目的 探究粉防己碱对三阴性乳腺癌（triple-negative breast cancer，TNBC）细胞的作用及作用机制，明确其是否通过调控NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor family pyrin domain containing 3，NLRP3）/半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）/Gasdermin D蛋白（Gasdermin D，GSDMD）通路诱导细胞焦亡，为TNBC的治疗提供新策略。方法 使用不同浓度粉防己碱处理MDA-MB-231细胞，采用MTT法检测细胞增殖抑制情况。运用GSDMD小干扰RNA（small interfering RNA，siRNA）转染技术沉默GSDMD基因，通过荧光显微镜观察转染效率，并利用Western blotting验证转染后GSDMD蛋白表达变化。设置对照组及粉防己碱（5、10、20 μg/mL）组和粉防己碱（20 μg/mL）＋GSDMD siRNA（siGSDMD）组，采用细胞形态观察、集落实验、膜联蛋白V（Annexin V）/碘化丙啶（propidium iodide，PI）双染色法、乳酸脱氢酶（lactate dehydrogenase，LDH）细胞毒性实验、划痕实验、Transwell实验、Western blotting及qRT-PCR，分别从细胞形态、克隆能力、焦亡率、膜通透性、迁移与侵袭能力以及蛋白和基因表达水平多个维度，系统评估粉防己碱对MDA-MB-231细胞的影响，并探究其是否通过NLRP3/Caspase-1/GSDMD通路发挥作用。结果 粉防己碱显著抑制MDA-MB-231细胞增殖，呈剂量相关性（P＜0.01），且能有效诱导细胞焦亡，表现为细胞形态改变、膜完整性破坏、细胞核固缩以及焦亡率和LDH释放量的显著增加（P＜0.01）。同时，随着粉防己碱给药浓度的增加，细胞的集落数目、划痕愈合率和侵袭细胞数显著降低（P＜0.01）。机制研究发现，粉防己碱上调NLRP3、Caspase-1、白细胞介素-18（interleukin-18，IL-18）和IL-1β的蛋白及mRNA表达（P＜0.01），促进pro-IL-1β向mature-IL-1β转化。而siGSDMD干预可显著逆转粉防己碱的调控作用（P＜0.01），表明GSDMD基因在粉防己碱诱导的焦亡过程中发挥关键作用。结论 粉防己碱通过调控NLRP3/Caspase-1/GSDMD通路诱导MDA-MB-231细胞焦亡，抑制细胞增殖、迁移和侵袭。

Objective To investigate the effect and mechanism of tetrandrine on triple-negative breast cancer (TNBC) cells, clarify whether it induces pyroptosis by regulating NOD-like receptor family pyrin domain containing 3 (NLRP3)/cystein-asparate protease-1 (Caspase-1)/Gasdermin D (GSDMD) pathway, thereby providing a novel therapeutic strategy for TNBC. Methods MDA-MB-231 cells were treated with varying concentrations of tetrandrine. Cell proliferation inhibition was assessed via MTT assay. GSDMD small interfering RNA (siRNA) transfection was used to silence GSDMD gene, transfection efficiency was observed by fluorescence microscopy, and the changes in GSDMD protein expression after transfection were verified by Western blotting. Control group, tetrandrine (5, 10, 20 μg/mL) groups and tetrandrine (20 μg/mL) + GSDMD siRNA (siGSDMD) group were set up. Cell morphology observation, colony assay, Annexin V/propidium iodide (PI) double staining method, lactate dehydrogenase (LDH) cytotoxicity assay, scratch assay, Transwell assay, Western blotting and qRT-PCR were used to systematically evaluate the effects of tetrandrine on MDA-MB-231 cells from multiple dimensions including cell morphology, cloning ability, pyroptosis rate, membrane permeability, migration and invasion ability, as well as protein and gene expression levels, and investigate whether tetrandrine exerts its effect through NLRP3/Caspase-1/GSDMD pathway. Results Tetrandrine significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner (P < 0.01), and induced pyroptosis, characterized by morphological changes, membrane rupture, nuclear condensation, elevated pyroptosis rates and increased LDH release (P < 0.01). At the same time, as the concentration of tetrandrine increased, the number of cell colonies, scratch healing rate and number of invasive cells were significantly decreased (P < 0.01). Mechanism studies found that tetrandrine up-regulated the protein and mRNA expressions of NLRP3, Caspase-1, interleukin-18 (IL-18) and IL-1β (P < 0.01), promoted the conversion of pro-IL-1β to mature-IL-1β. The intervention of siGSDMD could significantly reverse the regulatory effect of tetrandrine (P < 0.01), indicating that GSDMD gene played a key role in the process of tetrandrine induced pyroptosis. Conclusion Tetrandrine induces pyroptosis, inhibits cell proliferation, migration and invasion in MDA-MB-231 cells by regulating NLRP3/Caspase-1/GSDMD pathway.

R285.5

黑龙江省自然科学基金项目（PL2024H130）；2023年度哈尔滨商业大学镜湖学者支持计划；哈尔滨市科技计划自筹经费项目（2023ZCZJCG038）；黑龙江省中医药科研项目（ZHY2024-239）；2025年度黑龙江省省属本科高校基本科研业务费项目