BackgroundPremature ovarian failure (POF) is defined as the cessation of ovarian function before the age of 40 years, imposing a significant health burden on patients. However, effective etiological therapy for POF is scarce. Thus, we aimed to explore the protective role and targets of hydrogen-rich water (HRW) in POF.MethodsBased on cyclophosphamide (CTX)-induced POF rat models, the protective role of HRW treatment was mainly determined through serum 17-β-estradiol (E2), follicle-stimulating hormone (FSH), anti-mullerian hormone (AMH) levels, ovarian histomorphological analysis, and TUNEL assay. Tandem mass tag (TMT)-based quantitative proteomic analysis was then conducted on ovarian tissues, and the targets of HRW in POF were identified integrating differential expression analysis, functional enrichment analysis, and interaction analysis.ResultsIn HRW treatment of POF rats, the serum AMH and E2 levels significantly increased, and FSH level significantly reduced, indicating the protective role of HRW. After TMT quantitative proteomic analysis, a total of 16 candidate differentially expressed proteins (DEPs) were identified after the cross analysis of DEPs from POF vs. control and POF+HRW vs. POF groups, which were found to be significantly enriched in 296 GO terms and 36 KEGG pathways. The crucial targets, RT1-Db1 and RT1-Bb, were finally identified based on both protein-protein interaction network and GeneMANIA network.ConclusionsThe HRW treatment could significantly alleviate the ovarian injury of POF rats; RT1-Db1 and RT1-Bb are identified as two crucial targets of HRW treatment in POF rats.