Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway

Cyclophosphamide (CTX) is widely used as a first-line chemotherapeutic agent for various cancers, but it is associated with significant risks of ovarian dysfunction, even progressing to premature ovarian failure (POF), with granulosa cell senescence being a key phenotypic manifestation. hydroxychloroquine (HCQ) has demonstrated anti-senescence properties in the context of age-related diseases, but its efficacy in preventing CTX-induced ovarian damage has not been fully elucidated. We conducted a controlled animal study using a CTX-induced POF model in female C57BL/6 mice, comprising three groups: vehicle control, CTX-only, and CTX + HCQ. Complementary in vitro studies were conducted using the human ovarian granulosa cell line KGN, treated with phosphoramide mustard (PM, the active metabolite of CTX) and HCQ. HCQ partially reversed the CTX-induced decrease in ovarian index, normalized the estrous cycle, reduced follicle depletion, and improved serum hormone levels as well as reproductive outcomes. HCQ reduced the expression of granulosa cell senescence markers and the area of senescence-associated β-galactosidase(SA-β-gal)-positive cells, stabilized mitochondrial membrane potential, decreased reactive oxygen species(ROS) production and mitochondrial DNA(mtDNA) leakage, inhibited cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and suppressed senescence-associated secretory phenotype(SASP) factors. Collectively, HCQ protects CTX-induced POF by inhibiting activation of the mtDNA-cGAS signaling pathway, thereby alleviating granulosa cell senescence and preserving ovarian function.