[關(guān)鍵詞]
[摘要]
目的 探究芒柄花素抑制非小細(xì)胞肺癌(NSCLC) A549細(xì)胞的作用機(jī)制。方法 MTT法、細(xì)胞劃痕和克隆形成實(shí)驗(yàn)考察芒柄花素(20~100 μmol·L-1)對(duì)A549細(xì)胞增殖、遷移和克隆形成的抑制作用;A549細(xì)胞預(yù)孵育芒柄花素24 h后,使用Hoechst 33342細(xì)胞核染色定位,加入經(jīng)過Dil標(biāo)記的Jurkat細(xì)胞(人T淋巴細(xì)胞白血病細(xì)胞)共培養(yǎng)30 min,使用激光共聚焦拍攝2種細(xì)胞的共定位圖像;建立A549-Jurkat細(xì)胞共培養(yǎng)模型,MTT和結(jié)晶紫染色法檢測芒柄花素對(duì)A549細(xì)胞活性的影響;試劑盒法檢測A549細(xì)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(duì)線粒體應(yīng)激的誘導(dǎo)作用;Western blotting法考察芒柄花素對(duì)A549細(xì)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(dá)的影響。結(jié)果 與對(duì)照組比較,芒柄花素顯著抑制A549細(xì)胞的增殖、遷移和克隆形成能力(P<0.01、0.001),增強(qiáng)細(xì)胞共培養(yǎng)模型中T細(xì)胞的募集和殺傷作用(P<0.01、0.001);芒柄花素可誘導(dǎo)A549細(xì)胞線粒體應(yīng)激,誘導(dǎo)線粒體膜電位下降,顯著降低細(xì)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(dá)誘導(dǎo)細(xì)胞焦亡(P<0.01、0.001),同時(shí)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(xì)胞程序性死亡-配體1(PD-L1)蛋白表達(dá)抑制免疫逃逸(P<0.05、0.001)。結(jié)論 芒柄花素具有誘導(dǎo)NSCLC細(xì)胞焦亡并阻斷免疫逃逸的作用,其機(jī)制為一方面可通過誘導(dǎo)A549細(xì)胞線粒體應(yīng)激,促進(jìn)細(xì)胞焦亡招募T細(xì)胞,增強(qiáng)腫瘤免疫;另一方面抑制p-PI3K/Akt/NF-κB信號(hào)軸,進(jìn)而降低PD-L1蛋白表達(dá),抑制A549細(xì)胞免疫逃逸。
[Key word]
[Abstract]
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.
[中圖分類號(hào)]
R285.5
[基金項(xiàng)目]
國家自然科學(xué)基金資助項(xiàng)目(21775061);泰山學(xué)者青年專家計(jì)劃項(xiàng)目(tsqn202211136);濟(jì)南市“新高校20條”資助項(xiàng)目(202228085);山東省高等學(xué)校青創(chuàng)人才引育計(jì)劃項(xiàng)目(2021505031);山東中醫(yī)藥大學(xué)青年創(chuàng)新團(tuán)隊(duì)支持計(jì)劃項(xiàng)目(22202105)