Riboflavin alleviates fluoride-induced ferroptosis by IL-17A-independent system Xc−/GPX4 pathway and iron metabolism in testicular Leydig cells

Fluoride is widely found in groundwater, soil, animal and plant organisms. Excessive fluoride exposure can cause reproductive dysfunction by activating IL-17A signaling pathway. However, the adverse effects of fluoride on male reproductive system and the mechanisms remain elusive. In this study, the wild type and IL-17A knockout C57BL/6J mouse were treated with 24 mg/kg·bw·d sodium fluoride and/or 5 mg/kg·bw·d riboflavin-5′-phosphate sodium for 91 days. Results showed that fluoride caused dental fluorosis, increased the levels of ROS in testicular Leydig cells and GSSG in testicular tissue, and did not affect the iron and serum hepcidin levels in testicular tissue. Riboflavin alleviated above adverse changes, whereas IL-17A does not participate in the oxidative stress-mediated reproductive toxicity of fluoride. Based on this, TM3 cells were used to verify the injury of fluoride on Leydig cells. Results showed that fluoride increased mRNA levels of ferroptosis marker SLC3A2, VDAC3, TFRC, and SLC40A1 and decreased Nrf2 mRNA levels in TM3 cells. The ferroptosis inhibitor Lip-1 and DFO were used to further investigate the relationship between male reproductive toxicity and ferroptosis induced by fluoride. We found that the fluoride-induced decrease in cell viability, increase in xCT, TFRC, and FTH protein expression, and decrease in GPX4 protein expression, can all be rescued by Lip-1 and DFO. Similar results were also observed in the riboflavin treatment group. Moreover, riboflavin mitigated fluoride-induced increases in ROS levels and SLC3A2 protein levels. In all, our work revealed that riboflavin inhibited ferroptosis in testicular Leydig cells and improved the declined male reproductive function caused by fluoride. This study provides new perspectives for revealing new male reproductive toxicity mechanisms and mitigating fluoride toxicity damage.