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Midazolam activates caspase, MAPKs and endoplasmic reticulum stress pathways, and inhibits cell cycle and Akt pathway, to induce apoptosis in TM3 mouse Leydig progenitor cells

Authors Kang FC, Wang SC, Chang MM, Pan BS, Wong KL, Cheng KS, So EC, Huang BM

Received 18 October 2017

Accepted for publication 15 January 2018

Published 15 March 2018 Volume 2018:11 Pages 1475—1490


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 2

Editor who approved publication: Dr XuYu Yang

Fu-Chi Kang,1,* Shu-Chun Wang,2,* Ming-Min Chang,2 Bo-Syong Pan,3 Kar-Lok Wong,4 Ka-Shun Cheng,4,5 Edmund Cheung So,4,6 Bu-Miin Huang2,7

1Department of Anesthesia, Chi Mei Medical Center, Chiali, Tainan, Taiwan, Republic of China; 2Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China; 3Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, NC, USA; 4Department of Anesthesia, China Medical University, Taichung, Taiwan, Republic of China; 5Department of Anesthesiology, The Qingdao University Yuhuangding Hospital, Yantai, Shandong, China; 6Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan, Republic of China; 7Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan, Republic of China

*These authors contributed equally to this work

Background: Midazolam (MDZ) has powerful hypnosis, amnesia, anti-anxiety and anticonvulsant effects. Studies have shown that prenatally developmental toxicity of diazepam can be observed in many organs/tissues. However, it remains elusive in male reproductive system.
Materials and methods: TM3 mouse Leydig progenitor cell line was used to determine whether MDZ has any unfavorable effects.
Results: Midazolam significantly decreased cell viability in dose- and time-dependent manners in TM3 cells. In flow cytometry analysis, midazolam significantly increased subG1 phase cell numbers, and annexin V/PI double staining assay further confirmed that MDZ induced apoptosis in TM3 cells. Moreover, MDZ significantly induced the expression of caspase-8 and -3 proteins and the phosphorylation of JNK, ERK1/2 and p38. Besides, MDZ didn’t activate Akt pathway in TM3 cells. Furthermore, the expressions of p-EIF2α, ATF4, ATF3 and CHOP were induced by midazolam, suggesting that midazolam could induce apoptosis through endoplasmic reticulum (ER) stress in TM3 cells. Additionally, the expressions of cyclin A, cyclin B and CDK1 were inhibited by midazolam through the regulation of p53 in TM3 cells, indicating that midazolam could regulate cell cycle to induce apoptosis.
Conclusion: Midazolam could activate caspase, MAPKs and ER stress pathways and impede Akt pathway and cell cycle to induce apoptosis in TM3 mouse Leydig progenitor cells.

Keywords: midazolam, TM3, Leydig progenitor cells, apoptosis, caspase, MAPKs, ER stress, cell cycle

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