The up-regulation of RIPK3 mediated by ac4C modification promotes oxidative stress-induced granulosa cell senescence by inhibiting the Nrf2/HO-1 pathway.

Abnormality of granulosa cells (GCs) is the critical cause of follicular atresia in premature ovarian failure (POF). RIPK3 is highly expressed in GCs derived from atretic follicles. We focus on uncovering how RIPK3 contributes to ovarian GC senescence. Primary GCs were treated with H₂O₂ to induce senescence. ROS was detected via DCFH-DA staining. Levels of senescence-related molecules and SA-β-Gal activity were examined. Cyclophosphamide was administered to mice to induce POF. The impact of RIPK3 on atretic follicles and sex hormones was evaluated through HE staining and ELISA, respectively. The acRIP-qPCR analysis of RIPK3 ac4C levels, RIP detection for interaction between RIPK3 and NAT10, and actinomycin D treatment to detect RIPK3 degradation were conducted. In HO-treated GCs and POF mouse ovaries, levels of RIPK3, ROS, senescence-related molecules, as well as SA-β-Gal activity, were all up-regulated, and this effect was suppressed by RIPK3 inhibition. RIPK3 interference reduced atretic follicles and FSH levels while increasing AMH and E2 levels. Nrf2 and HO-1 content were diminished in the models, whereas si-RIPK3 facilitated their expression. The effect of si-RIPK3 on decreased levels of ROS and senescence-related molecules was reversed by ML385. HO decreased RIPK3 mRNA degradation and increased its ac4C modification. The ac4C modifying enzyme NAT10 was up-regulated in the models, and NAT10 enhanced RIPK3 mRNA stability through ac4C modification. NAT10 knockdown mitigated ovarian GC senescence by inhibiting RIPK3 expression. The promotion of RIPK3 mRNA stability through ac4C modification by NAT10, in turn, affects the Nrf2/HO-1 pathway and promotes ovarian GC senescence.