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Int. J. Mol. Sci. 2016, 17(6), 850; doi:10.3390/ijms17060850

Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism

Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
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Academic Editor: Paul B. Tchounwou
Received: 27 February 2016 / Revised: 16 May 2016 / Accepted: 17 May 2016 / Published: 2 June 2016
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Abstract

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. View Full-Text
Keywords: pekinenin C; rat intestinal crypt epithelial cell line (IEC-6 cells); apoptotic mechanism; cell viability analysis; mitochondrial pathway; death receptor pathway pekinenin C; rat intestinal crypt epithelial cell line (IEC-6 cells); apoptotic mechanism; cell viability analysis; mitochondrial pathway; death receptor pathway
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MDPI and ACS Style

Cao, Y.; Cheng, F.; Yao, W.; Bao, B.; Zhang, K.; Zhang, L.; Ding, A. Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism. Int. J. Mol. Sci. 2016, 17, 850.

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