鞘脂代谢重编程驱动小胶质细胞极化及黄连解毒汤干预研究

Nonresolving neuroinflammation initiated by polarization of microglia is the pivotal pathophysiological and biochemical process of central nervous system disease. Based on the metabolic reprogramming theory and the actions of Huang-lian Jie-du decoction on microglia phenotype and sphingolipid metabolism in our previous investigation, a scientific hypothesis is put forward that the metabolic reprogramming of sphingolipid might be the underlying biochemical mechanism of polarization of microglia and factor involving the nonresolving neuroinflammation, through which Huang-lian Jie-du decoction modulating the central neuroinflammation. Therefore, this project was designed to construct the integrate network and explore the regulating pattern of sphingolipid metabolism in microglia in vitro and in vivo (Cerk-/- and APP/PS1 mice) by targeted metabolomics, quantified transcription analysis, fluorescence-activated cell sorting, immunoblotting, immunofluorescence and pharmacological evaluation system. Following the annotation of the underlying interaction between the sphingolipid metabolism and microglia polarization, the role of sphingolipid metabolic reprogramming in microglia polarization and nonresolving neuroinflammation will be defined hopefully. On the other hand, the completion of this subject will further reveal the biochemical mechanism of Huang-lian Jie-du decoction on central neuroinflammation and promote development a novel formula on regulating sphingolipid metabolism, which will provide new therapeutic idea on central nervous system diseases.

小胶质细胞极化启动的非可控性神经炎症是中枢神经系统疾病重要的病理生化过程，基于代谢重编程理论和课题组前期关于黄连解毒汤调控小胶质细胞表型和干预鞘脂代谢的研究基础，我们提出“鞘脂代谢重编程是小胶质细胞表型极化和驱动非可控性中枢神经炎症的潜在生化机制，黄连解毒汤通过调节鞘脂代谢进而调控小胶质细胞表型，干预中枢神经炎症”的科学假说。本课题拟通过靶向代谢组学、定量转录水平分析、荧光激活细胞分选术、免疫印迹和免疫荧光等技术，结合药理效应评价体系，研究在特定病理因素框架和黄连解毒汤干预下，离体培养及在体(Cerk-/-和APP/PS1小鼠)小胶质细胞鞘脂代谢整合网络和调整规律，分析其和小胶质细胞表型和功能的潜在关系，以期揭示鞘脂代谢重编程在小胶质细胞表型极化和非可控性中枢神经炎症中的作用，阐明黄连解毒汤干预中枢神经炎症的潜在生化机制，并初步探索一种调控鞘脂代谢的组合物，为中枢神经系统疾病治疗提供参考。

代谢重编程是髓系细胞在特定病理条件和应激刺激下表型调控和功能转化的重要特征，差异性代谢产物及其代谢酶亦成为调控细胞表型功能的靶点。鞘脂代谢异常是中枢神经疾病如神经退行性疾病及脑血管病的典型特征，为揭示小胶质细胞的鞘脂代谢特点和规律，并研究黄连解毒汤通过鞘脂代谢调控小胶质细胞表型和功能的有效成分及潜在组合物。本项目的研究内容主要有，（1）特定病理因素诱导框架下小胶质细胞鞘脂代谢；（2）AD病理刺激小胶质细胞鞘脂代谢及黄连解毒汤的干预；（3）MCAO大鼠脑组织鞘脂代谢、小胶质细胞表型及黄连解毒汤的干预；（4）LPS诱导小胶质细胞鞘脂代谢伴随糖酵解的黄连解毒汤干预；（5）基于鞘脂代谢量丰成分组合物的效应评价。结果发现，（1）通过靶向鞘脂组学分析发现，D-半乳糖，LPS、IL-4、CoCl2处理细胞的差异鞘脂代谢物有共性也有特异性；黄连解毒汤化合物处理下以观察到该现象；（2）Aβ25-35刺激BV2细胞差异性鞘脂代谢物及相应代谢酶的变化可被HLJDD-CSF干预回调，其中BER、RUB、PAL、GEN作用比较明显；（3）体外神经细胞及MCAO大鼠的研究提示，IC和IP条件下Cer和SM增加、S1P降低可被BER逆转，同时伴随代谢酶水平的调整。（4）化合物COP改善LPS诱导的小胶质糖酵解，增加了PKM2磷酸化和四聚体，增加了PKM2酶活性。PKM2 F26A突变减弱COP的作用；（5）基于鞘脂代谢调控的特点，组合筛选发现BER与PAL组合物具有较佳抗炎作用，并可促进小胶质细胞的吞噬。利用外泌体载药体系，使脑组织中药物浓度增加2倍，并可通过抑制脑组织神经炎症并降低Aβ42水平显著改善APP/PS1小鼠学习记忆。鞘脂代谢重编程是多种病理条件下小胶质细胞典型代谢特征，基于对立功能的鞘脂成分的调控确立的BER和PAL组合物具有较佳的抗AD作用。