Study on podophyllotoxin induced rat epididymalepithelial cell apoptosis and its mechanism in vitro
10.3969/j.issn.1001-1978.2017.10.007
- VernacularTitle:鬼臼毒素诱导离体大鼠附睾上皮细胞凋亡机制研究
- Author:
Guoting LI
;
Ruihua ZHONG
;
Jieyun ZHOU
;
Shuwu XIE
;
Ping CHEN
;
Yueying QIAN
;
Yan ZHU
- Keywords:
podophyllotoxin;
fertility;
rat epididymal epithelial cell;
cell apoptosis;
mitochondria;
TNF-α;
caspase activation
- From:
Chinese Pharmacological Bulletin
2017;33(10):1357-1363
- CountryChina
- Language:Chinese
-
Abstract:
Aim To investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.Methods Primary epididymal epithelial cells were cultured in vitro.CCK-8 assay was used to analyze proliferation of epididymal epithelial cells induced by podophyllotoxin on 24, 48 and 72 h.The ultra structural changes of the epididymal epithelial cells were observed by transmission electron microscope.AnnexinV-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The TdTmediated dUTP nick end labeling(TUNEL) technique was applied to observe the morphological changes of apoptotic cells.The expression of tumor necrosis factor alpha(TNF-α) mRNA was investigated by real-time RT-PCR.The level of TNF-α in cell culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA) technology.Western blot was per-formed to determine the protein expression of cytochrome C, caspase-3, caspase-8, caspase-9.Results Podophyllotoxin significantly inhibited the activity of proliferation and induced apoptosis of epididymal epithelial cells in a dose-and time-dependent manner(P<0.05), with a 50％ inhibitory concentration(IC50) value and corresponding 95％ confidence intervals(CI) of 59.36(15.50～227.41), 0.37(0.080～1.70), 0.077(0.017～0.35) μmol·L-1 at 24, 48 and 72 h, respectively.Podophyllotoxin induced cell volume turned round and cell nuclear fragmentation and mitochondrial vacuolation.RT-PCR and ELISA results showed that podophyllotoxin improved the expression of TNF-α mRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C, caspase-3, caspase-8 and caspase-9.Conclusion Podophyllotoxin can induce rat epididymal epithelial cell apoptosis through both the mitochondria-regulated intrinsic pathway and the TNF receptor-mediated extrinsic pathway.
