【作者】 钱皎；

【导师】 苏定冯；

【作者基本信息】 第二军医大学 ， 药理学， 2009， 博士

【摘要】 目的：高血压发病率高，在中国成人高达18．8%，目前我国大约有2亿高血压病人。脑卒中是高血压病最重要的并发症，近些年脑卒中发生率呈持续上升趋势，且日趋年轻化，中国高血压病人脑卒中的发生率要远远高于欧美国家的高血压病人。脑卒中一旦发生，后果严重，病人非死即残。全球每年新发脑卒中1500万，其中500万人死亡，500万人永久性残废，要使幸存者恢复功能，耗时费力，因此，预防和治疗脑卒中已经成为科学研究迫切需要解决的问题。如何预防脑卒中的发生?血压水平是一个非常重要的心脑血管危险因素和预测因子，血压愈高，脑卒中的危险性就愈大，且80%的脑卒中和52%的脑梗死因高血压而引发，降低血压可以显著预防脑卒中的发生，但降压治疗并不是唯一的预防策略，特别是对于非高血压性脑卒中，并且，脑卒中发生的病理生理机制复杂，有必要对其进行进一步的深入研究，找出除了血压以外的其他关键因子或预防措施，针对这些因子或措施进行干预，作为防治脑卒中的新靶点。热量限制（caloric restriction，CR）是指在保证机体基本营养需求前提下，降低机体约30%～40%的能量摄入。1935年McCay第一次提出了热量限制可以延长啮齿类动物的寿命，随着科学研究不断进步，发现这一作用几乎可以在所有生物中起到延长寿命的作用。热量限制可以延缓很多疾病的发生，如肾病、肿瘤、自身免疫性疾病和糖尿病等。热量限制能降低年龄相关的神经退化、神经紊乱，如帕金森病或阿尔茨海默病。最近几年有不少关于热量限制延长低等生物和细胞寿命的报道，资料显示热量限制延长寿命可能与Sir基因有关，基因家族是一类调控寿命的重要基因，它主要通过参与染色质沉默以及能量代谢而调节细胞的衰老过程。Sir2相关酶类（Sirtuins）是最近发现的NAD⁺依赖的蛋白去乙酰化酶类，在哺乳动物包括SIRT1-7七种不同的同系物。其中SIRT1蛋白研究最多，功能也最为明确。哺乳动物SIRT1蛋白通过与不同的组蛋白和非组蛋白的相互作用来完成不同的功能，对细胞生存、衰老、凋亡等生理活动起到十分重要的调节作用。我们先前的初步结果显示，热量限制可以显著地延缓SHR-SP大鼠脑卒中发生时间，延长动物寿命，但其具体作用机制尚不清楚。阐明热量限制对脑卒中的预防作用途径对于寻找新的脑卒中预防靶点具有非常重要的意义。动脉压力感受性反射（arterial barloreflex，ABR）功能，是心血管系统活动最重要的自身调节机制之一，ABR功能强弱可以用压力感受性反射敏感性（Baroreflex sensitivity，BRS）这一指标来表示。近年来研究发现，ABR功能的异常，参与了多种心血管系统疾病的发生发展过程。因此，自上世纪80年代以来，对ABR功能的研究越来越得到人们的重视。1988年到1998年10年间，意大利学者La Rovere等用大量的临床数据证实，BRS高低可以预测急性心肌梗死（acute myocardial infarction，AMI）病人的预后，死亡患者的ABR功能明显低于幸存者。同时，在脑卒中动物模型以及慢性脑血管疾病的病人身上，也出现了BRS受损情况。Robinson及同事也发现，脑卒中病人，BRS功能低下者，其预后较BRS正常者明显要差。因此，ABR功能与脑卒中之间存在密切的联系，是影响脑卒中预后的一个非常重要的因素。前期的研究我们已经初步证实了ABR功能作为一个重要因子可以独立于其他诸如血压等因素之外来预测脑卒中的发生，改善ABR功能，可进一步起到预防或者延缓脑卒中的发生。那么热量限制预防脑卒中的作用是否与ABR功能相关，如果二者有关，又是如何发挥作用的，需要我们通过实验进行证实。2000年，Nature杂志报道乙酰胆碱（acetylcholine，ACh）与巨嗜细胞上尼古丁受体结合能够抑制内毒素诱导的炎性因子TNFα，IL-1β，IL-6，IL-18等分泌。进一步研究发现乙酰胆碱的作用主要是通过与巨嗜细胞上的α7尼古丁受体结合，通过细胞内一系列的信号传递，最终抑制了Jak2-STAT3和NF-κB信号的激活，从而抑制了炎症因子的产生。由于乙酰胆碱是迷走神经末梢分泌的主要神经递质，因此，他们把这条通路命名为“胆碱能抗炎通路”（cholinergic anti-inflammatory pathway，CAP）。它主要指中枢的免疫调节信号通过激活传出迷走神经，引起外周神经末梢释放乙酰胆碱，与免疫细胞上具有α7亚单位的N型ACh受体结合，通过细胞内信号传导抑制促炎因子的释放，调控炎症反应。传出迷走神经能够抑制外周炎症因子的产生，提示迷走神经与炎症细胞间存在信号传导机制。当机体受到伤害性刺激后，会产生炎症反应，并将冲动传到中枢。中枢再将信号投射到各个迷走神经核团（主要为背核，疑核，孤束核等），激活传出迷走神经纤维，引起外周神经末梢释放Ach，通过细胞内信号传导途径抑制促炎因子的释放来起到调整全身性炎性反应的作用。Shytle等首次报道发现脑单核巨噬细胞-小神经胶质细胞上存在含有α7亚单位的N型受体，体外实验中ACh和烟碱预处理能抑制内毒素引起的小神经胶质细胞释放TNF。这一作用可被亚单位拮抗剂所抑制。此研究结果提示中枢可能也存在与外周相似的胆碱能保护通路，那么热量限制与胆碱能通路是否也有一定的联系?本课题对以上内容进行了初步的探索。方法：实验一：利用SHR-SP，采用大脑中动脉栓塞方法建立脑梗塞模型，观察不同程度的热量限制对SHR-SP急性脑梗死损伤的保护作用差异。实验二，SHR-SP随机分为2组，分别进行去窦弓神经（SAD）手术以及假手术（Sham），术后再各自分为2组（自由饮食组以及热量限制组），观察ABR功能对热量限制预防脑卒中的作用影响。实验三，观察热量限制、ABR功能与乙酰胆碱及胆碱能通路的关系研究：通过阿托品对SAD大鼠血流动力学的影响，研究ABR功能和迷走神经的关系；利用电生理方法研究神经元细胞疑核放电情况并探索其与脑卒中发生的作用。实验四，观察胆碱能抗凋亡通路在急性脑梗死损伤中的作用：侧脑室外源性的给予新斯的明，观察其与脑卒中发生关系；实验五，胆碱能通路防治脑卒中抗凋亡通路的初步探讨：采用FCM观察Cch对H₂O₂诱导的Bend．3细胞凋亡作用；用荧光显微镜观察Cch对神经元细胞的凋亡作用；利用分子生物学技术研究了Cch对Bend．3中SIRT1蛋白表达的影响；利用RNA干扰技术制备SIRT1屏蔽的细胞株。结果：1、SHR-SP大鼠热量限制40周，AL、CR组都发生脑卒中，但CR组损伤较轻；CR80%、CR60%、CR40%均可改善脑卒中发生，CR60%改善最好；大鼠SAD后，削弱了CR对脑卒中保护作用，说明ABR功能参与了CR对脑卒中的保护作用；2、ABR功能缺损大鼠对阿托品引起的心动间期的反射减弱，并且通过单因素相关性分析，发现心动间期的差值和BRS成正相关关系；在检测疑核放电实验中，SAD组疑核神经元的放电活动显著降低，并且疑核放电低的易于发生脑卒中；3、SAD术后，α7和AchT蛋白的表达均显著降低。血清中炎症因子IL-1β、IL-6的含量显著升高；外源性的乙酰胆碱可延缓脑卒中的发生；4、卡巴胆碱能剂量依赖性的抑制Bend．3和神经元细胞的凋亡，并且这一过程有α7nAChR的参与；卡巴胆碱能剂量依赖性的增加SIRT1蛋白的表达。结论：1、热量限制可以显著地延缓SHR-SP大鼠脑卒中发生时间，延长大鼠寿命。2、ABR功能参与了热量限制延缓脑卒中发生的作用机制，ABR功能缺损削弱了热量限制延缓脑卒中发生的保护作用3、ABR功能缺损引起迷走神经功能减弱；而迷走神经功能减弱大鼠易发生脑卒中，提示我们ABR功能防治脑卒中的发生伴随着胆碱能通路调控机制；4、胆碱能通路在急性脑梗死损伤中具有一定的保护作用，其作用机制与乙酰胆碱抗神经元和脑血管内皮细胞的凋亡有关，并且这一过程有SIRT1蛋白的参与。更多还原

【Abstract】 Background and Objectives:In many countries including China, stroke is the third leading cause of death only preceded by heart disease and total cancer. According to recent estimates published by the World Health Organization, about 15 million people per year fall victim to stroke worldwidely, of whom 5 million die and another 5 million are left permanently disabled. Many stroke survivors become dependent, and require lifelong assistance. Therefore, prevention is the only possible way to curb the stroke pandemicly. Blood pressure level is one of the most consistent and powerful predictor of stroke, so blood pressure control is an important way to reduce the morbidity. However, blood pressure level is not the unique determinant for stroke. Here we propose other important determinants for stroke.Calorie restriction （CR） is the only experimental manipulation that is known to extend the lifespan of a number of organisms including yeast, worms, flies, rodents and perhaps non-human primates. In addition, CR has been shown to reduce the incidence of age-related disorders （for example, diabetes, cancer and cardiovascular disorders） in mammals. The mechanisms through which this occurs have been unclear. CR induces metabolic changes, improves insulin sensitivity and alters neuroendocrine function in animals. In addition to its effects on metabolism, CR can have profound effects on brain function. For example, CR can protect neurons against degeneration in animal models of Huntington’s disease, Alzheimer’s disease, Parkinson’s disease and stroke.ABR is one of the most important mechanisms in the regulation of cardiovascular activities. Since the end of 1980s, the pathological importance of ABR function has attracted the attention of many investigators. Baroreflex function, expressed as baroreflex sensitivity （BRS）, was found as an important determinant of cardiac death after acute myocardial infarction. There is also established evidence of abnormal BRS in animal models of stroke and patients with chronic cerebrovascular disease. Indeed, it was found that BRS was impaired after acute stroke. Post-stroke patients with impaired BRS had a poor prognosis. We have proved that BRS can be as a new predictor for stroke incident. And CR can protect neurons against degeneration in animal models of stroke. However, to our knowledge, there is no more report about the mechanisms how BRS works in this process. So the first aim of this study was to investigate the relationship of ABR, CR, and Stroke.It had been reported that acetylcholine inhibits the production of pro-inflammatory cytokines from endotoxin-stimulated macrophages through a mechanism dependent on the a7 nicotinic acetylcholine receptor subunit in 2000. Because acetylcholine is the principal vagus neurotransmitter, the central nervous system also regulates proinflammatory cytokine production through the efferent vagus nerve, termed the "cholinergic anti-inflammatory pathway". It had been reported that activation of this mechanism via vagus nerve stimulation can control the production of pro-inflammatory cytokines in experimental models of systemic inflammation, including lethal endotoxemia, hemorrhagic shock, and ischemia-reperfusion injury. Thus, the "cholinergic anti-inflammatory pathway" can directly modulate the systemic response to pathogenic invasion. In this process, we know that vagus nerve plays an important role in the regulation of various signals central neruron system sent out. So investigation of the relationship of ABR and vagal function makes it easy to find the effects of cholinergic pathway on the protection of stroke. Shytle et al. report for the first time that cultured microglial cells express alpha7nAChR subunit as determined, and their findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine’s reported neuroprotective properties.Many reports have shown the effect of caloric restriction （CR） on pretection of stroke, and this effect is related to the function of ABR. How dose ABR produce a marked effect on it? Though preliminary, we investigate the regulations of cholinergic pathway on the protection of stroke caused by the impaired BRS. It will develop a new way to find the effective factors involved in the pretection and treatment of stroke.Main Methods and Results:Male SHR-SP, at the age of 12 weeks, were fed ad libitum （AL） or CR diet （80%, 60%, 40%of AL）. We subjected the animals to middle cerebral artery （MCA） occlusion, and the infarct area and hemisphere areas of each section （both sides） were traced and quantified by an image analysis system. We found the ratio of the infarct area and hemisphere areas was significantly different in the four groups. SHR-SP fed CR diet （60% of AL） showed the best protective effect of CR. Sinoaortic denervation （SAD） was performed to destroy the peripheral baroreflex arc in SHR-SP, and after a month, they were fed ad libitum （AL） or CR diet （60% of AL）. The infarct area was traced and quantified. The ratio of the infarct area and hemisphere areas was decreased in both of the CR group, while sinoaortic denervation group decreased less than the sham-operated group. The survival time and stroke incidence were observed and was expressed by Kaplan-Meier survival curves. The effects of caloric restriction to the prevention of stroke were weekened by sinoaortic denervation. Sinoaortic denervation （SAD） rats were given Atropine to observe the different reaction to the stimulation. After the injection of Atropine, the decrease of heart period in sham-operated group is more obviously than sinoaortic denervation group. Also we detected the discharge of the nucleus ambiguous of the two groups. The level of the discharge of the nucleus ambiguous in sinoaortic denervation group was lower than that of sham-operated group. The group with low discharge level has the liability to the development of stroke. We used Western blotting to detect the protein expression ofα7 and AchT. The protein expression of a7 and AchT in brain was obviously decreased in sinoaortic denervation rats. Levels of interleukin （IL）-1βand IL-6 in serum were detected by ELISA. The levels of interleukin （IL）-1βand IL-6 were higher in sinoaortic denervation rats than that in sham-operated rats. After the injection of Carbachol to the lateral cerebral ventricle, we observed the influence of Carbachol to the treatment of stroke. The ratio of the infarct area and hemisphere areas was significantly decreased by the injection of Carbachol. Carbachol dose-independently inhibited the H₂O₂-induced apoptosis of Bend. 3 detected by flow cytometry. It also inhibited the H₂O₂-induced apoptosis of neuron cells observed by fluorescence microscope. Western blotting was used to measure the protein expression of SIRT1. Carbachol dose-independently increased the protein expression of SIRT1. Knock-down of SIRT1 was attained by siRNA.Conclusion:In conclusion, the present study provides evidence that caloric restriction is a new and important predictor for stroke death in hypertension rats. Restoration of ABR function is a new target for the prevention of stroke. ABR functional impairment decreases the prevention of stroke by CR. The cholinegic pathway is involved in the protective mechanisms of stoke, and this function is mediated by ABR. The regulation of cholinegic pathway maybe have two aspects: one is due to the increase of ectogenic inflammatory transmitter, and the other is for the improvement of the apoptosis of vascular endothelial cells and neuron cells in the brain. Furthermore, we find this improvement is mediated by the protein expression of SIRT1.更多还原