异甘草素通过调节DUSP6泛素化修饰阻断小胶质细胞ERK1/2通路改善阿尔茨海默病的机制研究

Neuroinflammation is one of the most important contributor to Alzheimer's disease(AD) pathogenesis. Microglia can be activated by MAPKs inflammatory pathways followed by releasing proinflammatory factors, and aggravating neurofibrillary tangles and memory deficits, which are regarded as potential therapeutic targets. Licorice root functions to detoxify and to tonify spleen and to calm the nerves according to the classics of Traditional Chinese Medicine. Our preliminary experiment’ results indicate that Isoliquiritigenin(ISL), an active ingredient from licorice root, can increase DUSP6 protein expression and inhibit the activity of ERK1/2 and proinflammatory factors in activated microglia, and improve the behavior of memory deficits in mice with AD. Based on the dephosphorylation of DUSP6 on ERK1/2, our hypothesis will be tested by studying its pharmacological mechanism from two specific aims in this proposal, 1) we will probe the targets of ISL on the inhibition of neuroinflammation in primary microglia, BV2 microglial cell line and transgenic mice with DUSP6 overexpress or knockout. Further, we should make clear that ISL probably block the ERK1/2 pathway in activated microglia by increasing the protein stability of DUSP6 through the adjustment of Ubiquitination-modified DUSP6, which in turn regulates the rate of M1/M2 polar transformation (polarization) after microglial activation and suppresses neuroinflammation. 2) Based on microglia and neuron co-culture system and PS1/PS2 double knockout AD model mice, we will investigate the mechanism of ISL on suppressing the neurotoxicity caused by neuroinflammation. Moreover, anti-neurotoxic effects of ISL will also be investigated by determining if ISL can improve tau protein hyperphosphorylation and cognitive impairment in AD via preventing the neurotoxic events including apoptosis, excitatory toxicity and S-nitrosylation.

神经炎症为阿尔茨海默病(AD)发病机制之一，经MAPKs等炎症通路激活小胶质细胞释放促炎因子，加剧纤维缠结和记忆缺陷，故成为潜在治疗靶点。甘草有解毒健脾安神功效，预实验发现其有效成分异甘草素提高DUSP6蛋白表达并抑制激活小胶质细胞ERK1/2活性和促炎因子，改善AD小鼠记忆缺陷。鉴于DUSP6对ERK1/2的去磷酸化作用，故本项目拟深入研究其药理机制：①利用原代小胶质细胞、DUSP6过表达或敲除BV2细胞株和转基因小鼠探讨异甘草素抑制神经炎症的靶点，明确异甘草素阻断小胶质细胞ERK1/2通路可能是通过调节DUSP6泛素化修饰提高蛋白稳定性实现的，并调控小胶质细胞M1/M2极化抑制神经炎症；②利用小胶质细胞和神经元共培养和PS1/PS2双敲除AD小鼠探讨异甘草素抑制神经炎症引发的神经毒性机制，明确异甘草素可能是通过阻止凋亡、兴奋性毒性和亚硝基化途径改善AD的tau蛋白过度磷酸化和记忆缺陷。

阿尔茨海默病(Alzheimer’s disease，AD)以认知障碍、精神行为异常等为特征的神经退行性疾病，迄今为止尚无根治AD的药物。小胶质细胞激活诱发的神经炎症在AD病程早期已显现，是导致突触功能损伤的关键因素，因此抑制小胶质细胞激活和神经炎症有望成为阻断AD病程进展的有效治疗手段。异甘草素(Isoliquiritigenin, ISL)为中药甘草的活性成分，本项目为研究证实ISL具有较好的抑制小胶质细胞激活并改善AD的神经保护作用，主要从以下两方面着手：一是利用原代小胶质细胞、DUSP6敲低的BV2细胞和小鼠证实ISL抑制神经炎症并阻断小胶质细胞ERK1/2通路与调节双特异性蛋白磷酸酶6（dual-specificity protein phosphatases，DUSP6）泛素化修饰提高蛋白稳定性相关；二是利用小胶质细胞和神经元共培养和早老素1/2双基因敲除（Presenilin 1/2 conditional double knockout, PS cDKO）的AD模式小鼠进一步验证ISL确实可抑制小胶质细胞激活诱发的神经炎症的同时阻止突触功能下降，从而改善AD的tau蛋白过度磷酸化和记忆缺陷。.本项目研究结果提示，DUSP6作为具有去磷酸化作用的磷酸酶，可阻断p-ERK活性并抑制AD小胶质细胞激活过程中发挥关键作用，并且已证实DUSP6的蛋白稳定性与其泛素化修饰有关，因此调节小胶质细胞DUSP6的蛋白稳定性有希望成为改善AD的新药研发的新靶点。同时我们还发现，ISL改善AD小鼠tau蛋白过度磷酸化、突触可塑性下降和记忆障碍的根本原因与其调节DUSP6泛素化修饰并阻断ERK1/2通路，从而抑制小胶质细胞激活诱发的神经炎症有关。由此可见，ISL可能成为改善AD等神经退行性疾病潜在的有效药物，其药理机制的深入研究将具有一定的社会价值和临床应用前景。.本项目在实施过程中部分研究成果在国际期刊共发表8篇，项目组成员参加学术会议并投稿会议论文和墙报展示共4篇，项目申请人（徐颖教授）作会议报告5次，申请发明专利2项（审查阶段），培养硕博士研究生共8名。