目的 探究白屈菜堿-延胡索乙素聯(lián)合阿霉素協(xié)同誘導(dǎo)人乳腺癌阿霉素耐藥細胞MCF-7/ADR細胞焦亡的作用機制。方法 以乳腺癌MCF-7/ADR細胞為研究對象，采用CCK-8法檢測白屈菜堿-延胡索乙素聯(lián)合阿霉素對MCF-7/ADR細胞增殖的影響；采用劃痕實驗和Transwell小室實驗檢測白屈菜堿-延胡索乙素聯(lián)合阿霉素對MCF-7/ADR細胞侵襲、遷移的影響；熒光顯微鏡測定白屈菜堿-延胡索乙素聯(lián)合阿霉素對MCF-7/ADR細胞線粒體膜電位的影響；采用分子對接技術(shù)考察各成分與焦亡相關(guān)蛋白核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體蛋白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）和白細胞介素-18（interleukin-18，IL-18）的結(jié)合能力；采用qRT-PCR、Western blotting檢測焦亡相關(guān)蛋白的表達水平。結(jié)果 白屈菜堿-延胡索乙素聯(lián)合阿霉素可顯著增強阿霉素對MCF-7/ADR細胞的增殖抑制作用，說明白屈菜堿-延胡索乙素對阿霉素具有協(xié)同效應(yīng)作用。與白屈菜堿-延胡索乙素、阿霉素單獨使用比較，白屈菜堿-延胡索乙素聯(lián)合阿霉素對MCF-7/ADR細胞侵襲、遷移的抑制作用更為顯著（P＜0.01）；同時，亦可顯著降低線粒體膜電位（P＜0.01）。分子對接結(jié)果顯示，白屈菜堿、延胡索乙素和阿霉素與焦亡相關(guān)蛋白NLRP3、Caspase-1、GSDMD和IL-18均具有良好的結(jié)合活性。qRT-PCR結(jié)果表明，與白屈菜堿-延胡索乙素、阿霉素單獨使用比較，白屈菜堿-延胡索乙素聯(lián)合阿霉素可顯著上調(diào)GSDMD、NLRP3和Caspase-1 mRNA表達水平（P＜0.01）。Western blotting結(jié)果表明，與白屈菜堿-延胡索乙素、阿霉素單獨使用比較，白屈菜堿-延胡索乙素聯(lián)合阿霉素可顯著上調(diào)NLRP3、GSDMD、Caspase-1和IL-18的蛋白表達水平（P＜0.01），與相應(yīng)的mRNA表達結(jié)果一致。結(jié)論 白屈菜堿-延胡索乙素可協(xié)同增強阿霉素對人乳腺癌MCF-7/ADR細胞的增殖抑制作用，并可通過激活NLRP3/ Caspase-1/GSDMD信號通路誘導(dǎo)耐藥細胞發(fā)生細胞焦亡。

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

黑龍江省自然科學基金項目（LH2022H001）