Identification of CA2 as a Potential Candidate Gene Involved in the Therapeutic Effect of Huatan Huoxue Decoction on Polycystic Ovary Syndrome: An Integrated Study of Network Pharmacology, Mendelian Randomization, and Experimental Validation

Purpose: Huatan Huoxue decoction (HTHXD) is effective in treating polycystic ovary syndrome (PCOS); however, its underlying mechanisms of action remain unclear. Methods: Gene targets were obtained through Mendelian randomization (MR) analysis using druggable genes from public databases and genome-wide association study data for PCOS from the FinnGen R12 database. These targets were integrated with the active ingredients and protein targets of HTHXD retrieved from the TCMSP and BATMAN-TCM databases for network pharmacology analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted. Core targets were validated using molecular docking. Experimental validation was performed using a letrozole-induced PCOS rat model treated with HTHXD, with key targets assessed via quantitative PCR (qPCR) and immunohistochemical staining. Results: MR analysis identified 131 proteins causally related to PCOS, of which 52 with strong associations remained after quality control. Network pharmacology analysis revealed 549 gene targets and 243 active ingredients in HTHXD. The intersection of these datasets identified four core targets for HTHXD in PCOS treatment (CA2, CASP3, SNCA, and TSPO). Molecular docking identified tanshindiol B, 4-methylenemiltirone, and danshenol B in Danshen and crocetin in leech as key active components. HTHXD substantially ameliorated polycystic ovarian morphology and reduced corpora lutea in PCOS model rats. CA2 expression was significantly upregulated in PCOS ovarian tissue (P < 0.0001), and markedly reduced after HTHXD treatment, consistent with immunohistochemical findings. Conclusion: CA2 may serve as a potential candidate gene associated with the therapeutic response to HTHXD in PCOS, with tanshindiol B and crocetin as major active components.