2ml／kg)、B1R抑制剂组(300μg／kg)和B2R抑制剂组(0.75 nmol／kg)。
测定指标包括：神经功能评分(NSS)、梗死体积(TTC染色)、血脑屏障通透性(伊文氏蓝染色)、超微结构(电镜)、炎症因子分泌(ELISA)、细胞凋亡(TUNEL)以及B1R和B2R受体的表达(westem-blot)。
结果：1、即刻干预组动物，缓激肽B1R和B2R的抑制剂均能显著改善脑缺血导致的神经功能障碍、减小梗死体积、降低血脑屏障通透性、改善超微结构损伤、抑制炎症因子的释放、减少细胞凋亡；上述作用，B2R抑制剂均优于B1R抑制剂。2、24h后干预组动物，两种抑制剂作用显著不同，B1R抑制剂能够抑制缺血后细胞凋亡、减小梗死体积、改善神经功能；而B2R抑制剂能够增加缺血区细胞凋亡、增大梗死体积、加重缺血后神经损伤。3、缓激肽两受体间还存在相互作用关系，B2R抑制剂的运用能明显抑制B1R的表达，而B1R抑制剂对B2R的表达无显著影响。
研究提示：1、脑缺血早期，缓激肽B1和B2受体均能加重缺血后组织损伤。2、脑缺血后期，缓激肽B2受体能够抑制缺血导致的细胞凋亡。3、B2受体激活可以增加B1受体的表达。
第三部分：缓激肽受体在缺糖、缺氧神经元细胞凋亡中的作用
本研究体内实验观察到缓激肽B1R和B2R对脑缺血后细胞凋亡的影响显著不同。为排除体内复杂微环境的影响，本部分采用缺氧、缺氧(OGD)—复糖、复氧神经元模型，利用缓激肽受体的特异性激动剂和抑制剂进行干预，以深入探讨两者在神经元细胞凋亡中的作用。
体外原代培养大鼠皮层神经元，以培养第7～10天，生长状态良好的神经元进行相关实验。OGD 1.5h后复糖、复氧，MTT测定细胞存活率，以确定复糖、复氧时间。免疫荧光双标测定缓激肽B1R和B2R在体外培养神经元中的表达。分别采用B1R激动剂(des-Arg~9-BK，1μmol／L)、B2R激动剂(BK，1μmol／L)、B1R抑制剂[Lys-(des-Arg~9-Leu~8)-BK，100nmol／L]、B2R抑制剂(HOE140，100nmol／L)在OGD前24h进行干预，复糖、复氧后4h采用TUNEL测定细胞凋亡。
结果：1、缓激肽B1R和B2R在体外培养的神经元中均有表达，缺糖、缺氧后两者的表达量有所提高。2、OGD 1.5h复糖、复氧4h，细胞存活率下降50％。3、B1R激动剂和B2R抑制剂能够促进OGD所导致的神经元凋亡；而B1R抑制剂和B2R激动剂能够对OGD所导致的神经元凋亡发挥抑制作用。
研究提示：1、B1受体可以增加OGD所致的神经元凋亡。2、B2受体能够拮抗OGD导致的神经元凋亡。
结论
1．缓激肽B1受体：脑缺血再灌流后表达迅速上调。主要分布于反应增生的星形胶质细胞，可增加血脑屏障通透性，促进炎性因子的释放，加重缺血导致的脑损伤；神经元上分布的B1受体激活可增加OGD引起的神经元凋亡。
2．缓激肽B2受体：脑缺血再灌流后表达缓慢上调。主要分布于缺血周边区皮层神经元，可起到拮抗神经元凋亡的作用；但在缺血早期B2受体的激活可通过增加B1受体的表达，加重缺血导致的脑损伤。
Cerebral arterial thrombosis is one of the most common diseases that cause unnatural death and disability. Kallikrein-kinin system as an agreed inflammation regulatory system plays an important role in the pathophysiologic process after cerebral ischemia. However, reseaches from recent years implied that this system may produce completely different effects during various stages of cerebral ischemia. Bradykinin as the terminal effective factor of this system can influence the cell apoptotic process after cerebral ischemia by activating its B1 and B2 receptors. In the present study, we first observed the time course of each component of KKS and cellular distribution of bradykinin receptors in ischemic brain tissue. Then specific bradykinin receptor antagonists were used at different time points after brain ischemic damage to further explore the function of KKS in various phases of cerebral ischemia. In order to avoid the influence of the complicated environment in vivo we cultured neurons which were then deprived of oxygen and glucose(OGD) followed by oxygen-glucose re-supply. Specific bradykinin receptor agonists and antagonists were used to intervene these neurons, then neuronal apoptosis was observed. Through this study, we wish to further understand the role of KKS on various stages of cerebral ischemia and look forward to providing a promising target for the treatment of cerebral infarction.
PartⅠThe expression of Kallikrein-Kinin System at different time points after cerebral ischemia
To study the time course of each components of KKS after cerebral ischemia is necessary for the exploration of its function in various phases of this pathologic process.
Rats were rendered to undergo 90 minutes of the left middle cerebral artery occlusion(MCAO) by the intraluminal thread technique before reperfusion. We tested the enzyme activity of tissue kallikrein(TK), the concentration of bradykinin(BK), the expression and distribution of bradykinin B1 and B2 receptors in the ischemic brain tissue before ischemia and at the different time points after reperfusion.
Brain tissue of the left hemisphere 1mm before and after bregma (equal to the ischemic core) was taken to be examined by bio-enzymatic methods and ELISA for the determination of the enzyme activity of tissue kallikrein and the concentration of bradykinin respectively. The time points were as follows: before ischemia, 2h, 4h, 6h, 12h, 24h and 48h after reperfusion. RT-PCR and Western-blot were employed to detect the expression of mRNA and protein levels of bradykinin receptors before ischemia and at the 3rd, 6th, 12th, 24th, 48th, 72th hour and the 4th, 7th day after reperfusion. According to the results of Western-blot, the distribution of bradykinin B1 and B2 receptors on neurons and astrocytes were observed by double fluorescent staining at the peak expressing time points of B1 and B2 receptors, that is, 12h and 72h after reperfusion respectively.
Results showed that: 1. Ischemic damage to the brain resulted in the up-regulation of the enzyme activity of tissue kallikrein and the concentration of bradykinin who had the same time course with the peak time points at the 2nd hour after reperfusion. 2. The mRNA and protein level of B1 receptor were rapidly up-regulated after ischemia, peaked at the 12th hour after reperfusion and returned to baseline at the 4th day after reperfusion with a snap decline. However, the mRNA and protein level of B2 receptor gradually increased to peak at the 48～72nd hour after reperfusion. This high level maintained for at least 7 days with a slow decline. 3. After cerebral ischemia, B1 receptor was mainly located on the reactive astrocytes. But B2 receptor was predominantly distributed on cortical neurons in ischemic marginal zone.
The above results implied: The time course and distribution of B1 and B2 receptors were totally different after ischemic damage which could explain the different function of KKS on various stages after cerebral ischemia.
PartⅡThe function of bradykinin receptors at different stages after cerebral ischemia
We observed that the time course and distribution of B1 and B2 receptors were totally different after ischemic damage. To further explore the functions of KKS in cerebral infarction especially the bradykinin B1 and B2 receptors, Lys-(des-Arg~9-Leu~8)-BK and bradyzide as the specific antagonist of B1 receptor and B2 receptor respectively were employed for the treatment of cerebral ischemia models. The influence of these two receptors on ischemic prognosis was anticipated to be revealed. The same MCAO modals as in partⅠwere used in this part.
The animal models were divided into immediate intervention group and intervention after 24h group. In immediate intervention group, bradykinin B1 or B2 receptor antagonists were administrated immediately through vena caudalis after reperfusion, then the animals were sacrificed 24h after reperfusion. In the intervention after 24h group, animals were not treated with the above drugs until the 24th hour after reperfusion. After 7 days intervention once a day the rats were executed at 7th day after reperfusion. As to the means of intervention each of the above group was subdivided into four subgroups as follows: sham-operated group, saline intervened group (with saline 2ml/kg), B1 receptor antagonist group (300μg/kg) and B2 receptor antagonist group (0.75 nmol/kg).
Neurological function was appraised by NSS, infarct volume was determined by TTC stain, permeability of BBB was measured by evans blue extravasation, ultrastructure of BBB was observed by transmission electron microscope, the concentrations of inflammatory factors in ischemic tissue were measured by ELISA, the apoptosis in ischemic core and penumbra were detected by TUNEL, and the expression of B1 and B2 receptor were inspected by Western-blot.
The results showed that: 1. In the immediate intervention group the antagonists of bradykinin B1 and B2 can both dramatically improve the neurological function, decrease the infarct volume and permeability of BBB, maintain the integrity of BBB, suppress the secretion of inflammatory factors and relieve the cell apoptosis with a more obvious effect for B2 receptor antagonist. 2. However, in the intervention after 24h group the two antagonists have distinguished disparation.. B1 receptor antagonist Lys-(des-Arg~9-Leu~8)-BK can reduce the cell apoptosis, decrease the infarct volume and ameliorate the neurological fuction. B2 receptor antagonist bradyzide can enhance the cell apoptosis in ischemic zone, increase the infarct volume, aggravate the neurologic injury induced by ischemia. 3. Furthermore, B2 receptor antagonist has an obviously inhibitory effects on the expression of B1 receptor. But the B1 receptor antagonist has no significant impact on the expression of B2 receptor.
The results implied: 1. At the earlier stage after reperfusion, both bradykinin B1 and B2 receptors can aggravate the tissue damage of cerebral ischemia. 2. At the later stage after reperfusion bradykinin B2 receptor can inhibit the cell apoptosis induced by ischemic damage. 3. The activation of B2 receptor can increase the expression of B1 receptor.
PartⅢThe effects of bradykinin receptors on the apoptosis of neurons undergoing oxygen and glucose deprivation
We observed that bradykinin B1 and B2 receptors have conspicuously different effect on cell apoptosis induced by ischemia. In order to avoid the influence of the complicated environment in vivo. In this section we cultured neurons which were then deprived of oxygen and glucose (OGD) followed by oxygen-glucose re-supply. The neurons were then intervened with specific bradykinin receptor agonists and antagonists to further illuminate the effects of bradykinin receptors on the apoptosis of neurons.
Primary rat cortical neurons in good condition were used for the successive experiment after cultured in vitro for 7-10 days. After endured 1.5h of oxygen and glucose deprivation followed by oxygen-glucose re-supply, cell survival rate were measured by MTT to determine the time course of oxygen-glucose re-supply. The expression of bradykinin B1 and B2 receptors on cultured neurons were detected by double fluorensent stain. The cultured neurons were intervened with B1 receptor agonist (des-Arg~9-BK, 1μmol/L), B2 receptor agonist (BK, 1μmol/L), B1 receptor antagonist [Lys-(des-Arg~9-Leu~8)-BK, 100nmol/L] and B2 receptor antagonist (HOE140, 100nmol/L) 24h before oxygen and glucose deprivation. Cell apoptosis of the neurons were inspected by TUNEL 4 hours after oxygen-glucose re-supply.
The results showed that: 1. Both bradykinin B1 and B2 receptors are expressed on cultured neurons. The expression of these two receptors could up-regulated after oxygen glucose deprivation. 2. The cell survival rate declined 50% after OGD 1.5h followed by oxygen-glucose re-supply 4h. 3. B1 receptor agonist and B2 receptor antagonist could prominently enhance the neuron apoptosis induced by OGD. B1 receptor antagonist and B2 receptor agonist could markedly suppress the neuron apoptosis.
The results implied that: 1. Bradykinin B1 receptor can increase neuron apoptosis induced by OGD. 2. Bradykinin B2 receptor can inhibit the neuron apoptosis.
Conclusions
1. Bradykinin B1 receptor mainly located on the reactive astrocytes after cerebral ischemia was up-regulated rapidly after reperfusion with the function of aggravate the ischemia induced brain damage by increasing the permeability of BBB and the secretion of inflammatory factors. The activation of B1 receptor distributed on neurons can enhance neuronal apoptosis induced by OGD.
2. Braykinin B2 receptor mainly distributed on neurons in the marginal zone of ischemia was up-regulated gradually after reperfusion. It has an anti-apoptotic function although at the early stage of cerebral ischemia B2 receptor can aggravate brain damage by increasing the expression of B1 receptor.
引文
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