白花蛇舌草-半枝莲药对组分诱导三阴性乳腺癌细胞凋亡的机制  

Mechanism of Active Fractions from Hedyotidis Herba-Scutellariae Barbatae Herba in Inducing Apoptosis of MDA-MB-231 Cells

作　　者：杜江洋 徐元 王楠 蒋义鑫 杨月 章丹丹 DU Jiang-yang;XU Yuan;WANG Nan;JIANG Yi-xin;YANG Yue;ZHANG Dan-dan(School of Chinese Materia Medica,Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学中药学院交叉科学研究院

出　　处：《中国实验方剂学杂志》2018年第17期99-107,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81773946,81573673,81001666);第九届上海中医药大学大学生科创项目(201610268066);上海教委创新项目(13YZ048);上海教委优青项目(SZY07029);上海卫计委青年项目(20144Y0143)

摘　　要：目的：考察白花蛇舌草一半枝莲药对的活性组分诱导三阴性乳腺癌细胞MDA-MB-231凋亡及其作用机制。方法：超高效液相色谱法对药对活性组分中化学成分进行测定，噻唑蓝（MTr）比色法检测细胞体外增殖力，克隆形成实验考察克隆形成的数量变化，流式细胞仪检测凋亡细胞的比例，蛋白质免疫印迹法检测蛋白及信号通路的变化。结果：制备药对以3种配比（1：1，1：2，2：1）进行水煎后脱脂浸膏的乙酸乙酯组分（YDWll，YDWl2，YDW21）。YDWll含有对羟基苯乙酮、野黄芩苷、木犀草素和芹菜素4种成分。通过比对这3个组分对乳腺正常上皮细胞10A1和三阴性乳腺癌细胞MDA-MB-231的体外增殖的影响，发现YDWll在25-50mg·L。对10A1细胞无细胞毒性，且抑制MDA-MB-231的体外增殖（P〈0．01），其抑制活性强于YDWl2，YDW21。YDWll呈质量浓度和时间依赖性抑制三阴性乳腺癌细胞株MDA-MB-231，HS578T，BT549的体外增殖（P〈0．05，P〈0．01），但对MCF-7体外增殖影响不大。YDWll呈浓度依赖性抑制MDA-MB-231的克隆形成（P〈0．01），并诱导其凋亡（P〈0．01）。YDWll提高p21mRNA和蛋白表达的同时，抑制增殖细胞核抗原（PCNA）的表达（P〈0．05，P〈0．01），抑制丝裂原活化蛋白激酶（MAPK）信号通路中p38，c-Jun氨基末端激酶（JNK），细胞外调节蛋白激酶（ERK）的磷酸化（P〈0．05）。YDWll处理MDA-MB-231后，VASP在Serl57位点和Ser239位点的磷酸化水平提高（P〈0．05，P〈0．01），提示激活环状核苷酸（cAMP）和环磷酸鸟苷（cGMP）信号通路的活化。结论：白花蛇舌草一半枝莲药对等比配伍的乙酸乙酯组分（YDWll）在对乳腺正常上皮细胞10A1无毒的剂量25，50mg·L-1，呈剂量和时间依赖性显著抑制三阴性乳腺癌细胞MDA-MB．231的体外增殖、克隆形成并诱导凋亡。呈浓度依赖性提高周期抑制蛋白p21在mRNA和蛋白Objective： To investigate the mechanisms of the active fractions from Hedyotidis Herba- Scutellariae Barbatae Herba （YDWll） in inducing apoptosis of triple negative breast cancer MDA-MB-231 cells. Method： Ultra high performance liquid chromatography （UPLC） was used to determine the chemical components in the active fractions, and methylthiazoletetrazolium （MTT） assay was used to evaluate the proliferation of cells in vitro. The colony formation assay was used to measure the changes of colony numbers. Flow cytometery combined with apoptosis detection kit was used to measure the ratio of apoptosis cells, and the changes in expression of proteins and signaling pathways were evaluated by Western blot. In addition, UPLC assay was carried out to identify the major constituents in YDWll. Result： The ethyl acetate fractions （YDWll, YDW12, YDW21） of the water extracts of Hedyotidis Herba-Scutellariae Barbatae Herba were prepared with three different 1：2 and 2 ： 1 ）. YDWll had four components, p-hydroxyacetophenone, scutellarin, luteolin and ratios （ 1 ： 1, comparing the effect of these three fractions on the in vitro proliferation of normal mammary epithelial apigenin. By cells （ 10A1 ） and triple negative breast cancer cells （MDA-MB-231）, it was found that YDWll at 25 -50 mg.L-1 had no cytotoxicity to normal breast epithelial cells 10A1, but inhibited the in vitro proliferation of MDA-MB-231 （p 〈 O. 01 ）, and the inhibitory effect was stronger than that of YDW12 and YDW21. YDW11 inhibited the proliferation of three triple negative breast cancer cell lines （MDA-MB-231, HS578T, BT549） in a dose and time dependent manner （p 〈 0.05, P 〈 0.01 ）, but it had little effect on the proliferation of MCF-7 in vitro. YDW11 inhibited the colony formation of MDA-MB-231 cells in a dose-dependent manner and induced cells apoptosis （p 〈 0.05, P 〈 0. 01 ）. YDW11 increased the mRNA and protein expression of p21, and simultaneously inhibited the expression of prolife

关 键 词：白花蛇舌草 半枝莲药对 三阴性乳腺癌 细胞凋亡 环状核苷酸(cAMP)信号通路 环磷酸鸟苷(cGMP)信号通路 丝裂原活化蛋白激酶(MAPK)信号通路 

分 类 号：R22[医药卫生—中医基础理论] R24[医药卫生—中医学]