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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
[Di-(2-ethylhexyl) phthalate activates the apoptosis Caspase pathway in rat Leydig cells in vitro]
Wei-Guang Wu1, Ya-Juan Tang, Zhao-Ling Sun
1Department of Obstetrics and Gynecology, The Affiliated Hospital of Logistics College of Chinese People's Armed Police Force, Tianjin 300162, China. weiguangwu02@163.com
Objective:
To investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on the expressions of Caspase-3 and Caspase-9 genes in rat Leydig cells and the apoptosis of the cells in vitro.
Methods:
Leydig cells were isolated from male SD rats, primarily cultured and treated with DEHP at a low (10 nmol/L), a medium (50 nmol/L) and a high dose (100 nmol/L) for 24 hours. Then the mRNA expressions of Caspase-3 and Caspase-9 genes in the Leydig cells were detected by real time PCR, their protein expressions determined by Western blot, and the apoptosis of the Leydig cells measured by flow cytometry.
Results:
Compared with the DMSO control group, the low-, medium- and high-dose DEHP groups showed significantly upregulated expressions of Caspase-3 mRNA (1.69 +/- 0.38 vs 3.82 +/- 0.39, 6.91 +/- 0.40 and 15.47 +/- 0.40, P < 0.05), Caspase-3 protein (0.18 +/- 0.0.09 vs 0.32 +/- 0.10, 0.61 +/- 0.08 and 0.89 +/- 0.09, P < 0.05), Caspase-9 mRNA (2.24 +/- 0.41 vs 5.16 +/- 0.43, 9.61 +/- 0.45 and 19.22 +/- 0.43, P < 0.05) and Caspase-9 protein (0.26 +/- 0.07 vs 0.40 +/- 0.08, 0.68 +/- 0.09 and 0.96 +/- 0.08, P < 0.05), as well as increased apoptosis rate of Leydig cells (4.36 +/- 1.11 vs 7.52 +/- 1.09, 12.72 +/- 1.10 and 24.59 +/- 1.11, P < 0.05), all in a dose-dependent manner.
Conclusion:
DEHP can induce the apoptosis of rat Leydig cells by activating the apoptosis Caspase pathway, and consequently affect the function of Leydig cells.
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