组织蛋白酶C（二肽肽酶Ⅰ）促进趋化因子产生和小胶质/巨噬细胞M1极化参与多发性硬化神经炎症的分子机制

Neuroinflammation is the key factor involved in pathogenesis and pathological changes of multiple sclerosis (MS). Microglia (MG)/macrophages as antigen-presenting and immune cells in the central nervous system play an important role in innate immune. In MS, MG/macrophages not only trigger immune activation, but also participate in the neuroinflammatory processes through M1/M2 polarization. Our previous work have found that cathepsin C (Cat C) expression was upregulated in the demyelinated areas in MS animal models, and Cat C could aggravate neuroinflammation through promoting the production of chemokines and M1 polarization in vitro. However, whether Cat C aggravates demyelination and axon loss in MS by the same mechanism is unclear, and the signal transduction mechanisms involved in Cat C promoting MG producing chemokines and M1 polarization have not been acknowledged. Based on our previous study, this project aims to clarify the functional role of Cat C in MS animal models which will be created in conditional Cat C gene overexpression and knockdown mice, and to detect whether Cat C is involved in aggravating demyelination and axon loss by promoting MG producing chemokines and M1 polarization. Furthermore, in order to clarify the signal transduction mechanisms involved in promoting MG producing chemokines and M1 polarization, we will screen differentially expressed genes in primary cultured microglia after Cat C stimulation by using microarray technology, and try to find out possible signal transduction pathways through which Cat C functions. Finally, in order to explore the correlation of Cat C expression and MS clinical course, the peripheral blood and cerebrospinal fluid from MS patients will be collected and used to detect Cat C expression then compared with clinical data, aims to provide new diagnosis and treatment strategy of MS from the point of view of controlling neuroinflammation.

神经炎症是参与多发性硬化（Multiple sclerosis，MS）发病机制和病理改变的关键因素。小胶质（microglia, MG）/巨噬细胞活化不仅参与MS免疫触发，并通过M1/M2极化参与MS神经炎症过程。课题组前期通过体内和体外实验发现，组织蛋白酶C（Cat C）不仅由MG/巨噬细胞在MS模型髓鞘脱失部位高表达，并且具有促MG/巨噬细胞产生趋化因子和M1极化的作用。然而Cat C在MS中是否通过上述作用加重髓鞘脱失和轴突损害还不清楚，特别是Cat C上述作用的信号转导机制及与MS临床病程关系不清。本课题拟接续上述工作，利用条件性Cat C基因敲减和过表达小鼠建立MS动物模型，阐明Cat C在MS动物模型中的功能角色；利用芯片筛选Cat C刺激小胶质细胞后差异表达基因，阐释Cat C上述作用的信号转导机制，并与MS临床样本相结合，从控制和检测神经炎症角度为MS诊治提供新策略。

神经炎症是参与多发性硬化（Multiple sclerosis，MS）发病机制和病理改变的关键因素。小胶质（microglia, MG）/巨噬细胞活化不仅参与MS免疫触发，并通过M1和M2两种功能状态影响MS神经炎症过程。组织蛋白酶C（Cathepsin C, CatC）是属于溶酶体内番木瓜蛋白酶超家族中的组织蛋白酶，是一种分泌性的、具有独特二肽肽酶活性的外切蛋白酶，参加蛋白质降解、蛋白质加工、抗原呈递过程、细胞凋亡以及肿瘤浸润和炎症反应等。我们前期研究发现，CatC由MG /巨噬细胞在cuprizone和EAE（experimental autoimmune encephalomyelitis）模型髓鞘脱失部位高表达，然而Cat C在上述模型髓鞘脱失与再生中的作用及其机制不清。为此，本项目利用Cat C基因敲减和条件性过表达小鼠建立cuprizone、LPC（lysophosphatidyl choline）和EAE三种髓鞘脱失动物模型，采用行为学分析、髓鞘染色、免疫组织化学、原位杂交、透射电镜、免疫流式细胞术、蛋白印迹、Q-PCR 和基因芯片等技术和方法，结合体内和体外实验，证明了1. CatC具有加重髓鞘脱失、影响髓鞘再生和加重神经功能损伤的作用；2. 证明了CatC通过作用于MG/巨噬细胞细胞膜NADM受体亚基NR2B/FAK，加重Ca2+内流，通过Ca2+依赖的PKC/p38 MAPK/NF-κB和FAK触发的p38 MAPK/NF-κB通路，促进MG和巨噬细胞向M1极化，影响固有免疫；3. 证明了CatC增加MG表达促Th17和Tfh分化的调控分子，即IL-6、IL-23a、IL-12b和PD-L1，促进Th17和Tfh细胞分化，增加致病细胞因子IL-17A和IL-21的表达和MOG特异性IgG抗体的产生，通过对适应性免疫的影响，加重EAE神经损伤；4. CatC通过促进MG和神经元产生CCL2、CCL5和CXCL2趋化因子，加重神经炎症。此外，我们将对CatC的研究延展到抑郁的炎性机制，发现CatC通过促进神经炎症，加重急、慢性应激诱导的抑郁小鼠抑郁行为和神经化学紊乱。上述结果提示，CatC通过影响固有、适应性免疫以及趋化作用，参与多种疾病的神经炎症机制。这不仅对中枢神经系统慢性和持续性炎症发生机制提供了理论基础，同时对MS和其他疾病治疗提供了新思路。

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