METH激活胶质细胞经NF-κB-LCN2/焦亡通路致脑神经毒性损伤作用机制研究

Methamphetamine (METH) abuse, in addition to addiction, can lead to the toxicity of brain and other organs, and the study of METH drug damage has been a key problem in the forensic medical field. Previous series study of brain injury found that METH not only lead to neuronal injury, also found that neurogliocyte, especially astrocytes and microglia can produce toxic injury by activating the NF-κB pathway. Its participation effect and mechanism has been not yet clarified. Explore the mechanism of neurogliocyte in the METH toxic injury and build the overall relationship between brain injury and overall behavior change are the science topic proposed to solve in this research, and proposes the idea: METH activates NF-κB pathway in neurogliocyte, and secrete LCN2 cause neurons apoptosis caused by changing the iron ion concentration. On the other hand, it induced pyroptosis through the NF-κB-Caspase11/1-GSDMD signal axis, causing sustained inflammatory response and participating in METH neurotoxic injury.In this study, we will use molecular biology, neurobiology, behavioral science technology and so on, using the experiments in vivo and in vitro, using two important pathway induced by NF-κB, to explore the role and mechanism of neurogliocyte induced by METH, and to provide a new basis and targets for the diagnosis and treatment of METH.

甲基苯丙胺（METH）滥用除造成成瘾外，还可以导致脑和其他器官的毒性损伤，对其损伤机制的研究一直是法医学界的重点问题。前期对脑损伤的研究发现METH不仅导致神经元损伤，还对神经胶质细胞产生作用，通过星形胶质和小胶质细胞激活NF-κB通路产生毒性损伤，但其参与损伤的机制仍未阐明。探索神经胶质细胞在METH毒性损伤中作用机制并建立形态学与行为学的关系是本课题拟解决的科学问题。本研究提出设想：METH激活星形胶质细胞后引发持续炎症反应，经NF-κB激活胶质细胞分泌LCN2途径致神经元凋亡；经NF-κB-Caspase11/1-GSDMD信号轴致细胞焦亡，从而发挥参与METH脑毒性损伤作用。本研究以分子生物学、神经生物学、行为学等技术为手段，利用体内外实验，以NF-κB-诱导下的两条重要反应途径为线索，探索胶质细胞参与METH所致脑组织损伤的机制，为METH的诊断和治疗提供新的依据和靶点。

本课题拟探究LCN2在METH诱导的神经毒性中的作用及METH诱导LCN2表达的机制，旨在为METH诱导的神经毒性损伤的防治提供新的靶点及理论依据。METH染毒的细胞和动物模型中可见LCN2高表达，LCN2主要表达在星形胶质细胞中、小胶质细胞。LCN2作为一种分泌蛋白，在动物脑脊流和血清以及原代培养的星形胶质细胞的上以及小胶质细胞系BV2细胞中均可见其表达升高。建立星形胶质细胞和神经元共培养METH染毒模型，METH处理原代星形胶质细胞后与神经元共培养，星胶释放的LCN2可与神经元表面的LCN2受体LCN2R相结合介导神经元死亡。METH处理神经元后与正常星形胶质细胞共培养，显著激活星形胶质细胞，促进星胶合成和释放LCN2。METH通过ROS-PERK-cIF2a通路诱导星形胶质细胞的激活及调节LCN2合成与释放。METH通过ROS-PERK-clF2a通路介导星形胶丽细胞来源的LCN2的合成与释放，LCN2与神经元表面受体LCN2R相结合介导神经元凋亡，受损的神经元可诱导星形胶质细胞合成和分泌LCN2增加，进一步促进METH神经毒性。此外，我们还发现LCN2在METH染毒的小胶质细胞系BV-2中高表达，CEBP/beta 在METH诱导的BV-2高表达，调节LCN2的表达，参与METH介导的BV-2细胞凋亡。此外我们进一步探讨METH对小胶质细胞焦亡的作用。细胞焦亡(pyroptosis)是由炎性小体引发的一种细胞程序性死亡，表现为细胞不断胀大直至细胞膜破裂，导致细胞内容物释放进而引起强烈的炎症反应。细胞焦亡是机体一种重要的天然免疫反应，在抵抗感染中发挥着重要作用。我们发现：1）枸杞多糖（LBP）可以保护METH诱导的小胶质细胞死亡。2）METH可以诱导小胶质细胞发生焦亡，呈浓度和时间依赖性增加炎性小体NLRP3和ASC激活，进一步促进caspase1介导的炎性通路的激活。3）LBP可以阻断上述METH诱导的炎性损伤作用，对METH诱导的胶质细胞损伤起一定保护作用。