炎症小体信号通路诱导的细胞焦亡在锰致机体认知损害中的作用

Pyroptosis is a newly discovered programmed cell death mediated by gasdermin, closely associated with the development of infectious diseases and nervous system diseases. Studies have shown that activation of inflammasome is the key role of pyroptosis induced by inflammatory Caspase. Furthermore, Aβ could activate inflammasome related signaling pathway thus induce neuronal pyroptosis through NLRP1 in Alzheimer's disease. What is more, other studies suggested that cognitive function could be impaired by the activation of NLRP3-Caspase-1 pathway in mice after manganese exposure. However, whether could manganese induce pyroptosis and the role of pyroptosis induced by inflammasome in manganese neurotoxicity have not been reported yet. Our previous study showed that the cognitive function of mice was impaired along with the increased expression of Aβ in the cerebral cortex due to manganese exposure. Besides, we also observed that manganese can induce pyroptosis in primary cortical hippocampal neurons as well as SHSY5Y cells. According to our results, we hypothesize that the pyroptosis induced by inflammasome may play an important role in manganese-induced cognitive impairment. Therefore, technologies such as qRT-PCR, Western-blot, gene knockout and cell transfection will be used to explore the mechanisms of pyroptosis induced by inflammasome signaling pathway involving in manganese-induced cognitive impairment. In conclusion, this project is the first to study the mechanism of pyroptosis in manganese neurotoxicity, which provides new perspectives.

细胞焦亡是近年新发现的由gasdermin介导的程序性细胞死亡，与感染性和神经系统疾病等的发生发展密切相关。研究表明炎症小体激活是炎性Caspase诱导细胞焦亡的关键环节；AD中Aβ可激活炎症小体信号通路，并经NLRP1诱导神经元焦亡；另有研究认为锰暴露激活NLRP3-Caspase-1通路降低小鼠认知功能，但锰暴露是否诱导细胞焦亡及炎症小体诱导的细胞焦亡在锰的神经毒性中的作用未见报道。我们前期研究表明锰暴露致小鼠认知能力下降，皮层Aβ表达增加；体外实验表明锰致小鼠原代皮层海马神经元和SHSY5Y细胞焦亡。由此提出：炎症小体诱导的细胞焦亡在锰致机体认知功能损害中发挥了重要作用？为此拟用qRT-PCR、WB、基因敲除和细胞转染技术等，从体内外探讨炎症小体信号通路诱导的细胞焦亡在锰致机体认知损害中的作用。本项目首次研究细胞焦亡在锰神经毒性中的作用机制，为了解和研究锰的神经毒性提供了全新的视野。

广西从事锰作业的职业人员众多，是我国锰中毒的高发地区之一。国内外大量研究指出锰可损害机体学习记忆功能，但其分子作用机制尚未十分清楚。细胞焦亡是近年新发现的一种由Gasdermin蛋白家族介导的程序性细胞死亡方式，与神经系统疾病的发生发展密切相关，但锰暴露能否诱导神经细胞焦亡、ROS介导的Casepase3-GSDME信号通路诱导细胞焦亡在锰的神经毒性中的作用尚未见报道。本研究通过开展体外细胞及体内动物实验，采用Morris水迷宫、WB、基因敲除和细胞转染等技术及方法探讨锰暴露对小鼠学习记忆功能的影响，研究ROS介导的Casepase3-GSDME信号通路诱导细胞焦亡在锰致小鼠学习记忆功能损害的作用。本项目采用BV2细胞（小鼠小胶质细胞）和N2a细胞（小鼠神经瘤母细胞）进行体外细胞实验，证实锰暴露能导致神经细胞发生焦亡，进而导致炎症因子的表达升高，而抑制Caspase3-GSDME通路和ROS的生成可以降低细胞焦亡的水平及炎症因子的表达，提示锰可能通过ROS-Caspase3-GSDME通路诱导神经细胞焦亡，为进一步验证我们的细胞实验结果，本项目进行了体内动物实验。本课题组对C57BL/6小鼠进行12周亚慢性灌胃染锰，同时开展腹腔注射ROS抑制剂和侧脑室注射GSDME siRNA进行反向验证Caspase3-GSDME诱导的细胞焦亡在锰致机体认知损害中的作用。体内动物实验结果表明，锰暴露可致小鼠学习记忆能力下降及诱导海马神经元细胞焦亡，并使海马炎症因子累积，使用了ROS抑制剂或是抑制了GSDME的表达之后可以有效改善小鼠的学习记忆能力下降，减少炎症因子的生成，初步验证了ROS介导的Casepase3-GSDME信号通路在锰诱导细胞焦亡中发挥了作用，该作用可能是锰致机体认知功能障碍的分子毒理作用机制之一。本项目通过体内外实验相结合研究细胞焦亡在锰神经毒性中的作用机制，为了解和研究锰的神经毒性提供了全新的视野。