Pro-Oxidant Anthocyanins-Enriched Fraction Inhibits Androgen Synthesis by Transcriptional Repression of Cyp17a1 Through Nr0b2

Anthocyanins are plant polyphenols widely regarded as antioxidants, yet they can exert concentration-dependent effects and act as pro-oxidants in specific contexts. Although their protective role in stressed testicular cells is established, their impact on Leydig cell steroidogenesis under non-pathological conditions remains poorly understood. Here, we investigated how an anthocyanin-enriched fraction from Callistemon citrinus (0–1.00 μg/mL) affects androgen synthesis in murine TM3 Leydig cells. Cell viability, intracellular ROS, antioxidant capacity, mitochondrial function, androstenedione production, steroidogenic gene expression, and the exometabolome by 1H-NMR were assessed. The fraction exhibited biphasic, dose-dependent effects. At 0.01 μg/mL, it induced a mitohormetic response, upregulating mitochondrial complexes III and V. Conversely, higher concentrations (0.10–1.00 μg/mL) reduced metabolic activity, increased intracellular ROS, and significantly suppressed androstenedione synthesis independently of Star. These concentrations also induced dose-dependent repression of Cyp17a1, concomitant with upregulation of Nr0b2, encoding the transcriptional repressor Small Heterodimer Partner (SHP). Overall, the data support a redox-dependent mechanism whereby elevated ROS promotes Nr0b2 expression, leading to Cyp17a1 suppression and impaired androstenedione production. These findings challenge the view of anthocyanins as uniformly beneficial for male fertility and identify Callistemon citrinus as a sustainable source of bioactive anthocyanins capable of modulating redox–endocrine homeostasis in a dose-dependent manner under basal conditions.