NLRP3炎性复合体是由nod样受体（NLR）家族成员NLRP3、接头蛋白凋亡相关斑点样蛋白ASC和pro-caspase-1形成的蛋白复合物，是先天免疫系统的重要组成部分。NLRP3炎性复合体的激活由两个关键步骤介导：在priming阶段，PAMPs或DAMPs与细胞膜上相应受体结合，通过激活NF-κB信号通路，上调NLRP3、pro-IL-1β蛋白表达；在activation阶段，当受到如尼日利亚菌素、ATP和MSU等刺激后，NLRP3炎性复合体各组分（NLRP3、ASC、pro-caspase-1）发生聚集与组装，导致caspase-1的自我激活。活化的caspase-1进一步促进IL-1β和IL-18的成熟与分泌，从而参与代谢性疾病、神经退行性疾病及自身免疫性疾病等的发生发展进程。旋覆花为菊科植物旋覆花（Inula japonica Thunb.）或欧亚旋覆花（Inula britannica L.）的干燥头状花序，具有降气化痰、降逆止呕、行水消肿、抗菌消炎等多种功效，可用于治疗咳嗽、咳痰和呕吐等炎症性疾病。倍半萜内酯作为旋覆花属植物中的特征活性成分而备受关注，具有多种药理作用，包括抗炎、抗肿瘤、抗氧化及免疫调节等。本研究以旋覆花内酯部位（XFHNZ）及其中的主要成分旋覆花素（inulicin）为研究对象，考察其抑制NLRP3炎性复合体的分子机制与抗炎作用。
       考虑到NLRP3炎性复合体激活产生IL-1β分为priming和activation两个阶段，本研究首先考察了两种常用鼠源巨噬细胞株（RAW264.7和J774A.1）在这两个阶段的适用性：在针对priming阶段的模型上，RAW264.7细胞经LPS刺激后，其胞内pro-IL-1β水平及上清液中NO、IL-6、TNF-α和CCL2等炎症因子的释放显著升高，而J774A.1细胞在LPS刺激后，不仅NO和CCL2的分泌水平未见显著变化，而且胞内pro-IL-1β以及上清TNF-α和IL-6的表达量也相对较低，表明J774A.1并不适合用于priming阶段的研究，故我们选择RAW264.7细胞进行priming阶段的研究。在针对activation阶段的模型上，J774A.1细胞经LPS联合尼日利亚菌素诱导后，可分泌大量成熟IL-1β，但RAW264.7细胞未检测到IL-1β的释放，这与RAW264.7细胞缺乏NLRP3活化所必须组件ASC的事实相符，故选择J774A.1细胞用于activation阶段的研究。
       随后，我们制备了旋覆花（Inula japonica Thunb.）90%乙醇提取物，再经过乙酸乙酯萃取获得内酯部位（XFHNZ），在三种常用诱导剂（尼日利亚菌素、ATP、MSU）诱导的NLRP3炎性复合体活化的巨噬细胞中考察其对NLRP3激活典型分泌物IL-1β的作用。结果显示，XFHNZ能显著降低3种诱导剂所诱导的J774A.1细胞上清IL-1β的水平。随后，我们采用高效液相色谱（HPLC）技术对所得的内酯部位进行成分鉴定，结果显示其中的旋覆花素（Inulicin）含量较高且尚无其作用报道，故我们后期以它为对象，围绕NLRP3炎性复合体的priming和activation阶段展开研究。
        在旋覆花素对NLRP3炎性复合体activation阶段的作用研究中，本研究首先测定了旋覆花素对三种诱导剂刺激的J774A.1细胞上清IL-1β的作用。结果显示，旋覆花素也对3种诱导剂所诱导的上清IL-1β升高有显著抑制作用，表明其的确可以抑制NLRP3炎性复合体。与IL-1β一样，caspase-1 p20同样可以被释放到细胞外，反应NLRP3炎性复合体的活化情况。故本实验还对caspase-1 p20的生成情况进行研究。Western blot结果显示，旋覆花素能减少NLRP3活化的巨噬细胞上清caspase-1 p20的产生。由于活性caspase-1p20会进一步裂解GSDMD蛋白导致细胞焦亡，随后我们又考察了旋覆花素对细胞焦亡标志物GSDMD和LDH的作用。结果发现它也能剂量依赖性的抑制GSDMD蛋白的裂解和LDH的释放。为进一步探讨其作用机制，我们聚焦到NLRP3组装过程中的标志——ASC寡聚化和斑点形成及其上游调控事件。Western blot和免疫荧光结果均表明，旋覆花素能够减少ASC寡聚化和斑点形成，但对NLRP3组装的关键上游信号事件，胞内K+外流和线粒体ROS的产生无影响。
      IL-1β的生成除了受NLRP3炎性复合体activation阶段的调控，也受到其priming阶段的影响。故本研究继续考察了旋覆花素对NLRP3炎性复合体priming阶段的作用。在LPS活化的RAW264.7巨噬细胞的体外模型中，旋覆花素不仅能显著减少胞内pro-IL-1β的含量，还能抑制其mRNA转录。由于胞内pro-IL-1β的表达主要受上游转录因子NF-κB和AP-1的调控，我们又考察了旋覆花素对这两种转录因子活化的影响。结果表明，旋覆花素一方面能减少IκBα的磷酸化并阻止其自身降解、以及压制随后NF-κB p65的核易位，从而抑制NF-κB信号；另一方面，旋覆花素能减少ERK、JNK的磷酸化水平，从而抑制AP-1信号。鉴于NF-κB和AP-1不仅调节pro-IL-1β的生成，还能调控如NO、IL-6、CCL2等其他促炎介质的生成。所以本研究也考察了旋覆花素对这些炎症因子的作用。不意外地，旋覆花素对NO、IL-6和CCL2也有显著的抑制作用。在LPS致内毒素血症小鼠模型中，单次腹腔给药（40 mg/kg及60 mg/kg）也能显著降低血清中IL-1β、CCL2和TNF-α炎症因子水平，并压制该模型小鼠腹腔灌流液中促炎因子CCL2、TNF-α和IL-6的增高。
       综上，本研究首次较全面地探究了旋覆花素的抗炎作用及抑制NLRP3炎性复合体的潜在分子机制，针对activation阶段，旋覆花素通过影响ASC寡聚化和斑点形成来抑制NLRP3炎性复合体的活化；针对priming阶段，旋覆花素能显著降低胞内pro-IL-1β的水平，该作用是来源于对NF-κB和AP-1两条核转录信号的抑制，这也使其对其他促炎因子（NO、IL-6和CCL2）也有抑制作用。本研究的发现为旋覆花素抑制NLRP3炎性复合体提供了理论依据，提示其在开发成为NLRP3炎性复合体抑制剂方面有良好的应用前景。

       The NLRP3 inflammasome is a protein complex composed of the NOD-like receptor (NLR) family member NLRP3, the adaptor protein apoptosis-associated speck-like protein (ASC), and pro-caspase-1, serving as a critical component of the innate immune system. Activation of the NLRP3 inflammasome is mediated by two key steps: During the priming phase, pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) bind to their respective receptors on the cell membrane, activating the NF-κB signaling pathway and upregulating the expression of NLRP3 and pro-IL-1β. In the activation phase, upon stimulation by agents such as nigericin, ATP, or monosodium urate (MSU), the components of the NLRP3 inflammasome (NLRP3, ASC, and pro-caspase-1) aggregate and assemble, leading to the autoactivation of caspase-1. Activated caspase-1 further promotes the maturation and secretion of IL-1β and IL-18, thereby contributing to the pathogenesis of metabolic disorders, neurodegenerative diseases, and autoimmune diseases.

        Inula japonica Thunb. or Inula britannica L., commonly known as Inula flower, is the dried capitulum of plants from the Asteraceae family. It exhibits multiple pharmacological effects, including resolving phlegm, suppressing nausea, promoting diuresis, and exerting antibacterial and anti-inflammatory actions, making it useful in treating inflammatory conditions such as cough, expectoration, and vomiting. Sesquiterpene lactones, the characteristic bioactive constituents of Inula species, have garnered significant attention due to their diverse pharmacological activities, including anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. This study focuses on the lactone-enriched fraction (XFHNZ) of Inula japonica Thunb. and its major constituent, inulicin, to investigate their molecular mechanisms in inhibiting the NLRP3 inflammasome and their anti-inflammatory effects.

Given that NLRP3 inflammasome activation and subsequent IL-1β production involve two distinct phases (priming and activation), we first evaluated the suitability of two murine macrophage cell lines (RAW264.7 and J774A.1) for each phase. In the priming phase model, RAW264.7 cells stimulated with LPS exhibited significantly elevated intracellular pro-IL-1β levels and increased secretion of inflammatory mediators, including NO, IL-6, TNF-α, and CCL2, in the supernatant. In contrast, J774A.1 cells showed no significant changes in NO or CCL2 secretion upon LPS stimulation, and their intracellular pro-IL-1β, TNF-α, and IL-6 levels remained relatively low, indicating their unsuitability for priming phase studies. Thus, RAW264.7 cells were selected for priming phase experiments. For the activation phase model, J774A.1 cells treated with LPS plus nigericin secreted substantial amounts of mature IL-1β, whereas RAW264.7 cells failed to release detectable IL-1β, consistent with their deficiency in ASC, an essential component for NLRP3 activation. Therefore, J774A.1 cells were chosen for activation phase studies.

      Subsequently, we prepared a 90% ethanol extract of Inula japonica Thunb., followed by ethyl acetate extraction to obtain the lactone-enriched fraction (XFHNZ). Its effects on NLRP3 inflammasome activation were assessed in macrophages stimulated with three common inducers (nigericin, ATP, and MSU). Results demonstrated that XFHNZ significantly reduced IL-1β secretion in J774A.1 cells induced by all three stimuli. High-performance liquid chromatography (HPLC) analysis identified inulicin as a major constituent of XFHNZ, with no prior reports on its biological activity. Hence, we further investigated its role in modulating both priming and activation phases of the NLRP3 inflammasome.

In studying the effects of inulicin on the activation phase, we first measured its impact on IL-1β secretion in J774A.1 cells stimulated with the three inducers. Inulicin significantly suppressed IL-1β release in all cases, confirming its inhibitory effect on NLRP3 inflammasome activation. Since caspase-1 p20, like IL-1β, is released extracellularly upon NLRP3 activation, we also examined its production. Western blot analysis revealed that inulicin reduced caspase-1 p20 levels in the supernatant of NLRP3-activated macrophages. Given that active caspase-1 cleaves gasdermin D (GSDMD) to induce pyroptosis, we further evaluated inulicin’s effects on GSDMD cleavage and lactate dehydrogenase (LDH) release. Inulicin dose-dependently inhibited GSDMD cleavage and LDH release. To elucidate the underlying mechanism, we focused on ASC oligomerization and speck formation, key events in NLRP3 assembly, as well as upstream regulatory signals. Both Western blot and immunofluorescence assays demonstrated that inulicin attenuated ASC oligomerization and speck formation but had no effect on upstream signals such as intracellular K⁺ efflux and mitochondrial ROS production.

       Since IL-1β production is regulated not only by the activation phase but also by the priming phase of the NLRP3 inflammasome, we next investigated inulicin’s role in the priming phase. In LPS-activated RAW264.7 macrophages, inulicin significantly reduced intracellular pro-IL-1β levels and suppressed its mRNA transcription. As pro-IL-1β expression is primarily regulated by the transcription factors NF-κB and AP-1, we examined inulicin’s effects on their activation. Inulicin inhibited NF-κB signaling by reducing IκBα phosphorylation, preventing its degradation, and suppressing nuclear translocation of NF-κB p65. Concurrently, it attenuated AP-1 signaling by decreasing the phosphorylation of ERK and JNK. Given that NF-κB and AP-1 also regulate the production of other proinflammatory mediators (e.g., NO, IL-6, CCL2), we assessed inulicin’s effects on these factors. As expected, inulicin markedly suppressed NO, IL-6, and CCL2 production. In an LPS-induced endotoxemia mouse model, a single intraperitoneal dose of inulicin (40 or 60 mg/kg) significantly lowered serum levels of IL-1β, CCL2, and TNF-α and attenuated the elevation of CCL2, TNF-α, and IL-6 in peritoneal lavage fluid.

       In summary, this study provides the first comprehensive exploration of inulicin’s anti-inflammatory effects and its mechanisms in inhibiting the NLRP3 inflammasome. During the activation phase, inulicin suppresses NLRP3 inflammasome assembly by interfering with ASC oligomerization and speck formation. During the priming phase, it reduces pro-IL-1β levels by inhibiting NF-κB and AP-1 signaling, thereby also suppressing other proinflammatory factors (NO, IL-6, CCL2). These findings establish a theoretical foundation for inulicin as an NLRP3 inflammasome inhibitor and highlight its potential for therapeutic development.