痫性发作诱导NLRP3炎症小体持续活化触发ADK高表达的编程机制

Adenosine kinase (ADK) expression is increased in patients with epilepsy and plays an important role in chronic epilepsy. ADK is a key enzyme that regulates adenosine, decreasing the concentration of extracellular adenosine and increasing nervous system excitability. However, the mechanism that leads to the up-regulation of ADK expression is currently unknown. Our preliminary studies have shown that status epilepticus (SE) induces sustained activation of astrocytes NLRP3 inflammasome, accompanying with continual up-regulation of ADK expression, whereas NLRP3 inhibitors reduce ADK activation. Previous studies demonstrated that persistent activation of NLRP3 inflammasome relies on adenosine A2a receptor activation at low concentrations of adenosine. Therefore, we speculate that SE-induced activation of NLRP3 inflammasome signaling promotes the up-regulation of ADK expression. This study intends to use NLRP3 knockout mice combined with molecular biotechnology and biological information such as ChIP, Co-IP methylation BSP detection and dual luciferase reporter systems to explore the regulatory pathways between SE induced NLRP3 inflammasome sustained activation and high expression of ADK, clarifying the programming mechanism that NLRP3 activation induces high ADK express. Which may provide a new target for the prevention and treatment for chronic epilepsy.

腺苷激酶（ADK）表达在癫痫患者中表达增加，并在慢性癫痫形成中发挥重要作用。ADK是 调控腺苷的关键酶，降低胞外腺苷浓度而增加神经系统兴奋性。但导致ADK表达上调的机制目 前未知。我们的预试验发现癫痫持续状态（SE）诱导星形胶质细胞NLRP3炎症小体持续活化， 且伴随ADK持续表达上调，然而NLRP3抑制剂能减少ADK活化。NLRP3炎症小体持续活化依赖低浓 度腺苷激活的腺苷A2a受体。据此我们推测SE诱导的NLRP3炎症小体持续活化信号可能促进ADK 表达上调。本项目拟用NLRP3基因敲除鼠，采用ChIP-PCR, Co-IP，甲基化BSP检测及双荧光素酶报告系统等技术及生物学信息，探索SE诱导NLRP3炎症小体持续活化与ADK高表达之间的调控 通路，阐明NLRP3活化诱导ADK高表达编程的机制。为防治慢性癫痫提供新靶标。

异常免疫信号在慢性癫痫形成中发挥重要作用。临床中多种难治性癫痫对抗癫痫药物反应欠佳，却对丙种球蛋白、糖皮质激素及ACTH等免疫调节药物有较好反应。本课题研究了痫性发作后NLRP3炎症小体活化、细胞焦亡、腺苷激酶ADK的活化在慢性癫痫形成中的作用，探讨了SE发作诱导NLRP3炎症小体持续活化的机制、以及NLRP3与ADK活化的内在调节机制。得到以下研究结果：.1. 海人藻酸KA诱导SE后，海马区ADK表达明显增加。腺苷 A2α 受体被激活，诱导海马神经元丢失和促炎性因子 IL⁃18 和 IL⁃1β 释放。.2. SE发作后，焦亡主要指标Pro-caspase1及活化形式Cleaved-caspase1表达增加，同时焦亡分子标志物ASC、GSDEMD复合物生成，表明痫性发作后细胞焦亡过程启动。同时炎症小体NLRP3形成，蛋白表达增加。.3. SE促进ADK和炎症小体NLRP3活化，抑制NLRP3活化和焦亡，可保护海马神经元丢失和抑制胶质细胞ADK活化，表明SE后ADK表达增加依赖NLRP3活化。SE后焦亡标志物caspase1活化及ADK表达增加，抑制NLRP3及Caspase1活性可减少ADK活化。SE后海马神经元丢失，抑制NLRP3及caspase1活化可保护海马神经元。抑制NLRP3和caspase1活化减少IL-18和IL-1β的表达。SE诱导星形胶质细胞ADK表达，抑制NLRP3活化减少星形胶质细胞活化和ADK表达。.4. ADK活化受级联信号CREB/REST/SP1调控。 SP1作为潜在调控ADK表达的转录因子靶点，为了探讨SE发作后ADK高表达的信号转导机制，我们检测了CREB/REST/SP1级联信号变化对ADK表达的影响。SP1干扰沉默后，ADK表达增加。表明SP1可能存在负性调节SP1表达的效应。而干扰抑制元件1沉默转录子REST表达后，SP1表达增加，ADK表达明显受抑制。表明转录因子受REST的负向调控。而干扰CREB的表达后，SP1表达增加，REST及ADK表达下降。表明ADK活化受级联信号CREB/REST/SP1的多点调控。.5. SE促进炎症小体NLRP3活化和细胞焦亡，抑制NLRP3活化和焦亡，促进微血管生成。抑制SE后细胞焦亡可以减少SRS形成和改善行为学。SE诱导嘌呤受体P2X7R活化是NLRP3活化的上游信号机制。