目的 探究芒柄花素抑制非小細胞肺癌(NSCLC) A549細胞的作用機制。方法 MTT法、細胞劃痕和克隆形成實驗考察芒柄花素(20~100 μmol·L^-1)對A549細胞增殖、遷移和克隆形成的抑制作用;A549細胞預(yù)孵育芒柄花素24 h后,使用Hoechst 33342細胞核染色定位,加入經(jīng)過Dil標記的Jurkat細胞(人T淋巴細胞白血病細胞)共培養(yǎng)30 min,使用激光共聚焦拍攝2種細胞的共定位圖像;建立A549-Jurkat細胞共培養(yǎng)模型,MTT和結(jié)晶紫染色法檢測芒柄花素對A549細胞活性的影響;試劑盒法檢測A549細胞線粒體膜電位(JC-1染色)和谷胱甘肽(GSH)水平探究芒柄花素對線粒體應(yīng)激的誘導作用;Western blotting法考察芒柄花素對A549細胞線粒體應(yīng)激后非經(jīng)典焦亡相關(guān)蛋白和PD-L1蛋白表達的影響。結(jié)果 與對照組比較,芒柄花素顯著抑制A549細胞的增殖、遷移和克隆形成能力(P<0.01、0.001),增強細胞共培養(yǎng)模型中T細胞的募集和殺傷作用(P<0.01、0.001);芒柄花素可誘導A549細胞線粒體應(yīng)激,誘導線粒體膜電位下降,顯著降低細胞GSH水平(P<0.05、0.01),顯著上調(diào)bcl-2相關(guān)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蛋白表達誘導細胞焦亡(P<0.01、0.001),同時顯著下調(diào)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)、細胞程序性死亡-配體1(PD-L1)蛋白表達抑制免疫逃逸(P<0.05、0.001)。結(jié)論 芒柄花素具有誘導NSCLC細胞焦亡并阻斷免疫逃逸的作用,其機制為一方面可通過誘導A549細胞線粒體應(yīng)激,促進細胞焦亡招募T細胞,增強腫瘤免疫;另一方面抑制p-PI3K/Akt/NF-κB信號軸,進而降低PD-L1蛋白表達,抑制A549細胞免疫逃逸。

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.

R285.5

國家自然科學基金資助項目(21775061);泰山學者青年專家計劃項目(tsqn202211136);濟南市“新高校20條”資助項目(202228085);山東省高等學校青創(chuàng)人才引育計劃項目(2021505031);山東中醫(yī)藥大學青年創(chuàng)新團隊支持計劃項目(22202105)