Integrating network pharmacology, genetically informed multi-omics and granulosa-cell experiments identifies dauricine and oleanolic acid as candidate CASP3-modulating compounds of Zhi-Bai-Di-Huang-Wan in female pubertal timing

Abstract

Zhi-Bai-Di-Huang-Wan (ZBDHW) is a traditional Chinese medicine formula clinically used for precocious puberty, but its molecular basis remains unclear. Earlier age at menarche (AAM) reflects advanced female pubertal timing and provides a genetically tractable endpoint relevant to precocious puberty. This study aimed to identify ZBDHW-associated molecular targets relevant to pubertal timing by integrating target prediction, Mendelian randomization-based methylation-expression-protein analyses, Bayesian colocalization, network analysis, and granulosa-cell experiments. ZBDHW ingredients and candidate targets were retrieved from BATMAN-TCM 2.0. Summary data-based Mendelian randomization and Bayesian colocalization integrated methylation, expression, and protein quantitative trait loci with the largest European AAM genome-wide association study. Protein-protein interaction analysis assessed network connectivity. KGN human granulosa cells were treated with dauricine or oleanolic acid, and CASP3 mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction. CASP3 was the only ZBDHW-associated target showing convergent evidence across gene expression and protein abundance levels, with supportive methylation evidence at cg06796944. Higher genetically predicted CASP3 expression and protein abundance were associated with later AAM, and CASP3 was a central node in the ZBDHW-related protein-protein interaction network. In KGN cells, dauricine increased CASP3 mRNA at 40 and 80 µM, whereas oleanolic acid increased CASP3 mRNA at 200 µM. These findings support a CASP3-centered methylation-expression-protein target axis as a candidate mechanism linking ZBDHW-associated compounds to female pubertal timing, providing genetically informed multi-omics evidence and preliminary granulosa-cell support relevant to the adjunctive use of ZBDHW in precocious puberty.