基于神经胶质细胞IDO1介导色氨酸代谢途径探讨“从肝论治”癫痫共病抑郁的分子机制

Epilepsy-associated depression is common and seriously affect the prognosis of epilepsy. The mechanism of epilepsy comorbid depression is unclear. Current research focuses on neurotransmitter related mechanisms .Our studies showed that the activation of indoleamine 2,3-dioxygenase 1 (IDO1) in the tryptophan metabolic pathway is closely related to the occurence of epilepsy comorbid depression, but its specific regulatory mechanisms is still unclear. The research hypothesis that IDO1 activation is mainly mediated by astrocytes/microglia and regulates “tryptophan depletion”, expression of neuroinflammation, neurotransmitter disorders, and oxidative stress, affecting the excitability of neurons and the occurrence of epilepsy comorbid depression.Liver related prescriptions can inhibit the epilepsy comorbid depression state by inhibiting the overexpression of IDO1 in astrocytes/microglia. In this project, conditional knockout mice were used to establish animal model of epilepsy comorbid depression, and a coculture model of astrocytes/microglia and neurons, we will use electrophysiology, protein and nucleic acid detection, liquid color Mass spectrometry, immunohistochemistry and other techniques to study the molecular mechanisms involved in epilepsy comorbid depression, and to enrich the scientific connotation of Chinese medicine “liver associated treatments” on epilepsy comorbid depression.

癫痫患者共病抑郁障碍的发生率明显高于普通人群，严重影响癫痫患者预后。癫痫共病抑郁的机制尚不明了，目前研究多集中于神经递质相关改变。申请者的前期研究表明，色氨酸代谢途径中的吲哚胺2,3-双加氧酶1（IDO1）活化与癫痫共病抑郁发病关系密切，但其具体调控机制尚未见报道。因此申请者提出研究假说：其可能的途径是，IDO1的活化主要由星形胶质细胞/小胶质细胞介导，并通过调控“色氨酸消耗”、神经炎症表达、递质紊乱及氧化应激等途径，影响神经元兴奋性及癫痫共病抑郁的发生，“从肝论治”方药可通过抑制星形胶质细胞/小胶质细胞IDO1过度表达，干预癫痫共病抑郁状态。本项目拟采用条件性基因敲除小鼠，建立癫痫共病抑郁动物模型、星形胶质细胞/小胶质细胞与神经元共培养模型，利用神经电生理学、蛋白和核酸检测、液相色谱质谱联用、免疫组化等技术研究癫痫共病抑郁的相关分子机制，丰富中医“从肝论治”癫痫共病抑郁的科学内涵。

癫痫患者共病抑郁障碍的发生率明显高于普通人群，严重影响癫痫患者预后。癫痫共病抑郁的机制尚不明了，目前研究多集中于神经递质相关改变。申请者的前期研究表明，色氨酸代谢途径中的吲哚胺2,3-双加氧酶1（IDO1）活化与癫痫共病抑郁发病关系密切，但其具体调控机制尚未见报道。本项目拟采用条件性基因敲除小鼠，建立癫痫共病抑郁动物模型、星形胶质细胞/小胶质细胞与神经元共培养模型，利用神经电生理学、蛋白和核酸检测、液相色谱质谱联用、免疫组化等技术研究癫痫共病抑郁的相关分子机制，丰富中医“从肝论治”癫痫共病抑郁的科学内涵。本研究成功构建了氯化锂－匹罗卡品慢性颞叶癫痫小鼠模型，通过强迫游泳、蔗糖消耗实验筛选癫痫共病抑郁小鼠模型，并且利用IDO1基因敲除小鼠构建氯化锂—匹罗卡品慢性癫痫小鼠模型，评估慢性癫痫小鼠模型抑郁行为的变化，液相色谱-质谱法检测血清和海马组织IDO1、犬尿酸、色氨酸及下游代谢产物的浓度，Elisa法检测血清和海马组织IL-1β、IL-6、TNF-α、抗氧化酶活性的表达，免疫组化和免疫荧光技术检测慢性癫痫模型中海马区神经元损伤情况和胶质细胞的变化。结果显示IDO1基因敲除减轻癫痫小鼠抑郁样行为表现，减少癫痫小鼠海马CA1和CA3区神经元的丢失和减轻海马组织中胶质细胞的活化，降低了血清和海马组织中炎症因子IL-6和TNF-α的水平。提示IDO1基因缺失对神经元具有一定的保护作用和能够减轻癫痫的炎症反应。说明IDO1可能通过介导色氨酸代谢、炎症反应及氧化应激影响癫痫发作。这可能为IDO1基因缺失对发作的保护作用提供了依据。柴胡疏肝汤中、高剂量能够缓解癫痫共病抑郁小鼠模型的抑郁样行为，并且明显改善癫痫动物模型海马CA1和CA3区病理损伤，并且抑制QUIN的产生，进而改善胶质细胞活化，降低促炎因子水平和氧化应激水平，从而减轻癫痫发作的严重程度。柴胡疏肝汤通过抑制IDO1活化，使神经元丢失减少，改善炎症反应和氧化应激状态，起到抑制癫痫发作的作用，这为IDO1作为癫痫共病抑郁治疗的靶点提供了可靠的实验证据。