Huyang yangkun formula protects against 4-Vinylcyclohexene diepoxide-induced premature ovarian insufficiency in rats via the Hippo–JAK2/STAT3 signaling pathway

Huyang Yangkun Formula (HYF) has been prescribed for premature ovarian insufficiency (POI) for decades in the clinical setting. Little is known regarding its underlying molecular mechanism. This study was conducted to elucidate the possible mechanism of the protective potential of HYF against POI induced by the industrial chemical 4-vinylcyclohexene diepoxide (VCD) in rats. Quality control of HYF was conducted via HPLC and UPLC-MS. Female rats were injected with VCD (160 mg/kg) daily for 15 days. Then, 1.35 g/kg (low dose) or 0.235 g/kg (high dose) HYF was administered once/day for 25 days. Serum AMH, FSH, E2, ALT, AST, BUN and Cr levels were detected through ELISA and HE-stained follicles were counted in ovarian sections. Additionally, RNA-seq profiling analysis and functional assays were used to screen for differentially expressed genes and key regulators with potentially important roles associated with HYF. The ovaries of POI rats contained fewer antral and maturing follicles (p < 0.05) than those of control rats, whereas atretic follicles were increased significantly (p < 0.05), and AMH levels were significantly lower in the VCD group than in the control group (p < 0.05). These conditions showed some improvement after low- and high-dose HYF treatment. Low- and high-dose HYF increased AMH levels by 42.4% and 25.9% and decreased FSH levels by 17.5% and 24.1%, respectively, in comparison to the VCD group. The two HYF dosage groups showed significantly increased numbers of antral and maturing follicles but a reduced number of atretic follicles (p < 0.05). HYF down-regulation of JAK, Lats2 and YAP mRNA expression gene expression (p < 0.05) compared with the VCD group. HYF resulted in a strongly attenuated VCD-induced phosphorylation of JAK2 and STAT3 (p < 0.01) and YAP (p < 0.001), but induced an increase in protein levels of LATS2 (p < 0.05). Our findings demonstrated the treatment efficacy of HYF in POI rats and showed that HYF repairs the dysfunction and enhances the ovarian function of POI rats through the Hippo–JAK2/STAT3 signaling pathway.