黄芪活性成分芒柄花素靶向GSK-3β蛋白调控巨噬细胞/小胶质细胞极化抗心梗合并抑郁的作用及分子机制

The incidence of myocardial infarction (MI) combined with depression was as high as 45%, which seriously affected the prognosis of patients. And there are no ideal drugs for treatment in clinical. Radix Astragali (RA) is widely used for treating cardiac and brain diseases with clinical efficacy. In our pilot study, experimental data showed that fomononetin has potential effect on improving myocardial ischemia-reperfusion injury and the behavior of depression. Preliminary mechanism study showed that formononetin promoted macrophage M2 polarization and directly bound to GSK-3β, inhibiting its activity. And M2 macrophage and microglia play important roles in the pathological processes of myocardial infarction and depression. Thus, we proposed the scientific hypothesis that RA and formononetin regulates macrophage/microglia M1/M2 polarization against myocardial infarction combined with depression targeting glycogen synthase kinase-3β. We will confirm the effect of RA and formononetin in treatment of MI combined depression by using the mouse myocardial ischemia model plus the chronic mild unpredictability stimulation model after MI; using gene knockout and overexpression technology to study the role of GSK-3β in regulating macrophage and microglia cell polarization; using brain tissue culture and macrophage/microglia scavenging methods to study the effect of M2 macrophage/microglia on improving myocardial ischemia combined with depression. This study will illustrate the efficacy and material base of RA on treating MI combined with depression, and also pave the avenue for the development of novel macrophage/microglia-targeting drugs for the treatment of MI combined with depression.

心梗合并抑郁症发生率高达45%，严重影响患者预后，临床上尚无理想治疗药物。前期实验发现心脑疾病常用中药黄芪活性成分芒柄花素有潜在抗心梗和抑郁行为作用。初步机制研究发现芒柄花素可促进M2型巨噬细胞极化，并直接作用GSK-3β蛋白，抑制其活性。而M2型巨噬细胞和小胶质细胞在抗心梗合并抑郁症中发挥重要作用。因此，提出科学假设：黄芪活性成分芒柄花素可通过抑制GSK-3β调控巨噬细胞和小胶质细胞极化而抗心梗合并抑郁症。本课题拟利用小鼠心肌缺血合并抑郁症模型评价黄芪及芒柄花素抗心梗合并抑郁症作用；借助基因敲除、过表达等手段，研究GSK-3β在黄芪活性成分芒柄花素调控巨噬细胞和小胶质细胞极化中的作用；利用脑片培养和细胞清除方法研究M2型巨噬细胞和小胶质细胞在抗心梗合并抑郁症的作用。本课题为阐明黄芪抗心梗合并抑郁症的药效物质基础提供依据，也为开发以调控巨噬细胞/小胶质细胞抗心梗合并抑郁的药物提供新思路。

心梗合并抑郁症发生率高，且严重影响心梗患者预后和增加死亡风险，目前无理想治疗药物。基于前期发现中药黄芪活性成分芒柄花素有潜在抗心梗和抑郁行为作用。本课题旨在揭示芒柄花素改善心肌梗死合并抑郁药效及潜在分子机制。首先，我们采用心梗合并抑郁模型，发现芒柄花素在40mg/kg下可显著改善小鼠心梗中抑郁状态，上调BDNF的表达和脑组织中5-HT的含量，并且显著提高模型小鼠心功能，抑制心脏组织炎性细胞浸润和心肌细胞凋亡。其次，我们发现芒柄花素可显著抑制M1型巨噬细胞/小胶质细胞极化，促进M2巨噬细胞/小胶质细胞极化。芒柄花素可抑制心脏组织CD68+细胞数量，提高CD206+细胞数量；抑制脑组织中Iba+ iNOS+细胞数量，提高Iba+ Arg1+细胞数量。重要的是，我们发现心梗小鼠血液中IL-6和IL-17A含量升高，且该血清可刺激诱导M1型小胶质细胞，而芒柄花素可有效抑制M1型，促进M2型。并且单纯合用IL-6和IL-17A也可刺激诱导M1型小胶质细胞，芒柄花素可有效抑制其激活。第三，进一步机制研究表明芒柄花素可抑制GSK-3β活性和及其下游Notch1和C/EBPα信号通路。分子对接表明芒柄花素可与GSK-3β的Cys199结合。SPR结果表明芒柄花素对GSK-3β具有较高的亲和力（Kd=1.48μM）。当Cys199突变时，芒柄花素对巨噬细胞/小胶质细胞中GSK-3β活性和M1极化的抑制作用也被阻断。我们还与靶向Cys199的GSK3β抑制剂Tideglusib进行了对比，发现芒柄花素的抑制活性优于Tideglusib，且毒性远小于Tideglusib。第四, 我们发现心梗病人和心梗小鼠中IL-6，IL-17A含量升高，采用IL-6/IL-17A联用可导致小鼠出现抑郁行为，且存在神经炎症，从而揭示心肌梗死后产生的IL-6和IL-17A可引起神经炎症，可能是心梗后抑郁发生的作用机制。本课题不仅揭示了芒柄花素通过靶向GSK-3β调节巨噬细胞/小胶质细胞极化来改善心肌梗死合并抑郁症小鼠的心功能和抑郁行为，也为调控M2型巨噬细胞/小胶质细胞作为治疗心梗合并抑郁的策略提供实验依据。总之，中药黄芪活性成分芒柄花素可能是一种潜在的治疗心肌梗死合并抑郁症的药物，且毒副作用小，具有一定应用前景。