两种药用灵芝真菌中抑制小胶质细胞过度活化的三萜类物质的发现及作用机制研究

An important strategy of preventing neuroinflammation and neurodegenerative diseases is to inhibit the microglia overactivation. Ganoderma were recorded to be “relieving uneasiness”, “enhancing intelligence”, “improving memory” in ancient herbal medicine books, and now were confirmed to be definitely neuroprotective, and anti-inflammatory in clinic. In our previous work, we found that, for the first time, the ethanol extracts of G. curtisii and G. applanatum could inhibit the release of NO from the BV-2 microglia induced with LPS. We purified 27 triterpenoids from the ethanol extract of G. curtisii, and 9 triterpenoids among them were found to be strong inhibitors of microglia overactivation, their IC50 values were all less than 10μM. Therefore, we speculated that the triterpenoids from G. curtisii and G. applanatum were the exciting mines of the inhibitors of microglia overactivation, which can regulate the immune inflammatory reaction mediated by microglia. In this grant application, we want to carry on the systematically purification of triterpenoids from G. curtisii and G. applanatum, to collect more triterpenoids to support the screening of their activities on microglia overactivation inhibition, and to study their Structure-Activity-Relationships (SARs), and furthermore, to study the mechanism of their activities, and elucidate its bioactive components of “relieving uneasiness” and “enhancing intelligence” from the perspective of anti-neuroinflammation. Our work will present the fundamental data for the drug discovery of neuroinflammation mediated by microglia overactivation.

抑制小胶质细胞过度活化及其介导的炎症反应是防治脑内炎症及神经退行性疾病的策略之一。灵芝在历代本草上均记载有“安神”、“增智慧”、“不忘”等功效，在临床上具有确切的神经保护、抗炎功效。前期工作中，我们首次发现弱光泽灵芝及树舌灵芝的乙醇提取物能浓度依赖性的抑制LPS诱导的BV-2小胶质细胞释放NO，从弱光泽灵芝中分离得到27个三萜类化合物，其中9个化合物具有较强的抑制小胶质细胞活化的能力(IC50值＜10μM)，因此，我们推测上述两种灵芝中富含能高效抑制小胶质细胞过度活化的三萜类化合物。本研究拟继续对上述两种灵芝的三萜类成分进行全面的系统研究，纯化出尽可能多的三萜类成分，以LPS活化的BV-2小胶质细胞建立炎症模型评价其活性，开展构效关系研究，并深入研究活性化合物的作用机制，从抑制神经炎症角度阐述其“安神”、“增智慧”等的物质基础，为来源于天然产物的小胶质细胞异常活化的抑制剂的发现提供参考。

抑制小胶质细胞过度活化及其介导的炎症反应是防治脑内炎症及神经退行性疾病的策略之 一。灵芝在历代本草上均记载有“安神”、“增智慧”、“不忘”等功效，在临床上具有确切 的神经保护、抗炎功效。本项目通过LPS诱导BV-2小胶质细胞释放一氧化氮的细胞炎症模型为分离活性导向，深入研究弱光泽灵芝及树舌灵芝具有免疫调节活性的单体物质，从免疫细胞、分子水平基本阐明了其“安神”、“增智慧”等的物质基础。    . 通过LPS诱导BV-2小胶质细胞炎症模型活性为分离导向，从两种灵芝的85%乙醇提取部位分离并鉴定了68个灵芝三萜类单体化合物，其中有6个新化合物。采用LPS诱导BV-2小胶质细胞释放NO的炎症模型，结果表明大部分灵芝三萜化合物有不同程度的抑制NO释放活性。对树舌灵芝三萜新化合物applanone F （compound 3）的抗炎作用机制开展初步研究，结果表明applanone F能够抑制LPS诱导的BV-2小胶质细胞内NO和PGE2的释放，同时抑制iNOS和Cox-2的蛋白表达，表明applanone F可通过抑制NO和PGE2的合成而发挥抗炎作用。另外，applanone F不仅降低了炎症因子IL-6、TNF-α的释放量以及ROS的蓄积，还下调了异常活化的小胶质细胞MAPK和NF-κB信号通路p-p38、p-JNK、p-ERK、p-NF-κB p65和p-IκBα的蛋白表达，提示MAPK和NF-κB信号通路可能是applanone F减轻神经炎症作用的主要途径。. 利用LPS诱导BV-2小胶质细胞活化后的细胞培养上清液作用于HT22小鼠海马神经元细胞，JC-1、TUNEL、Heochst 33258染色法检测凋亡细胞，ELISA测定细胞内MAP2水平，探讨applanone F的抗炎保护神经元作用。结果显示，applanone F处理后的LPS诱导BV-2细胞的条件培养基作用于HT22细胞，发现能够升高HT22细胞下降的线粒体膜电位、减少凋亡细胞的产生以及改善细胞膜的通透性，增加细胞内MAP2的水平。研究结果证实了，灵芝三萜类化合物通过减轻小胶质细胞的异常活化保护神经元细胞，是灵芝发挥了其“安神”、“增智慧”等的物质基础。