七氟烷后处理对窒息性心搏骤停后脑损伤的保护作用及机制的探究
发布时间：2018-09-17 16:55
【摘要】：心搏骤停(cardiac arrest,CA)是世界范围内的主要致死原因,能够造成全脑严重的缺氧缺血性损害。虽然心肺复苏(cardio-pulmonary resuscitation,CPR)技术的提高和急救医疗体系的完善能挽回部分患者的自主呼吸和循环,提高最初的复苏成功率,但后期的存活率及生存质量却令人沮丧。研究表明,仅不足50%的患者在心搏骤停后得以长期生存,而其中大多患者会遗留有不同程度的认知和行为功能障碍。尽管人们进行了大量的基础研究和临床实践,迄今为止仍没有找到能够有效减少复苏后脑损伤的理想脑保护药物,这已经成为一个亟待解决的临床医学难题。近年来,有研究表明七氟烷后处理能够显著改善局灶性脑损伤的神经缺损,而对心肺复苏后的全脑损伤的保护作用却鲜有报道。磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidyl inositol 3-kinase/protein kinase B,PI3K/Akt)通路为体内最为重要的信号通路之一,参与细胞的生长、存活、代谢及凋亡,在药物处理的动物局灶性脑缺血损伤或全脑缺血损伤模型中发挥了重要的作用。糖原合成激酶-3β(glycogen synthase kinase-3β,GSK-3β)是PI3K/Akt通路的重要下游因子之一,它除了广泛参与体内细胞的生长和发育,还兼有调节血糖和促进肿瘤的形成的作用。研究表明,GSK-3β不仅在四血管栓塞所致的全脑损伤中具有保护作用,而且在关于胰岛素受体的大量研究中发现,GSK-3β还可以通过多种机制来实现体内糖代谢的调节,涉及糖代谢的各个环节,如影响胰岛β细胞的功能、糖原合成等。因此,本实验建立大鼠窒息性心搏骤停模型,在复苏即刻给大鼠吸入不同浓度七氟烷,通过观察复苏后大鼠血糖值、血清神经元特异性烯醇化酶(neuron specific enolase,NSE)值、海马凋亡蛋白P53表达情况、组织病理损伤情况以及大鼠整体认知行为能力的变化,研究七氟烷后处理对窒息性心搏骤停后脑损伤的保护作用;在上述实验的基础上,静脉给予PI3K特异性拮抗剂,评价其对七氟烷后处理神经保护作用的影响,揭示PI3K/Akt通路在七氟烷后处理脑保护中的作用,探究其与抗凋亡和糖代谢调节之间的关系,为心肺复苏脑损伤的救治提供新的思路和方法。实验一:不同浓度七氟烷后处理对窒息性心搏骤停大鼠脑损伤的保护作用目的研究表明,在局灶性脑损伤模型中,七氟烷后处理能够显著改善神经缺损,而对心肺复苏后的全脑损伤的保护作用研究却较少。本实验旨在评价不同浓度七氟烷后处理对大鼠心搏骤停后全脑缺血损伤的保护作用。方法将80只成年雄性SD大鼠随机分为五组:假手术组(sham组)、对照组(control组)、0.5倍最低肺泡有效浓度(minimum alveolar concentration,MAC)、1.0 MAC和1.5 MAC七氟烷后处理组(0.5SP组、1.0SP组及1.5SP组)。Control组、0.5SP组、1.0SP组及1.5SP组建立8min窒息性心搏骤停模型,后给予心肺复苏抢救;各七氟烷后处理组在复苏即刻给予间断吸入七氟烷2次,每次5min,间隔10min,使用气体分析仪确保浓度分别为1.3%、2.5%和3.8%,而control组大鼠仅吸入100%氧气。记录各组大鼠复苏成功率、复苏后72h血糖值、血清神经元特异性烯醇化酶(NSE)含量、海马CA1区神经元形态学变化、海马区P53的表达、复苏后24、72h和7d的神经功能缺损评分(neurological deficit score,NDS),及复苏后7d至11d的大鼠空间学习记忆能力。结果各处理组大鼠复苏成功率没有统计学差异,1.0SP组及1.5SP组在复苏后72h的血糖值、NSE值明显较control组低(P0.05),海马CA1区有较多的存活神经元、较少的凋亡蛋白P53表达(P0.05),神经功能和空间学习记忆能力也较control组水平高(P0.05),而0.5SP组并未表现出明显的神经保护作用。结论较高浓度(1.0MAC和1.5MAC)的七氟烷后处理可以显著改善大鼠窒息性心搏骤停所致的全脑损伤。实验二:七氟烷后处理减轻窒息性心搏骤停脑损伤过程中PI3K/Akt的作用研究目的PI3K/Akt通路是体内最为重要的信号通路之一,参与细胞的生长、存活、代谢及凋亡。而其下游因子GSK-3β可以从糖原合成等多方面参与机体糖代谢调节过程,本文旨在探索七氟烷的脑保护作用是否与PI3K/Akt通路介导的抗凋亡及GSK-3β参与的糖代谢调节作用有关。方法80只健康雄性SD大鼠随机均分为五组:假手术组(sham组)、对照组(control组)、1.0MAC七氟烷后处理组(SP组)、PI3K特异性抑制剂Wortmannin+七氟烷后处理组(W+SP组)和单纯Wortmannin组(W组)。Sham组、control组和SP组具体处理方法同实验一;W+SP组于窒息前30min经股静脉给予Wortmannin,并按实验一方法建立心搏骤停模型,进行心肺复苏,吸入七氟烷并确保呼气末浓度为2.5%;而W组仅于窒息前30min经股静脉给予Wortmannin、并建立心搏骤停模型。记录各组大鼠复苏成功率、复苏后72h血糖值、海马CA1区神经元形态学变化,并观察海马区凋亡相关蛋白Bcl-2、Bax,通路相关蛋白p Akt/Akt、pGSK-3β/GSK-3β等的表达。结果各组大鼠复苏成功率没有统计学差异;较control组,SP组在复苏后72h有较低的血糖值(P0.05),海马CA1区有较多的存活神经元(P0.05),较多的抗凋亡蛋白Bcl-2和较少的凋亡蛋白Bax(P0.05),以及上调了下游通路磷酸化蛋白p Akt及p GSK-3β(P0.05),而对总蛋白没有显著影响;而W+SP组较SP组各结果均有部分拮抗,如血糖值略有升高(P0.05),存活神经元有所减少(P0.05),凋亡相关蛋白也有显著的变化(P0.05),并且部分降低了p Akt和pGSK-3β蛋白的表达(P0.05)。结论在窒息性心搏骤停模型中,七氟烷对全脑损伤的保护作用不仅与PI3K/Akt介导的抑制凋亡作用有关,可能还与GSK-3β参与的糖代谢调节作用密切相关。
[Abstract]:Cardiac arrest (CA) is the leading cause of death worldwide and can cause severe hypoxic-ischemic damage to the whole brain. Studies have shown that fewer than 50% of patients survive long-term after cardiac arrest, and most of them suffer from cognitive and behavioral impairments of varying degrees. Despite extensive basic research and clinical practice, no effective reduction has been found so far. In recent years, studies have shown that sevoflurane postconditioning can significantly improve the neurological deficit of focal brain injury, but the protective effect of sevoflurane postconditioning on whole brain injury after cardiopulmonary resuscitation is rarely reported. Phosphatidylinositol 3-kinase/protein kinase B (p Hosphatidyl inositol 3-kinase / protein kinase B (PI3K / Akt) pathway is one of the most important signaling pathways in vivo. It participates in cell growth, survival, metabolism and apoptosis, and plays an important role in drug-treated animal models of focal cerebral ischemia injury or global cerebral ischemia injury. E-3beta, GSK-3beta) is one of the important downstream factors of PI3K/Akt pathway. It not only participates in the growth and development of cells in vivo, but also regulates blood glucose and promotes tumor formation. Studies have shown that GSK-3beta not only plays a protective role in brain injury induced by four-vessel embolism, but also has a great deal of research on insulin receptors. It was found that GSK-3 beta can also regulate glucose metabolism in vivo through a variety of mechanisms, involving various aspects of glucose metabolism, such as affecting the function of islet beta cells, glycogen synthesis and so on. Serum neuron specific enolase (NSE), expression of apoptotic protein P53 in hippocampus, histopathological damage and changes of cognitive and behavioral abilities in rats were measured to investigate the protective effect of sevoflurane postconditioning on brain injury after asphyxiated cardiac arrest. Specific antagonists were used to evaluate the neuroprotective effect of PI3K/Akt pathway on sevoflurane postconditioning, reveal the role of PI3K/Akt pathway in brain protection after sevoflurane postconditioning, explore the relationship between PI3K/Akt pathway and anti-apoptosis and glucose metabolism regulation, and provide new ideas and methods for the treatment of brain injury after cardiopulmonary resuscitation (CPR). Objective To study the protective effects of sevoflurane postconditioning on brain injury in rats with asphyxiated cardiac arrest.The results showed that sevoflurane postconditioning could significantly improve the nerve defect in the model of focal brain injury,but the protective effects of sevoflurane postconditioning on brain injury after cardiopulmonary resuscitation were seldom studied. Methods 80 adult male SD rats were randomly divided into five groups: sham group, control group, 0.5 times minimum alveolar concentration (MAC), 1.0 MAC and 1.5 MAC sevoflurane postconditioning group (0.5 SP group, 1.0 SP group and 1.5 SP group). Control group, 0.5 SP group, 1.0 SP group and 1.5 SP group. Each sevoflurane treatment group was given intermittent inhalation of sevoflurane 2 times, 5 minutes each time, 10 minutes interval, using gas analyzer to ensure the concentration of 1.3%, 2.5% and 3.8% respectively, while the control group rats only inhaled 100% oxygen. Results The successful rate of resuscitation in each treatment group was 7 to 11 days after resuscitation. There was no significant difference in blood glucose, NSE, P53 expression, neurological function and spatial learning and memory ability between 1.0SP group and 1.5SP group at 72 hours after resuscitation (P 0.05), and there were more surviving neurons in CA1 area of hippocampus, less apoptotic protein P53 expression (P 0.05), but no significant neurological protection in 0.5SP group. Conclusion Sevoflurane postconditioning at high concentrations (1.0MAC and 1.5MAC) can significantly improve the whole brain injury induced by asphyxiated cardiac arrest in rats. Experiment 2: Effect of sevoflurane postconditioning on reducing PI3K/Akt in brain injury induced by asphyxiated cardiac arrest Objective PI3K/Akt pathway is one of the most important signaling pathways in vivo and participates in it. Cell growth, survival, metabolism and apoptosis. The downstream factor GSK-3beta may participate in the regulation of glucose metabolism from glycogen synthesis and other aspects. The aim of this study was to explore whether the brain protective effect of sevoflurane was related to PI3K/Akt pathway-mediated anti-apoptosis and GSK-3beta-mediated regulation of glucose metabolism. They were divided into five groups: sham group, control group, 1.0 MAC sevoflurane post-treatment group (SP group), PI3K specific inhibitor Wortmannin + sevoflurane post-treatment group (W + SP group) and pure Wortmannin group (W group). The specific treatment methods of Sham group, control group and SP group were the same as those of experiment 1. In addition, the cardiopulmonary resuscitation was performed by inhaling sevoflurane and ensuring 2.5% end-expiratory concentration. In group W, Wortmannin was administered via femoral vein 30 minutes before asphyxia, and the cardiac arrest model was established. Results There was no significant difference in the success rate of resuscitation among the groups. Compared with control group, SP group had lower blood glucose level 72 hours after resuscitation (P 0.05), hippocampal CA1 area had more surviving neurons (P 0.05), more anti-apoptotic proteins Bcl-2 and less. Apoptotic protein Bax (P 0.05), and up-regulation of phosphorylated protein P Akt and P GSK-3 beta (P 0.05) in the downstream pathway had no significant effect on total protein, but W+SP group had some antagonistic effects on the results of SP group, such as a slight increase in blood sugar (P 0.05), a decrease in survival neurons (P 0.05), and a significant change in apoptosis-related proteins (P 0.05). Conclusion The protective effect of sevoflurane on brain injury in asphyxiated cardiac arrest model is not only related to PI3K/Akt-mediated inhibition of apoptosis, but also closely related to GSK-3 beta-mediated regulation of glucose metabolism.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
[Abstract]:Cardiac arrest (CA) is the leading cause of death worldwide and can cause severe hypoxic-ischemic damage to the whole brain. Studies have shown that fewer than 50% of patients survive long-term after cardiac arrest, and most of them suffer from cognitive and behavioral impairments of varying degrees. Despite extensive basic research and clinical practice, no effective reduction has been found so far. In recent years, studies have shown that sevoflurane postconditioning can significantly improve the neurological deficit of focal brain injury, but the protective effect of sevoflurane postconditioning on whole brain injury after cardiopulmonary resuscitation is rarely reported. Phosphatidylinositol 3-kinase/protein kinase B (p Hosphatidyl inositol 3-kinase / protein kinase B (PI3K / Akt) pathway is one of the most important signaling pathways in vivo. It participates in cell growth, survival, metabolism and apoptosis, and plays an important role in drug-treated animal models of focal cerebral ischemia injury or global cerebral ischemia injury. E-3beta, GSK-3beta) is one of the important downstream factors of PI3K/Akt pathway. It not only participates in the growth and development of cells in vivo, but also regulates blood glucose and promotes tumor formation. Studies have shown that GSK-3beta not only plays a protective role in brain injury induced by four-vessel embolism, but also has a great deal of research on insulin receptors. It was found that GSK-3 beta can also regulate glucose metabolism in vivo through a variety of mechanisms, involving various aspects of glucose metabolism, such as affecting the function of islet beta cells, glycogen synthesis and so on. Serum neuron specific enolase (NSE), expression of apoptotic protein P53 in hippocampus, histopathological damage and changes of cognitive and behavioral abilities in rats were measured to investigate the protective effect of sevoflurane postconditioning on brain injury after asphyxiated cardiac arrest. Specific antagonists were used to evaluate the neuroprotective effect of PI3K/Akt pathway on sevoflurane postconditioning, reveal the role of PI3K/Akt pathway in brain protection after sevoflurane postconditioning, explore the relationship between PI3K/Akt pathway and anti-apoptosis and glucose metabolism regulation, and provide new ideas and methods for the treatment of brain injury after cardiopulmonary resuscitation (CPR). Objective To study the protective effects of sevoflurane postconditioning on brain injury in rats with asphyxiated cardiac arrest.The results showed that sevoflurane postconditioning could significantly improve the nerve defect in the model of focal brain injury,but the protective effects of sevoflurane postconditioning on brain injury after cardiopulmonary resuscitation were seldom studied. Methods 80 adult male SD rats were randomly divided into five groups: sham group, control group, 0.5 times minimum alveolar concentration (MAC), 1.0 MAC and 1.5 MAC sevoflurane postconditioning group (0.5 SP group, 1.0 SP group and 1.5 SP group). Control group, 0.5 SP group, 1.0 SP group and 1.5 SP group. Each sevoflurane treatment group was given intermittent inhalation of sevoflurane 2 times, 5 minutes each time, 10 minutes interval, using gas analyzer to ensure the concentration of 1.3%, 2.5% and 3.8% respectively, while the control group rats only inhaled 100% oxygen. Results The successful rate of resuscitation in each treatment group was 7 to 11 days after resuscitation. There was no significant difference in blood glucose, NSE, P53 expression, neurological function and spatial learning and memory ability between 1.0SP group and 1.5SP group at 72 hours after resuscitation (P 0.05), and there were more surviving neurons in CA1 area of hippocampus, less apoptotic protein P53 expression (P 0.05), but no significant neurological protection in 0.5SP group. Conclusion Sevoflurane postconditioning at high concentrations (1.0MAC and 1.5MAC) can significantly improve the whole brain injury induced by asphyxiated cardiac arrest in rats. Experiment 2: Effect of sevoflurane postconditioning on reducing PI3K/Akt in brain injury induced by asphyxiated cardiac arrest Objective PI3K/Akt pathway is one of the most important signaling pathways in vivo and participates in it. Cell growth, survival, metabolism and apoptosis. The downstream factor GSK-3beta may participate in the regulation of glucose metabolism from glycogen synthesis and other aspects. The aim of this study was to explore whether the brain protective effect of sevoflurane was related to PI3K/Akt pathway-mediated anti-apoptosis and GSK-3beta-mediated regulation of glucose metabolism. They were divided into five groups: sham group, control group, 1.0 MAC sevoflurane post-treatment group (SP group), PI3K specific inhibitor Wortmannin + sevoflurane post-treatment group (W + SP group) and pure Wortmannin group (W group). The specific treatment methods of Sham group, control group and SP group were the same as those of experiment 1. In addition, the cardiopulmonary resuscitation was performed by inhaling sevoflurane and ensuring 2.5% end-expiratory concentration. In group W, Wortmannin was administered via femoral vein 30 minutes before asphyxia, and the cardiac arrest model was established. Results There was no significant difference in the success rate of resuscitation among the groups. Compared with control group, SP group had lower blood glucose level 72 hours after resuscitation (P 0.05), hippocampal CA1 area had more surviving neurons (P 0.05), more anti-apoptotic proteins Bcl-2 and less. Apoptotic protein Bax (P 0.05), and up-regulation of phosphorylated protein P Akt and P GSK-3 beta (P 0.05) in the downstream pathway had no significant effect on total protein, but W+SP group had some antagonistic effects on the results of SP group, such as a slight increase in blood sugar (P 0.05), a decrease in survival neurons (P 0.05), and a significant change in apoptosis-related proteins (P 0.05). Conclusion The protective effect of sevoflurane on brain injury in asphyxiated cardiac arrest model is not only related to PI3K/Akt-mediated inhibition of apoptosis, but also closely related to GSK-3 beta-mediated regulation of glucose metabolism.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
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