Mechanism of Xiao-ai-fei Honey Ointment, a Traditional Uyghur Multi-Ingredient Medicinal Preparation, Against Cervical Cancer Based on Network Pharmacology and In Vitro Evaluation of Anti-Cancer Activity

Background/Objectives: Cervical cancer, primarily driven by persistent high-risk HPV infection, remains a major global health issue. Xiao-ai-fei honey ointment, a traditional Uyghur multi-ingredient preparation, has shown clinical promise in cancer treatment, but its mechanisms against cervical cancer are not fully understood. This study aimed to investigate the potential molecular mechanisms of ethanolic extract of Xiao-ai-fei honey ointment (XAFHO) in cervical cancer using network pharmacology, single-cell RNA sequencing, and experimental validation. Methods: Differentially expressed genes (DEGs) in cervical cancer were identified from TCGA database. Active components and corresponding targets of XAFHO were retrieved from the TCMSP database, and disease targets were obtained from GeneCard, OMIM, and the TTD. Intersection targets were subjected to multivariate Cox and LASSO regression to construct a prognostic model. Immune infiltration, TMB, and MSI were compared between risk groups. Single-cell RNA-seq data were analyzed to determine cellular origins and inter-cellular communication. In vitro assays were performed on HeLa and SiHa cells to assess the anti-cancer activity of XAFHO. Molecular docking evaluated binding affinities between active compounds and core targets. The expression and functional roles of FASN and SPP1 were further validated by RT-qPCR, Western blotting, and siRNA transfection. Results: Sixty-three potential XAFHO targets were identified, and an 11-gene prognostic model was established, effectively stratifying patients into high- and low-risk groups with significantly different overall survival (AUC > 0.7). The high-risk group exhibited an immunosuppressive microenvironment and higher TMB. Single-cell analysis revealed that FASN and ACACA were predominantly expressed in tumor cells, while SPP1 was enriched in macrophages/monocytes. Tumor cells communicated with immune cells via the TGFB1–TGFβR1/R2 axis, promoting immune evasion. In vitro, XAFHO significantly inhibited proliferation, colony formation, migration, and invasion of cervical cancer cells. Molecular docking confirmed the strong binding of quercetin, kaempferol, and isorhamnetin to FASN and SPP1 (binding energy < –6.0 kcal/mol). Functional validation indicated that upregulated FASN and SPP1 contribute to malignant behaviors in cervical cancer cells. Conclusions: This study integrates network pharmacology with single-cell and experimental approaches to demonstrate that XAFHO exerts multi-target and multi-cell anti-cervical cancer effects, potentially by modulating lipid metabolism and immune-related pathways via FASN and SPP1. These findings provide a scientific basis for the therapeutic application of XAFHO in cervical cancer.