Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.
Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.
复制标题
大戟中北京素C对大鼠小肠隐窝上皮细胞的毒性及其凋亡机制
DOI：
10.3390/ijms17060850
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发表时间：
2016-06-02
影响因子：
5.6
通讯作者：
Ding A
中科院分区：
文献类型：
--
作者：
Cao Y;Cheng F;Yao W;Bao B;Zhang K;Zhang L;Ding A
Pekinenin C是从中药大戟的根中分离出来的卡班二萜类化合物，用作治疗水肿、腹水、胸水的药物。尽管 Pekinenin C 表现出严重的细胞毒性，但确切的毒性机制尚不清楚。在本研究中，检测了pekinenin C对细胞抑制、细胞周期和细胞凋亡的影响，以解释其毒性机制。与不同浓度的 Pekinenin C 孵育后，通过 MTT 比色法检测 IEC-6 细胞的增殖情况。用流式细胞仪分析经 Pekinenin C 处理的 RNase/PI 和 Annexin V/PI 标记的 IEC-6 细胞，分别评估细胞周期分布和细胞凋亡。通过酶联免疫吸附试验（ELISA）检测caspase-3、8、9的活性，Western-blot检测Bax、Bcl-2、凋亡诱导因子（AIF）、Apaf-1、Fas相关死亡结构域（FADD）和1型相关死亡结构域（TRADD）的蛋白表达，Fas受体（FasR）、Fas配体的mRNA表达，探讨PEKENIN C对IEC-6的凋亡机制。 (FasL)、肿瘤坏死因子受体 (TNFR1) 和 NF-κB（通过 RT-PCR 检测）。结果表明，京黄素C对IEC-6细胞具有明显的毒性，IC50值为2.1 μg·mL−1。透射电镜下观察到典型的凋亡特征，发现pekinenin C能够以剂量依赖的方式引起IEC-6细胞G0/G1期阻滞，诱导IEC-6细胞凋亡。此外，pekinenin C还可增加Bax、AIF、Apaf-1、FasR、FasL、TNFR1和NF-κB的表达，抑制Bcl-2、FADD和TRADD的表达，进而激活caspase-3、8、9级联反应，最终导致细胞凋亡。这些结果表明，pekinenin C有效促进细胞凋亡，并通过线粒体和死亡受体途径诱导IEC-6细胞凋亡。
Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL−1. Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways.