摘要
目的探讨夏枯草(Prunella vulgaris L.)水提物中活性成分对人甲状腺乳头状癌细胞株K1自噬性死亡的影响。方法通过高效液相色谱串联质谱分析夏枯草水提物中的活性成分,采用CCK-8观察海藻糖、山柰酚、原儿茶酸、丹酚酸B、金丝桃苷、虫草素、木犀草素及水飞蓟宾8种活性成分对K1细胞及人正常甲状腺滤泡上皮细胞HUM-CELL-0097的作用。使用平板克隆实验观察IC_(50)组(120 mmol/L海藻糖)和IC_(50)组(110μmol/L山柰酚)对K1细胞增殖能力的影响。海藻糖分为对照组、0.5 IC_(50)(60 mmol/L)和IC_(50)组(120 mmol/L),山柰酚分为对照组、0.5 IC_(50)(55μmol/L)和IC_(50)组(110μmol/L),分别处理细胞24 h,Western Blot测定K1细胞内自噬相关蛋白(Beclin-1、LC3Ⅱ、P62)的表达量,透射电镜观察自噬小体形成情况,以上试验均重复3次。结果夏枯草水提物中具有潜在抗肿瘤作用的物质包括海藻糖(3.16%)、山柰酚(0.11%)、原儿茶酸(0.25%)、丹酚酸B(2.11%)、金丝桃苷(0.18%)、虫草素(0.10%)、木犀草素(0.084‰)及水飞蓟宾(0.092%)。海藻糖、木犀草素、水飞蓟宾、虫草素及山柰酚可抑制K1细胞增殖(P<0.05),在受试浓度范围内海藻糖与山柰酚没有对HUM-CELL-0097细胞活力产生影响(P>0.05)。海藻糖及山柰酚抑制K1细胞增殖的IC_(50)分别为120 mmol/L和110μmol/L,IC_(50)浓度的海藻糖及山柰酚处理K1细胞10 d,K1细胞的增殖克隆能力显著降低。使用0.5 IC_(50)及IC_(50)浓度的海藻糖及山柰酚处理K1细胞24 h,胞内Beclin-1及LC3Ⅱ蛋白表达增加(P<0.05),P62蛋白表达下降(P<0.05),均以IC_(50)组变化最为明显,可见自噬小体形成。IC_(50)海藻糖组Beclin-1、LC3Ⅱ、P62分别是对照组的1.35、2.06、0.42倍(P<0.05);IC_(50)山柰酚组Beclin-1、LC3Ⅱ、P62分别是对照组的1.60、1.96、0.28倍(P<0.05)。结论夏枯草水提物中海藻糖与山柰酚可促进K1细胞自噬性死亡。
Objective To investigate effects of active ingredients in aqueous extract of Prunella vulgaris L.(PV)on autophagic death of human thyroid papillary carcinoma(PTC)cells.Methods The active ingredients in PV aqueous extract were analyzed by high-performance liquid chromatography-tandem mass spectrometry.CCK-8 was used to observe the effects of the effects of 8 active ingredients including trehalose,kaempferol,protocatechuic acid,salvianolic acid B,hyperoside,cordycepin,luteolin and silybin on K1 cells and human normal thyroid follicular epithelial cells HUM-CELL-0097.The effects of trehalose in IC_(50) group(120 mmol/L)and kaempferol in IC_(50) group(110μmol/L)on the proliferation of K1 cells were observed by plate cloning experiment.Trehalose was divided into control group,0.5 IC_(50) group(60 mmol/L)and IC_(50) group(120 mmol/L),and kaempferol was divided into control group,0.5 IC_(50)(55μmol/L)and IC_(50) group(110μmol/L),respectively,and the cells were treated for 24 h,Western Blot was used to determine the expression of autophagy-related proteins(Beclin-1,LC3Ⅱ,P62)in K1 cells,and the formation of autophagosome was observed by transmission electron microscopy,the above experiments were repeated three times.Results The active ingredients in PV aqueous extract with potential anti-tumor activity included trehalose(3.16%),kaempferol(0.11%),protocatechuic acid(0.25%),salvianolic acid B(2.11%),hyperoside(0.18%),cordycepin(0.10%),luteolin(0.084‰)and silybin(0.092%).Trehalose,luteolin,silybin,cordycepin and kaempferol could inhibit the proliferation of K1 cells(P<0.05),trehalose and kaempferol had no effect on the viability of HUM-Cell-0097 cells in the tested concentration range(P>0.05).The IC_(50) values of trehalose and kaempferol were 120 mmol/L and 110μmol/L,respectively,in K1 cells.K1 cells were treated with trehalose and kaempferol at IC_(50) concentration for 10 days,and the proliferation and cloning ability of K1 cells was significantly reduced.After treatments with trehalose or kaempferol at the concen
作者
黄海斌
张朵
宋佳
万宇
苏桦
黎姿茵
林秀琴
杨杏芬
HUANG Hai-bin;ZHANG Duo;SONG Jia;WAN Yu;SU Hua;LI Zi-yin;LIN Xiu-qin;YANG Xing-fen(Food Safety and Health Research Center,Key Laboratory of Cosmetics Safety Evaluation of National Medical Products Administration,School of Public Health,Southern Medical University,Guangzhou,510515;Guangdong Provincial Center for Disease Control and Prevention,Guangzhou,511430)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2022年第7期856-862,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
广州市科技计划项目(No.201803010107)
广东省医学科学技术研究基金项目(No.A2019106)。
关键词
夏枯草
海藻糖
山柰酚
K1细胞
自噬
Prunella vulgaris L.
trehalose
kaempferol
K1 cells
autophagy
