AMBRA1 activation alleviates zearalenone-induced swine testicular cell ferroptosis by facilitating mitophagy

Background: Mycotoxin contamination poses a major challenge to public health and has garnered increasing attention across the world in recent decades. Zearalenone (ZEA), as one of the most prevalent contaminants, induces reproductive toxicity and then poses potential threats to animal health. Autophagy/beclin-1 regulator 1 (AMBRA1) is a protein critical for Autophagy induction, and can enhance Mitophagy by co-localizing with LC3. However, the potential health risk caused by ZEA in male germ cells of Animals is unclear. This study aimed to investigate the underlying mechanisms of ZEA-induced swine testicular (ST) cell injury and to clarify the role of AMBRA1 in this process.

Methods: We established ST cell models to explore the effects of AMBRA1 on Ferroptosis induced by ZEA. Multiple experimental approaches were applied to assess cell viability, mitochondrial dysfunction, oxidative stress, iron accumulation, and Mitophagy. Mechanistic insights were further validated using AMBRA1 overexpression, RNA-seq, molecular docking, western blotting, immunofluorescence, and qRT-PCR analyses.

Results: In this study, ZEA induced mitochondrial structural damage and impaired mitochondrial function, leading to excessive ROS generation and loss of mitochondrial membrane potential. We also found that ZEA disrupted the iron homeostasis and thus led to the accumulation of ferrous iron, which further induce Ferroptosis. In addition, ZEA reduced Autophagy activity and autophagic flux, ultimately suppressing Mitophagy. Of note, AMBRA1 overexpression effectively relieved ZEA-induced Ferroptosis through restoration of Mitophagy in ST cells.

Conclusions: In conclusion, our study demonstrated that ZEA targeted the AMBRA1, leading to down-regulation of AMBRA1 expression, which in turn inhibited Mitophagy and thus resulted in Ferroptosis in ST cells. Given the potential role of AMBRA1 in ST cells, our results uncover a previously unrecognized mechanism in which AMBRA1-mediated Mitophagy functions as a crucial defense target against Ferroptosis in testicular cells. Importantly, our results propose a unique insight which AMBRA1 as a promising therapeutic target for counteracting mycotoxin-induced testicular injury in Animals.

AMBRA1; Ferroptosis; Mitophagy; Swine testicular cells; Zearalenone.