不同氧化状态HMGB1在动物抑郁样行为中的作用及受体机制研究

发布时间：2018-05-04 01:25

本文选题：HMGB1 + 氧化状态　；参考：《第二军医大学》2017年硕士论文

【摘要】：抑郁症是一种常见的心理障碍,但发病机制尚未完全阐明。基于细胞因子学说,我们对高迁移率蛋白族1(High-mobility group box 1,HMGB1)在抑郁症发病中的作用及可能机制进行研究。HMGB1作为晚期炎性介质,广泛参与了自身免疫疾病的发病过程。最近的研究发现HMGB1在一些神经炎性疾病中也有关键作用。我们的前期研究证实,在腹腔注射低剂量细菌脂多糖(lipopolysaccharide,LPS)诱导的抑郁模型中,中枢HMGB1呈主动释放。HMGB1是一种非组蛋白染色体结合蛋白,细胞处于稳态时主要位于细胞核;当遭受到适当的信号刺激时,则经过一定的化学修饰分泌到细胞外,这是HMGB1的主动释放。在此过程中,由于其所处的环境不同,其第23位、45位及106位半胱氨酸所连接的巯基会发生氧化形成三种不同形式的HMGB1:完全还原型HMGB1(fully reduced HMGB,fr-HMGB1),二硫键型HMGB1(disulfide HMGB1,ds-HMGB1),完全氧化型HMGB1(fully oxidized HMGB1,ox-HMGB1)。Fr-HMGB1主要与细胞膜表面的糖基化终产物受体(receptor for advanced glycation end-products,RAGE)及CXC趋化因子受体4(CXC-chemokine receptor 4,CXCR4)受体结合发挥趋化作用;ds-HMGB1主要与Toll样受体家族(toll-like family of receptors,TLRs)部分成员结合促进炎症反应;ox-HMGB1无免疫活性。最近的研究表明,细胞外HMGB1的生物活性是由其氧化状态决定的。本研究的目的主要在于明确HMGB1在抑郁症发病中的作用,并在此基础上探讨导致抑郁的HMGB1的氧化状态及受体机制。通过慢性不可预知应激(chronic unpredictable mild stress,CUMS)构建动物模型,4周后进行抑郁行为学检测,包括悬尾实验(tail suspension test,TST)、糖水偏爱度检测(sucrose preference test,SPT)及开场实验(open field test,OPT)。采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白质免疫印迹(western blot,WB)的方法对外周和中枢HMGB1的蛋白含量进行检测。对导致抑郁的HMGB1的氧化状态及机制进行研究的部分,采用侧脑室注射的方法,将三种商品化的重组HMGB1:ds-HMGB1,fr-HMGB1及非氧化型HMGB1(non-oxidizable chemokine-HMGB1,nonoxid-HMGB1)分别给予小鼠进行研究。与fr-HMGB1相比,nonoxid-HMGB1也具有趋化作用,但在体内不会发生氧化状态的改变。通过中枢将这三种HMGB1以200ng/5μL/只的剂量给予小鼠,20h后对小鼠进行抑郁行为学检测;为了进一步研究其致抑郁行为的受体机制,在给予HMGB1之前半小时分别用相应的受体拮抗剂TLR4/RAGE/CXCR4(TAK-242/FPS-ZM1/AMD3100)(3mg/kg)进行拮抗,检测其对小鼠抑郁行为的影响。最后,通过测定中枢海马炎性因子肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和髓鞘形成相关蛋白-髓鞘碱性蛋白(myelin basic protein,MBP)的表达初步探究HMGB1与受体结合后致抑郁的下游机制。我们的研究表明:在CUMS构建的抑郁模型中,与对照组相比,CUMS组外周及中枢HMGB1的表达均显著增高。中枢分别给予ds-HMGB1与fr-HMGB1均可诱导小鼠出现抑郁样行为;相应的受体拮抗剂能改善抑郁样行为。TAK-242也可以改善fr-HMGB1诱导的抑郁样行为。而non-oxidHMGB1不能诱导小鼠出现抑郁样行为。经检测两种氧化状态的HMGB1均能引起TNF-α表达的增加,且与抑郁行为学指标相一致。在进一步检测中,我们发现中枢给予ds-HMGB1可以引起小鼠海马组织MBP表达量的降低;而TLR4拮抗剂对ds-HMGB1引起的效应具有MBP保护作用。综上所述:两种氧化状态HMGB1在抑郁症的发病中起重要作用,这种作用可能是通过激活炎症通路诱导中枢炎症,进而导致髓鞘损伤引起的。
[Abstract]:Depression is a common mental disorder, but the pathogenesis has not been fully elucidated. Based on the cytokine theory, we study the role and possible mechanism of high mobility protein family 1 (High-mobility group box 1, HMGB1) in the pathogenesis of depression..HMGB1 is a late inflammatory medium and is widely involved in the pathogenesis of autoimmune diseases. Recent studies have found that HMGB1 also plays a key role in some neuroinflammatory diseases. Our previous study confirmed that in the depressive model induced by low dose lipopolysaccharide (LPS) intraperitoneal injection of bacterial lipopolysaccharide (LPS), central HMGB1 actively releases.HMGB1 as a non histone chromosome binding protein, the main position of the cell at the time of homeostasis. In the nucleus; when a proper signal is stimulated, a certain chemical modification is secreted to the extracellular, which is the active release of HMGB1. In this process, the sulfhydryl groups connected by its twenty-third, 45 and 106 - bit cysteine will be oxygenation to form three different forms of HMGB1: completely prototype HMGB1 (f Ully reduced HMGB, fr-HMGB1), two sulfur bond HMGB1 (disulfide HMGB1, ds-HMGB1), completely oxidized HMGB1 (fully oxidized HMGB1), mainly with the receptor of the cell membrane surface, and chemokine receptor 4 receptor. In combination with chemotaxis, ds-HMGB1 is mainly associated with some members of the Toll like receptor family (Toll-like family of receptors, TLRs) to promote inflammatory response; ox-HMGB1 has no immune activity. Recent studies have shown that the biological activity of extracellular HMGB1 is determined by its oxidation state. The purpose of this study is to identify HMGB1 in the pathogenesis of depression. On this basis, we explored the oxidative status and receptor mechanism of HMGB1 causing depression. The animal model was constructed by chronic unpredictable stress (chronic unpredictable mild stress, CUMS), and the depressive behavior was detected after 4 weeks, including the tail suspension test (tail suspension test, TST), sugar water preference test (sucrose preference) EST, SPT) and the opening experiment (open field test, OPT). Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) and protein immunoblotting (Western blot) were used to detect the protein content of the peripheral and central nervous systems. The part of the study of the oxidative state and mechanism of depression, using the lateral brain Three commercialized recombinant HMGB1:ds-HMGB1, fr-HMGB1 and non oxidative HMGB1 (non-oxidizable chemokine-HMGB1, nonoxid-HMGB1) were given to mice respectively. Compared with fr-HMGB1, nonoxid-HMGB1 also had chemotaxis, but there was no change in the oxidative state in the body. The three HMGB1 were 20 in the center. The dose of 0ng/5 mu L/ was given only to mice, and the mice were tested for depressive behavior after 20h. In order to further study the receptor mechanism of their depressive behavior, the corresponding receptor antagonist TLR4/RAGE/CXCR4 (TAK-242/FPS-ZM1/AMD3100) (3mg/kg) was used to detect the effect of the receptor antagonist on the depressive behavior of the mice at half an hour before the HMGB1. Later, through the determination of the expression of Tumor necrosis factor- alpha (TNF- alpha) and myelin formation related protein myelin basic protein (myelin basic protein, MBP) in the central hippocampal inflammatory factors, the downstream mechanism of depression after the combination of HMGB1 and receptor is preliminarily explored. Our study showed that in the depressive model constructed by CUMS, and the control The expression of peripheral and central HMGB1 in the group CUMS was significantly higher than that in the group. Both ds-HMGB1 and fr-HMGB1 could induce depressive behavior in mice, and the corresponding receptor antagonists could improve the depressive behavior.TAK-242 and improve the depressive behavior induced by fr-HMGB1, while non-oxidHMGB1 could not induce depressive behavior in mice. HMGB1, which was detected in two oxidation states, could cause an increase in the expression of TNF- alpha and was in accordance with the depressive behavioral index. In further detection, we found that the central administration of ds-HMGB1 could cause a decrease in the expression of MBP in the hippocampus of the mice; and the TLR4 antagonist had the effect of MBP protection on the effect of ds-HMGB1. To sum up, two kinds of oxygen are described. The chemical state HMGB1 plays an important role in the pathogenesis of depression, which may be caused by the activation of the inflammatory pathway to induce central inflammation, which may lead to myelin injury.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.4

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