Hydrogen sulfide and ferroptosis inhibition underlies the dietary restriction-induced protection against cyclophosphamide cystitis

Dietary restriction (DR) has emerged as a potential therapeutic intervention for various pathological conditions. This study investigated the effects of DR on cyclophosphamide-induced cystitis in mice. Animals were subjected to controlled food restriction for 1 week prior to cyclophosphamide administration. We evaluated changes in body weight, bladder pathology, redox status, and ferroptotic parameters. DR significantly attenuated cyclophosphamide-induced cystitis severity, as evidenced by reduced bladder weight, decreased lipid peroxidation, and diminished ferroptotic markers in bladder tissue. Mechanistic investigations revealed that DR upregulated hepatic hydrogen sulfide (H 2 S)-synthesizing enzymes and enhanced H 2 S production. Inhibition of H 2 S-synthesizing enzymes with DL-propargylglycine (PAG) and aminooxyacetic acid (AOAA) exacerbated cyclophosphamide-induced cystitis, whereas administration of diallyl trisulfide (DATS), an H 2 S donor, markedly ameliorated bladder pathology. In vitro studies demonstrated that H 2 S donors, NaHS and DATS, protected against cyclophosphamide metabolite acrolein (ACR)-induced urothelial cell death by suppressing oxidative stress, as indicated by reduced p38 MAPK activation and protein carbonylation. These findings suggest that DR confers protection against cyclophosphamide-induced cystitis through the induction of endogenous H 2 S production and inhibition of ferroptosis. Our study provides additional evidence supporting the health-promoting effects of DR as well as novel mechanistic insights into the beneficial effects of DR. Given H 2 S has anti-inflammatory and anti-oxidative properties and that oxidative stress and ferroptosis underlie various diseases, our finding could have broader implications.