Icariin alleviates triptolide-induced testicular vacuolization via modulating germline ferroptosis and blood-testis barrier integrity.

Introduction: Environmental factors mediated testicular vacuolization injury is a prevalent occurrence, and its etiology and mitigation strategies have long remained inadequately elucidated. Methods: A testicular injury model was established using Triptolide (TP), with Icariin (ICA) employed as a rescue compound. Function and mechanism investigations primarily employed testicular coefficient and sperm concentration, H&E staining, immunofluorescence staining, and western blot analysis. Single-cell RNA-sequencing (scRNA-seq) predominantly examined the impacts of TP and ICA on various cell populations within the testis at a single-cell resolution. Results: We successfully established a TP-induced model of testicular injury and identified ICA as a protective agent in alleviating testicular vacuolization. Moreover, we delineated a comprehensive single-cell transcriptome profile of ICA in the repair of testicular injury, revealing the pivotal role of Sertoli-germline communications in the genesis of testicular vacuolization and the reparative process mediated by ICA. Furthermore, our investigation unveiled that ICA mitigated TP-induced damage to niche integrity through signatures associated with the blood-testis barrier (BTB), thereby averting substantial germ cell loss via the germline associated-ferroptosis signatures, potentially a key factor in the occurrence of testicular vacuolization injury. Additionally, we also identified ferroptosis-related molecules for testicular vacuolization injury. Discussion: We suggest that TP-induced testicular injury disrupts the spermatogenic microenvironment mediated by the BTB, leading to the formation of testicular vacuoles through the ferroptosis pathway in germ cells. Our findings offer fresh perspectives for ICA on mitigating this process.