ATP/P2X7R-NLRP3炎性小体通路介导的神经可塑受损在肝疏泄失常怒情志中的作用及机制研究

Anger is one of the most important negative emotions in seven human emotions of TCM, harming to human health seriously. The biological basis of "anger injured liver (in TCM)" is the important scientific problems of basis of TCM and clinical practice. However, the neurobiological mechanism of its occurrence is not clear. In international reports and early stage, we found that anger lead to increased glutamate and inflammatory factors, neuroplastic damage and aggressive behavior. Tiaogan formula can reverse this neurobiological change. We hypothesize that anger stress increase neuronal glutamate release, increase release of ATP in astrocytes, activate P2X7R in microglia, further activate of NLRP3 inflammasome, increase parainflammation and damage neural plasticity, leading to mental and behavioral abnormalities, which is the initiating factors of "anger injured liver (in TCM)". We will employ the whole animal and block P2X7R and NLRP3 inflammatory pathways, and NLRP3-/- mice, verificating the function of NLRP3 in anger. In vitro experiment, We will block and activate NLRP3 inflammasome pathway, detecting neuronal morphology and electrophysiological signals of neural plasticity, exploring the effects of Tiaogan formula Baixiangdan capsule, Shu Suyu capsule and their active components on NLRP3 inflammasome function. We aim to verify the scientific hypothesis of “abnormal function of liver (in TCM)” related to CNS parainflammation.

怒作为七情最重要的负性情志之一，严重危害人类健康！“怒伤肝”致病的生物学基础是中医学基础与临床重要的科学问题。但其发生的神经生物学机制不清。国际报道及我们前期发现，怒情志导致谷氨酸及炎症因子升高、神经可塑损伤并伴有攻击行为，调肝方药可逆转这一神经生物学改变。由此我们推测怒情志应激增加神经元谷氨酸释放，导致星形胶质细胞ATP释放增多，激活小胶质细胞P2X7R，进一步激活NLRP3炎症小体，导致神经亚炎症升高，损害神经可塑，表现精神及行为异常，是“怒伤肝”的始动因素。本研究拟从整体动物及阻断P2X7R和NLRP3炎症通路，并利用NLRP3-/-小鼠，验证NLRP3在怒情志失常中的作用；离体实验拟进行阻断及激活NLRP3炎症小体通路，检测神经元形态及其神经可塑性电生理信号，阐明调肝方药白香丹胶囊、舒郁胶囊及其活性成分对于NLRP3炎症小体的作用。验证“肝疏泄失常与中枢神经亚炎症相关”的科学假说。

怒作为七情最重要的负性情志之一，严重危害人类健康！“怒伤肝”致病的生物学基础是中医学基础与临床重要的科学问题。但其发生的神经生物学机制不清。本项目主要进行了：1.建立和拓展适合白香丹胶囊调控攻击行为药效的筛选与评价方法。2.明确ATP/P2X7R-NLRP3炎性小体通路在怒情志攻击行为中的作用。3.发现调肝方药白香丹胶囊通过ATP/P2X7R-NLRP3炎性小体通路改善怒情志攻击行为的作用机制。行为学结果显示：怒情志小鼠旷场总路程、中央区总路程以及中央区停留时间均显著升高，高架十字迷宫实验进入开放臂的次数百分比、活动总路程与平均速度均明显升高，攻击行为测试潜伏期显著缩短，攻击行为得分显著升高；白香丹胶囊、氟西汀、A804598及IL-1Ra可逆转上述行为学改变。蛋白表达结果显示：怒情志小鼠NLRP3蛋白表达显著升高；白香丹胶囊、氟西汀、A804598、IL-1Ra及NLRP3-/-组NLRP3蛋白表达均显著降低。回归分析表明怒情志攻击行为与NLRP3有强相关性。HE染色结果显示：怒情志小鼠脑组织皮层及海马CA1、CA3区神经细胞排列紊乱，细胞体积变小且细胞着色较深，胞体呈不规则形；白香丹胶囊及NLRP3-/-组小鼠可逆转上述形态学改变。免疫组化结果显示：怒情志小鼠脑组织皮层及海马小胶质细胞阳性细胞显著增多，胞体变大，多为圆形，分支变粗变短；白香丹胶囊及NLRP3-/-组小鼠脑可逆转上述形态学改变。白香丹胶囊、丹皮酚及A804598显著降低培养小胶质细胞NLRP3表达。ATP诱导的小胶质细胞上清液IL-1β显著升高，电生理活动无明显差异。表明：①怒情志的发生机制与外周血及海马脑区NLRP3、IL-1β的含量改变存在密切关联，白香丹胶囊可以降低攻击行为及上述病理过程。怒情志诱导神经炎症、小胶质细胞活化及突触损伤，白香丹胶囊能改善神经炎症；②白香丹胶囊及其有效成分通过抑制ATP/P2X7R-NLRP3炎性小体通路的激活，从而抑制NLRP3、IL-1β的表达，进而起到调控怒情志攻击行为的作用。