Abstract：
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 resis-tance 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.
摘要：
本研究旨在探讨紫花提取物(SCL)对乳腺癌多药耐药(MDR)的影响及其机制。实验采用人三阴性乳腺癌细胞株MDA-MB-231及其阿霉素耐药细胞株MDA-MB-231/ADR。通过甲基噻唑基四唑（MTT）测定法检测细胞活力，DAPI染色和Annexin-V/Pi双重染色检测细胞凋亡。Westernblot(WB)检测Keap1、Nrf2、HO-1、Bcl-2、Bax、caspase-9和caspase-3。免疫荧光染色观察Nrf2在细胞中的分布,流式细胞术检测细胞内活性氧(ROS)水平。结果表明，SCL的因子为0.69，阿霉素和紫杉醇的因子分别为8.40和16.36。DAPI染色显示SCL可引起乳腺癌细胞核收缩和碎裂。Annexin-V/Pi双重染色显示耐药细胞的平均凋亡率为32.64%和50.29%,分别在中等和高剂量的SCL。WB结果显示，SCL可以显着降低抗凋亡蛋白Bcl-2，caspase-9和caspase-3的表达水平，并显着增加促凋亡蛋白Bax的表达水平。进一步研究表明，SCL能显著促进Keap1的表达，显著抑制Nrf2和HO-1的表达，显著降低Nrf2在细胞核中的表达水平。相应地,流式细胞仪显示细胞内ROS水平显著升高。总之,SCL能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,机制与抑制Keap1/Nrf2信号通路有关,导致ROS在耐药细胞中积累并增加凋亡相关蛋白的表达。
