吴茱萸碱联合自噬抑制剂氯喹抗肝癌作用的研究  

Effect of Evodiamine Combined with Chloroquine on Hepatocarcinoma

作　　者：沈星星 时乐[1] 姚亮宇 牛玉珍 梁涛[1] SHEN Xing-xing;SHI Le;YAO Liang-yu;NIU Yu-zhen;LIANG Tao(School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China;The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]南京医科大学第一附属医院,江苏南京210029

出　　处：《南京中医药大学学报》2021年第3期400-403,共4页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金青年基金(81703835)。

摘　　要：目的研究吴茱萸碱(EVO)联合自噬抑制剂氯喹(CQ)的抗肝癌作用。方法用CCK8法检测EVO及EVO联合CQ对HepG2细胞活性的影响,Western blot法检测微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)/微管相关蛋白1轻链3-Ⅰ(LC3-Ⅰ)(LC3-Ⅱ/LC3-Ⅰ)、P62、血管内皮生长因子(VEGFA)蛋白表达,ELISA法检测HepG2细胞上清液中VEGFA的表达水平,细胞侵袭实验检测条件培养液对人脐静脉血管细胞HUVECs侵袭能力的影响。结果不同浓度的EVO在作用24 h后能明显抑制HepG2细胞的活性且呈剂量依赖性,当EVO与CQ联合使用时其抑制细胞活性能力更强(P<0.01)。Western blot结果显示EVO能上调LC3Ⅱ/LC3Ⅰ的表达(P<0.05),降低P62的表达(P<0.05),对VEGFA的表达没有明显作用,但有下降的趋势。丹酰戊二胺(MDC)染色结果显示EVO能促进HepG2细胞自噬小体增加,EVO+CQ组细胞内自噬小体聚集明显增多。ELISA结果显示EVO能降低VEGFA的表达(P<0.01),并且EVO联合CQ作用效果更加明显(P<0.01)。HUVECs侵袭实验表明,EVO能有效抑制HUVECs细胞侵袭(P<0.01),同时EVO联合CQ抑制HUVECs细胞侵袭能力更为显著(P<0.01)。结论EVO能抑制HepG2细胞活性,并且能提高其自噬水平,降低血管生成能力。EVO与自噬抑制剂CQ联合使用时,对HepG2细胞活性和血管生成的抑制作用更为显著。OBJECTIVE To study the anti-liver cancer effect of evodiamine(EVO)combined with the autophagy inhibitor chloroquine(CQ).METHODS CCK8 method was used to detect the EVO and EVO+CQ on HepG2 cell vitality.Western blot method was used to detect LC3Ⅱ/LC3Ⅰ,P62,vascular endothelial growth factor(VEGFA)protein expressions.ELISA method was applied to detect VEGFA expression level in the supernatant fluid of HepG2 cells.The effect of cell conditioned medium on HUVEC cell invasion ability was tested.RESULTS Different concentrations of EVO could significantly inhibit the activity of HepG2 cells in a dose-dependent manner after 24 hours.When combined with CQ,the inhibitory activity was stronger(P<0.01).Western blot results showed that EVO could up-regulate the expression of LC3Ⅱ/LC3Ⅰ(P<0.05)and reduce that of P62(P<0.05),but show no significant effect on VEGFA,although a downward trend was observed.The results of MDC staining showed that EVO could increase autophagosomes in HepG2 cells.When combining EVO and CQ,the accumulation of autophagosomes in HepG2 cells increased significantly.ELISA results showed that EVO reduced the expression of VEGFA(P<0.01),and much significantly when combining EVO and CQ(P<0.01).The HUVECs invasion experiments showed that EVO effectively inhibited the invasion of HUVECs(P<0.01),and much significantly when combining EVO and CQ(P<0.01).CONCLUSION Evodiamine can inhibit the activities of HepG2 cells,increase the autophagy level and reduce the angiogenesis.Evodiamine shows more potent inhibitory activities against HepG2 and angiogensis,when it is combined with chloroquine.

关 键 词：吴茱萸碱 氯喹 HEPG2细胞 自噬 血管生成 

分 类 号：R285.5[医药卫生—中药学]