Jinfeng Pills alleviate letrozole-induced PCOS by correcting CYP17A1/CYP19A1 steroidogenic imbalance and PI3K/Akt/mTOR-related signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinfeng Pills (JFP) is a classical Chinese patent medicine originating as an imperial court prescription during the Qianlong reign of the Qing dynasty (1735-1796), in continuous use in southern China and Southeast Asia for over two centuries (NMPA approval No. Z20064400). Comprising nine ingredients - Panax ginseng C.A.Mey., Cervus elaphus Linnaeus, Ligustrum lucidum W.T.Aiton, Epimedium brevicornu Maxim., Reynoutria multiflora (Thunb.) Moldenke, Leonurus japonicus Houtt., Cinnamomum cassia (L.) J.Presl, Equus asinus Linnaeus, and Curculigo orchioides Gaertn. - formulated as a honey-based pill and traditionally indicated for kidney-yang deficiency with blood stasis, its clinical presentations of oligomenorrhea and anovulatory infertility closely mirror PCOS hallmarks, yet its multi-constituent pharmacological mechanisms remain unresolved. AIM OF THE STUDY: This study aimed to evaluate the effects of JFP in a letrozole-induced PCOS rat model and explore associated molecular pathways using network pharmacology and multi-layer molecular profiling. MATERIALS AND METHODS: PCOS was induced in SD rats via letrozole, followed by 21-day JFP treatment (160, 240, 320 mg/kg) or metformin. Estrous cyclicity, serum hormones, and ovarian morphology were evaluated. Network pharmacology, ovarian proteomics, qPCR, Western blot, serum metabolomics, and 16S rRNA gut microbiota profiling were performed as complementary analyses. RESULTS: Compared with the model group, JFP at 320 mg/kg, the most effective dose among the tested doses, significantly reduced testosterone and LH, restored estradiol levels, and improved the LH/FSH ratio mainly through LH suppression, while FSH levels remained unchanged. JFP also increased corpus luteum number (P = 0.0020) and reduced atretic follicles (P = 0.0152), with no detectable hepatorenal toxicity. Network pharmacology and multi-layer molecular profiling, supported by qPCR and Western blot validation, suggested that JFP-associated improvements in reproductive endocrine disturbance and follicular developmental abnormalities were accompanied by changes in CYP17A1/CYP19A1-mediated steroidogenesis, PI3K/Akt/mTOR-related signaling, and IL-17A/COX2- and MAPK/ERK-p53-associated inflammatory-apoptotic pathways. CONCLUSION: JFP improved endocrine and ovarian dysfunction in letrozole-induced PCOS rats, with its effects associated with modulation of CYP17A1/CYP19A1-mediated steroidogenesis, PI3K/Akt/mTOR-related signaling, and inflammatory-apoptotic pathways. These findings provide preclinical evidence and mechanistic clues for further investigation of JFP in PCOS-related reproductive endocrine disturbance and ovarian histomorphological abnormalities, while direct ovulation and fertility outcomes require further validation.