Article

Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response

Authors: Yu WANG1, Su-han GUO1, Xue-jun SHANG2, Li-sha YU1, Jian-wei ZHU1, Ang ZHAO1, Yan-fen ZHOU1, Guo-hua AN3, Qi ZHANG1, Bo MA1
1 School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China
2 Department of Andrology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China
3 Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, Iowa City, Iowa, USA
Corresponding to: Qi ZHANG: nancyzhang03@hotmail.com, Bo MA: mabo201012@njtech.edu.cn,
DOI: 10.1038/aps.2017.95
Received: 15 January 2017
Accepted: 19 June 2017
Advance online: 14 September 2017

Abstract

Abstract
Triptolide (TP), an oxygenated diterpene, has a variety of beneficial pharmacodynamic activities but its clinical applications are restricted due to severe testicular injury. This study aimed to delineate the molecular mechanisms of TP-induced testicular injury in vitro and in vivo. TP (5–50000 nmol/L) dose-dependently decreased the viability of TM4 Sertoli cells with an IC50 value of 669.5– 269.45 nmol/L at 24 h. TP (125, 250, and 500 nmol/L) dose-dependently increased the accumulation of ROS, the phosphorylation of JNK, mitochondrial dysfunction and activation of the intrinsic apoptosis pathway in TM4 cells. These processes were attenuated by co-treatment with the antioxidant N-acetyl cysteine (NAC, 1 mmol/L). Furthermore, TP treatment inhibited the translocation of Nrf2 from cytoplasm into the nucleus as well as the expression of downstream genes NAD(P)H quinone oxidoreductase1 (NQO1), catalase (CAT) and hemeoxygenase 1 (HO-1), thus abrogating Nrf2-mediated defense mechanisms against oxidative stress. Moreover, siRNA knockdown of Nrf2 significantly potentiated TP-induced apoptosis of TM4 cells. The above results from in vitro experiments were further validated in male mice after oral administration of TP (30, 60, and 120 mg·kg-1·d-1, for 14 d), as evidenced by the detected indexes, including dose-dependently decreased SDH activity, increased MDA concentration, altered testicle histomorphology, elevated caspase-3 activation, apoptosis induction, increased phosphorylation of JNK, and decreased gene expression of NQO1, CAT and HO-1 as well as nuclear protein expression of Nrf2 in testicular tissue. Our results demonstrate that TP activates apoptosis of Sertoli cells and injury of the testis via the ROS/JNK-mediated mitochondrial-dependent apoptosis pathway and down-regulates Nrf2 activation.
Keywords: triptolide; Sertoli cells; MAPKs; apoptosis; ROS; Nrf2; NAC