炎症小体激活小胶质细胞促进异常新生神经元形成在锂-毛果芸香碱致痫大鼠中的作用机制

Mechanism of action of inflammasome in promoting the formation of abnormal neonatal neurons by activating microglial cells in rats with lithium-pilocarpine-induced epilepsy

摘要:

目的 在锂-毛果芸香碱（Lithium-Pilocarpine）致痫大鼠模型中，检测炎症小体的激活，观察小胶质细胞的活化和异常新生神经元的形成，探索炎症小体激活小胶质细胞促进异常新生神经元形成在癫痫发生发展中的作用机制。方法 通过腹腔注射氯化锂和毛果芸香碱建立癫痫大鼠模型，将大鼠分为Sham组（生理盐水对照组，n=12）、Pilo组（锂-毛果芸香碱致痫组，n=20）和Pilo+MCC950组[锂-毛果芸香碱致痫后给予核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）抑制剂组，n=20]，在急性期观察记录大鼠癫痫的行为学表现，通过病理切片确认脑组织损伤情况和细胞激活情况，借助分子检测确认炎症小体和其他分子的表达情况。采用体外细胞模型挽救实验验证炎症小体的作用。结果 Pilo组大鼠有6只出现V级，5只出现Ⅳ级，2只出现III级的癫痫发作情况。相较之下，Pilo+MCC950组大鼠则没有出现V级，6只出现Ⅳ级，8只出现III级，2只出现Ⅱ级的癫痫发作情况。Pilo组强直痉挛发作的潜伏期短于Pilo+MCC950组，III级以上癫痫发作的次数和持续时间大于Pilo+MCC950组。Pilo组NLRP3和pro-caspase-1的含量、4种促炎细胞因子的含量以及脑源性神经营养因子（BNDF）和细胞外调节蛋白激酶（ERK）均高于Pilo+MCC950组。免疫荧光检测显示，Pilo组小胶质细胞大量活化，出现大量异常新生神经元。体外细胞实验后，ELISA结果验证了炎症小体的作用。结论 在锂-毛果芸香碱致痫大鼠模型中，炎症小体NLRP3接受外界炎症刺激而活化聚集，进而大量产生多种促炎细胞因子，使得小胶质细胞活化，分泌BDNF并激活ERK信号通路，调控异常新生神经元的形成和聚集等生物学行为，参与癫痫的发生发展。 [国际神经病学神经外科学杂志, 2024, 51(4): 28-35]

Abstract:

Objective To investigate the activation of inflammasome and microglial cells and the formation of abnormal neonatal neurons in a rat model of lithium-pilocarpine-induced epilepsy， as well as the mechanism of action of inflammasome in the development and progression of epilepsy by activating microglial cells and promoting the formation of abnormal neonatal neurons.Methods Rats were given intraperitoneal injection of lithium and pilocarpine to establish a model of epilepsy. Rats were divided into Sham group （saline control group， n=12）， Pilo group （lithium-pilocarpine epileptogenic group， n=20） and Pilo+MCC950 group （lithium-pilocarpine epileptogenic followed by administration of NLRP3 inhibitor group， n=20）. The behavioral manifestations of epilepsy were observed in the acute stage； pathological sections were used to observe brain tissue injury and cell activation； molecular assays were used to measure the expression of inflammasome and other molecules； in vitro cell model rescue experiments were used to verify the effect of inflammasome.Results In the Pilo group， there were six rats with grade V seizures， five with grade IV seizures， and two with grade III seizures， while in the Pilo+MCC950 group， there were no rats with grade V seizures， and there were six rats with grade IV seizures， eight with grade III seizures， and two with grade II seizures. Compared with the Pilo+MCC950 group， the Pilo group had a significantly shorter latency period of myotonic seizures and significantly higher number and longer duration of grade ≥III seizure. The Pilo group had significantly higher levels of NLRP3， pro-caspase-1， four inflammatory cytokines， BNDF， and ERK than the Pilo+MCC950 group. Immunofluorescence assay showed the activation of a large number of microglial cells and the presence of a large number of abnormal neonatal neurons in the Pilo group. After in vitro cell experiments， the results of ELISA validated the role of inflammasome.Conclusion In the rat model of lithium-pilocarpine-induced epilepsy， inflammasome NLRP3 is activated by external inflammatory stimuli and then produces a large number of inflammatory cytokines， thereby activating microglial cells to secrete BDNF and activate the ERK signaling pathway， and thus it regulates the formation and aggregation of abnormal neonatal neurons and participates in the development and progression of epilepsy. ［Journal of International Neurology and Neurosurgery, 2024, 51(4): 28-35］