Zishen Qingre Lishi Huayu recipe improves polycystic ovary syndrome by restoring granulosa cell homeostasis via C/EBPβ/STAT3 signaling.

UNLABELLED: Polycystic ovary syndrome (PCOS) is a multifactorial endocrine disorder characterized by ovulation disorders. Zishen Qingre Lishi Huayu Recipe (ZQLHR), a traditional Chinese medicine (TCM) formula, has shown clinical efficacy by effectively increasing the ovulation rate and menstrual cycle regularity in patients with PCOS and is widely used in practice; however, its underlying pharmacological mechanism has not been fully elucidated.

AIM OF THE STUDY: Aim of the study: This study aimed to elucidate how ZQLHR modulates the proliferation-apoptosis balance of ovarian granulosa cells (GCs) in PCOS via the C/EBPβ/STAT3 signaling axis.

METHODS

Human granulosa cells (hGCs) from PCOS and non-PCOS patients were analyzed for proliferation, apoptosis, and C/EBPβ/STAT3 expression. In vivo, letrozole-induced PCOS mice were subjected to scAAV-mediated C/EBPβ knockdown, followed by administration of ZQLHR to evaluate treatment outcomes. In vitro, C/EBPβ or STAT3 was silenced in Human granulosa-like tumor cell line (KGN) cells to explore their regulatory relationship and roles in proliferation and apoptosis. Additionally, ZQLHR effects were evaluated in a dose- and time-dependent manner. Notably, CETSA was employed to confirm for the first time direct binding of ZQLHR to C/EBPβ, and C/EBPβ-silenced cells were subsequently treated with ZQLHR to examine whether its effects involve C/EBPβ-dependent STAT3. The pharmacological mechanism of ZQLHR was further evaluated through histological and molecular biology experiments.

RESULTS

PCOS patient-derived GCs exhibited reduced proliferation, elevated apoptosis, and decreased C/EBPβ/STAT3 expression. In LET-induced PCOS mice, ZQLHR treatment significantly improved estrous cycle regularity, hormonal balance, follicular development, and GC apoptosis, accompanied by modulation of the C/EBPβ/STAT3 pathway. C/EBPβ knockdown exacerbated ovarian dysfunction and KGN apoptosis, whereas ZQLHR attenuated these effects and partially restored pathway activity. In vitro, C/EBPβ and STAT3 influenced each other and CFTR expression, associated with changes in proliferation and apoptosis. ZQLHR increased proliferation markers (PCNA, cyclin D1) and decreased apoptosis markers (Caspase-3, PARP) in a dose- and time-dependent manner, with CETSA confirming direct binding to C/EBPβ. Notably, despite C/EBPβ knockdown, ZQLHR partially restored KGN cell proliferation and modestly modulated the C/EBPβ/STAT3 signaling pathway.

CONCLUSION

ZQLHR improves GC homeostasis and promotes follicular development in PCOS by modulating the C/EBPβ/STAT3 pathway.