Mechanistic Insights into the Therapeutic Effects of Zishen Yutai Pillon Premature Ovarian Insufficiency via the RIPK1/RIPK3/MLKLNecroptosis Pathway

INTRODUCTION/OBJECTIVE: Zishen Yutai Pill (ZYP) has shown clinical efficacy in the treatment of Premature Ovarian Insufficiency (POI). However, its underlying mechanisms remain unclear. This study aimed to investigate the therapeutic effects and underlying mechanism of ZYP in cyclophosphamide (CTX)-induced POI in rats. METHODS: A total of 119 female rats were divided into two groups: blank controls (n=17) and POI rat model (n=102). POI rats were randomly assigned (n = 17 per group) to six groups: POI model controls or five treatments, including low-dose ZYP, high-dose ZYP, caspase inhibitor, RIPK1 inhibitor, or estradiol valerate (positive control). Sex hormone levels of follicle-stimulating hormone (FSH), estradiol (E2), and Anti-Müllerian Hormone (AMH) were assessed by enzyme-linked immunosorbent assay. Ovarian morphology was assessed by Hematoxylin and Eosin (H&E) staining and Transmission Electron Microscopy (TEM). The expression of apoptosis- and necroptosis-related proteins, including caspase-3, caspase-8, tumor necrosis factor-alpha (TNF-α), RIPK1, RIPK3, and MLKL, was assessed by immunohistochemistry, western blot (WB), and immunofluorescence (IF). RESULTS: ZYP improved general condition and stabilized body weight in POI rats. It significantly decreased FSH while increasing E2 and AMH (p<0.001). High-dose ZYP increased primary (p<0.05) and antral follicles (p<0.001), and reduced atretic follicles (p<0.001). Ultrastructural damage was alleviated. ZYP downregulated apoptosis- and necroptosis-related proteins (caspase- 8, caspase-3, TNF-α, RIPK1, RIPK3, MLKL) (p<0.001), comparable to the RIPK1 inhibitor. WB and IF further showed that ZYP high-dose group significantly downregulated RIPK3 and MLKL expression (p<0.001). DISCUSSION: These findings provide mechanistic insight into the protective role of ZYP and support its potential as a therapeutic strategy for POI. CONCLUSION: ZYP ameliorates CTX-induced POI by regulating hormones, improving ovarian morphology, and inhibiting the RIPK1/RIPK3/MLKL pathway.