NLRP3炎症体介导的小胶质细胞炎症反应在锰神经毒性中的作用

发布时间：2018-06-12 06:43

本文选题：锰 + 神经毒性　；参考：《第四军医大学》2016年博士论文

【摘要】：背景锰是人体所必须的微量元素。参与人体免疫反应,ATP的生成,骨骼生长等生理反应。此外,锰还可以作为机体许多酶的辅助因子,保障其发挥正常的生理作用。然而,当机体摄入过多的锰则会引起锰中毒的发生,其临床表现主要为类似帕金森氏病病症。中枢神经系统是锰作用人体的主要靶器官,黑质纹状体通路更是锰损伤神经系统的关键核团。形成了锰能够引起黑质纹状体内多巴胺能神经元功能减弱的经典理论。随着人们对锰神经毒性的进一步关注,锰对海马脑区调控的空间记忆能力损伤也渐渐被人们所证实。研究证实锰在大脑中的蓄积所造成的神经毒性作用与阿尔兹海默症(Alzheimer’s disease,AD)、帕金森症(Parkinson's disease,PD)、等多种神经退行性疾病都有着密切的关系。在中枢神经系统中,小胶质细胞是免疫反应的关键细胞,主要来源于脑膜,脉络丛以及血管周围。其与巨噬细胞的功能相似,能通过一系列的模式识别受体(Pattern-Recognition Receptors,PRR)实时监测中枢神经系统的内环境。当组织受损或有害物质入侵的情况下,小胶质细胞发生活化反应,一方面发挥吞噬作用,一方面释放大量炎症因子,诱导外周固有免疫细胞及适应性免疫细胞迁移至受损或入侵部位,发挥免疫防御作用。因此,小胶质细胞活化及炎性因子释放在中枢神经系统的免疫应答防御反应中发挥了重要作用。小胶质细胞活化后能够快速诱导出多种炎性因子,其中包括白介素-1β(interleukin-1beta,il-1β)、肿瘤坏死因子-α(tumornecrosisfactor-alpha,tnf-α)和il-18等。本课题组以往研究发现,锰暴露能够诱导小胶质细胞活化,活化后释放的炎性因子可能是锰暴露导致神经元损伤的关键因素,研究结果为锰神经毒性机制阐明及锰中毒防护提供了重要线索。炎症体是近年来炎性疾病领域关注的重点,在炎性因子成熟、释放过程中发挥重要作用。nlrp3炎症体是调控il-1β、il-18等炎性因子成熟释放的重要途径。在神经炎症中,小胶质细胞和巨噬细胞内的nlrp3炎症体能被β淀粉样蛋白(amyloid-beta,aβ),α-共核蛋白(α-synuclein,α-syn)所激活。活化的nlrp3发生寡聚化,募集接头蛋白asc,通多card结构域与pyd结构域相互作用,进一步激活caspase-1,进而加工pro-il-1β为成熟的il-1β,并释放于细胞外发挥作用。调控nlrp3炎症体活化成熟的因素很多,细胞内ros的增多、k+浓度改变、atp水平降低以及自噬等均能够影响nlrp3炎症体的活化。自噬是细胞维持内环境稳定重要的生理过程。自噬的形成过程包括自噬的启动、延伸、与溶酶体的融合和降解几个重要阶段。当自噬形成的某一过程受到抑制时则会导致自噬功能处于紊乱状态。研究表明,许多神经系统疾病的发生都与自噬功能的异常相关,pd、ad等均发现体内自噬降解过程异常。自噬与nlrp3炎症体之间的关系较为复杂,一方面当nlrp3炎症体处于激活状态时,则会促进自噬的发生;另一方面,自噬的过度活化则能够抑制nlrp3炎症体的活化。许多研究表明,自噬的抑制能够活化nlrp3炎症体,促进pro-il-1β(36kda)向成熟il-1β转变。虽然自噬能够调控nlrp3炎症体的活化,但其具体机制至今尚未阐明,这也成为当今该领域研究的热点和难点。目的研究锰暴露诱导的小胶质细胞活化及其释放的炎性因子在锰介导的学习记忆损伤中的作用;揭示nlrp3炎症体活化是锰诱导小胶质细胞活化释放炎性因子il-1β和il-18的关键环节;阐明自噬溶酶体功能紊乱与nlrp3炎症体活化之间的关系;揭示锰暴露诱导的自噬功能紊乱调控nlrp3炎症体活化的具体机制,为锰神经毒性的防护和治疗提供关键靶点和理论依据。方法1.通过皮下注射氯化锰的方法,构建锰暴露的小鼠体内模型,采用原子荧光光谱法检测小鼠血锰和脑锰的浓度,运用恐惧条件箱以及电生理实验评估小鼠锰暴露后学习和记忆能力的改变;2.通过免疫荧光化学法检测锰暴露后nlrp3炎症体在小鼠海马区以及bv2细胞(之后简称为bv2)中的表达改变。westernblot检测nlrp3炎症体相关蛋白nlrp3、cleavedcaspase-1,炎性因子il-1β的表达改变;3.通过elisa检测锰暴露后炎性因子il-1β、il-18和tnf-α的改变,以及qrt-pcr检测nlrp3和炎性因子il-1β、il-18mrna水平的改变。4.通过免疫荧光化学法检测自噬相关蛋白lc3的表达影响,westernblot检测自噬相关蛋白beclin1、atg5、lc3、p62、组织蛋白酶b(cathepsinb)蛋白的变化。透射电镜观察bv2自噬亚细胞结构的改变;,5.分别运用atg5sirna、bafa1以及nh4cl处理bv2,westernblot检测锰暴露后nlrp3炎症体相关蛋白nlrp3、cleavedcaspase-1,炎性因子il-1β的表达改变,elisa检测锰暴露后炎性因子il-1β、il-18的改变。结果1.皮下注射氯化锰7天后,小鼠血锰和脑锰与对照组相比明显升高,恐惧条件箱检测以及ltp实验发现锰暴露后能够引起小鼠学习记忆能力下降;2.海马脑片以及bv2免疫荧光化学染色表明,锰暴露能够引起nlrp3炎症体表达增多。westernblot结果显示,体内外锰暴露均能够引起nlrp3炎症体相关蛋白nlrp3、cleavedcaspase-1表达的增加;3.elisa实验检测发现锰暴露能够诱导炎性因子il-1β、il-18表达增加,并且在mrna水平检测上也得以证实;4.免疫细胞化学荧光染色结果提示,锰暴露可导致bv2内lc3标记的自噬体大量聚集。westernblot发现与对照组相比,锰暴露后自噬体相关蛋白beclin1、atg5、lc3ii、p62、组织蛋白酶bbiao’da表达显著增加。p62升高提示锰暴露能够引起自噬溶酶体降解功能出现障碍。组织蛋白酶b的表达增加以及电镜检测所发现的溶酶体形态异常提示锰暴露能够引起bv2溶酶体功能异常。5.Atg5 siRNA和Baf A1分别抑制自噬体的启动、延伸和与溶酶体的融合后,并不能抑制锰暴露所诱导的NLRP3炎症体的活化及IL-1β、IL-18的释放。NH4Cl作用BV2后,Western blot检测发现其能够降低组织蛋白酶B的表达,抑制锰暴露所诱导的NLRP3炎症体的活化以及炎性因子IL-1β、IL-18的释放。结论1.体内实验证实锰能够降低海马脑区所调控的学习和记忆能力。2.锰暴露对学习记忆能力的影响可能与其所诱导的小胶质细胞活化后释放的促炎性因子(IL-1β和IL-18等)有关。3.锰暴露激活NLRP3炎症体通路是其介导炎性因子释放的关键因素。4.自噬溶酶体功能紊乱参与了锰诱导的NLRP3炎症体活化,自噬体启动、延伸及与溶酶体融合的异常不是调控NLRP3炎症体活化的重要环节.5.锰暴露导致的溶酶体功能异常及cathepsin B的释放是引起NLRP3炎症体活化的关键因素。
[Abstract]:Background manganese is a necessary trace element in human body. It participates in human immune response, ATP formation, bone growth and other physiological responses. In addition, manganese can also be used as an auxiliary factor for many enzymes in the body to ensure its normal physiological function. However, excessive manganese intake may lead to the occurrence of manganese poisoning, and its clinical manifestations are mainly similar. Parkinson's disease. The central nervous system is the main target organ of manganese in the human body, and the nigrostriatal pathway is the key nucleus of the manganese damaged nervous system. The classical theory that manganese can cause the dysfunction of dopaminergic neurons in the substantia nigra is a classic theory. With the further attention to the toxicity of manganese, manganese has been used in the hippocampus The neurotoxicity of manganese in the brain has been confirmed. The neurotoxicity of manganese in the brain is closely related to Alzheimer 's disease (AD), Parkinson's disease (PD), and other neurodegenerative diseases. In the central nervous system, small glue is found. The cell is the key cell of the immune response, mainly from the meninges, choroid plexus, and around the blood vessels. It is similar to the function of macrophages and can monitor the internal environment of the central nervous system in real time through a series of pattern recognition receptors (Pattern-Recognition Receptors, PRR). On the one hand, the cell acts as a phagocytosis. On the one hand, it releases a large number of inflammatory factors, and induces the migration of immune cells and adaptive immune cells to the damaged or invasive sites, and plays an immune defense role. Therefore, the activation of microglia and inflammatory factors are released in the immune response defense response of the central nervous system. A variety of inflammatory factors can be induced quickly after the activation of microglia, including -1 beta (interleukin-1beta, IL-1 beta), tumor necrosis factor - alpha (tumornecrosisfactor-alpha, tnf- alpha), and IL-18, etc. this group has previously found that manganese exposure can induce microglia activation and release after activation. Inflammatory factors may be the key factors of neuronal damage caused by manganese exposure. The results provide an important clue to the clarification of manganese neurotoxicity and the protection of manganese poisoning. The inflammatory body is the focus of attention in the field of inflammatory diseases in recent years. The inflammatory factors are mature and play an important role in the release process. The.Nlrp3 inflammatory body is the regulation of IL-1 beta, IL-18 and so on. An important route to mature release of inflammatory factors. In neuropathy, NLRP3 inflammation in microglia and macrophages can be activated by beta amyloid (amyloid-beta, a beta), alpha -synuclein (alpha -synuclein, alpha -syn). Activated NLRP3 oligomerization, recruitment of the head protein ASC, and the interaction of the multi card domain with the PYD domain. One step activates caspase-1, and then processes pro-il-1 beta as the mature IL-1 beta and releases it out of the cell. There are many factors regulating the activation of the NLRP3 inflammatory body, the increase of ROS in the cell, the change of k+ concentration, the decrease of ATP level and autophagy, which can affect the activation of NLRP3 inflammatory body. Autophagy is an important source of the cell maintenance of the internal stability. Process. The process of autophagy consists of the initiation, extension, fusion and degradation of the lysosomes. The autophagy is in disorder when a certain process of autophagy is inhibited. The study shows that the occurrence of many nervous system diseases is associated with autophagic function, PD, ad and so on. The process of autophagic degradation is abnormal. The relationship between autophagy and NLRP3 inflammatory body is more complex. On one hand, when the NLRP3 inflammatory body is activated, autophagy can be promoted; on the other hand, the excessive activation of autophagy can inhibit the activation of the NLRP3 inflammatory body. Many studies have shown that the inhibition of autophagy can activate the NLRP3 inflammatory body and promote the pro- IL-1 beta (36kDa) changes to mature IL-1 beta. Although autophagy can regulate the activation of NLRP3 inflammatory body, its specific mechanism has not yet been elucidated. This has also become a hot and difficult point in this field. The activation of NLRP3 inflammatory body is a key link in the activation and release of inflammatory factors IL-1 beta and IL-18 by manganese induced microglia, and clarifies the relationship between the dysfunction of autophagic lysosome and the activation of NLRP3 inflammatory body, and reveals the body mechanism of the regulation of the dysfunction of autophagy induced by manganese exposure, which regulates the activation of NLRP3 inflammatory bodies, and provides the protection and treatment of manganese neurotoxicity. For the key target and theoretical basis. Method 1. the model of manganese exposed mice was constructed by subcutaneous injection of manganese chloride. The concentration of manganese and manganese in mice was detected by atomic fluorescence spectrometry. The changes of learning and memory ability of mice after manganese exposure were evaluated by using the fear condition box and electrophysiological experiments. 2. by immunofluorescence. The expression changes of NLRP3 inflammatory body in the hippocampus and BV2 cells (later called BV2) after manganese exposure were detected by.Westernblot to detect the changes in the expression of NLRP3 inflammatory proteins NLRP3, cleavedcaspase-1, and inflammatory factor IL-1 beta; 3. the changes of IL-1 beta, IL-18 and tnf- alpha after manganese exposure were detected by ELISA, and the changes in IL-18 and tnf- alpha were detected by ELISA. Detection of NLRP3 and inflammatory factor IL-1 beta, il-18mrna level changes.4. by immunofluorescence chemical assay of autophagy related protein LC3 expression effect, Westernblot detection of autophagy related proteins Beclin1, ATG5, LC3, p62, cathepsin B (CathepsinB) protein changes. Transmission electromicroscope observation of autophagy subcellular structure changes; 5. G5sirna, bafa1 and NH4Cl treated BV2, Westernblot detected the expression of NLRP3 inflammatory body associated protein NLRP3, cleavedcaspase-1, and inflammatory factor IL-1 beta after manganese exposure. ELISA detected the IL-1 beta and IL-18 changes after manganese exposure. Results 1. after 7 days of subcutaneous injection of manganese chloride, the blood manganese and brain manganese in mice were significantly higher than those of the control group. Condition box test and LTP experiment found that manganese exposure could cause the decrease of learning and memory ability in mice. 2. hippocampal slices and BV2 immunofluorescence staining showed that manganese exposure could cause increased expression of NLRP3 inflammatory body.Westernblot, and manganese exposure in vivo and in vivo could lead to NLRP3 inflammation related protein NLRP3, cleavedcaspase-1 3.elisa test found that manganese exposure could induce inflammatory factors IL-1 beta, IL-18 expression increased, and confirmed on mRNA level detection; 4. immunocytochemical fluorescence staining showed that manganese exposure could lead to a large amount of.Westernblot in the autophagic body of LC3 labeled BV2 within BV2 and the manganese exposure was compared with the control group. The expression of phagocytic related proteins Beclin1, ATG5, lc3ii, p62, cathepsin bbiao 'Da expressed a significant increase in.P62, suggesting that manganese exposure can cause the dysfunction of autophagic lysosome degradation. The increase in the expression of cathepsin B and the abnormalities of lysosome morphology found by electron microscopy detection suggest that manganese exposure can cause BV2 lysosome dysfunction.5.At. G5 siRNA and Baf A1 inhibit the activation of autophagosomes, extending and merging with lysosomes, which do not inhibit the activation of NLRP3 inflammatory bodies induced by manganese exposure and IL-1 beta. After the release of.NH4Cl in IL-18, Western blot detection found that the Western blot can reduce the expression of cathepsin B, and inhibit the activation of inflammatory bodies induced by manganese exposure. And the release of inflammatory factors IL-1 beta, IL-18. Conclusion 1. in vivo real manganese can reduce the learning and memory ability of the hippocampus, the effects of.2. manganese exposure on learning and memory ability may be related to the proinflammatory factors (IL-1 beta and IL-18, etc.) released by the induced microglia activation (IL-1 beta and IL-18, etc.), which are related to the activation of NLRP3 inflammation by.3. manganese exposure. The pathway is a key factor in the release of inflammatory factors,.4. autophagic lysosome dysfunction participates in the activation of manganese induced NLRP3 inflammatory bodies, autophagosome initiation, extension, and the abnormality of fusion with lysosomes are not important links to regulate the activation of NLRP3 inflammatory bodies. The dysfunction of the lysosome caused by.5. manganese exposure and the release of cathepsin B is the cause of NLR The key factor in the activation of P3 inflammatory body.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R135.1

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