STUDY ON THE REGULATORY MECHANISM OF TCDD INHIBITING PROLIFERATION OF RAT LEYDIG CELLS AND INDUCING APOPTOSIS

Objective: To investigate the effects of TCDD on proliferation and apoptosis of rat Leydig cells and its possible regulatory mechanisms, and to provide an entry point for intervention in reproductive toxicity. Methods: Rat Leydig cells were isolated and cultured, treated with TCDD and divided into control groups, including a low dose group, medium dose group and high dose getup. The proliferation and apoptosis of Leydig cells were assessed by MTT assay and flow cytometry. The Fas/FasL, caspase-8 and ROS levels in each group were detected by western blot and DCFH-DA fluorescent probes. Results: The proliferation of Leydig cells was detected by MTT assay in the control, low dose, medium dose and high dose groups. At the same time, the absorbance values of the four groups decreased with the increase of TCDD concentration (P<0.05). Except for the control group, the absorbance values of the cultures at 24 h, 48 h and 72 h were gradually decreased under the same TCDD concentration (P<0.05). As well, the apoptosis of Leydig cells was detected by flow cytometry in the control group, low dose group, middle dose group and high dose group, while the apoptosis rate of the four groups increased with the increase of TCDD concentration at the same time point (P<0.05). At the same TCDD concentration, the apoptosis rate of cells was increased at 24 h, 48 h and 72 h (P<0.05). Further, the western Blot results showed that the expression of Fas/FasL and caspase-8 in the Leydig cells were significantly increased in the low dose, medium dose and high dose groups, and were the highest in the high-dose group (P<0.05). At the same TCDD concentration, the apoptosis rate of cells was increased at 24 h, 48 h and 72 h (P<0.05). At the same time point, the intracellular ROS content of the four groups increased with the increase of TCDD concentration (P<0.05). However, at the same TCDD concentration, the intracellular ROS content increased gradually at 24 h,48 h and 72 h (P<0.05). Conclusion: TCDD may induce apoptosis of the Leydig cells through a ROS-mediated mitochondrial pathway and death receptor pathway, and inhibit its proliferation. Moreover, it has a certain time effect and dose-effect relationship.