芹菜素对人肺鳞癌细胞NCI-H520增殖、转移、凋亡及自噬的影响  

Effect of apigenin on proliferation,metastasis,apoptosis and autophagy of human lung squamous cell carcinoma NCI-H520 cells

作　　者：刘萍 耿亚迪 刘云霄 谢彦博 张欣格 张蕾 魏伟[1] Liu Ping;Geng Yadi;Liu Yunxiao;Xie Yanbo;Zhang Xinge;Zhang Lei;Wei Wei(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-Inflammatory and Immune Medicine,Ministry of Education,Ministry of Education Rheumatoid Arthritis Research Center,Anhui Medical University,Hefei 230032;Dept of Pharmacy,The First Affiliated Hospital of University of Science and Technology of China,Anhui Provincial Hospital,Hefei 230001)

机构地区：[1]安徽医科大学临床药理研究所、抗炎免疫药物教育部重点实验室、安徽医科大学类风湿关节炎研究中心,合肥230032 [2]中国科学技术大学附属第一医院(安徽省立医院)药剂科,合肥230001

出　　处：《安徽医科大学学报》2022年第7期1009-1015,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年项目(编号:81803774);安徽省重点研究与开发计划项目(编号:201904a07020092);安徽省自然科学基金(编号:2108085MH311)。

摘　　要：目的研究芹菜素对人肺鳞癌细胞NCI-H520增殖、转移、凋亡及自噬的影响。方法体外培养人肺鳞癌细胞NCI-H520,采用CCK-8法检测不同浓度芹菜素(2.5、5、10、20μmol/L)或顺铂(2.5、5、10、20μmol/L)对细胞活力的影响;采用细胞增殖示踪荧光探针(CFDA SE)检测芹菜素或顺铂对细胞分裂能力的影响;采用划痕实验和Transwell实验检测芹菜素对细胞迁移和侵袭能力的影响;采用Annexin V/PI双染法、Hoechst 33258核染色法检测芹菜素对细胞凋亡的影响;透射电镜观察细胞内自噬嚢泡的产生情况;吖啶橙(AO)染色观察细胞内酸性细胞器的变化;Western blot检测微管相关蛋白1轻链3B-Ⅱ(LC3B-Ⅱ)和p62蛋白表达情况。结果与对照组相比,CCK-8法和CFDA SE结果显示芹菜素或顺铂使NCI-H520细胞的增殖水平下降(P<0.05);划痕实验和Transwell实验表明芹菜素使细胞的迁移和侵袭水平下降(P<0.01);Annexin V/PI双染结果显示芹菜素可使细胞凋亡率增加(P<0.05);Hoechst 33258核染色显示芹菜素可促进细胞出现细胞核聚缩和浓染;AO染色荧光值增大、电镜下细胞出现自噬双分子结构、Western blot中LC3B-Ⅱ和p62蛋白表达水平上升的结果表明芹菜素增加了细胞NCI-H520的自噬水平(P<0.05);加入氯喹(CQ)后,未能增加芹菜素作用细胞中LC3B-Ⅱ和p62的蛋白水平。结论不同浓度芹菜素能抑制人肺鳞癌细胞NCI-H520增殖、迁移、侵袭,其抑制细胞的生长转移可能与诱导细胞凋亡和自噬有关,且自噬水平的升高可能是阻断自噬体的降解所致。Objective To investigate the effect of apigenin on the proliferation, migration, invasion, apoptosis and autophagy of human lung squamous carcinoma NCI-H520 cells. Methods Human lung squamous carcinoma NCI-H520 cells were cultured in vitro, and the CCK-8 method was used to detect the effects of different concentrations of apigenin(2.5, 5, 10, 20 μmol/L) or cisplatin(2.5, 5, 10, 20 μmol/L) on cell viability. Carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) was used to detect the effect of apigenin or cisplatin on cell division. Scratch test and Transwell test were used to detect the effect of apigenin on cell migration and invasion. Annexin V/PI double staining method and Hoechst 33258 nuclear staining method were used to detect the effect of apigenin on cell apoptosis. Transmission electron microscopy was used to observe the generation of autophagic vesicles in cells. Acridine orange(AO) staining was used to observe the changes of acidic organelles in cells. Western blot was used to detect microtubule-associated protein 1 light chain 3 B-Ⅱ(LC3 B-Ⅱ) and p62 protein expression. Results Compared with the control group, CCK-8 assay and CFDA SE showed that apigenin or cisplatin reduced proliferation of NCI-H520 cells(P<0.05). Scratch test and Transwell test showed that apigenin reduced the migration and invasion levels of cells(P<0.01). Annexin V/PI double staining showed that apigenin increased apoptosis rate(P<0.05). Hoechst 33258 nuclear staining showed that apigenin promoted nuclear condensation and hyperchromatism of cells. AO staining fluorescence value enhanced, the autophagy bimolecular structure appeared under transmission electron microscopy, and the results of LC3 B-Ⅱ and p62 protein expression levels in Western blot showed that apigenin increased the autophagy level of cell NCI-H520(P<0.05). Chloroquine(CQ) failed to increase the protein levels of LC3 B-Ⅱ and p62 in apigenin treated cells. Conclusion Apigenin at different concentrations can inhibit the proliferation, migration and invasi

关 键 词：芹菜素 NCI-H520 增殖 转移 凋亡 自噬 

分 类 号：R734.2[医药卫生—肿瘤]