瑞香狼毒通过Nrf2抗三阴性乳腺癌MDA-MB-231细胞多药耐药的研究  

Stellera chamaejasme against multidrug resistance of triple-negative breast cancer MDA-MB-231 cell through Nrf2

作　　者：曾蕊 王晓萱 崔羲和 杨庆[1] 朱晓新[1] 王娅杰[1] ZENG Rui;WANG Xiao-xuan;CUI Xi-he;YANG Qing;ZHU Xiao-xin;WANG Ya-jie(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院中药研究所,北京100700

出　　处：《中国中药杂志》2024年第8期2222-2229,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82174030);中国中医科学院科技创新工程项目(CI2021B015);重大新药创制“科技重大专项”(2017ZX09301012002)。

摘　　要：该研究将探讨瑞香狼毒提取物(SCL)抑制乳腺癌多药耐药的作用及机制。实验以人三阴性乳腺癌细胞亲本株MDA-MB-231,及其阿霉素耐药细胞株MDA-MB-231/ADR为研究对象,通过噻唑蓝(MTT)法检测细胞活力;DAPI染色和Annexin-V/Pi凋亡双染检测细胞凋亡,Western blot(WB)检测Kelch样环氧氯丙烷相关蛋白1(Keap1)、核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱天冬酶(caspase)-9、caspase-3的表达水平,免疫荧光染色观察Nrf2细胞内分布,流式细胞术检测细胞内活性氧(ROS)水平。实验结果显示SCL的耐药因子是0.69,对应地,阿霉素和紫杉醇的耐药因子分别是8.40、16.36;DAPI染色结果显示SCL能够导致乳腺癌细胞核皱缩、碎裂;Annexin-V/Pi凋亡双染显示,在中、高剂量SCL的作用下,耐药细胞的平均凋亡率分别为32.64%、50.29%;WB检测结果提示:SCL能够明显降低抗凋亡蛋白Bcl-2、caspase-9和caspase-3表达水平,明显升高促凋亡蛋白Bax表达水平,进一步研究发现,SCL能够显著促进Keap1的表达,并明显抑制Nrf2和HO-1的表达,同时能够明显减少Nrf2的入核表达水平;相应地,流式检测细胞内ROS水平明显升高。综上所述,瑞香狼毒能够显著抑制三阴性乳腺癌MDA-MB-231多药耐药细胞的增殖,引起细胞凋亡,其机制与抑制Keap1/Nrf2信号通路,导致耐药细胞内ROS累积,并增加凋亡相关蛋白的表达有关。This study aims to investigate the effect and mechanism of Stellera chamaejasme extract(SCL)on multidrug resistance(MDR)in breast cancer.Human triple-negative breast cancer cell line MDA-MB-231 and its adriamycin-resistant cell line MDA-MB-231/ADR were used in the experiment.Cell viability was detected by methyl thiazolyl tetrazolium(MTT)assay,and cell apoptosis was detected by DAPI staining and Annexin-V/Pi double staining.Western blot(WB)was used to detect the expression levels of Keap1,Nrf2,HO-1,Bcl-2,Bax,caspase-9,and caspase-3.Immunofluorescence staining was used to observe the distribution of Nrf2 in the cell,and flow cytometry was used to detect the level of reactive oxygen species(ROS)in the cell.The results showed that the resistance factor of SCL was 0.69,and that of adriamycin and paclitaxel was 8.40 and 16.36,respectively.DAPI staining showed that SCL could cause nuclear shrinkage and fragmentation of breast cancer cells.Annexin-V/Pi double staining showed that the average apoptosis rate of the drug-resistant cells was 32.64%and 50.29%,respectively under medium and high doses of SCL.WB results showed that SCL could significantly reduce the expression levels of anti-apoptotic proteins Bcl-2,caspase-9,and caspase-3 and significantly increase the expression level of pro-apoptotic protein Bax.Further studies showed that SCL could significantly promote the expression of Keap1,significantly inhibit the expression of Nrf2 and HO-1,and significantly reduce the expression level of Nrf2 in the nucleus.Correspondingly,flow cytometry showed that the intracellular ROS level was significantly increased.In conclusion,SCL can significantly inhibit the proliferation of MDA-MB-231 multidrug-resistant cells of triple-negative breast cancer and cause cell apoptosis,and the mechanism is related to inhibiting Keap1/Nrf2 signaling pathway,leading to ROS accumulation in drug-resistant cells and increasing the expression of apoptosis-related proteins.

关 键 词：瑞香狼毒提取物 乳腺癌多药耐药 凋亡 NRF2 抗氧化 

分 类 号：R285[医药卫生—中药学]