从炎症反应和氧化应激探究麻醉剂影响老龄大鼠认知功能的分子机制

发布时间：2019-05-29 01:10

【摘要】：由血管损伤和炎症引起的认知障碍影响着大脑功能。研究已经证实,认知障碍与血管性损伤、炎症直接关联,而损伤程度与炎症、血管性损伤呈正相关。研究表明,大脑在认知功能上发挥着自已的特殊作用,全面的大脑保护可能在急性心肌缺血再灌注损伤的情况下,有助于降低患者的认知障碍。七氟醚对抗心肌和脑损伤能起到积极的作用,因为它有助于降低心肌耗氧量,抑制心肌钙离子的运动,激活线粒体信号传导通路,并且阻止炎症因子的产生。目前对于血管损伤的治疗主要依赖于手术与麻醉的联合治疗,然而,关于七氟醚在急性心肌缺血再灌注损伤导致的认知障碍治疗方面的效果,没有直接的和系统化的验证。在本研究中,我们设法确定麻醉剂七氟醚和芬太尼对于大鼠脑组织长期认知功能的影响,以及和炎性因子如血管内皮生长因子(VEGF)、白介素-1β (IL-1β)和肿瘤坏死因子-α (TNF-α)潜在的相关性。我们使用穿梭箱和水迷宫测试去研究Wistar大鼠的认知功能。结果表明,经过七氟醚或芬太尼治疗的大鼠同急性心肌缺血再灌注大鼠模型相比,电击次数更少并且有更多的主动逃生次数。麻醉剂的治疗也缩短学习和记忆的周期,这表明了麻醉剂在认知功能方面的保护作用。此外,我们的研究结果揭示在使用麻醉药的大鼠头部组织中TNF-α和IL-1β表达降低而VEGF表达升高。这些发现强调了通过调节炎症因子的表达,七氟醚和芬太尼在急性心肌缺血再灌注损伤老龄大鼠的认知功能恢复中发挥着改善作用。与微血管损伤相关的缺血组织再灌注是炎症反应活化的直接后果。临床治疗血管损伤的主要方法是手术结合麻醉,常规的麻醉剂如丙泊酚可能引起自主认知功能障碍,不利于血管损伤后认知障碍的康复。所以我们计划通过动物实验的方法,对于七氟醚和芬太尼对急性心肌缺血再灌注损伤的老龄大鼠大脑的保护作用进行调查。前期研究中,我们认为七氟醚和芬太尼在上调VEGF表达,降低TNF-α和IL-1β表达水平时,可能有助于老龄大鼠术后认知功能损害的康复。本研究拟从分子机制方面探讨七氟醚和芬太尼对老龄大鼠认知功能的影响,探讨其对认知功能的保护机制。120只Wistar大鼠随机分为四组,A组大鼠接受假手术,B组大鼠为急性心肌缺血再灌注模型,C组为B组的基础上吸入七氟醚,D组在B组的基础上静脉注射芬太尼。采用TUNEL检测Wistar大鼠海马神经元细胞凋亡指数,采用流式细胞术测定海马神经元的凋亡率和胞浆内钙离子水平。再利用荧光探针测定鼠海马神经元胞浆中线粒体的膜电位情况。酶联免疫吸附试验(ELISA)测定大鼠心尖血液中炎症因子的含量。测定各组经前述方法分离提纯的海马神经元细胞匀浆内线粒体呼吸链复合物活性。使用麻醉药的大鼠血液中TNF一α和IL-1β表达降低而VEGF表达升高。麻醉剂的治疗降低了神经元凋亡率和凋亡指数,减轻了缺血时钙离子向神经元细胞内的转移,提高了神经元胞浆内线粒体膜电位活性和线粒体呼吸链复合物Ⅰ～Ⅳ活性。麻醉剂可以有效减轻老龄大鼠因急性心肌缺血再灌注损伤造成的认知功能障碍,其机制与提高VEGF表达水平,促进新生血管形成过程,减轻炎症因子所参与的对海马神经元细胞所造成的炎性损伤有关。此外,麻醉剂还能够减轻海马神经元内氧化应激过程,从而减少了海马神经元细胞凋亡,减轻了认知功能障碍的发生。
[Abstract]:Cognitive disorders caused by vascular injury and inflammation affect the brain function. The study has confirmed that cognitive impairment is directly related to vascular injury and inflammation, and the degree of injury is positively related to inflammation and vascular injury. The study shows that the brain plays a special role in the cognitive function, and the comprehensive brain protection can help to reduce the cognitive disorder of the patient under the condition of acute myocardial ischemia-reperfusion injury. Sevoflurane can play a positive role in the fight against myocardial and brain damage, as it helps to reduce the oxygen consumption of the myocardium, inhibit the movement of the calcium ions in the myocardium, activate the mitochondrial signaling pathway, and prevent the generation of inflammatory factors. At present, the treatment of vascular injury mainly depends on the combination of operation and anesthesia. However, the effect of sevoflurane on the treatment of cognitive impairment caused by reperfusion injury of acute myocardial ischemia is not directly and systematically verified. In this study, we managed to determine the effects of anesthetic sevoflurane and fentanyl on the long-term cognitive function of brain tissue in rats, as well as the potential correlation of inflammatory factors such as vascular endothelial growth factor (VEGF), interleukin-1 (IL-1), and tumor necrosis factor-1 (TNF-1). We used the shuttle box and the water maze test to study the cognitive function of the Wistar rats. The results showed that compared with the model of acute myocardial ischemia-reperfusion in rats treated with sevoflurane or fentanyl, the number of electric shock was less and the number of active escape was more. The treatment of the anesthetic also shortens the cycle of learning and memory, which shows the protective effect of the anesthetic in the cognitive function. In addition, our findings reveal a decrease in the expression of TNF-1 and IL-1 in the head tissue of a rat using an anesthetic, while the expression of VEGF is increased. These findings highlight the improvement in the recovery of cognitive function in aged rats by modulating the expression of inflammatory factors, sevoflurane and fentanyl. Ischemia-reperfusion associated with microvascular injury is a direct consequence of inflammatory response activation. The main method of the clinical treatment of vascular injury is to combine the operation with the anesthesia, and the conventional anesthetic such as propofol may cause the self-cognitive dysfunction, which is not conducive to the rehabilitation of the cognitive disorder after the vascular injury. So we plan to investigate the protective effect of sevoflurane and fentanyl on the brain of aged rats with acute myocardial ischemia-reperfusion injury through the method of animal experiment. In the earlier study, we think that sevoflurane and fentanyl may be helpful to the recovery of cognitive function impairment in aged rats at the time of up-regulation of the expression of VEGF, the reduction of the level of TNF-1 and IL-1. In this study, the effects of sevoflurane and fentanyl on the cognitive function of aged rats were discussed from the aspects of molecular mechanism, and the protection mechanism for cognitive function was discussed.120 Wistar rats were randomly divided into four groups. In group C, the group B was given a group B, and the group B was given fentanyl on the basis of group B. TUNEL was used to detect the apoptosis index of the rat hippocampal neurons, and the apoptosis rate and the intracellular calcium ion level of the hippocampal neurons were measured by flow cytometry. The membrane potential of the mitochondria in the rat hippocampal neurons was determined by using the fluorescence probe. Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of inflammatory factors in the apical blood of the rat. The activity of the mitochondrial respiratory chain complex in the homogenate of the cultured hippocampal neurons was determined by the above method. The expression of TNF-1 and IL-1 in the blood of rats using the anesthetic was decreased and the expression of VEGF was increased. The treatment of the anesthetic reduces the neuron apoptosis rate and the apoptosis index, reduces the transfer of the calcium ions to the neuron cells during the ischemia, and improves the mitochondrial membrane potential activity and the mitochondrial respiratory chain complex I-IV activity in the neuron cytoplasm. The anesthetic can effectively relieve the cognitive dysfunction caused by acute myocardial ischemia-reperfusion injury in the aged rats, and the mechanism is related to the improvement of the expression level of the VEGF, the promotion of the formation of the new blood vessel and the reduction of the inflammatory injury caused by the inflammatory factors involved in the hippocampal neuronal cells. In addition, the anesthetic can reduce the oxidative stress in the hippocampal neurons, thereby reducing the apoptosis of the hippocampal neurons and reducing the occurrence of cognitive dysfunction.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R614.2

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