摘要
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mt ROS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 peroxidase4,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,Cyt C)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
Objective To explore the effect and mechanism of quercetin on proliferation of human non-small cell lung cancer A549cells 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
作者
李畅
王浩
贺千羽
郭向宇
姜彤伟
郭焱
LI Chang;WANG Hao;HE Qian-yu;GUO Xiang-yu;JIANG Tong-wei;GUO Yan(School of Clinical Medicine,Changchun University of Chinese Medicine,Changchun 130117,China;Ji’an Hospital,Ji’an 134200,China)
出处
《中草药》
CAS
CSCD
北大核心
2022年第22期7112-7120,共9页
Chinese Traditional and Herbal Drugs
基金
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)。
关键词
非小细胞肺癌
槲皮素
铁死亡
细胞增殖
线粒体活性氧
脂质过氧化物
non-small cell lung
quercetin
ferroptosis
cell proliferation
mitochondrial reactive oxygen species
lipid peroxides
