目的 探究芒柄花素抑制非小细胞肺癌(NSCLC) A549细胞的作用机制。方法 MTT法、细胞划痕和克隆形成实验考察芒柄花素(20~100 μmol·L^-1)对A549细胞增殖、迁移和克隆形成的抑制作用;A549细胞预孵育芒柄花素24 h后,使用Hoechst 33342细胞核染色定位,加入经过Dil标记的Jurkat细胞(人T淋巴细胞白血病细胞)共培养30 min,使用激光共聚焦拍摄2种细胞的共定位图像;建立A549-Jurkat细胞共培养模型,MTT和结晶紫染色法检测芒柄花素对A549细胞活性的影响;试剂盒法检测A549细胞线粒体膜电位(JC-1染色)和谷胱甘肽(GSH)水平探究芒柄花素对线粒体应激的诱导作用;Western blotting法考察芒柄花素对A549细胞线粒体应激后非经典焦亡相关蛋白和PD-L1蛋白表达的影响。结果 与对照组比较,芒柄花素显著抑制A549细胞的增殖、迁移和克隆形成能力(P<0.01、0.001),增强细胞共培养模型中T细胞的募集和杀伤作用(P<0.01、0.001);芒柄花素可诱导A549细胞线粒体应激,诱导线粒体膜电位下降,显著降低细胞GSH水平(P<0.05、0.01),显著上调bcl-2相关x蛋白(BAX)、裂解的半胱氨酸-天冬氨酸蛋白酶-3(cleaved-Caspase-3)、穿孔素蛋白E (Gasdermin E)-N蛋白表达诱导细胞焦亡(P<0.01、0.001),同时显著下调p-磷酸化磷脂酰肌醇3激酶(p-PI3K)、蛋白激酶B (Akt)、核因子κB (NF-κB)、细胞程序性死亡-配体1(PD-L1)蛋白表达抑制免疫逃逸(P<0.05、0.001)。结论 芒柄花素具有诱导NSCLC细胞焦亡并阻断免疫逃逸的作用,其机制为一方面可通过诱导A549细胞线粒体应激,促进细胞焦亡招募T细胞,增强肿瘤免疫;另一方面抑制p-PI3K/Akt/NF-κB信号轴,进而降低PD-L1蛋白表达,抑制A549细胞免疫逃逸。

Objectives To investigate the mechanism of inhibition of non-small cell lung cancer (NSCLC) A549 cells by formononetin (formononetin). Methods MTT assay, cell scratch and clone formation assays were performed to investigate the inhibitory ability of formononetin (20-100 μmol·L^-1) on proliferation, migration and clone formation of A549 cells. A549 cells were pre-incubated with formononetin for 24 h, and then the nuclear staining was performed by Hoechst 33342, and Dil-labeled Jurkat cells (human T-lymphocyte leukemia cells) were added to co-culture for 30 min, and the co-localization images of the two cells were captured by laser confocal microscopy. Mitochondrial membrane potential (JC-1 staining) and glutathione (GSH) of A549 cells were detected by kit method to investigate the induction effect of onondine on mitochondrial stress. The effects of formononetin on non-classical pyroptosis-associated proteins and PD-L1 proteins after mitochondrial stress in A549 cells were examined by Western blotting. Results Formononetin inhibited the proliferation, migration, and clone-forming ability of A549 cells (P < 0.01, 0.001), and enhanced the recruitment and killing of T cells in the cell co-culture model (P < 0.01, 0.001). Formononetin induced mitochondrial stress, induced a decrease in the mitochondrial membrane potential, lowered the level of cellular GSH (P < 0.05, 0.01), and up-regulated Bax, cleaved-Caspase-3 in A549 cells, Gasdermin E-N protein expression induced cellular pyroptosis (P < 0.01, 0.001), while down-regulating p-PI3K, Akt, NF-κB, PD-L1 protein expression inhibited immune escape (P < 0.05, 0.001). Conclusion Formononetin has the effect of inducing focal death of NSCLC cells and blocking immune escape. The mechanism is that formononetin can enhance tumor immunity by inducing mitochondrial stress in A549 cells and promoting cellular pyroptosis to recruit T cells on the one hand; on the other hand, it inhibits the p-PI3K/Akt/NF- κB signaling axis, which in turn reduces the expression of PD-L1 protein and inhibits immune escape from A549 cells.

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国家自然科学基金资助项目(21775061);泰山学者青年专家计划项目(tsqn202211136);济南市“新高校20条”资助项目(202228085);山东省高等学校青创人才引育计划项目(2021505031);山东中医药大学青年创新团队支持计划项目(22202105)