He’s Yangchao recipe ameliorates premature ovarian insufficiency by remodeling intestinal microbiota to promote granulosa cell glycolysis

Abstract

Objective: To investigate the molecular mechanism by which He’s Yangchao recipe improves ovarian function in premature ovarian insufficiency (POI) mice through intestinal flora modulation. Methods: Forty female ICR mice (aged 6–8 weeks) were intraperitoneally injected with cyclophosphamide (150 mg/kg) to establish a POI model, while 10 untreated mice served as the blank control. The cyclophosphamide-injected mice were randomly divided into four groups with 10 mice in each group: low-dose He’s Yangchao recipe (crude herb, 6 g/kg), high-dose He’s Yangchao recipe (crude herb, 25 g/kg), estradiol (positive control), and model control (distilled water). Treatments were administered daily by gavage for 6 weeks. Vaginal exfoliated cells were stained with Wright-Giemsa solution to monitor estrous cycles. Serum estradiol and follicle-stimulating hormone (FSH) levels were measured by enzyme-linked immunosorbent assay. Ovarian FSH receptor (FSHR) expression was assessed by immunohistochemistry. Fecal samples from blank control, model control, and high-dose groups underwent metagenomic sequencing to evaluate intestinal microbiota diversity and composition. Glycolysis-related proteins pyruvate kinase M2 (PKM2) and glucose transporter 4 (GLUT4) were analyzed by Western blotting and immunofluorescence. Results: He’s Yangchao recipe restored estrous cyclicity, increased serum estradiol (P<0.05), decreased serum FSH (P<0.05), and upregulated FSHR expression in granulosa cells (P<0.05). Metagenomic analysis revealed significant structural differences in intestinal flora among blank control, model control, and high-dose groups (P<0.05). The high-dose group showed reduced abundance of opportunistic pathogens (e.g., Alistipes, Prevotella, Odoribacter, Blautia, Rikenella) compared to the model control (all P<0.05). Functional enrichment analysis indicated involvement of glycolysis-related pathways. In the model control, PKM2 expression was downregulated (P<0.05), and GLUT4 expression showed a decreasing trend. Both of them were upregulated in the high-dose He’s Yangchao recipe group (both P<0.05). Conclusions: He’s Yangchao recipe ameliorates POI in mice by remodeling intestinal flora structure, enhancing glycolytic activity, improving ovarian sex hormone secretion, increasing granulosa cell FSHR expression, and restoring estrous cyclicity.