ETHNOPHARMACOLOGICAL RELEVANCE: Qilin Pills (QLP) is a classic proprietary Chinese medicine derived from Wuziyanzong Pills, a traditional formula originating from the Tang Dynasty. It serves to tonify the kidneys and replenish essence, and is used to enhance fertility. QLP is widely used to treat diminished ovarian reserve (DOR), a condition characterised by ovulatory dysfunction and reduced fertility. However, its mechanism of action remains unclear.
AIM OF THE STUDY: To investigate the protective effects of QLP against DOR and elucidated the underlying mechanisms.
MATERIALS AND METHODS: We established a DOR mouse model by cyclophosphamide (CTX) and concurrently administered oral QLP at low, medium, and high doses, with oestradiol valerate (EV) as the positive control. We evaluated the ovarian function by measuring ovarian index, follicular count, and serum anti-Müllerian hormone (AMH). UHPLC-QTOF-MS and network pharmacology techniques were used to identify therapeutic targets. KGN cells were treated with 4-hydroperoxy cyclophosphamide (4-HC) to mimic in vitro chemotherapy toxicity. Mitochondrial function was assessed by Seahorse XF analysis, JC-1 and MitoSOX Red staining, DHE fluorescence, mtDNA quantification, and transmission electron microscopy. Key gene in PINK1/Parkin related mitophagy were detected by western blotting and immunofluorescence in both ovarian tissue and KGN cells. PINK1 overexpression validated this mechanism. 2
RESULTS: In vivo, QLP increased the ovarian reserve of DOR mice by enhancing growing follicles and AMH levels while reducing atrophic follicles and GC apoptosis, with the medium dose showing superior effects to EV. Network pharmacology analysis of compounds identified by UHPLC-QTOF-MS revealed 166 targets enriched in mitochondrial bioenergetic metabolism and mitophagy pathways. QLP mitigated mitochondrial dysfunction, oxidative stress, and excessive mitophagy in GC. In vitro, QLP medicated serum restored mitochondrial respiratory function and membrane potential while reducing ROS. Mechanistically, CTX/4-HC upregulated PINK1/Parkin and induced excessive mitophagy. Notably, QLP exhibited a non-monotonic dose-response: the medium dose optimally rebalanced this overactivation to protect GC, whereas the high dose appeared to induce mild cellular stress, triggering a compensatory rebound in mitophagy. PINK1 overexpression abrogated QLP's protection, confirming PINK1/Parkin inhibition as essential for its therapeutic action. 2
CONCLUSION: QLP prevents DOR by rebalancing the pathological overactivation of PINK1/Parkin-mediated mitophagy, thereby restoring mitochondrial homeostasis. These findings provide evidence for QLP in protecting ovarian function.
