Jiawei Qingxin Zishen Decoction mitigates granulosa cell apoptosis and enhances ovarian reserve through Netrin/UNC5B signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Diminished ovarian reserve (DOR) is a leading cause of female infertility, characterized by a decline in ovarian function and follicular pool. Jiawei Qingxin Zishen Decoction (JWQZD) is a modernized formulation rooted in the classical theory of 'Heart-Kidney Interaction' from Fu Qingzhu's Gynecology. It is derived from the empirical formula Qingxin Zishen Decoction (QZD), which was traditionally used for perimenopausal syndrome. JWQZD has been specifically optimized to tonify the kidney, clear heart fire, and regulate the heart-kidney-uterus axis, thereby addressing the modern clinical need for treating infertility associated with DOR. It has been clinically used to improve ovarian function and menstrual regularity in women with DOR-related infertility. However, the precise mechanisms through which JWQZD exerts its therapeutic effects remain incompletely elucidated.

AIM OF THE STUDY: To reveal the mechanism by which JWQZD mitigates granulosa cell apoptosis through the Netrin/UNC5B signaling pathway and thereby alleviates DOR.

MATERIALS AND

METHODS

Components of JWQZD were analyzed using UPLC-Q-Exactive Plus tandem mass spectrometry. Network pharmacology identified core targets and pathways via HERB, DisGeNET, and the HPA databases. For in vivo validation, a cyclophosphamide-induced DOR rat model was used (n = 6 per group). Groups included Control, Model, JWQZD-low (16 g/kg), and JWQZD-high (32 g/kg). Hormone levels (AMH, E2, FSH, LH), apoptosis (TUNEL, Bcl-2, Bax, and cleaved caspase-3), and pathway proteins (Netrin/UNC5B, PI3K/AKT/mTOR) were assessed.

RESULTS

Chemical analysis identified 316 components in JWQZD, with network pharmacology revealing 10 core targets (including TP53) and significant enrichment of the Netrin/UNC5B pathway. In vivo studies demonstrated that JWQZD treatment exerted significant therapeutic effects: serum AMH levels increased from 2.35 ± 1.00 to 7.12 ± 2.28 ng/mL (P < 0.01) and E2 levels rose from 40.44 ± 7.06 to 85.83 ± 18.28 pg/mL (P < 0.01), while simultaneously decreasing FSH (50.74 ± 6.12 to 19.00 ± 5.36 mIU/mL, P < 0.01) and LH levels (33.07 ± 4.86 to 13.89 ± 4.44 mIU/mL, P < 0.01). JWQZD treatment reduced granulosa cell apoptosis by approximately 40 % (P < 0.01) and elevated the Bcl-2/Bax ratio 2.1-fold (P < 0.001). Mechanistically, JWQZD activated Netrin/UNC5B signaling, leading to a 65 % reduction in p53 expression (P < 0.001) and a 3.2-fold increase in AKT phosphorylation (P < 0.001), indicating potent stimulation of the PI3K/AKT/mTOR pathway.

CONCLUSION

This pioneering study established that JWQZD mitigates granulosa cell apoptosis and enhances ovarian reserve by activating the Netrin/UNC5B signaling pathway, offering a solid pharmacological foundation for the practical use of JWQZD in clinical settings.