Combining network pharmacology and transcriptomics to validate and explore the efficacy and mechanism of Huayu Wan in treating non-small cell lung cancer.

Ethnopharmacological Relevance: Huayu Wan (HYW), a traditional Chinese medicine prescription widely used in the clinical treatment of advanced lung cancer, has been clinically proven to effectively inhibit the progression of pulmonary tumors and improve patients' quality of life. However, its specific components and potential anti-cancer molecular mechanisms remain unclear.

Aim Of The Study: To explore the active ingredients of HYW and predict its effective targets and pathways against non-small cell lung cancer (NSCLC) using a combination of network pharmacology and transcriptomics. These predictions were subsequently validated through in vitro and in vivo experiments, providing a theoretical basis for its anti-cancer mechanism.

Materials And Methods: We first established a LEWIS tumor-bearing mouse model to evaluate the dose-response relationship and inhibitory effect of HYW in NSCLC. Using Ultra-High Performance Liquid Chromatography-Quadrupole-Orbitrap-High Resolution Mass Spectrometry (UHPLC-Q-Orbitrap-HRMS), we comprehensively explored the material basis of HYW's therapeutic effect on lung cancer. Combining network pharmacology and transcriptomics, we further verified the potential molecular targets and pathways of HYW. Finally, in vitro and in vivo molecular biological experiments were conducted to validate the predicted results.

Results: HYW exhibited a dose-dependent tumor inhibitory effect in the LEWIS tumor-bearing mouse model. Comprehensive qualitative analysis of the chemical components of HYW through UHPLC-Q-Orbitrap HRMS identified 39 major active ingredients, including geniposide, quercetin, taurine, and paeoniflorin. The constructed HYW active compound-NSCLC target network revealed 48 core targets, which may play a critical role in HYW's anti-NSCLC therapeutic effects. Combining transcriptomic data from mouse tumor tissues, four core targets-Pik3ca, Akt1, Pdk1, and VEGFA-were identified, along with the key signaling pathway PI3K/AKT/VEGFA. Immunofluorescence results indicated that HYW dose-dependently inhibited the positive expression of Ki67 in mouse tumor tissues. In vitro experiments showed that HYW significantly suppressed the proliferation, migration, and invasion abilities of H1299 and A549 cells. qRT-PCR and Western blot analyses demonstrated that HYW treatment downregulated the expression of Pik3ca, Akt1, Pdk1, and VEGFA, and inhibited the protein expression levels of p-PI3K/PI3K, p-AKT/AKT, and VEGFA.

Conclusion: HYW effectively inhibits the malignant proliferation of NSCLC cells. The mechanism of its anti-cancer effects is likely mediated by the suppression of the PI3K/AKT/VEGFA signaling pathway. This finding provides new molecular insights into the potential therapeutic application of HYW in the treatment of lung cancer.