藁本内酯通过干预Gasdermin D介导的IL-1β分泌缓解炎性肠病的作用及机制研究

The etiology of inflammatory bowel disease (IBD) remains unclear, which has received tremendous attention presently. Nowadays, the existing anti-TNF-α therapy doesn’t work for about 30% IBD patients and the TNF-α monoclonal antibody is applied clinically with high risk of drug resistance, and cause serious side effects including systemic immune response during long-term medication. It can be urgent to develop effective drugs for the treatment of IBD. Literature reports have shown that traditional Chinese medicine has good efficacy on IBD with low side effect. Our previous systems pharmacology and experiment studies have shown that Ligustilide (LIG) can relieve the symptoms of IBD model mice, protect intestinal mucosa and inhibit IL-1β secretion of macrophages. Mechanically, it relates to the intervention of Gasdermin D (GSDMD), which is a key protein regulating IL-1β secretion. However, the exact mechanisms of the inhibitory effect of LIG on IBD and the secretion of IL-1β by targeting GSDMD remain unknown. This project aims to establish cell co-culture system, pyroptosis and hyperactivated macrophage models, explore the binding effect of LIG and GSDMD by BioLayer Interferometry and fluorescent probes, and confirm the inhibitory effect of LIG on IBD. Especially, focusing on the secretion process of IL-1β regulated by GSDMD to elaborate the mechanisms of LIG. Importantly, we would like to provide theoretical basis for the clinical application of LIG and new strategy for developing novel anti-IBD drugs targeting inflammatory cytokines.

炎性肠病（IBD）至今病因未明，是当前国内外研究的热点之一。现有抗TNF-α疗法对30%患者无效，TNF-α单抗易耐药，副作用多，急需寻找安全有效的抗IBD药物。文献资料显示传统中药对IBD疗效良好且副作用低。我们前期用系统药理学方法筛选并用实验证明，中药活性单体藁本内酯能改善IBD模型小鼠肠炎症状，保护肠黏膜，抑制巨噬细胞分泌IL-1β，其机制与调节IL-1β分泌的关键蛋白Gasdermin D（GSDMD）有关。但关于藁本内酯如何通过GSDMD抑制IL-1β分泌，进而发挥抗IBD作用的确切机制国内外文献未见报道。本项目旨建立共培养、焦亡及过度激活的巨噬细胞等模型，利用荧光探针标记、光学干涉等技术研究藁本内酯与GSDMD的结合作用，全面确证藁本内酯的抗IBD活性，重点围绕GSDMD调控IL-1β分泌，阐明藁本内酯抗IBD的机理，为该药的临床应用及靶向炎症因子的抗IBD药物研发提供新思路。

研究背景：由于炎性肠病（IBD）的具体病因尚不明确，目前仍无有效治疗方法，开发有效的抗IBD 药物迫在眉睫。文献资料显示传统中药对IBD疗效良好且副作用低，从中草药来源的单体化合物中寻找可用于治疗 IBD 的活性物质具有广泛的应用前景。我们前期用系统药理学方法筛选并用实验证明，中药活性单体藁本内酯能改善IBD模型小鼠肠炎症状，其机制可能与调节IL-1β分泌的关键蛋白GSDMD有关，但关于藁本内酯如何通过GSDMD抑制IL-1β分泌，进而发挥抗IBD作用的确切机制国内外文献未见报道。.研究内容：本项目运用系统药理学方法发现具有抗IBD 潜能的中药活性单体，并预测中药活性单体藁本内酯抗IBD的可能作用靶标；采用DSS及TNBS诱导的经典肠炎模型体内评价藁本内酯的体内抗IBD活性及其对IL-1β表达及分泌的体内抑制作用，构建细胞三维共培养体系完成了LIG对肠粘膜的保护作用研究，构建巨噬细胞焦亡及过度活化的细胞模型，体外全面证实藁本内酯对IL-1β表达及分泌的抑制作用；利用荧光探针标记等技术研究藁本内酯对GSDMD及其上游NLRP3炎症小体及NF-κB信号通路的抑制作用。.研究结果：藁本内酯在体内外均具有良好的缓解IBD疾病症状的作用，藁本内酯通过GSDMD 结合等作用方式抑制重要促炎症因子IL-1β的表达及分泌，同时LIG还能抑制GSDMD上游NLRP3炎症小体的生成及NF-κB信号通路的激活。.科学意义：本项目揭示了藁本内酯抑制IL-1β分泌抗 IBD的分子机理，为研发靶向IL-1β的新型抗IBD药物提供新的思路；从整体动物及细胞分子水平验证系统药理学研究结果，为开发有效的中草药活性单体提供新的方法和策略，也为复杂性重大疾病的新药研发提供新思路。