[摘 要] 目的：探究蟾毒灵（Buf）对卵巢癌细胞A2780和SKOV3的毒性作用及其机制，评估其与顺铂（DDP）联合应用的协同 毒性。方法：常规培养A2780和SKOV3细胞。用CCK-8法、克隆形成实验检测Buf对A2780和SKOV3细胞增殖的影响及半数 抑制浓度（IC50 ）。用不同浓度的Buf处理细胞，显微镜下观察细胞形态变化，比色法检测乳酸脱氢酶（LDH）释放水平，流式细胞 术分析检测 Annexin Ⅴ+ PI + 细胞比例，qPCR 和 WB 法检测细胞焦亡相关分子 mRNA 及蛋白表达水平，通过小干扰 RNA 敲减 A2780 和 SKOV3 细胞中的 GSDMD 并进行后续功能实验进行验证。通过 SynergyFinder 分析 Buf 与 DDP 对于杀伤 A2780 和 SKOV3细胞的协同效应，并通过流式细胞术、qPCR、WB进行机制探索，进一步利用流式细胞术检测Buf与DDP联用对DDP耐药 A2780和SKOV3细胞的影响。结果：与对照组相比，Buf显著抑制卵巢癌细胞增殖及迁移能力（均P < 0.05），诱导LDH释放和细 胞焦亡（均P < 0.05），上调核苷酸结合寡聚化结构样受体热蛋白结构域相关蛋白3（NLRP3）、半胱氨酸天冬氨酸蛋白水解酶1 （CASP1）、gasdermin家族成员D（GSDMD）、白细胞介素-1β（IL-1β）及IL-18的mRNA及蛋白的表达（均P < 0.05），Buf与DDP联 用具有协同抑制A2780和SKOV3细胞增殖的作用，并可进一步增强DDP耐药的A2780和SKOV3细胞对DDP的敏感性（P < 0.01 或 P < 0.001）。结论：Buf 可通过激活 NLRP3/CASP1/GSDMD 信号通路诱导 A2780 和 SKOV3 细胞焦亡，并增强 A2780 和 SKOV3细胞及DDP耐药A2780和SKOV3细胞对DDP的敏感性。

[Abstract] Objective: To investigate the cytotoxic effects and underlying mechanisms of Bufalin (Buf) on ovarian cancer cells (A2780 and SKOV3) and to evaluate its synergistic cytotoxicity with cisplatin (DDP). Methods: A2780 and SKOV3 cells were routinely cultured. The effect of Buf on cell proliferation and its half-maximal inhibitory concentration (IC50) were determined using CCK-8 assay and colony formation assay. Following treatments with various concentrations of Buf, morphological changes of tumor cells were observed under a microscope, lactate dehydrogenase (LDH) release was measured using colorimetric assay, and the proportion of Annexin Ⅴ?PI? cells was analyzed using flow cytometry. The mRNA and protein expression levels of pyroptosis-related molecules were detected using qPCR and WB, respectively. Small interfering RNA was used to knock down gasdermin D (GSDMD) in A2780 and SKOV3 cells to validate its functional role. The synergistic cytotoxic effects of Buf and DDP against A2780 and SKOV3 cells were analyzed using the SynergyFinder platform, and the underlying mechanisms were further explored using flow cytometry, qPCR, and WB. Additionally, the synergistic effect of Buf and DDP on DDP-resistant A2780 and SKOV3 cells was evaluated using flow cytometry. Results: Compared with the control group, Buf significantly inhibited proliferation and migration of ovarian cancer cells (both P < 0.05), induced LDH release and cell pyroptosis (both P < 0.05), and upregulated the mRNA and protein expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3), cysteine-aspartic acid protease 1 (CASP1), gasdermin D (GSDMD), interleukin(IL) -1β, and IL-18 (all P < 0.05). The combination of Buf and DDP synergistically inhibited the proliferation of A2780 and SKOV3 cells and further enhanced the sensitivity of DDP-resistant A2780 and SKOV3 cells to DDP. Conclusion: Buf induces pyroptosis in A2780 and SKOV3 cells by activating the NLRP3/CASP1/GSDMD signaling pathway and enhances the sensitivity of DDP-resistant A2780 and SKOV3 cells to DDP.

[基金项目] 国家自然科学基金（82172717）