Abstract
BACKGROUND: The development and progression of bladder cancer are closely linked to its complex tumor microenvironment. Hedyotis diffusa and Scutellaria barbata (HD-SB) are commonly used as a prominent herbal pair for treating bladder cancer. However, the pharmacological targets and molecular mechanisms by which HD-SB impacts bladder cancer require further elucidation. Additionally, it remains uncertain whether this herbal pair affects the prognosis and treatment efficacy of bladder cancer. METHODS: We employed network pharmacology to predict the targets of HD-SB and bladder cancer, identifying overlaps with prognostic genes linked to the overall survival of bladder cancer patients in the TCGA dataset. Subsequently, we utilized least absolute shrinkage and selection operator (LASSO) and Cox regression analyses to pinpoint a prognostic signature and construct a prognostic model. We further explored the correlations between risk scores, immune cells, immune checkpoint genes, and treatment efficacy. Single-cell RNA-sequencing (scRNA-seq) was used to profile the expression of prognostic genes across various cell types, and immunohistochemistry validated the protein levels of these targets. Molecular docking studies were conducted to clarify the interactions between HD-SB components and the identified genes, and in vitro experiments demonstrated the effects of HD-SB on bladder cancer cells. RESULTS: Venn diagram analysis identified 497 common targets shared between HD-SB and bladder cancer. LASSO and Cox regression identified a 15-gene prognostic signature, including VEGFA, EGFR, MYC, PDGFRA, JUN, FN1, PTPN6, PTGER3, MAP2, CALM1, CTSV, CES1, ADRA1D, PYGL, and PLA2G1B. Kaplan-Meier analysis showed better overall survival in the low-risk group (median 19.8 months) versus the high-risk group (median 15.9 months). Linear regression analysis revealed a significant correlation between risk scores and specific immune cell types, as well as the dysregulated expression of immune checkpoint genes across different groups. The prognostic gene-based risk score was also found to correlate with the efficacy of both immunotherapy and chemotherapy. Six key targets—VEGFA, MYC, JUN, FN1, PTPN6, and CALM1—were validated through scRNA-seq and immunohistochemistry. Molecular docking analysis demonstrated that components of HD-SB bind with high affinity to these signature targets. In vitro experiments showed that HD-SB effectively inhibited bladder cancer cell viability, colony formation, and migration. CONCLUSION: This is the first study to explore the potential of HD-SB in enhancing the prognosis and treatment outcomes of bladder cancer through network pharmacology, bioinformatics, and experimental approaches. While the focus is primarily on tumor microenvironment-related factors, the findings provide valuable insights into the molecular mechanisms of HD-SB and identify potential novel targets for bladder cancer therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12906-026-05279-5.