高迁移率族蛋白1在蛛网膜下腔出血后早期脑损伤中的作用研究

发布时间：2018-08-30 10:15

【摘要】：蛛网膜下腔出血(Subarachnoid hemorrhage, SAH)是高病死率和病残率的三大脑血管疾病之一。蛛网膜下腔出血后蛛网膜下腔及脑实质内发生一系列复杂的病理生理改变,包括血肿形成、脑水肿、氧化应激、炎性反应、细胞毒性物质等。这些病理因素一起作用导致神经细胞的死亡,并进一步引起神经功能损伤。高迁移率族蛋白1(HMGB1)是一种内源性的危险因子,可以诱导与外源性病原微生物相似的免疫炎性反应,在损伤相关分子模式(DAMP)炎性反应中发挥着关键作用。HMGB1是一种高度保守的DNA结合蛋白,作为染色体的组件,通常存在于细胞核内。在特定情况下,HMGB1释放至细胞外,通过TLR-Myd88-NF-κB途径启动DAMP型炎性反应,并起到级联放大作用,同时伴随HMGB1的释放,其表达水平也相应升高。因此,我们提出假设：HMGB1可能是SAH后启动DAMP型炎性反应的核心因子,并在SAH后早期脑损伤中发挥着极其重要的作用。本研究中,首先是要确定HMGB1在SAH后早期是否从细胞核中转移出。因此我们采用Sprague-Dawley(SD)大鼠作为动物模型,予以蛛网膜下腔注入自体动脉血制作SAH模型,使用western blot、real-time PCR及免疫荧光在不同时间点观察大鼠脑组织内HMGB1的位相及表达水平的变化,使用real-time PCR的方法观察下游炎症因子的改变,探讨]HMGB1是否早期释放。研究发现,蛛网膜下腔出血早期,HMGB1即释放至细胞外,伴随其基因及蛋白表达水平被上调。绝大部分释放出的HMGB1来自神经元细胞。为进一步明确神经元释放的HMGB1能否启动胶质细胞的炎性反应,采用Hb刺激神经元,去除Hb后,将神经元的培养液置换给混合胶质细胞培养,观察胶质细胞炎性反应的程度。结果提示Hb能够刺激神经元释放HMGB1至细胞培养基中,而去除了Hb的神经元细胞培养基能够上调胶质细胞的炎性反应。当给予HMGB1特异性抑制剂甘草酸铵盐(GA)干预后,能够逆转上调的炎性反应,这个结果提示神经元释放的HMGB1能够发挥促炎反应的作用。为进一步明确HMGB1在早期脑损伤中的作用,因此设计实验给予蛛网膜下腔注射重组人HMGB1 (rHMGB1),使用western blot观察不同时间点脑组织核蛋白内NF-κB(P65)的含量,real-time PCR测定下游炎性因子的基因表达水平,明确HMGB1的促进炎性反应的性质,并用cleaved-caspase 3与神经元免疫共染色评价组织凋亡程度,反应早期脑损伤情况。研究发现细胞外的HMGB1能够激活脑组织细胞内的NF-κB,引起下游炎症因子的升高,加重神经细胞的凋亡。本课题的另一个目标是研究HMGB1在神经系统中通过何种信号通路引起炎性反应。我们引入TLR4受体的拮抗剂TAK242,在蛛网膜下腔给予rHMGB1后半小时通过侧脑室给药TAK242,比较1天后各分组内P65含量及炎性因子基因表达量。研究发现,TLR4受体拮抗剂TAK242能够逆转HMGB1导致的NF-κB (P65)含量的上升及炎性因子的增加,研究结果表明TLR4在HMGB1引起的神经系统炎性反应中起着重要的作用。为进一步以HMGB1为治疗靶点,寻找潜在性治疗药物,我们引入HMGB1特异性的抑制剂甘草酸铵盐(GA),能够通过血脑屏障,并抑制HMGB1在细胞外的活性。在自体血SAH造模后半小时通过静脉给予甘草酸治疗,比较不同治疗组及未治疗组脑组织内炎症因子的变化,脑水肿的程度以及脑组织细胞凋亡及坏死的程度。研究结果表明使用甘草酸治疗能够抑制HMGB1向细胞外的释放,减轻炎性反应及脑水肿,减轻神经细胞的凋亡及死亡,达到减轻脑损伤的作用。综上所述,HMGB1在SAH后早期由神经元释放,释放出的HMGB1能够影响周边的胶质细胞,主要通过TLR4受体介导的炎性反应在蛛网膜下腔出血后的早期脑损伤中起着至关重要的作用,HMGB1可能是SAH后炎性反应的重要起始因素之一。甘草酸通过抑制细胞外HMGB1的活性以及抑制HMGB1的释放,起着重要的神经保护作用。HMGB1可能成为SAH干预的新的靶点。
[Abstract]:Subarachnoid hemorrhage (SAH) is one of the three major cerebrovascular diseases with high mortality and disability. After SAH, a series of complex pathophysiological changes occur in the subarachnoid space and brain parenchyma, including hematoma formation, brain edema, oxidative stress, inflammatory reaction, cytotoxic substances and so on. High mobility group protein 1 (HMGB1) is an endogenous risk factor that can induce similar immuno-inflammatory responses to exogenous pathogenic microorganisms and plays a key role in the inflammatory response of damage-associated molecular model (DAMP). Degree-conserved DNA-binding proteins, as components of chromosomes, are usually present in the nucleus. Under certain conditions, HMGB1 is released into the extracellular space to initiate DAMP-type inflammatory response via the TLR-Myd88-NF-kappa B pathway, which acts as a cascade amplifier. At the same time, the expression level of HMGB1 increases with the release of HMGB1. In this study, the first step was to determine whether HMGB1 was transferred from the nucleus early after SAH. So we used Sprague-Dawley (SD) rats as animal models and injected subarachnoid space into autologous arteries. The changes of HMGB1 phase and expression level in rat brain tissues were observed by Western blot, real-time PCR and immunofluorescence at different time points. The changes of downstream inflammatory factors were observed by real-time PCR to explore whether HMGB1 was released in early stage of SAH. Most of the HMGB1 released from neurons came from neurons. To further determine whether the HMGB1 released by neurons could initiate the inflammatory reaction of glial cells, Hb was used to stimulate neurons. After removal of Hb, the cultured neurons were replaced by mixed glial cells to observe the glial fineness. The results suggest that Hb can stimulate neurons to release HMGB1 into the cell culture medium, while the neutrophil culture medium without Hb can up-regulate the inflammatory response of glial cells. In order to further clarify the role of HMGB1 in early brain injury, a subarachnoid injection of recombinant human HMGB1 (rHMGB1) was designed to observe the content of NF-kappa B (P65) in the nucleoprotein of brain tissues at different time points by Western blot and determine the gene table of downstream inflammatory factors by real-time PCR. It was found that extracellular HMGB1 could activate NF-kappa B in brain tissue cells, induce the increase of downstream inflammatory factors and aggravate the apoptosis of nerve cells. Another goal was to investigate the signaling pathways through which HMGB1 causes inflammatory responses in the nervous system. We introduced TAK242, an antagonist of TLR4 receptor, and then subarachnoid rHMGB1 was administered to TAK242 via the lateral ventricle half an hour later to compare the levels of P65 and the expression of inflammatory factor genes in each group one day later. AK242 can reverse the increase of NF-kappa B (P65) and inflammatory factors induced by HMGB1. The results indicate that TLR4 plays an important role in the inflammatory response of the nervous system induced by HMGB1. In order to find potential therapeutic drugs for HMGB1, ammonium glycyrrhizinate (GA), a specific inhibitor of HMGB1, can be introduced. Glycyrrhizic acid was administered intravenously half an hour after autologous blood SAH modeling. The changes of inflammatory factors, the degree of brain edema and the degree of apoptosis and necrosis of brain tissue in different treatment groups and untreated groups were compared. The results showed that glycyrrhizic acid could be used to treat SAH. To sum up, HMGB1 released by neurons in the early stage after SAH can affect peripheral glial cells, mainly through TLR4 receptor-mediated inflammatory response in the subarachnoid space. HMGB1 may be one of the important initiating factors of inflammation after SAH. Glycyrrhizin plays an important neuroprotective role by inhibiting the activity of extracellular HMGB1 and inhibiting the release of HMGB1. HMGB1 may become a new target of SAH intervention.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.35

【相似文献】