The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency

Background Premature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. Ningxin-Tongyu-Zishen formula (NTZF), developed based on the "simultaneous heart-kidney treatment" principle, demonstrates efficacy in treating POI, potentially through regulating proBDNF/mBDNF balance. This study aimed to elucidate the molecular mechanism by which NTZF treats POI via proBDNF/mBDNF modulation. Methods POI rat models were established using cyclophosphamide (CTX). The therapeutic effects of NTZF were evaluated by analyzing estrous cycles, ovarian indices, follicular development, serum sex hormone levels (FSH, E ₂ , AMH), and ovarian granulosa cells (OGCs) apoptosis. Following immunofluorescence staining to localize BDNF receptors, proBDNF/mBDNF protein expression was quantified in brain and ovarian tissues. CTX's active metabolite, Phosphoramide mustard (PM), was used to induce KGN cell damage. The regulatory effect of NTZF on proBDNF/mBDNF was investigated and compared with recombinant mBDNF protein. tPA and PAI-1 was screened, and their interactions with NTZF were analyzed. mRNA and protein expression of tPA, PAI-1, and tPA-PAI-1 complexes were assessed via q-PCR and Western Blot. Results NTZF composition was characterized and shown to improve ovarian function in POI rats. Its mechanism involves correcting proBDNF/mBDNF imbalance in both brain and ovarian tissues. NTZF achieved this correction through the PAI-1/tPA signaling pathway, thereby inhibiting apoptosis in damaged KGN cells. Conclusion Our findings demonstrate that NTZF inhibits PAI-1, reduces tPA-PAI-1 complex synthesis, and enhances tPA-mediated proteolytic conversion of proBDNF to mBDNF. This restores proBDNF/mBDNF balance, suppresses OGCs apoptosis, and ultimately ameliorates POI.