【作者】 黄峰；

【导师】 朱鹏立； 林帆；

【作者基本信息】 福建医科大学 ， 老年医学， 2010， 硕士

【摘要】 研究背景:高血压病是一种严重危害人类健康的多发病、常见病。高血压所致的脑血管损害是高血压并发症之一。长期持续的高血压可引起小动脉硬化,内膜及弹性纤维增生,中膜平滑肌细胞增生、肥大并伴不同程度的胶原纤维和弹力纤维增生,血管壁增厚,管腔狭窄。脑动脉硬化造成脑局部组织缺血,毛细血管通透性增高,脑部可发生一系列神经生理学和脑组织结构的改变。大量的临床和实验表明,氧化应激与高血压及并发症的发生发展密切相关。高血压状态下,不仅活性氧(ROS)产生增多,同时高血压还能对有清除自由基作用的各种抗氧化剂酶的活性产生影响,使自由基的产生和抗氧化防御之间严重失衡,脑、心、肾等靶器官氧化应激反应增强,从而导致高血压靶器官损害。晚期糖基化终产物(AGEs)是一组在蛋白质、脂肪酸或核酸的氨基基团与还原糖的醛基之间发生非酶性糖基化反应(又称Maillard反应)所形成的一系列具有高度活性终产物的总称。组织中AGEs的聚集能引起很多毒性反应,它对细胞的毒性作用通过与其特异性受体——晚期糖基化终产物受体(RAGE)的结合来实现的。AGEs-RAGE系统通过信号传导产生氧化应激反应和炎症反应参与高血压靶器官损害的进程。通过阻滞AGEs的形成就可以阻断AGEs-RAGE的信号传导系统,减少氧化应激反应所致的靶器官损害。目的:1.观察高血压脑组织氧化应激指标的变化。2.观察羧甲基赖氨酸(CML)(AGEs主要成分之一)、RAGE以及与AGEs-RAGE信号传导途径相关的炎症因子核转录因子kappa B(NF-κB)、血管细胞粘附分子-1(VCAM-1)、细胞间粘附分子-1(ICAM-1)在高血压脑组织中的表达。3.探讨替米沙坦、吡哆胺及两者联合干预后对氧化应激指标及AGEs-RAGE信号传导通路的影响。4.探讨AGEs-RAGE信号传导通路及氧化应激在高血压脑损害中的作用及可能机制,探寻高血压脑损害的早期有效的防治方法。方法:选用22周龄、雄性自发性高血压大鼠(SHR)48只,威斯塔京都大鼠(WKY)大鼠13只,适应性喂养2周,SHR随机分为4组,每组12只。高血压对照组(HC组)每天用蒸馏水2ml灌胃;替米沙坦组(T组)每天用6mg/kg的替米沙坦溶解于2ml蒸馏水灌胃;吡哆胺组(P组)每天用200mg/kg的盐酸吡哆胺溶解于2ml蒸馏水灌胃;联合治疗组(TP组)每天用6mg/kg的替米沙坦加上200mg/kg的盐酸吡哆胺溶解于2ml蒸馏水灌胃。12只WKY大鼠作为正常对照组(NC组)每天用蒸馏水2ml灌胃。干预16周后测量以下指标:1.测定各组大鼠的体重和收缩压。2.比色法测定脑组织中SOD活性和MDA含量。3.免疫组化检测脑组织RAGE的表达。4.Western Blot检测CML、RAGE、VCAM-1、ICAM-1蛋白的表达。5.荧光实时定量PCR法检测RAGE、NF-κB mRNA的表达。结果:1.干预16周后各组大鼠血压的变化实验开始前,HC组、T组、P组和TP组血压明显高于NC组;干预16周后,T组和TP组与HC组比较血压明显降低(P<0.01),而P组与HC组比较血压无明显统计学差异(P>0.05)。2.各组大鼠海马病理学检测HE染色显示NC组大鼠海马CAI区锥体细胞形态规则,排列整齐,数量丰富,核膜清晰,核仁明显;HC组大鼠海马CAI区锥体细胞层变薄、紊乱,神经元变性坏死明显,排列稀疏,严重脱失,残留的锥体细胞胞核变浅,基质疏松,可见微空泡形成,部分可见核固缩;T组、TP、P组锥体细胞数量介于两者之间,排列较整齐,神经元形态结构接近正常并且明显减轻了海马部分细胞核固缩现象。3.MDA含量、SOD活性检测结果与NC组比较,HC组MDA含量明显升高、SOD活性明显减低(P<0.05);与HC组比较T组、P组、TP组MDA含量明显减低,SOD活性明显升高(P<0.05)。4.免疫组化检测RAGE的表达结果NC组大鼠海马CAI区可见极少量RAGE在细胞膜和胞浆的表达,表达较弱,表达强度+;HC组在细胞膜及胞浆中可见明显RAGE的表达,表达强度+++;与HC组比较,T组、TP组表达强度减弱,阳性细胞数量减少,表达强度+。5.Western Blotting检测结果药物干预16周后,5组大鼠CML、RAGE、VCAM-1、ICAM-1蛋白水平的表达变化如下:(1)NC组仅见极少量CML蛋白表达,HC组CML蛋白表达与NC组比明显增加(P<0.01);药物干预的T组、P组、TP组CML的表达下调与HC组比较有明显差异(P<0.01)。(2)HC组RAGE蛋白表达与NC组比明显增加(P<0.01);药物干预的T组、TP组RAGE的表达与HC组比较下降有显著性差异(P<0.01);而P组与HC组比较无显著性差异(P>0.05)。(3)NC组ICAM-1、VCAM-1蛋白表达量极少,HC组的表达较NC组显著上调(P<0.01);T组、P组和TP组ICAM-1、VCAM-1蛋白表达较HC组呈显著下调(P<0.01)。6.荧光实时定量PCR检测结果荧光PCR检测NF-κB、RAGE mRNA表达检测结果示:与NC组比较HC组NF-κB、RAGE mRNA表达显著上调(P<0.05),T组、P组、TP组NF-κB mRNA表达显著下降(P<0.05);RAGE mRNA仅在T组、TP组表达下调(P<0.05),而P组与HC组比较RAGE mRNA表达无显著性差异(P>0.05)。结论:1.高血压脑组织氧化应激增强,可能是高血压脑损害途径之一。2.高血压脑组织AGEs-RAGE系统表达增加,微炎症相关因子NF-κB、VCAM-1、ICAM-1表达也明显增加。3.替米沙坦可能通过抑制RAS系统、AGEs-RAGE系统减轻高血压脑损害;吡哆胺通过抑制AGEs的形成保护脑组织;但两者联合应用无显著的协同作用。4.AGEs-RAGE系统的激活可能是高血压脑损害的发病机制之一。5.使用替米沙坦和吡哆胺可能有利于预防早期高血压脑损害。更多还原

【Abstract】 Background:Today hypertension has become a commonly and frequently encountered disease.The impairment of the cerebral vessel is one of the most important complication of hypertension.It is well known to all that persistent hypertension causes pathological change of systemic anteriola,which is represented by the hyperplasy and fibrosis of vessel intermedial smooth muscles,the thickening vessel wall,and stenosis of lumens. The eventual results of the above mentioned pathogenesis are brain atrophy and functional deterioration.Oxidative stress is important in the pathogenesis of hypertension complication. High level active oxygen(ROS)can affect the activity and expression of antioxidases which can clear free radicals in hypertension.There is evidence that hytertensive stimuli,such as high salt and angiotensionⅡ,promote the production of ROS contributes either to hypertension or to the complication of hypertension.Advanced glycation end products (AGEs) develop through complex, sequential reactions, collectively called the Maillard reaction. Accumulation of AGEs in tissues has been implicated in the progression of chronic diseases, these compounds have also been shown to play a pivotal role in acute and chronic atherosclerotic disease, causing structural protein changes in the vascular wall, as well as activating cellular receptors, such as the receptor for AGEs(RAGE),which in turn leads to activation of several oxidative and inflammatory pathways .Hypertension is one of atherosclerotic disease.AGEs and its receptors (RAGE) also play an important role in pathological terms of hypertension complication. Engagement of RAGE by AGEs activates its downstream signaling and subsequently evokes oxidative stress and inflammatory responses,thus contributing to the development and progression of hypertension. Therefore,inhibition of the AGEs-RAGE expression may be a promising target for therapeutic intervention in this devastating disorder.Objective:1.To explor the condition of oxidative stress in the brain tissue of SHR.2.To observe the expression of AGEs-RAGE system (CML、RAGE、NF-κB、VCAM-1、ICAM-1)in the brain tissue of the SHR model rats.3. To investigate changs of AGEs-RAGE system, oxidative stress in brain tissues of SHR model rat interferd with Telmisartan and Pridoxamine4.To research the effect and role of AGEs-RAGE system, oxidative stress on brain damage caused by hypertension, and to explore the possible effective treatment of it in the early stage.Methods:48 SHR male rats with 24 weeks’ages were divided averagely into four groups at random:the spontaneously hypertensive rat group(HC group, lavaged with water 2ml), the Telmisartan treatment group(T group lavaged with Telmisartan 8mg/kg),the Pridoxamine treatment group(P group lavaged with Pridoxamine 200mg/kg), the Telmisartan and Pridoxamine treatment group(TP group lavaged with Telmisartan 8mg/kg and Pridoxamine 200mg/kg). The homongenous WKY rats were 12 Wistar-kyoto rats,served as the normal control group(NC group, lavaged with water 2ml).All the rats had been fed for 16 weeks.At the end of the 16th week ,the weight and blood pressure were valued.Activities of superoxide dismutase(SOD),maleic dialdehyde (MDA)were measured by chromatometry.The expression of RAGE、NF-κB were detected by Quantitative real-time PCR.The expression of RAGE was detected by immunohistochemistry. Western Blot was performed to detect the expression of CML、RAGE、NF-κB、VCAM-1、ICAM-I.Rusults:1.Effect of Telmisartan and Pridoxamine on the Blood Pressure of SHRThe blood pressure was obviously higher in the HC、T、TP group than that in NC group(P<0.01);after drug interference for 16 weeks,the T group and TP group was obviously reduced compared with HC group(P<0.01).There were no obvious difference between P group and HC group(P>0.05).2.The Pathological changes of hippocamps in groupsHE staining displayed that the pyramidal neuron of the hippocamp CAI in NC group had regulated shape and quantity with clear caryotheca, conspicuous nucleolus and lined up in order;However,the pyramidal neuron of the hippocamp CA1 in HC group had unregulated shape and the less quantity with loose matris and pyknosis.The shape and quantity of the pyramidal neuron of the hippocamp CAI in T group、P group and TP group were between NC groups and HC group, pyknosis were less than it in HC group.3.The activities of SOD and the level of MDACompared with NC group,the level of MDA in HC group significantly increased,the activities of SOD in HC group significantly decreased (P<0.05);Compared with HC group,the level of MDA in T group、P group and TP group significantly decreased,the activities of SOD in T group、P group and TP group significantly increased (P<0.05).4. ImmunohistochemistryThe expression of RAGE was mainly on the cellular membrane and kytoplasm of the pyramidal neuron of the hippoeamp CAI in NC group,the expression was fairly Poor;The expression of RAGE in the hippocamp CA1 was strongly positive;Compared with HC group,the expression of RAGE significantly decreased,the quantity of positive cell decreased.5.Consequence Western blotting (1)Compared with NC group, the expression of CML significantly increased in HC group (P<0.01),Compared with HC group,the level of CML in T group、P group and TP group significantly decreased (P <0.01);(2)Compared with NC group, the expression of RAGE in HC group significantly increased (P<0.01),Compared with HC group,the level of RAGE in T group and TP group significantly decreased (P<0.01). There were no obvious difference between P group and HC group(P>0.05).(3) Compared with NC group, the expression of VCAM-1、ICAM-1 significantly increased in HC group (P<0.01),Compared with HC group,the level of VCAM-1、ICAM-1 in T group、P group and TP group significantly decreased (P<0.01).6.The mRNA expression of RAGE、NF-κBThe mRNA expression of RAGE、NF-κB in HC group was significantly higher compared to those in NC group(P<0.01);Compared with HC group,the mRNA expression of NF-κB significantly desceased in T group、P group and TP group;the mRNA expression of RAGE was no significantly difference between P group and HC group.Conclusion:1.Hypertension can up-regulate the expression of ROS and decrease the activity of antioxidase in brain,it is hypothesized that increased the level of oxidative stress might lead to the brain damage of hypertension.2.Hypertension can up-regulate the expression of AGEs-RAGE、activate NF-κB and VCAM-1,ICAM-1.3. Telmisartan and Pridoxamine may inhibit the expression RAS and AGEs-RAGE, decrease oxidative stress ,delay progression of brain damage in hypertension.4. AGEs-RAGE might play a part in the harm of the brain caused by hypertension.5. Telmisartan and Pridoxamine might prevent brain harm caused by hypertension the early stage.更多还原