Gao Jing, Gao Lan, Zhang Li, Yao Weifeng, Cao Yudan, Bao Beihua, Ding Anwei
Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China.
Nanjing Jiangning Hospital of Chinese Medicine, Teaching Hospital of Nanjing University of Chinese Medicine, Nanjing 211100, China.
J Ethnopharmacol. 2015 Nov 4;174:331-8. doi: 10.1016/j.jep.2015.08.036. Epub 2015 Aug 28.
Euphorbia kansui is a traditional Chinese medicine widely used for the treatment of edema, ascite and asthma in China for centuries. However, its serious gastrointestinal toxicity restricted its safe clinical application. 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), a diterpenoid compound derived from kansui, has obvious gastrointestinal cytotoxicity in cells. Until now, its gastrointestinal cytotoxic mechanism is mostly unknown. This study focused on elucidating the cytotoxic mechanism of 3EZ,20Ac-ingenol in intestinal epithelial cells of rats (IEC-6 cells) to guide safer application of this herb in clinic.
3EZ,20Ac-ingenol was isolated from the EtOAc extract of kansui. Cell morphology was detected by inverted phase contrast microscope and transmission electron microscope (TEM). Cell apoptosis was examined by Annexin V-FITC/PI dual-staining or Hoechst staining. ROS generation was detected with DCFH-DA staining by laser scanning confocal microscope. MMP change was examined with JC-1 staining by high content screening (HCS). Further, the release of cytochrome c, the expressions of Bax, Bcl-2, AIF and Apaf-1 were analyzed by western blot and the activities of caspase-3, 8, 9 were determined by ELISA. Additionally, cell cycle analysis was performed to detect the effects of 3EZ,20Ac-ingenol on cell cycle in IEC-6 cells by flow cytometry.
The study showed that 3EZ,20Ac-ingenol significantly reduced IEC-6 cells viability in a dose-dependent manner and the IC50 value was 5.74 μg/mL. Consistently, 3EZ,20Ac-ingenol could elevate reactive oxygen species (ROS), disrupt mitochondrial membrane potential (MMP), induce the release of cytochrome c from mitochondria to cytosol, enhance the expressions of Bax, AIF and Apaf-1, suppress the expression of Bcl-2, then activate caspase-3, 8, 9 cascade, and subsequently result in apoptosis. Additionally, 3EZ,20Ac-ingenol also could cause G2/M phase arrest in IEC-6 cells.
The results indicated that 3EZ,20Ac-ingenol induced the cytotoxicity of IEC-6 cells depends on induction of cell apoptosis via mitochondrial pathway and cell cycle arrest.
甘遂是一种传统中药，在中国已有数百年用于治疗水肿、腹水和哮喘的历史。然而，其严重的胃肠道毒性限制了它在临床上的安全应用。3-O-(2'E,4'Z-癸二烯酰基)-20-O-乙酰大戟醇（3EZ,20Ac-大戟醇）是一种从甘遂中提取的二萜类化合物，在细胞中具有明显的胃肠道细胞毒性。到目前为止，其胃肠道细胞毒性机制大多未知。本研究聚焦于阐明3EZ,20Ac-大戟醇在大鼠肠上皮细胞（IEC-6细胞）中的细胞毒性机制，以指导该草药在临床上更安全地应用。
从甘遂的乙酸乙酯提取物中分离出3EZ,20Ac-大戟醇。通过倒置相差显微镜和透射电子显微镜（TEM）检测细胞形态。采用Annexin V-FITC/PI双染法或Hoechst染色法检测细胞凋亡。用DCFH-DA染色通过激光扫描共聚焦显微镜检测活性氧（ROS）的产生。用JC-1染色通过高内涵筛选（HCS）检测线粒体膜电位（MMP）的变化。此外，通过蛋白质免疫印迹法分析细胞色素c的释放、Bax、Bcl-2、AIF和Apaf-1的表达，并通过酶联免疫吸附测定法（ELISA）测定caspase-3、8、9的活性。另外，通过流式细胞术进行细胞周期分析，以检测3EZ,20Ac-大戟醇对IEC-6细胞周期的影响。
研究表明，3EZ,20Ac-大戟醇以剂量依赖性方式显著降低IEC-6细胞活力，IC50值为5.74μg/mL。同样，3EZ,20Ac-大戟醇可升高活性氧（ROS）、破坏线粒体膜电位（MMP）、诱导细胞色素c从线粒体释放到细胞质中、增强Bax、AIF和Apaf-1的表达、抑制Bcl-2的表达，进而激活caspase-3、8、9级联反应，随后导致细胞凋亡。此外，3EZ,20Ac-大戟醇还可使IEC-6细胞发生G2/M期阻滞。
结果表明，3EZ,20Ac-大戟醇诱导IEC-6细胞的细胞毒性取决于通过线粒体途径诱导细胞凋亡和细胞周期阻滞。
