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 folliclestimulating
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.