血管紧张素-（1-7）在高血压及脑缺血损伤中的作用及机制研究
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摘要
血管紧张素-(1-7)在高血压及脑缺血损伤中的作用及机制研究
缺血性脑卒中严重危害人类的健康，其发病率高，致死致残率高，给家庭和社会带来了沉重的经济负担。高血压作为脑卒中的独立危险因素，在卒中的发生发展过程中发挥关键的作用。研究表明，全世界约54%的卒中患者死因与高血压直接相关。在控制其它危险因素后，收缩压每升高10mmHg，卒中发病的相对危险增加49%；舒张压每升高5mmHg，卒中发病的相对危险增加46%。因此，加强高血压的防治，探索脑卒中的神经保护机制，具有重大的意义。
肾素-血管紧张素系统(RAS)是机体重要的循环调节系统。经典的RAS通路是血管紧张素原(AGT)在肾素作用下生成血管紧张素I(Ang I)，而Ang I在血管紧张素转化酶(ACE)作用下生成血管紧张素II(Ang II)，与血管紧张素受体1(AT1R)结合构成ACE-Ang II-AT1R轴，在血压调节、水电解质的平衡中发挥重要作用。高血压患者长期存在RAS的过度激活，而Ang II的增加是促使患者发生致死及致残性临床转归终点事件的核心环节。研究表明，Ang II可通过多条交错复杂的途径诱导氧化应激，促使炎症反应，加速细胞凋亡，损伤血管内皮，刺激交感兴奋，最终导致卒中的发生。近年来，RAS的旁路途径逐步受到重视，血管紧张素-(1-7)[Ang-(1-7)]作为RAS新成员，主要由Ang II在ACE2作用下水解生成，通过与其特异性Mas受体结合组成ACE2-Ang-(1-7)-Mas途径，与经典途径ACE-Ang II-AT1R既相互抗衡又存在交互作用，共同维持机体内环境的稳定。Ang-(1-7)在多个方面发挥着与Ang II相反的功效，如舒张血管、降低血压、抑制平滑肌增生等，在心、脑、肾等系统的发育及功能修复中具有重要的作用。本课题组前期研究结果表明，Ang-(1-7)显著降低大鼠脑缺血再灌注后氧化应激水平，缩小梗死体积，改善神经功能评分，保护脑缺血再灌注损伤。然而，Ang-(1-7)对高血压脑损伤的病理生理改变有何影响及其在脑缺血损伤中发挥保护作用的机制目前尚不清楚。
鉴于上述，本研究第一部分工作采用自发性高血压大鼠(SHRs)及与之匹配的正常Wistar-Kyoto大鼠(WKYs)，通过微型渗透泵在大鼠侧脑室持续输注Ang-(1-7)，研究不同剂量Ang-(1-7)对高血压大鼠脑内经典RAS通路代谢的影响，及其对脑内氧化应激、神经元凋亡及细胞自噬水平的影响及可能的机制，剖析Ang-(1-7)对高血压大鼠脑内一系列病理生理环节的作用。第二部分建立大鼠永久性脑缺血模型(pMCAO)，探索Ang-(1-7)在脑缺血损伤中发挥保护作用尤其是对缺血后炎症反应的影响及可能的分子机制，深化我们对Ang-(1-7)保护脑缺血损伤的认识，为RAS在脑缺血发生发展过程中的作用提供直接的实验依据。
目前，针对脑卒中的防治，控制高血压等危险因素已引起重视，及时有效的溶栓已经得到认可，神经保护剂的研制也在不断探索中。近年来，脑卒中的内源性保护作用受到越来越多的关注。缺血预适应(IPC)是指组织在遭受一次或多次短暂亚致死性缺血再灌注后，对随后更长时间的严重缺血再灌注损伤的抵抗能力提高。目前IPC是外科干预中研究最为广泛的一种方法，对器官的保护作用得到了大多学者的认可。然而由于缺血事件的不可预见性，限制了其在临床的应用。缺血后适应(IPOC)是组织在经历长时间缺血后，再灌注起始时给予短暂重复的缺血再灌注，可诱导器官的保护作用。IPOC发生在缺血后再灌注的起始阶段，如血管成形术或溶栓治疗时，此时多为临床医生所控制，具有较高的临床应用价值。目前IPOC在心肌缺血介入治疗领域已逐步得到应用，IPOC对脑缺血的保护作用在动物实验中也已得到验证，然而迄今为止，IPOC的机制仍未完全阐明。因此，本文第三部分建立大鼠大脑中动脉阻塞模型，从脑缺血内源性治疗学的角度，探索IPOC在脑缺血损伤中发挥保护作用的机制，为脑卒中的临床治疗学提供新的思路和策略。
第一部分血管紧张素-(1-7)在自发高血压大鼠脑损伤的
作用及机制研究目的：研究血管紧张素-(1-7)对自发高血压大鼠脑内病理生理改变的影响及机制。
方法：通过微型渗透泵对16周龄的SHRs及WKYs侧脑室持续输注aCSF及低、中、高剂量的Ang-(1-7)。4周后，采用ELISA测定脑内Ang II的含量；Westernblotting测定ACE、AT1R、AT2R、iNOS、gp91phox、凋亡及自噬相关蛋白的表达；分光光度计法测定MDA的含量和SOD的活性；TUNEL检测大鼠皮层、海马CA1区和DG区的细胞凋亡数；免疫荧光观察大鼠皮层LC3的荧光表达；透射电镜观察神经元亚显微结构及自噬小体形成。
结果：(1)相对于WKYs，SHRs脑内Ang II水平升高，ACE表达上调，AT1R的表达增加而AT2R表达减少。侧脑室持续输注中、高剂量的Ang-(1-7)显著降低脑内Ang II的水平及AT1R的表达，而对AT2R的表达及ACE的表达和活性无明显影响。(2) SHRs脑内MDA含量升高，SOD活性下降，iNOS及gp91phox的表达增多；中、高剂量的Ang-(1-7)可在不同程度上降低SHRs脑内MDA的含量，上调SOD的活性，减少iNOS及gp91phox的表达。(3) SHRs脑内Bcl-2表达升高，Bax表达降低，TUNEL阳性细胞数较WKYs明显增多；中、高剂量的Ang-(1-7)可显著上调SHRs脑内Bcl-2的表达，下调Bax的表达，减少TUNEL阳性细胞数。(4) SHRs脑内自噬相关蛋白LC3及Beclin1表达较WKYs明显升高，p62表达下降，LC3荧光细胞数增多。电镜结果显示，SHRs脑内细胞核膜破裂，染色质固缩，双层膜结构的自噬小体增多，细胞损伤较重；中、高剂量的Ang-(1-7)显著降低脑内LC3及Beclin1的表达，上调p62，减少LC3荧光细胞数，改善SHRs脑内神经元结构损伤，抑制自噬小体形成。(5)然而，Ang-(1-7)在SHRs脑内的上述效应可被Mas受体拮抗剂A-779取消。
结论： SHRs脑内存在组织RAS的激活，同时氧化应激水平升高，神经元凋亡增多，细胞自噬激活；Ang-(1-7)通过作用于Mas受体在不同程度上减轻SHRs脑内氧化应激损伤，减少神经元凋亡，抑制细胞自噬激活，调节脑内局部RAS的代谢，参与高血压脑损伤病理生理的调节，为进一步阐明RAS在高血压靶器官损伤中的作用奠定了一定的学术基础。
第二部分血管紧张素-(1-7)对大鼠脑缺血损伤后炎症反应的影响
目的：探索血管紧张素-(1-7)对大鼠局灶性脑缺血后炎症反应的影响及可能的机制。
方法：建立大鼠大脑中动脉永久性闭塞模型，术前48h通过微型渗透泵在不同分组大鼠侧脑室持续输注aCSF、Ang-(1-7)或A-779。缺血后24h行神经功能缺损评分及TTC染色检测脑损伤程度；分光光度计法测定MDA的含量和SOD的活性；Western blotting检测各组大鼠缺血侧皮层I-κB、NF-κB p65及COX-2蛋白的表达；免疫组织化学检测NF-κB p65空间分布以及阳性细胞数；ELISA测定脑组织中TNF-、IL-1β含量。
结果：相对于永久性脑缺血大鼠，侧脑室内给予Ang-(1-7)可减小大鼠脑梗死体积，改善神经功能缺损评分，降低脑内MDA的含量，提高SOD的活性，抑制缺血侧皮层细胞NF-κB p65由胞浆向胞核移位，降低NF-κB p65阳性细胞数，并减少COX-2的表达及炎症因子TNF-、IL-1β的含量。Mas受体拮抗剂A-779可加剧大鼠脑缺血损伤，诱导氧化应激，上调NF-κB p65的表达，促进炎症反应，并且取消Ang-(1-7)对脑缺血损伤的保护作用。
结论：Ang-(1-7)通过Mas受体显著改善脑缺血后神经功能障碍，减小脑梗死体积，减轻氧化应激损伤，抑制NF-κB介导的炎症反应，提示NF-κB介导的炎症途径是Ang-(1-7)抑制缺血后炎症反应发挥保护作用的重要靶点，这也为进一步研究RAS在脑缺血损伤中的作用提供了直接的实验证据。
第三部分缺血后适应对大鼠局灶性脑缺血损伤的保护作用及机制研究
目的：研究缺血后适应对局灶性脑缺血大鼠脑保护作用及机制。
方法：通过电凝法建立大鼠局灶性脑缺血后适应模型，自噬激动剂雷帕霉素、自噬抑制剂3-MA在不同分组大鼠侧脑室给药，缺血后24h行TTC染色及脑含水量检测脑损伤程度；Western blotting检测各组大鼠缺血侧皮层LC3、Beclin1、p62及Bcl-2蛋白的表达；免疫荧光检测LC3及Beclin1的荧光细胞数；透射电镜观察神经元亚显微结构及自噬小体形成。
结果：(1) Western blotting结果提示，缺血后1h脑内LC3及Beclin1的表达开始上调，6h差异显著，24h达峰，缺血后48h表达开始下降；而p62的表达则在缺血后1h开始下降，24h最低，持续至缺血后48h。(2)以缺血后24h为观察时间点，IPOC显著减小大鼠脑梗死体积，减轻脑水肿程度，并降低LC3及Beclin1的表达，上调p62的表达，减少LC3和Beclin1的荧光细胞数；同时，电镜的结果显示大鼠脑缺血后皮层神经元出现坏死及凋亡的形态特征，胞浆内自噬小体增多，而IPOC大鼠脑内神经元核膜相对完整，细胞器结构损伤较轻，自噬小体形成减少。(3)然而，相对于IPOC，侧脑室给予雷帕霉素则显著增大了大鼠脑梗死体积，加重脑水肿，并上调LC3及Beclin1的表达，下调p62的表达，同时LC3和Beclin1的荧光细胞数也增加，神经元损伤加重。(4)与单纯缺血组相比，缺血后再灌前给予3-MA能显著减小大鼠脑梗死体积，减轻脑水肿，抑制自噬激活，同时上调抗凋亡蛋白Bcl-2的表达，与IPOC发挥相似的保护作用。
结论：大鼠局灶性脑缺血损伤后存在自噬的激活，且随着缺血时间的不同激活的程度不同。IPOC保护脑缺血损伤，抑制自噬激活，而雷帕霉素可能通过进一步诱导自噬激活阻断IPOC的保护作用。3-MA能够减轻大鼠脑缺血损伤，模拟IPOC的内源性保护作用，为进一步研究脑缺血内源性保护的临床治疗提供新的靶点。
综上所述，本研究工作的主要创新之处在于：
1.血管紧张素-(1-7)在高血压脑损伤的一系列病理生理环节中发挥多靶点保护作用Ang-(1-7)通过作用于Mas受体在不同程度上减轻SHRs脑内氧化应激损伤，减少神经元凋亡，抑制细胞自噬激活，影响脑组织经典RAS通路的代谢，参与高血压脑损伤病理生理的调节，进一步阐明了Ang-(1-7)在高血压发病机制中的重要作用，为靶向于RAS调节药物应用于高血压靶器官损伤的临床治疗提供了理论依据。
2.血管紧张素-(1-7)参与缺血后炎症反应促进神经功能修复脑缺血损伤后存在炎症反应的级联放大效应，Ang-(1-7)通过作用于Mas受体抑制缺血后NF-κB介导的炎症反应，减轻氧化应激损伤，减小梗死体积，促进神经功能修复，诱导缺血后神经保护作用，深化了我们对Ang-(1-7)在脑缺血损伤中作用的认识，为RAS在脑缺血损伤发生发展中的作用提供直接的支持证据。
3.缺血后适应抑制缺血再灌注损伤发挥内源性保护作用IPOC抑制神经元凋亡，抑制缺血后细胞自噬激活，改善神经功能障碍，减轻脑缺血再灌注损伤，启动内源性保护机制，为脑缺血神经保护临床治疗学提供新的思路和靶点。
The neuroprotective role of Ang-(1-7) in hypertension andischemic injury and its related mechanisms
Stroke is one of the main causes of morbidity and mortality worldwidely andpresents as a great burden to family and society. As the most powerful risk factor,hypertension plays a vital role in stroke onset. It has been reported approximately54%of strokes can be attributed to hypertension. Apart from other risk factors, therelative risk of stroke increases by49%and46%when systolic and diastolic bloodpressure is increased by10mmHg and5mmHg, respectively. Therefore, it is of greatsignificance to strengthen hypertension prevention and explore mechanisms ofneuroprotection.
The renin-angiotensin system (RAS) is an important circulative system inenvioromental homeostasis regulation. Classically, angiotensinogen (AGT) ishydrolyzed by renin to form the decapeptide Angiotensin I (Ang I), which is thenconverted by angiotensinconverting enzyme (ACE) into the biologically activepeptide Angiotensin I (Ang II). The Ang II receptor type (AT1R) is the primaryreceptor of Ang II and the ACE-Ang-II-AT1R axis has long been thought to be themain path in blood pressure control and renal sodium and water reabsorption.Overactivation of RAS is always closely associated with the development andmaintenance of hypertension, and the increase of Ang II has been found to be the keytarget of hypertension-related disasters. By modulating different redox signaling, AngII could elevate oxidative stress, promote inflammatory response, accelerate cellapoptosis, induce endothelial dysfunction and stimulate sympathetic tone, thusincreasing the risk of stroke onset. Angiotensin-(1-7)[Ang-(1-7)] is a newlyestablished bioactive fragment of RAS and generated predominately from Ang II byACE2. Through acting with the receptor Mas, the ACE2-Ang-(1-7)-Mas axiscounteract with ACE-Ang-II-AT1R axis and play a vital role in envioromentalhomeostasis. As a protective component of RAS, Ang-(1-7) is reported to oppose thedeleterious effects of Ang II, such as blood pressure downregulation, vasodilatory promotion, vascular smooth muscle cell growth inhibition, and provides beneficialeffects on hypertension-related end-organ damage. Moreover, in a previous studyfrom our group, central administration of Ang-(1-7) was found to ameliorateoxidative stress, minimize the size of cerebral infarction and improve neurologicalfunctions in a rat model of middle cerebral artery occlusion (MCAO). However, themechanisms of Ang-(1-7) on pathophysiology of hypertension and neuroprotection incerebral ischemia are still unknown.
Therefore, in the first part of present study, we adopted spontaneouslyhypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs), to investigate the effects ofAng-(1-7) on classic components of RAS in SHR brain, and to explore whetherAng-(1-7) attenuates brain oxidative stress, prevents neurons from apoptosis andinfluences autophagic activation in SHRs. In the second part, we employedpermanent middle cerebral artery occlusion model (pMCAO) to investigate whetherAng-(1-7) exerts anti-inflammatory effects and contributes to neuroprotectionfollowing cerebral ischaemia.
In the present, blood pressure control has got more and more attention to theprevention and treatment of stroke. Meanwhile, timely thrombolysis has beenrecognized and neuroprotactants are also being constantly explored. In recent years,attention has been focused on the endogenous neuroprotectiond. Ischemicpreconditioning (IPC) is the phenomenon whereby a brief non-injurious episode ofischemic stress renders the organ resistant to a subsequent lethal ischemic insult.Although extensive research has demonstrated that IPC reduces cerebral ischemicdamage, it is clinically feasible only when the occurrence of stroke is predictable.Ischemic postconditioning (IPOC) is induced by a repetitive series of briefinterruptions of reperfusion, which is applied at the onset of reperfusion after aprolonged period of ischemia. It has translational relevance to reperfusion andthrombolitic therapy in acute cerebral ischemia, and represents a promising strategyto reduce ischemia/reperfusion injury. In the research field of myocardial ischemia,the intensive research of IPOC has even led to clinical trials. Moreover, theneuroprotective effects of IPOC also have been proved in animal studies in cerebralischemia. However, the mechanism has not been fully understood. Thus, in the third part, we intended to explore the mechanism of IPOC-induced tolerance to cerebralischemia, and to provide a novel therapeutic strategy for the treatment of stroke.
Part I Angiotensin-(1-7) modulates renin-angiotensin systemassociated with reducing oxidative stress and attenuatingneuronal apoptosis in the brain of hypertensive rats
Objectives: To investigate the effects of Ang-(1-7) on the pathophysiologicchanges caused by hypertension in brain of spontaneously hypertensive rats SHRs.
Methods: Sixteen-week-old male normotensive WKYs and SHRs were chosenin this study. Different doses of Ang-(1-7) or artificial CSF were continuousadministrated by implanted Alzet osmotic minipumps into lateral cerebral ventricle.After4weeks, brain tissues were collected and Ang II was detected by ELISA. Theexpressions of ACE, AT1R, AT2R, iNOS, gp91phox, and some others related toapoptosis and autophagy were detected by western blotting. The MDA content andSOD activity was determined by spectrophotometric assays. TUNEL staining wasperformed to detect the apoptotic neurons in frontal cortex and hippocampus. Inaddition, the immunofluorescence of LC3was analysed with a fluorescencemicroscopy and the morphological changes of neurons were observed under TEM.
Results:(1) Compared with WKYs, the SHRs exhibited a significant increase inAng II, ACE, AT1R expression and a marked reduction in AT2R level. Medium-andhigh-dose of Ang-(1-7) significantly reduced the content of Ang II and expression ofAT1R in SHRs, but it didn’t affect the expressions of AT2R and ACE.(2) In the brainof SHRs, the level of MDA and total SOD activity increased, which is accompaniedby upregulations of NADPH oxidase subunit gp91phoxand iNOS. Medium-andhigh-dose of Ang-(1-7) inhibited the oxidative stress by downregulating MDAcontent and total SOD activity, and decreasing the expressions of gp91phoxand iNOS.(3) The hypertension-induced increase in the percentage of TUNEL-positive neuronsand Bax to Bcl-2ratio in SHRs brain was also attenuated by medium-and high-doseof Ang-(1-7).(4) Compared with WKYs, autophagy was activated with theupregulation of LC3/Beclin1and downregulation of p62in SHRs. Meanwhile, theimmunostaining of LC3increased and the cortical neurons were vacuolated with disrupted cell structure and double-membraned autophagosomes. Medium-andhigh-dose of Ang-(1-7) attenuated autophagy induction by decreasing the expressionsof LC3/Beclin1, increasing p62level and inhibiting autophagosomes formation.(5)All the aforementioned effects induced by Ang-(1-7) were abolished by Mas inhibitorA-779.
Conclusion: Our findings demonstrated that treatment with Ang-(1-7) for4weeks modulated the component of RAS accompanied by reducing oxidative stress,inhibiting neuronal apoptosis and attenuating autophagic activation in the brain ofSHRs. These findings indicated that chronic treatment with Ang-(1-7) may bebeneficial to attenuate hypertension-induced physiopathologic changes in brain andprovide a novel therapeutic foundation to demonstrate the role of RAS inhypertension related disease.
Part II Suppressing inflammation by inhibiting the NF-κBpathway contributes to the neuroprotective effect ofangiotensin-(1-7) in rats with permanent cerebral ischaemia
Objectives: To explore whether Ang-(1-7) exerts anti-inflammatory effect andcontributes to the neuroprotection in a rat model of permanent middle cerebral arteryocclusion.
Methods: We infused aCSF, Ang-(1-7) or A-779into the right lateral ventricleof male Sprague-Dawley rats from48h before onset of pMCAO until the rats werekilled. Twenty-four hours after ischemia, the neuroprotective effect of Ang-(1-7) wasanalysed by evaluating infarct volume and neurological deficits. The MDA contentand SOD activity were determined by spectrophotometric assays. The expression ofNF-κB p65subunit in cell nuclei of the ischemic cortex were determined by westernblotting analysis and the spatial distribution of NF-κB p65subunit was detected byimmunohistochemical assay. The level of COX-2was tested by Western blotting andconcentrations of pro-inflammatory cytokines such as TNF-、IL-1β were measuredby ELISA.
Results: Infusion of Ang-(1-7), i.c.v., significantly reduced infarct volume andimproved neurological deficits. It decreased the level of MDA, increased total SOD activity and suppressed NF-κB activity, which was accompanied by a reduction ofpro-inflammatory cytokines and COX-2in the peri-infarct regions. These effects ofAng-(1-7) were reversed by A-779. Additionally, infusion of A-779alone increasedoxidative stress levels and enhanced NF-κB activity, which was accompanied by anup-regulation of pro-inflammatory cytokines and COX-2.
Conclusion: Our findings indicate that Ang-(1-7) significantly improvedneurological deficits, reduced infarct volume, inhibited oxidative stress andinflammatory response via Mas receptor. Suppressing NF-κB dependent pathwaymay represent one mechanism that contributes to the anti-inflammatory effects ofAng-(1-7) in rats with pMCAO, which offers a direct evidence to study the role ofRAS in stroke onset and exacerbation.
Part III Inhibition of Autophagy Contributes to IschemicPostconditioning-Induced Neuroprotection AgainstFocal Cerebral Ischemia in Rats
Objectives: To determine the role of autophagy in IPOC-inducedneuroprotection against focal cerebral ischemia in rats.
Methods: A focal cerebral ischemic postconditiong model was established withpermanent MCA occlusion plus transient CCA occlusion and postconditioning wasperformed at the onset of reperfusion. Rapamycin and3-MA was infuced into the leftlateral ventricle of different group rats respectively. The expressions of LC3, Beclin1,and p62were evaluated by Western blotting. The immunofluorescence of LC3andBeclin1was analysed with a fluorescence microscopy and the morphologicalchanges of neurons were observed under TEM.
Results: We found that autophagy was markedly induced with the upregulationof LC3/Beclin1and downregulation of p62in the penumbra at various time intervalsfollowing ischemia. IPOC reduced infarct size, attenuated brain edema, inhibited theupregulation of LC3/Beclin1, and reversed the reduction of p62simultaneously.Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effectsinduced by IPOC. Conversely, autophagy inhibitor3-MA attenuated the ischemicinsults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.
Conclusions: The present study suggests that autophagy is induced in cerebralischemia and varied with the exent and time. Inhibition of the autophagic pathwayplays a key role in IPOC-induced protection against focal cerebral ischemia, whichcould be simulated by3-MA. Thus, pharmacological inhibition of autophagy mayprovide a novel therapeutic strategy for the treatment of stroke.
In summary, our present study has the following new concerns:
1. Ang-(1-7) exerts multi-target neuroprotective effects on pathophysiologyof hypertension. By interacting with Mas receptor, chronic treatment with Ang-(1-7)remarkably reduced oxidative stress levels, attenuated neuronal apoptosis,downregulated autophagic induction, and modulated RAS activity in SHRs brain. Allof these findings provide a theoretical basis to further elucidate the importantance ofAng-(1-7) in hypertension and target RAS in clinical treatment ofhypertension-related end-organ damage.
2. Ang-(1-7) participates in inflammatory response and contributes toneuroprotection after stroke. With an interaction with Mas receptor, Ang-(1-7)significantly reduced infarct volume and improved neurological deficits, which wasaccompanied by a reduction of oxidative stress and inflammation suppression. Thesefindings deepen our understanding of the role of Ang-(1-7) in stroke and may offerdirect evidence to recognize the mechanism of RAS in stroke onset and exacerbation.
3. IPOC exerts endogenous neuroprotection and attenuatedischemia/reperfusion injury. IPOC inhibited neuronal apoptosis, downregulatedautophagic induction, attenuated ischemia/reperfusion injury, and then initiatedendogenous neuroprotection after stroke, which may provide a new perspective forthe development of novel options for stroke treatment.
缺血性脑卒中严重危害人类的健康，其发病率高，致死致残率高，给家庭和社会带来了沉重的经济负担。高血压作为脑卒中的独立危险因素，在卒中的发生发展过程中发挥关键的作用。研究表明，全世界约54%的卒中患者死因与高血压直接相关。在控制其它危险因素后，收缩压每升高10mmHg，卒中发病的相对危险增加49%；舒张压每升高5mmHg，卒中发病的相对危险增加46%。因此，加强高血压的防治，探索脑卒中的神经保护机制，具有重大的意义。
肾素-血管紧张素系统(RAS)是机体重要的循环调节系统。经典的RAS通路是血管紧张素原(AGT)在肾素作用下生成血管紧张素I(Ang I)，而Ang I在血管紧张素转化酶(ACE)作用下生成血管紧张素II(Ang II)，与血管紧张素受体1(AT1R)结合构成ACE-Ang II-AT1R轴，在血压调节、水电解质的平衡中发挥重要作用。高血压患者长期存在RAS的过度激活，而Ang II的增加是促使患者发生致死及致残性临床转归终点事件的核心环节。研究表明，Ang II可通过多条交错复杂的途径诱导氧化应激，促使炎症反应，加速细胞凋亡，损伤血管内皮，刺激交感兴奋，最终导致卒中的发生。近年来，RAS的旁路途径逐步受到重视，血管紧张素-(1-7)[Ang-(1-7)]作为RAS新成员，主要由Ang II在ACE2作用下水解生成，通过与其特异性Mas受体结合组成ACE2-Ang-(1-7)-Mas途径，与经典途径ACE-Ang II-AT1R既相互抗衡又存在交互作用，共同维持机体内环境的稳定。Ang-(1-7)在多个方面发挥着与Ang II相反的功效，如舒张血管、降低血压、抑制平滑肌增生等，在心、脑、肾等系统的发育及功能修复中具有重要的作用。本课题组前期研究结果表明，Ang-(1-7)显著降低大鼠脑缺血再灌注后氧化应激水平，缩小梗死体积，改善神经功能评分，保护脑缺血再灌注损伤。然而，Ang-(1-7)对高血压脑损伤的病理生理改变有何影响及其在脑缺血损伤中发挥保护作用的机制目前尚不清楚。
鉴于上述，本研究第一部分工作采用自发性高血压大鼠(SHRs)及与之匹配的正常Wistar-Kyoto大鼠(WKYs)，通过微型渗透泵在大鼠侧脑室持续输注Ang-(1-7)，研究不同剂量Ang-(1-7)对高血压大鼠脑内经典RAS通路代谢的影响，及其对脑内氧化应激、神经元凋亡及细胞自噬水平的影响及可能的机制，剖析Ang-(1-7)对高血压大鼠脑内一系列病理生理环节的作用。第二部分建立大鼠永久性脑缺血模型(pMCAO)，探索Ang-(1-7)在脑缺血损伤中发挥保护作用尤其是对缺血后炎症反应的影响及可能的分子机制，深化我们对Ang-(1-7)保护脑缺血损伤的认识，为RAS在脑缺血发生发展过程中的作用提供直接的实验依据。
目前，针对脑卒中的防治，控制高血压等危险因素已引起重视，及时有效的溶栓已经得到认可，神经保护剂的研制也在不断探索中。近年来，脑卒中的内源性保护作用受到越来越多的关注。缺血预适应(IPC)是指组织在遭受一次或多次短暂亚致死性缺血再灌注后，对随后更长时间的严重缺血再灌注损伤的抵抗能力提高。目前IPC是外科干预中研究最为广泛的一种方法，对器官的保护作用得到了大多学者的认可。然而由于缺血事件的不可预见性，限制了其在临床的应用。缺血后适应(IPOC)是组织在经历长时间缺血后，再灌注起始时给予短暂重复的缺血再灌注，可诱导器官的保护作用。IPOC发生在缺血后再灌注的起始阶段，如血管成形术或溶栓治疗时，此时多为临床医生所控制，具有较高的临床应用价值。目前IPOC在心肌缺血介入治疗领域已逐步得到应用，IPOC对脑缺血的保护作用在动物实验中也已得到验证，然而迄今为止，IPOC的机制仍未完全阐明。因此，本文第三部分建立大鼠大脑中动脉阻塞模型，从脑缺血内源性治疗学的角度，探索IPOC在脑缺血损伤中发挥保护作用的机制，为脑卒中的临床治疗学提供新的思路和策略。
第一部分血管紧张素-(1-7)在自发高血压大鼠脑损伤的
作用及机制研究目的：研究血管紧张素-(1-7)对自发高血压大鼠脑内病理生理改变的影响及机制。
方法：通过微型渗透泵对16周龄的SHRs及WKYs侧脑室持续输注aCSF及低、中、高剂量的Ang-(1-7)。4周后，采用ELISA测定脑内Ang II的含量；Westernblotting测定ACE、AT1R、AT2R、iNOS、gp91phox、凋亡及自噬相关蛋白的表达；分光光度计法测定MDA的含量和SOD的活性；TUNEL检测大鼠皮层、海马CA1区和DG区的细胞凋亡数；免疫荧光观察大鼠皮层LC3的荧光表达；透射电镜观察神经元亚显微结构及自噬小体形成。
结果：(1)相对于WKYs，SHRs脑内Ang II水平升高，ACE表达上调，AT1R的表达增加而AT2R表达减少。侧脑室持续输注中、高剂量的Ang-(1-7)显著降低脑内Ang II的水平及AT1R的表达，而对AT2R的表达及ACE的表达和活性无明显影响。(2) SHRs脑内MDA含量升高，SOD活性下降，iNOS及gp91phox的表达增多；中、高剂量的Ang-(1-7)可在不同程度上降低SHRs脑内MDA的含量，上调SOD的活性，减少iNOS及gp91phox的表达。(3) SHRs脑内Bcl-2表达升高，Bax表达降低，TUNEL阳性细胞数较WKYs明显增多；中、高剂量的Ang-(1-7)可显著上调SHRs脑内Bcl-2的表达，下调Bax的表达，减少TUNEL阳性细胞数。(4) SHRs脑内自噬相关蛋白LC3及Beclin1表达较WKYs明显升高，p62表达下降，LC3荧光细胞数增多。电镜结果显示，SHRs脑内细胞核膜破裂，染色质固缩，双层膜结构的自噬小体增多，细胞损伤较重；中、高剂量的Ang-(1-7)显著降低脑内LC3及Beclin1的表达，上调p62，减少LC3荧光细胞数，改善SHRs脑内神经元结构损伤，抑制自噬小体形成。(5)然而，Ang-(1-7)在SHRs脑内的上述效应可被Mas受体拮抗剂A-779取消。
结论： SHRs脑内存在组织RAS的激活，同时氧化应激水平升高，神经元凋亡增多，细胞自噬激活；Ang-(1-7)通过作用于Mas受体在不同程度上减轻SHRs脑内氧化应激损伤，减少神经元凋亡，抑制细胞自噬激活，调节脑内局部RAS的代谢，参与高血压脑损伤病理生理的调节，为进一步阐明RAS在高血压靶器官损伤中的作用奠定了一定的学术基础。
第二部分血管紧张素-(1-7)对大鼠脑缺血损伤后炎症反应的影响
目的：探索血管紧张素-(1-7)对大鼠局灶性脑缺血后炎症反应的影响及可能的机制。
方法：建立大鼠大脑中动脉永久性闭塞模型，术前48h通过微型渗透泵在不同分组大鼠侧脑室持续输注aCSF、Ang-(1-7)或A-779。缺血后24h行神经功能缺损评分及TTC染色检测脑损伤程度；分光光度计法测定MDA的含量和SOD的活性；Western blotting检测各组大鼠缺血侧皮层I-κB、NF-κB p65及COX-2蛋白的表达；免疫组织化学检测NF-κB p65空间分布以及阳性细胞数；ELISA测定脑组织中TNF-、IL-1β含量。
结果：相对于永久性脑缺血大鼠，侧脑室内给予Ang-(1-7)可减小大鼠脑梗死体积，改善神经功能缺损评分，降低脑内MDA的含量，提高SOD的活性，抑制缺血侧皮层细胞NF-κB p65由胞浆向胞核移位，降低NF-κB p65阳性细胞数，并减少COX-2的表达及炎症因子TNF-、IL-1β的含量。Mas受体拮抗剂A-779可加剧大鼠脑缺血损伤，诱导氧化应激，上调NF-κB p65的表达，促进炎症反应，并且取消Ang-(1-7)对脑缺血损伤的保护作用。
结论：Ang-(1-7)通过Mas受体显著改善脑缺血后神经功能障碍，减小脑梗死体积，减轻氧化应激损伤，抑制NF-κB介导的炎症反应，提示NF-κB介导的炎症途径是Ang-(1-7)抑制缺血后炎症反应发挥保护作用的重要靶点，这也为进一步研究RAS在脑缺血损伤中的作用提供了直接的实验证据。
第三部分缺血后适应对大鼠局灶性脑缺血损伤的保护作用及机制研究
目的：研究缺血后适应对局灶性脑缺血大鼠脑保护作用及机制。
方法：通过电凝法建立大鼠局灶性脑缺血后适应模型，自噬激动剂雷帕霉素、自噬抑制剂3-MA在不同分组大鼠侧脑室给药，缺血后24h行TTC染色及脑含水量检测脑损伤程度；Western blotting检测各组大鼠缺血侧皮层LC3、Beclin1、p62及Bcl-2蛋白的表达；免疫荧光检测LC3及Beclin1的荧光细胞数；透射电镜观察神经元亚显微结构及自噬小体形成。
结果：(1) Western blotting结果提示，缺血后1h脑内LC3及Beclin1的表达开始上调，6h差异显著，24h达峰，缺血后48h表达开始下降；而p62的表达则在缺血后1h开始下降，24h最低，持续至缺血后48h。(2)以缺血后24h为观察时间点，IPOC显著减小大鼠脑梗死体积，减轻脑水肿程度，并降低LC3及Beclin1的表达，上调p62的表达，减少LC3和Beclin1的荧光细胞数；同时，电镜的结果显示大鼠脑缺血后皮层神经元出现坏死及凋亡的形态特征，胞浆内自噬小体增多，而IPOC大鼠脑内神经元核膜相对完整，细胞器结构损伤较轻，自噬小体形成减少。(3)然而，相对于IPOC，侧脑室给予雷帕霉素则显著增大了大鼠脑梗死体积，加重脑水肿，并上调LC3及Beclin1的表达，下调p62的表达，同时LC3和Beclin1的荧光细胞数也增加，神经元损伤加重。(4)与单纯缺血组相比，缺血后再灌前给予3-MA能显著减小大鼠脑梗死体积，减轻脑水肿，抑制自噬激活，同时上调抗凋亡蛋白Bcl-2的表达，与IPOC发挥相似的保护作用。
结论：大鼠局灶性脑缺血损伤后存在自噬的激活，且随着缺血时间的不同激活的程度不同。IPOC保护脑缺血损伤，抑制自噬激活，而雷帕霉素可能通过进一步诱导自噬激活阻断IPOC的保护作用。3-MA能够减轻大鼠脑缺血损伤，模拟IPOC的内源性保护作用，为进一步研究脑缺血内源性保护的临床治疗提供新的靶点。
综上所述，本研究工作的主要创新之处在于：
1.血管紧张素-(1-7)在高血压脑损伤的一系列病理生理环节中发挥多靶点保护作用Ang-(1-7)通过作用于Mas受体在不同程度上减轻SHRs脑内氧化应激损伤，减少神经元凋亡，抑制细胞自噬激活，影响脑组织经典RAS通路的代谢，参与高血压脑损伤病理生理的调节，进一步阐明了Ang-(1-7)在高血压发病机制中的重要作用，为靶向于RAS调节药物应用于高血压靶器官损伤的临床治疗提供了理论依据。
2.血管紧张素-(1-7)参与缺血后炎症反应促进神经功能修复脑缺血损伤后存在炎症反应的级联放大效应，Ang-(1-7)通过作用于Mas受体抑制缺血后NF-κB介导的炎症反应，减轻氧化应激损伤，减小梗死体积，促进神经功能修复，诱导缺血后神经保护作用，深化了我们对Ang-(1-7)在脑缺血损伤中作用的认识，为RAS在脑缺血损伤发生发展中的作用提供直接的支持证据。
3.缺血后适应抑制缺血再灌注损伤发挥内源性保护作用IPOC抑制神经元凋亡，抑制缺血后细胞自噬激活，改善神经功能障碍，减轻脑缺血再灌注损伤，启动内源性保护机制，为脑缺血神经保护临床治疗学提供新的思路和靶点。
The neuroprotective role of Ang-(1-7) in hypertension andischemic injury and its related mechanisms
Stroke is one of the main causes of morbidity and mortality worldwidely andpresents as a great burden to family and society. As the most powerful risk factor,hypertension plays a vital role in stroke onset. It has been reported approximately54%of strokes can be attributed to hypertension. Apart from other risk factors, therelative risk of stroke increases by49%and46%when systolic and diastolic bloodpressure is increased by10mmHg and5mmHg, respectively. Therefore, it is of greatsignificance to strengthen hypertension prevention and explore mechanisms ofneuroprotection.
The renin-angiotensin system (RAS) is an important circulative system inenvioromental homeostasis regulation. Classically, angiotensinogen (AGT) ishydrolyzed by renin to form the decapeptide Angiotensin I (Ang I), which is thenconverted by angiotensinconverting enzyme (ACE) into the biologically activepeptide Angiotensin I (Ang II). The Ang II receptor type (AT1R) is the primaryreceptor of Ang II and the ACE-Ang-II-AT1R axis has long been thought to be themain path in blood pressure control and renal sodium and water reabsorption.Overactivation of RAS is always closely associated with the development andmaintenance of hypertension, and the increase of Ang II has been found to be the keytarget of hypertension-related disasters. By modulating different redox signaling, AngII could elevate oxidative stress, promote inflammatory response, accelerate cellapoptosis, induce endothelial dysfunction and stimulate sympathetic tone, thusincreasing the risk of stroke onset. Angiotensin-(1-7)[Ang-(1-7)] is a newlyestablished bioactive fragment of RAS and generated predominately from Ang II byACE2. Through acting with the receptor Mas, the ACE2-Ang-(1-7)-Mas axiscounteract with ACE-Ang-II-AT1R axis and play a vital role in envioromentalhomeostasis. As a protective component of RAS, Ang-(1-7) is reported to oppose thedeleterious effects of Ang II, such as blood pressure downregulation, vasodilatory promotion, vascular smooth muscle cell growth inhibition, and provides beneficialeffects on hypertension-related end-organ damage. Moreover, in a previous studyfrom our group, central administration of Ang-(1-7) was found to ameliorateoxidative stress, minimize the size of cerebral infarction and improve neurologicalfunctions in a rat model of middle cerebral artery occlusion (MCAO). However, themechanisms of Ang-(1-7) on pathophysiology of hypertension and neuroprotection incerebral ischemia are still unknown.
Therefore, in the first part of present study, we adopted spontaneouslyhypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs), to investigate the effects ofAng-(1-7) on classic components of RAS in SHR brain, and to explore whetherAng-(1-7) attenuates brain oxidative stress, prevents neurons from apoptosis andinfluences autophagic activation in SHRs. In the second part, we employedpermanent middle cerebral artery occlusion model (pMCAO) to investigate whetherAng-(1-7) exerts anti-inflammatory effects and contributes to neuroprotectionfollowing cerebral ischaemia.
In the present, blood pressure control has got more and more attention to theprevention and treatment of stroke. Meanwhile, timely thrombolysis has beenrecognized and neuroprotactants are also being constantly explored. In recent years,attention has been focused on the endogenous neuroprotectiond. Ischemicpreconditioning (IPC) is the phenomenon whereby a brief non-injurious episode ofischemic stress renders the organ resistant to a subsequent lethal ischemic insult.Although extensive research has demonstrated that IPC reduces cerebral ischemicdamage, it is clinically feasible only when the occurrence of stroke is predictable.Ischemic postconditioning (IPOC) is induced by a repetitive series of briefinterruptions of reperfusion, which is applied at the onset of reperfusion after aprolonged period of ischemia. It has translational relevance to reperfusion andthrombolitic therapy in acute cerebral ischemia, and represents a promising strategyto reduce ischemia/reperfusion injury. In the research field of myocardial ischemia,the intensive research of IPOC has even led to clinical trials. Moreover, theneuroprotective effects of IPOC also have been proved in animal studies in cerebralischemia. However, the mechanism has not been fully understood. Thus, in the third part, we intended to explore the mechanism of IPOC-induced tolerance to cerebralischemia, and to provide a novel therapeutic strategy for the treatment of stroke.
Part I Angiotensin-(1-7) modulates renin-angiotensin systemassociated with reducing oxidative stress and attenuatingneuronal apoptosis in the brain of hypertensive rats
Objectives: To investigate the effects of Ang-(1-7) on the pathophysiologicchanges caused by hypertension in brain of spontaneously hypertensive rats SHRs.
Methods: Sixteen-week-old male normotensive WKYs and SHRs were chosenin this study. Different doses of Ang-(1-7) or artificial CSF were continuousadministrated by implanted Alzet osmotic minipumps into lateral cerebral ventricle.After4weeks, brain tissues were collected and Ang II was detected by ELISA. Theexpressions of ACE, AT1R, AT2R, iNOS, gp91phox, and some others related toapoptosis and autophagy were detected by western blotting. The MDA content andSOD activity was determined by spectrophotometric assays. TUNEL staining wasperformed to detect the apoptotic neurons in frontal cortex and hippocampus. Inaddition, the immunofluorescence of LC3was analysed with a fluorescencemicroscopy and the morphological changes of neurons were observed under TEM.
Results:(1) Compared with WKYs, the SHRs exhibited a significant increase inAng II, ACE, AT1R expression and a marked reduction in AT2R level. Medium-andhigh-dose of Ang-(1-7) significantly reduced the content of Ang II and expression ofAT1R in SHRs, but it didn’t affect the expressions of AT2R and ACE.(2) In the brainof SHRs, the level of MDA and total SOD activity increased, which is accompaniedby upregulations of NADPH oxidase subunit gp91phoxand iNOS. Medium-andhigh-dose of Ang-(1-7) inhibited the oxidative stress by downregulating MDAcontent and total SOD activity, and decreasing the expressions of gp91phoxand iNOS.(3) The hypertension-induced increase in the percentage of TUNEL-positive neuronsand Bax to Bcl-2ratio in SHRs brain was also attenuated by medium-and high-doseof Ang-(1-7).(4) Compared with WKYs, autophagy was activated with theupregulation of LC3/Beclin1and downregulation of p62in SHRs. Meanwhile, theimmunostaining of LC3increased and the cortical neurons were vacuolated with disrupted cell structure and double-membraned autophagosomes. Medium-andhigh-dose of Ang-(1-7) attenuated autophagy induction by decreasing the expressionsof LC3/Beclin1, increasing p62level and inhibiting autophagosomes formation.(5)All the aforementioned effects induced by Ang-(1-7) were abolished by Mas inhibitorA-779.
Conclusion: Our findings demonstrated that treatment with Ang-(1-7) for4weeks modulated the component of RAS accompanied by reducing oxidative stress,inhibiting neuronal apoptosis and attenuating autophagic activation in the brain ofSHRs. These findings indicated that chronic treatment with Ang-(1-7) may bebeneficial to attenuate hypertension-induced physiopathologic changes in brain andprovide a novel therapeutic foundation to demonstrate the role of RAS inhypertension related disease.
Part II Suppressing inflammation by inhibiting the NF-κBpathway contributes to the neuroprotective effect ofangiotensin-(1-7) in rats with permanent cerebral ischaemia
Objectives: To explore whether Ang-(1-7) exerts anti-inflammatory effect andcontributes to the neuroprotection in a rat model of permanent middle cerebral arteryocclusion.
Methods: We infused aCSF, Ang-(1-7) or A-779into the right lateral ventricleof male Sprague-Dawley rats from48h before onset of pMCAO until the rats werekilled. Twenty-four hours after ischemia, the neuroprotective effect of Ang-(1-7) wasanalysed by evaluating infarct volume and neurological deficits. The MDA contentand SOD activity were determined by spectrophotometric assays. The expression ofNF-κB p65subunit in cell nuclei of the ischemic cortex were determined by westernblotting analysis and the spatial distribution of NF-κB p65subunit was detected byimmunohistochemical assay. The level of COX-2was tested by Western blotting andconcentrations of pro-inflammatory cytokines such as TNF-、IL-1β were measuredby ELISA.
Results: Infusion of Ang-(1-7), i.c.v., significantly reduced infarct volume andimproved neurological deficits. It decreased the level of MDA, increased total SOD activity and suppressed NF-κB activity, which was accompanied by a reduction ofpro-inflammatory cytokines and COX-2in the peri-infarct regions. These effects ofAng-(1-7) were reversed by A-779. Additionally, infusion of A-779alone increasedoxidative stress levels and enhanced NF-κB activity, which was accompanied by anup-regulation of pro-inflammatory cytokines and COX-2.
Conclusion: Our findings indicate that Ang-(1-7) significantly improvedneurological deficits, reduced infarct volume, inhibited oxidative stress andinflammatory response via Mas receptor. Suppressing NF-κB dependent pathwaymay represent one mechanism that contributes to the anti-inflammatory effects ofAng-(1-7) in rats with pMCAO, which offers a direct evidence to study the role ofRAS in stroke onset and exacerbation.
Part III Inhibition of Autophagy Contributes to IschemicPostconditioning-Induced Neuroprotection AgainstFocal Cerebral Ischemia in Rats
Objectives: To determine the role of autophagy in IPOC-inducedneuroprotection against focal cerebral ischemia in rats.
Methods: A focal cerebral ischemic postconditiong model was established withpermanent MCA occlusion plus transient CCA occlusion and postconditioning wasperformed at the onset of reperfusion. Rapamycin and3-MA was infuced into the leftlateral ventricle of different group rats respectively. The expressions of LC3, Beclin1,and p62were evaluated by Western blotting. The immunofluorescence of LC3andBeclin1was analysed with a fluorescence microscopy and the morphologicalchanges of neurons were observed under TEM.
Results: We found that autophagy was markedly induced with the upregulationof LC3/Beclin1and downregulation of p62in the penumbra at various time intervalsfollowing ischemia. IPOC reduced infarct size, attenuated brain edema, inhibited theupregulation of LC3/Beclin1, and reversed the reduction of p62simultaneously.Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effectsinduced by IPOC. Conversely, autophagy inhibitor3-MA attenuated the ischemicinsults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.
Conclusions: The present study suggests that autophagy is induced in cerebralischemia and varied with the exent and time. Inhibition of the autophagic pathwayplays a key role in IPOC-induced protection against focal cerebral ischemia, whichcould be simulated by3-MA. Thus, pharmacological inhibition of autophagy mayprovide a novel therapeutic strategy for the treatment of stroke.
In summary, our present study has the following new concerns:
1. Ang-(1-7) exerts multi-target neuroprotective effects on pathophysiologyof hypertension. By interacting with Mas receptor, chronic treatment with Ang-(1-7)remarkably reduced oxidative stress levels, attenuated neuronal apoptosis,downregulated autophagic induction, and modulated RAS activity in SHRs brain. Allof these findings provide a theoretical basis to further elucidate the importantance ofAng-(1-7) in hypertension and target RAS in clinical treatment ofhypertension-related end-organ damage.
2. Ang-(1-7) participates in inflammatory response and contributes toneuroprotection after stroke. With an interaction with Mas receptor, Ang-(1-7)significantly reduced infarct volume and improved neurological deficits, which wasaccompanied by a reduction of oxidative stress and inflammation suppression. Thesefindings deepen our understanding of the role of Ang-(1-7) in stroke and may offerdirect evidence to recognize the mechanism of RAS in stroke onset and exacerbation.
3. IPOC exerts endogenous neuroprotection and attenuatedischemia/reperfusion injury. IPOC inhibited neuronal apoptosis, downregulatedautophagic induction, attenuated ischemia/reperfusion injury, and then initiatedendogenous neuroprotection after stroke, which may provide a new perspective forthe development of novel options for stroke treatment.
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