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

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.