脑缺血缺氧中缺氧诱导因子HIF-1α对小胶质细胞自噬调节的机制研究
发布时间：2018-09-06 17:59
【摘要】：缺血性脑卒中是一种由于脑血流中断并导致神经功能障碍的疾病。缺血性脑卒中的发病率在急性脑血管中占有很高的比例，并且具有很高的伤残率，已严重危害了人类的健康。根据相关研究调查，我国老年患者每年新发缺血性脑卒中的人数达到120-150万左右，其中死亡人数达到80-100万左右。在幸存患者中，半数以上伴有不同程度的神经功能残疾。随着我国的老龄人口的逐渐增加，缺血性脑卒中的发病人数将进一步增加。因此，缺血性脑卒中成为人类健康的头号敌人，深入研究缺血性脑卒中的发病机制及治疗策略具有重要的科学意义和临床价值。 当前对于缺血性脑卒中的发病机制及治疗策略提出了全新的概念-血管神经网络学说。该学说研究认为：血管内皮细胞、神经元、胶质细胞以及周围细胞外基质等成分有机组成了血管神经单元复合体。复合体内的各种细胞成分及复合体之间互相影响和调控，形成了网络结构。该学说认为缺血性脑卒中不是单纯的血管事件，也不是单存的神经事件，而是血管与神经之间，实质细胞与基质之间的相互影响和对话，互相调节和平衡的作用过程。因此，在缺血性脑卒中后，所有的神经细胞和细胞成分都应该受到重视和保护，而不是单纯保护某种细胞。在以前的缺血性脑卒中的大量相关研究都关注神经元细胞和血管内皮细胞，而缺乏对胶质细胞的深入研究。目前逐渐认为：小胶质细胞介导的炎症反应在缺血性脑卒中后发挥重要的损伤作用。 小胶质细胞与正常细胞一样，在缺氧环境下均能感受低氧信号的刺激，并能活化相应的信号通路，启动相应基因的表达，适应低氧微环境对细胞的有害作用，并维持细胞内环境的稳定。当细胞受到低氧信号刺激后，有多种基因诱导表达。其中低氧诱导因子(HIF-1)发挥重要的作用。研究表明HIF-1可以在缺氧环境下迅速启动下游相关基因的活化，调节细胞内的能量代谢水平和对氧的利用，在机体器官或局部组织发挥对低氧适应能力中具有重要作用。在营养缺乏、损伤等外界有害环境下，特别是缺氧应激下，细胞能通过启动HIF-1的分子开关作用，使下游基因的表达做到准确的精细调控，并使细胞维持基本的能量代谢。 自噬(Autophagy)是生物在进化过程中保留的一种特殊的现象。本质是细胞在生存、发育、分化及代谢过程中的一种溶酶体降解途径。其主要的作用是保护机体内环境的稳定，并避免受到外界各种因素的影响。新近研究表明，自噬除了维持细胞自身环境的平衡以外，还作为一种重要的分子调控机制，参与了众多的基因的调节和多种疾病的发生，如代谢病、心脏病、遗传病、肿瘤、炎症及神经疾病等。研究报道在缺氧过程中，自噬变化明显，并影响细胞的病理及生理学效应。 然后在脑缺血缺氧环境下，小胶质细胞的病理生理学特点是怎样的？HIF-1α的在小胶质细胞上的表达情况如何？小胶质细胞是否有自噬发生？其具体发生及调节机制又是怎样的？干扰上述环节是否对脑缺血的神经功能具有保护作用？为了证实上述的疑问，我们在本研究中设计系列实验，以明确在脑缺血缺氧环境下HIF-1α的表达对小胶质细胞的自噬影响及生物学意义。 研究内容：①在体外应用氧糖剥夺模型，在0-48小时的不同时间点，应用PI和MTT检测小胶质细胞的生存率；②体外应用氧糖剥夺模型，在0-48小时的不同时间点，应用RT-PCR和ELISA检测小胶质细胞的炎症细胞因子IL-8和TNF-α表达量；③体外应用氧糖剥夺模型，在0-48小时的不同时间点，应用western blot检测小胶质细胞的HIF-1α表达量；④体外应用氧糖剥夺模型，分别用HIF-1α的抑制剂2ME和YC-1以及HIF-1α的RNA阻断HIF-1α的表达，应用PI和MTT检测小胶质细胞的生存率；⑤体外应用氧糖剥夺模型，分别用HIF-1α的抑制剂2ME和YC-1以及HIF-1α的RNA阻断HIF-1α的表达，应用ELISA检测小胶质细胞的炎症细胞因子IL-8和TNF-α表达量；⑥体外应用氧糖剥夺模型，在0-48小时的不同时间点，应用western blot检测检测小胶质细胞自噬相关蛋白LC3的表达变化；应用激光共聚焦显微镜观察小胶质细胞的GFP-LC3质粒表达变化；应用MDC (单丹磺酰尸胺)及丫啶橙染色，流式细胞仪分析小胶质细胞自噬相关的情况；电子显微镜检测小胶质细胞自噬囊泡的形成；⑦体外应用氧糖剥夺模型，分别用HIF-1α的抑制剂2ME和YC-1以及HIF-1α的RNA阻断HIF-1α的表达，通过上述手段检测对小胶质细胞自噬的影响；⑧体外应用氧糖剥夺模型，分别用自噬的抑制剂3-MA和BafA1，以及促进剂Rapa作用小胶质细胞，检测小胶质细胞的存活率；运用自噬关键调节蛋白Beclin1的RNAi作用小胶质细胞，检测小胶质细胞的存活率；⑨建立小鼠大脑中动脉栓塞模型，检测缺血区小胶质细胞的自噬及小鼠神经功能情况；⑩建立小鼠大脑中动脉栓塞模型，侧脑室注射自噬的抑制剂3-MA，检测抑制自噬后，缺血区小胶质细胞的自噬及小鼠神经功能情况。 我们的研究得出了以下结果：①在体外氧糖剥夺模型中，随着时间延长，小胶质细胞的死亡率逐渐增加，存活率逐渐下降；②在体外氧糖剥夺模型中，随着时间延长，炎症细胞因子IL-8和TNF-α表达量逐渐增加；③体外氧糖剥夺模型型中，阻断HIF-1α的表达，小胶质细胞的死亡率下降，存活率增加；⑤体外氧糖剥夺模型中，阻断HIF-1α的表达，小胶质细胞的炎症细胞因子IL-8和TNF-α表达量减少；⑥体外氧糖剥夺模型中，，小胶质细胞自噬增加；⑦体外氧糖剥夺模型中，阻断HIF-1α的表达，小胶质细胞自噬减少；⑧体外氧糖剥夺模型中，抑制自噬及Beclin1蛋白，小胶质细胞的死亡率减少，存活率增加；促进自噬，小胶质细胞的死亡率增加，存活率减少；⑨体内小鼠大脑中动脉栓塞模型中，缺血区的小胶质细胞自噬增加，小鼠神经功能受损；自噬抑制剂3-MA能减轻缺血区的小胶质细胞自噬及小鼠神经功能缺损情况。 研究结论：脑缺血缺氧环境诱导小胶质细胞HIF-1α表达上调，并通过Beclin1信号通路启动自噬途径，导致了细胞的炎症和死亡效应，并进一步加重了神经功能损伤。干扰自噬途径，可以抑制小胶质细胞的炎症和死亡效应，为缺血性脑卒中提供了理想的脑保护策略。
[Abstract]:Ischemic stroke is a neurological disorder caused by interruption of cerebral blood flow. The incidence of ischemic stroke is high in acute cerebrovascular diseases, and has a high disability rate. It has seriously endangered human health. More than half of the survivors are accompanied by varying degrees of neurological disability. With the gradual increase of the elderly population in China, the number of ischemic stroke patients will further increase. Therefore, ischemic stroke has become the number one enemy of human health. It is of great scientific significance and clinical value to study the pathogenesis and treatment strategy of ischemic stroke.
Vascular nerve network theory, a new concept for the pathogenesis and treatment strategy of ischemic stroke, has been proposed. It is believed that vascular endothelial cells, neurons, glial cells and peripheral extracellular matrix constitute the vascular nerve unit complex. The theory holds that ischemic stroke is not a simple vascular event, nor a single neurological event, but a process of interaction and dialogue between blood vessels and nerves, between parenchymal cells and matrix, and of mutual regulation and balance. Neurons and cell components should be valued and protected rather than simply protecting certain cells. Previous studies on ischemic stroke have focused on neurons and vascular endothelial cells, but lack of in-depth study of glial cells. After stroke, it plays an important role in injury.
Microglia, like normal cells, can sense the stimulation of hypoxic signals under hypoxic conditions, activate the corresponding signaling pathways, activate the expression of corresponding genes, adapt to the harmful effects of hypoxic microenvironment on cells, and maintain the stability of the cellular environment. When cells are stimulated by hypoxic signals, a variety of genes are induced to express. Intermediate hypoxia inducible factor-1 (HIF-1) plays an important role in hypoxic adaptation. It has been shown that HIF-1 can activate downstream related genes rapidly in hypoxic environment, regulate intracellular energy metabolism and oxygen utilization, and play an important role in organs or local tissues. HIF-1 can play an important role in hypoxic adaptation, such as nutritional deficiency, injury, etc. Under harmful environment, especially under hypoxia stress, cells can regulate the expression of downstream genes accurately and precisely by activating the molecular switch of HIF-1, and maintain basic energy metabolism.
Autophagy is a special phenomenon that organisms retain in their evolutionary process. It is essentially a lysosomal degradation pathway in the process of cell survival, development, differentiation and metabolism. Its main role is to protect the stability of the body's internal environment and avoid being influenced by various external factors. Recent studies have shown that autophagy is not only fine-grained but also fine-grained. In addition to the homeostasis of cellular environment, it is also an important molecular mechanism involved in the regulation of many genes and the occurrence of many diseases, such as metabolic diseases, heart diseases, genetic diseases, tumors, inflammation and neurological diseases.
Then what are the pathophysiological characteristics of microglia in hypoxic and cerebral ischemia environment? How is the expression of HIF-1a in microglia? Is microglia autophagy occurring? What are the specific mechanisms of its occurrence and regulation? Does interfering with the above-mentioned links have a protective effect on the neurological function of cerebral ischemia? To confirm these doubts, we designed a series of experiments in this study to clarify the effect of HIF-1a expression on microglia autophagy and its biological significance in hypoxic and ischemic environment.
The contents of this study were as follows: 1) The survival rate of microglia was measured by PI and MTT at different time points of 0-48 hours after oxygen-glucose deprivation in vitro; 2) The expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by RT-PCR and ELISA at different time points of 0-48 hours after oxygen-glucose deprivation in vitro. Western blot was used to detect the expression of HIF-1a in microglia at different time points of 0-48 hours with oxygen-glucose deprivation model. In the model of glucose deprivation, the expression of HIF-1a was blocked by 2ME, YC-1 and RNA of HIF-1a respectively, and the expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by ELISA. _Oxygen-glucose deprivation model was used in vitro, and the autophagy correlation of microglia was detected by Western blot at different time points of 0-48 hours. The expression of protein LC3, the expression of GFP-LC3 plasmid in microglia was observed by laser confocal microscopy, the autophagy-related condition of microglia was analyzed by MDC and acridine orange staining, the autophagy vesicle formation of microglia was detected by electron microscopy, and_Oxyglucose was used in vitro. In the deprivation model, HIF-1a inhibitors 2ME, YC-1 and HIF-1a RNA were used to block the expression of HIF-1a respectively, and the effects of HIF-1a on microglia autophagy were detected by these methods. _Oxygen-glucose deprivation model was used in vitro, autophagy inhibitors 3-MA and BafA1, and accelerator Rapa were used to treat microglia respectively to detect the survival of microglia. Rate; The survival rate of microglia was detected by using the RNAi of autophagy key regulatory protein Beclin1; _The middle cerebral artery embolization model was established in mice to detect the autophagy of microglia in ischemic area and the neurological function of mice; _The middle cerebral artery embolization model was established in mice, and autophagy inhibitor 3-M was injected into lateral ventricle. A, to detect autophagy, microglia autophagy and neurological function in mice after inhibition of autophagy.
Our results are as follows: 1) In the oxygen-glucose deprivation model in vitro, the mortality of microglia gradually increased and the survival rate gradually decreased with the prolongation of time; 2) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased with the prolongation of time; 3) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased. Blocking the expression of HIF-1a resulted in a decrease in microglial mortality and an increase in survival rate. _Blocking the expression of HIF-1a resulted in a decrease in the expression of inflammatory cytokines IL-8 and TNF-alpha. _Increased microglial autophagy in the oxygen-glucose deprivation model in vitro and_Blocking HIF-1 in the oxygen-glucose deprivation model in vitro. _in vitro oxygen-glucose deprivation model, inhibition of autophagy and Beclin-1 protein reduced the mortality of microglia and increased the survival rate; promote autophagy, microglia mortality increased, the survival rate decreased; _in vivo mouse middle cerebral artery embolism model, the ischemic area of microglia autophagy Autophagy inhibitor 3-MA can alleviate microglia autophagy and neurological deficit in ischemic area.
Conclusion: HIF-1a expression in microglia is up-regulated by hypoxic-ischemic environment, and autophagy pathway is activated by Beclin-1 signaling pathway, which leads to inflammation and death of microglia, and further aggravates neurological impairment. It provides ideal brain protection strategy.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3
本文编号：2227101
[Abstract]:Ischemic stroke is a neurological disorder caused by interruption of cerebral blood flow. The incidence of ischemic stroke is high in acute cerebrovascular diseases, and has a high disability rate. It has seriously endangered human health. More than half of the survivors are accompanied by varying degrees of neurological disability. With the gradual increase of the elderly population in China, the number of ischemic stroke patients will further increase. Therefore, ischemic stroke has become the number one enemy of human health. It is of great scientific significance and clinical value to study the pathogenesis and treatment strategy of ischemic stroke.
Vascular nerve network theory, a new concept for the pathogenesis and treatment strategy of ischemic stroke, has been proposed. It is believed that vascular endothelial cells, neurons, glial cells and peripheral extracellular matrix constitute the vascular nerve unit complex. The theory holds that ischemic stroke is not a simple vascular event, nor a single neurological event, but a process of interaction and dialogue between blood vessels and nerves, between parenchymal cells and matrix, and of mutual regulation and balance. Neurons and cell components should be valued and protected rather than simply protecting certain cells. Previous studies on ischemic stroke have focused on neurons and vascular endothelial cells, but lack of in-depth study of glial cells. After stroke, it plays an important role in injury.
Microglia, like normal cells, can sense the stimulation of hypoxic signals under hypoxic conditions, activate the corresponding signaling pathways, activate the expression of corresponding genes, adapt to the harmful effects of hypoxic microenvironment on cells, and maintain the stability of the cellular environment. When cells are stimulated by hypoxic signals, a variety of genes are induced to express. Intermediate hypoxia inducible factor-1 (HIF-1) plays an important role in hypoxic adaptation. It has been shown that HIF-1 can activate downstream related genes rapidly in hypoxic environment, regulate intracellular energy metabolism and oxygen utilization, and play an important role in organs or local tissues. HIF-1 can play an important role in hypoxic adaptation, such as nutritional deficiency, injury, etc. Under harmful environment, especially under hypoxia stress, cells can regulate the expression of downstream genes accurately and precisely by activating the molecular switch of HIF-1, and maintain basic energy metabolism.
Autophagy is a special phenomenon that organisms retain in their evolutionary process. It is essentially a lysosomal degradation pathway in the process of cell survival, development, differentiation and metabolism. Its main role is to protect the stability of the body's internal environment and avoid being influenced by various external factors. Recent studies have shown that autophagy is not only fine-grained but also fine-grained. In addition to the homeostasis of cellular environment, it is also an important molecular mechanism involved in the regulation of many genes and the occurrence of many diseases, such as metabolic diseases, heart diseases, genetic diseases, tumors, inflammation and neurological diseases.
Then what are the pathophysiological characteristics of microglia in hypoxic and cerebral ischemia environment? How is the expression of HIF-1a in microglia? Is microglia autophagy occurring? What are the specific mechanisms of its occurrence and regulation? Does interfering with the above-mentioned links have a protective effect on the neurological function of cerebral ischemia? To confirm these doubts, we designed a series of experiments in this study to clarify the effect of HIF-1a expression on microglia autophagy and its biological significance in hypoxic and ischemic environment.
The contents of this study were as follows: 1) The survival rate of microglia was measured by PI and MTT at different time points of 0-48 hours after oxygen-glucose deprivation in vitro; 2) The expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by RT-PCR and ELISA at different time points of 0-48 hours after oxygen-glucose deprivation in vitro. Western blot was used to detect the expression of HIF-1a in microglia at different time points of 0-48 hours with oxygen-glucose deprivation model. In the model of glucose deprivation, the expression of HIF-1a was blocked by 2ME, YC-1 and RNA of HIF-1a respectively, and the expression of inflammatory cytokines IL-8 and TNF-alpha in microglia was detected by ELISA. _Oxygen-glucose deprivation model was used in vitro, and the autophagy correlation of microglia was detected by Western blot at different time points of 0-48 hours. The expression of protein LC3, the expression of GFP-LC3 plasmid in microglia was observed by laser confocal microscopy, the autophagy-related condition of microglia was analyzed by MDC and acridine orange staining, the autophagy vesicle formation of microglia was detected by electron microscopy, and_Oxyglucose was used in vitro. In the deprivation model, HIF-1a inhibitors 2ME, YC-1 and HIF-1a RNA were used to block the expression of HIF-1a respectively, and the effects of HIF-1a on microglia autophagy were detected by these methods. _Oxygen-glucose deprivation model was used in vitro, autophagy inhibitors 3-MA and BafA1, and accelerator Rapa were used to treat microglia respectively to detect the survival of microglia. Rate; The survival rate of microglia was detected by using the RNAi of autophagy key regulatory protein Beclin1; _The middle cerebral artery embolization model was established in mice to detect the autophagy of microglia in ischemic area and the neurological function of mice; _The middle cerebral artery embolization model was established in mice, and autophagy inhibitor 3-M was injected into lateral ventricle. A, to detect autophagy, microglia autophagy and neurological function in mice after inhibition of autophagy.
Our results are as follows: 1) In the oxygen-glucose deprivation model in vitro, the mortality of microglia gradually increased and the survival rate gradually decreased with the prolongation of time; 2) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased with the prolongation of time; 3) In the oxygen-glucose deprivation model in vitro, the expression of inflammatory cytokines IL-8 and TNF-a gradually increased. Blocking the expression of HIF-1a resulted in a decrease in microglial mortality and an increase in survival rate. _Blocking the expression of HIF-1a resulted in a decrease in the expression of inflammatory cytokines IL-8 and TNF-alpha. _Increased microglial autophagy in the oxygen-glucose deprivation model in vitro and_Blocking HIF-1 in the oxygen-glucose deprivation model in vitro. _in vitro oxygen-glucose deprivation model, inhibition of autophagy and Beclin-1 protein reduced the mortality of microglia and increased the survival rate; promote autophagy, microglia mortality increased, the survival rate decreased; _in vivo mouse middle cerebral artery embolism model, the ischemic area of microglia autophagy Autophagy inhibitor 3-MA can alleviate microglia autophagy and neurological deficit in ischemic area.
Conclusion: HIF-1a expression in microglia is up-regulated by hypoxic-ischemic environment, and autophagy pathway is activated by Beclin-1 signaling pathway, which leads to inflammation and death of microglia, and further aggravates neurological impairment. It provides ideal brain protection strategy.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3
【参考文献】
相关期刊论文 前3条
1 GE Jiao;LIU Yan;LI Qiang;GUO Xia;GU Ling;MA Zhi Gui;ZHU Yi Ping;;Resveratrol Induces Apoptosis and Autophagy in T-cell Acute Lymphoblastic Leukemia Cells by Inhibiting Akt/mTOR and Activating p38-MAPK[J];Biomedical and Environmental Sciences;2013年11期
2 彭雯;刘艺;徐卫娟;夏清华;;Role of Beclin 1-dependent Autophagy in Cardioprotection of Ischemic Preconditioning[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2013年01期
3 Hai-Hu Hao;Li Wang;Zhi-Jian Guo;Lang Bai;Rui-Ping Zhang;Wei-Bing Shuang;Yi-Jia Jia;Jie Wang;Xiao-Yu Li;Qiang Liu;;Valproic acid reduces autophagy and promotes functional recovery after spinal cord injury in rats[J];Neuroscience Bulletin;2013年04期
本文编号：2227101
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