中药活性成分吴茱萸碱增强微管蛋白乙酰化和炎症小体活化的药理作用及机制研究

Evodia is a vital traditional Chinese medicine for “wen zhong xia qi” exhibiting special therapeutic action on infectious diseases, but the underlying immunopharmacological mechanism is largely unclear. Our preliminary study showed that evodiamine, one of the major active ingredients of Evodia, significantly promoted the activation of NLRP3 inflammasome and pyroptosis in macrophages, and markedly upregulated the acetylation of α-tubulin. Based on these previously unreleased findings and literature review of acetylated α-tubulin in promoting vesicle trafficking and NLRP3 inflammasome assembly, we hypothesize that upregulation of α-tubulin acetylation by evodiamine may accelerate the translocation of NLRP3 inflammasome adapter ASC and other components to the perinuclear region, thereby promoting the assembly and activation of NLRP3 inflammasome. This project will use in vitro cellular models, by Western blotting, immunofluorescence microscopy, gene knockdown and other techniques to study evodiamine’s effects on promoting NLRP3 inflammasome activation and pyroptosis, and to explore the role of the increased acetylation of α-tubulin by evodiamine in promoting the NLRP3 inflammasome activation and pyroptosis, as well as the underlying action mechanism. Finally, we will also confirm these pharmacological effects of evodiamine in NLRP3 inflammasome-related and infectious animal models, thus uncovering the action mechanism underlying its anti-infectious activity. This study will help to expand the clinical application of traditional Chinese medicine Evodia and its active ingredients.

吴茱萸是中医温中下气之要药，对感染相关炎症性疾病有特效，但其免疫药理学机制不清。我们前期研究发现，其主要活性成分之一吴茱萸碱可明显促进巨噬细胞NLRP3炎症小体的活化和细胞焦亡，显著上调α-微管蛋白乙酰化修饰。基于这些前期新发现，并综合文献关于乙酰化α-微管蛋白促进细胞囊泡运输和NLRP3炎症小体组装的报道，我们提出如下假设：吴茱萸碱通过上调α-微管蛋白乙酰化，促进NLRP3炎症小体接头蛋白ASC等组分向细胞核周围运输，从而促进炎症小体的组装与活化，加强机体抗感染固有免疫应答。本项目将利用体外细胞模型，通过免疫印迹、免疫荧光显微术、基因敲低等多种技术手段，研究吴茱萸碱对NLRP3炎症小体活化和细胞焦亡的促进作用，探索吴茱萸碱上调α-微管蛋白乙酰化修饰对上述活性的影响机制，并在相关动物模型中印证其药理效应，从而阐明其抗感染药理作用机制。这将有助于拓展传统中药吴茱萸及其有效成分的临床应用范围。

吴茱萸是常用中药材，其性热，味辛、苦。归肝、脾、胃、肾经。主治：“温中下气，止痛，除湿血痹，逐风邪，开腠理，咳逆寒热”（《本经》）；是中医下气之要药。从主治活性来看，吴茱萸针对的是炎症相关疾病，其中有很大一部分涉及病毒、细菌、真菌等各种病原体引起的局部感染或慢性迁延性感染。因此，吴茱萸在中医实践中显示了明确的抗炎效应。.吴茱萸碱是从吴茱萸中提取的主要天然化合物之一。在本项目中，我们利用细胞模型和动物模型，结合免疫印迹、免疫荧光、基因敲低、流式细胞术等多种研究方法和技术，深入研究了吴茱萸碱对巨噬细胞NLRP3炎症小体活化和细胞焦亡的影响。在其作用机制的探索中，研究了吴茱萸碱对α-Tubulin乙酰化修饰的影响及其与炎症小体组装之间的关系，分析了吴茱萸碱对α-Tubulin乙酰基转移酶（TAT）、去乙酰化酶调控α-Tubulin乙酰化修饰的影响；在小鼠感染模型中验证了吴茱萸碱的抗感染效应及其作用机制。.结果发现：吴茱萸碱可通过TAT、去乙酰化酶调控α-Tubulin乙酰化修饰，从而促进NLRP3炎症小体的组装和活化，并促进细胞焦亡。通过小鼠感染模型发现，吴茱萸碱可增强小鼠的抗感染能力，抑制α-Tubulin乙酰化可拮抗吴茱萸碱的抗感染效应。研究还发现，紫杉醇可以类似的作用机制，促进NLRP3炎症小体活化和细胞焦亡。.通过本项目的研究，我们发现吴茱萸碱可以一种独特的作用机制、增强NLRP3炎症小体活化和细胞焦亡。炎症小体活化和细胞焦亡是机体抗感染和炎症细胞退出的重要机制之一。吴茱萸碱增强巨噬细胞的功能、促进NLRP3炎症小体活化和细胞焦亡，有助于解释吴茱萸“温中下气”的功效，为拓展吴茱萸及吴茱萸碱的临床应用提供免疫药理学的理论基础。