目的 基于铁死亡信号通路探究槲皮素对人非小细胞肺癌A549细胞增殖的影响及其作用机制。方法 CCK-8法筛选槲皮素作用于A549细胞的实验浓度;平板克隆法检测槲皮素对A549细胞集落形成能力的影响;利用试剂盒检测槲皮素对A549细胞内谷胱甘肽(glutathione,GSH)水平的影响;采用Western blotting检测槲皮素对A549细胞铁死亡相关蛋白及线粒体凋亡蛋白表达的影响;采用流式细胞仪检测槲皮素对A549细胞内线粒体活性氧(mitochondrial reactive oxygen species,mtROS)、脂质过氧化物水平及细胞凋亡的影响。联合铁死亡抑制剂(Ferrostatin-1)或ROS清除剂N-乙酰半胱氨酸(Nacetylcysteine,NAC)检测槲皮素对A549细胞内GSH水平及铁死亡相关蛋白表达的影响。结果 与对照组比较,槲皮素显著抑制A549细胞存活率,且呈时间和剂量相关性(P<0.01);槲皮素呈剂量相关性地抑制A549细胞集落形成(P<0.01),显著降低A549细胞内GSH水平(P<0.01),上调细胞内mtROS及脂质过氧化物水平(P<0.05、0.01),诱导细胞凋亡(P<0.01);显著促进铁死亡相关蛋白p53表达(P<0.05、0.01),并抑制谷胱甘肽过氧化物酶4(recombinant glutathione peroxidase 4,GPX4)及胱氨酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)蛋白表达(P<0.01);显著促进线粒体凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、Caspase-9、细胞色素C(cytochrome C,CytC)和B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)相关X蛋白(Bcl-2 associated X protein,Bax)蛋白表达(P<0.05、0.01),并抑制抗凋亡因子Bcl-2蛋白表达(P<0.01)。与槲皮素组比较,槲皮素+NAC组与槲皮素+Ferrostatin-1组均不同程度恢复槲皮素引起的细胞存活率下降(P<0.05、0.001),Ferrostatin-1可显著上调GPX4及SLC7A11蛋白表达水平(P<0.05),并回调GSH水平(P<0.05)。结论 槲皮素能够抑制A549细胞增殖并诱导铁死亡,进而导致细胞凋亡,具有诱导A549细胞铁死亡的生物学效应。

Objective To explore the effect and mechanism of quercetin on proliferation of human non-small cell lung cancer A549 cells based on ferroptosis signaling pathway. Methods CCK-8 method was used to screen the experimental concentration of quercetin on A549 cells; The effect of quercetin on colony forming ability of A549 cells was detected by plate cloning method; The effect of quercetin on glutathione (GSH) level in A549 cells was detected by kit; Western blotting was used to detect the effect of quercetin on the expressions of ferroptosis related protein and mitochondrial apoptosis protein in A549 cells; The effects of quercetin on mitochondrial reactive oxygen species (mtROS), lipid peroxide level and apoptosis in A549 cells were detected by flow cytometry.Combined with ferroptosis inhibitor (Ferrostatin-1) or ROS scavenger N-acetylcysteine (NAC), the effects of quercetin on GSH level and expressions of ferroptosis related proteins in A549 cells were detected. Results Compared with control group, quercetin significantly inhibited the survival rate of A549 cells, with a time-dose correlation (P< 0.01). Quercetin inhibited the colony formation of A549 cells in a dose-dependent manner (P< 0.01), significantly decreased GSH level in A549 cells (P< 0.01), upregulated mtROS and lipid peroxide levels in A549 cells (P< 0.05, 0.01), and induced apoptosis (P< 0.01); Quercetin significantly promoted the expression of ferroptosis related protein p53 (P< 0.05, 0.01), and inhibited the expressions of glutathione peroxidase 4 (GPX4) and soluble carrier family 7 member 11 (SLC7A11) (P< 0.01); Quercetin significantly promoted expressions of mitochondrial apoptosis related proteins such as cysteine-aspartate protease-3 (Caspase-3), Caspase-9, cytochrome c (Cyt C) and B cell lymphoma-2 (Bcl-2) related X protein (Bax) (P< 0.05, 0.01), and inhibited the protein expression of anti-apoptotic factor Bcl-2 (P< 0.01). Compared with quercetin group, quercetin + NAC group and quercetin + Ferrostatin-1 group recovered the decrease of cell survival rate caused by quercetin to varying degrees (P< 0.05, 0.001), and ferrostatin-1 could significantly up-regulated GPX4 and SLC7A11 protein expressions (P< 0.05), and regulated the level of GSH (P< 0.05). Conclusion Quercetin can inhibit the proliferation of A549 cells and induce ferroptosis, and then lead to cell apoptosis, which has the biological effect of inducing ferroptosis in A549 cells.

R285.5

吉林省自然科学基金资助项目（YDZJ202201ZYTS151）；国家自然科学基金国际合作项目（3191101705）；吉林省科学技术厅（20210204029YY）