目的 探究白屈菜碱-延胡索乙素联合阿霉素协同诱导人乳腺癌阿霉素耐药细胞MCF-7/ADR细胞焦亡的作用机制。方法 以乳腺癌MCF-7/ADR细胞为研究对象，采用CCK-8法检测白屈菜碱-延胡索乙素联合阿霉素对MCF-7/ADR细胞增殖的影响；采用划痕实验和Transwell小室实验检测白屈菜碱-延胡索乙素联合阿霉素对MCF-7/ADR细胞侵袭、迁移的影响；荧光显微镜测定白屈菜碱-延胡索乙素联合阿霉素对MCF-7/ADR细胞线粒体膜电位的影响；采用分子对接技术考察各成分与焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白3（nucleotide-binding oligomerization domain-like receptor protein 3，NLRP3）、半胱氨酸天冬氨酸蛋白酶-1（Cystein-asparate protease-1，Caspase-1）、消皮素D（gasdermin D，GSDMD）和白细胞介素-18（interleukin-18，IL-18）的结合能力；采用qRT-PCR、Western blotting检测焦亡相关蛋白的表达水平。结果 白屈菜碱-延胡索乙素联合阿霉素可显著增强阿霉素对MCF-7/ADR细胞的增殖抑制作用，说明白屈菜碱-延胡索乙素对阿霉素具有协同效应作用。与白屈菜碱-延胡索乙素、阿霉素单独使用比较，白屈菜碱-延胡索乙素联合阿霉素对MCF-7/ADR细胞侵袭、迁移的抑制作用更为显著（P＜0.01）；同时，亦可显著降低线粒体膜电位（P＜0.01）。分子对接结果显示，白屈菜碱、延胡索乙素和阿霉素与焦亡相关蛋白NLRP3、Caspase-1、GSDMD和IL-18均具有良好的结合活性。qRT-PCR结果表明，与白屈菜碱-延胡索乙素、阿霉素单独使用比较，白屈菜碱-延胡索乙素联合阿霉素可显著上调GSDMD、NLRP3和Caspase-1 mRNA表达水平（P＜0.01）。Western blotting结果表明，与白屈菜碱-延胡索乙素、阿霉素单独使用比较，白屈菜碱-延胡索乙素联合阿霉素可显著上调NLRP3、GSDMD、Caspase-1和IL-18的蛋白表达水平（P＜0.01），与相应的mRNA表达结果一致。结论 白屈菜碱-延胡索乙素可协同增强阿霉素对人乳腺癌MCF-7/ADR细胞的增殖抑制作用，并可通过激活NLRP3/ Caspase-1/GSDMD信号通路诱导耐药细胞发生细胞焦亡。

Objective To investigate the mechanism of chelidonine (CHE)-tetrahydropalmatine (THP) combined with adriamycin (ADR) in synergically inducing pyroptosis of adriamycin-resistant human breast cancer MCF-7/ADR cells. Methods CCK-8 method was used to detect the effect of CHE-THP combined with ADR on the proliferation of MCF-7/ADR cells. The impact of CHE-THP combined with ADR on the invasion and migration of MCF-7/ADR cells was detected using scratch assay and Transwell chamber assay. The mitochondrial membrane potential was examined by fluorescence microscopy. Molecular docking was applied to detect the binding ability of each component to the pyroptosis-related proteins, including nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), Cystein-asparate protease-1 (Caspase-1), gasdermin D (GSDMD) and interleukin-18 (IL-18). The expressions of pyroptosis-related proteins were detected by Western Blotting. The expressions of pyroptosis-related genes were detected by qRT-PCR. Results CHE-THP in combination with ADR can significantly enhance the inhibitory effect on proliferation of ADR on MCF-7/ADR cells, indicating that CHE-THP has a synergistic effect on ADR. Compared with CHE-THP and ADR treatment alone, the inhibition effect of the combined administration on MCF-7/ADR invasion and migration was more significant (P < 0.01). Meanwhile, combined administration could also significantly reduce the mitochondrial membrane potential of MCF-7/ADR cells (P < 0.01). The molecular docking showed that the CHE, THP and ADR had excellent binding activity with pyroptosis-related proteins including NLRP3, Caspase-1, GSDMD and IL-18. The results of qRT-PCR revealed that compared with ADR or CHE-THP alone, the combined administration could markedly up-regulate the mRNA expression of GSDMD, NLRP3 and Caspase-1 (P < 0.01). Western blotting results also showed that compared with ADR or CHE-THP treatment alone, the protein expression levels of NLRP3, GSDMD, Caspase-1 and IL-18 were significantly up-regulated by combined administration of CHE-THP and ADR (P < 0.01), which was consistent with the corresponding mRNA expression results. Conclusion CHE-THP can synergistically enhance the proliferation inhibition effect of ADR on human breast cancer MCF-7/ADR cells, as well as induce pyroptosis in ADR-resistant cells by activating NLRP3/Caspase-1/GSDMD signalling pathway.

R285.5

黑龙江省自然科学基金项目（LH2022H001）