Exploring the mechanisms of Hu-gu-xiao-ji formula and its components for treatment on non-small cell lung cancer using UPLC-QTOF-MS/MS, network pharmacology, and experiments.

Ethnopharmacological relevance: Non-small cell lung cancer (NSCLC) represents the leading cause of cancer-related morbidity and mortality worldwide, where effective and safe therapeutic options for advanced stages remain limited. The Hu-gu-xiao-ji (HGXJ) formula, derived from traditional formulations including Si-jun-zi decoction and Bu-gu pill, has demonstrated efficacy in alleviating bone pain in metastatic NSCLC. Nevertheless, its active components and underlying mechanisms against primary NSCLC are not fully elucidated.

Aim of the study: To explore the therapeutic effects, chemical compositions, and molecular mechanisms of HGXJ formula against NSCLC through integrated in vitro and in vivo approaches.

Materials and methods: The antitumor efficacy of HGXJ formula was assessed using subcutaneous xenograft models and NSCLC cell lines. Serum-absorbed bioactive compounds were identified by ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). Potential targets and signaling pathways were predicted through integrated network pharmacology, bioinformatics analysis, and molecular biology experiments. Molecular docking, cellular assays and in vivo validation were subsequently employed to verify antitumor effects and compound-target interactions.

Results: HGXJ formula exhibited significant in vivo and in vitro antitumor activity by promoting apoptosis and inhibiting proliferation. UPLC-QTOF-MS/MS analysis identified 30 blood-entry constituents. Subsequent investigations revealed that HGXJ formula disrupted the kelch-like ECH-associated protein 1 (KEAP1) -nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway, triggering reactive oxygen species (ROS) accumulation and consequent cell death. Three bioactive compounds, isobavachin, bavachalcone, and 18β-glycyrrhetinic acid were screened out and validated to induce ROS-mediated cytotoxicity and target antioxidant pathway. In vivo studies further substantiated the antitumor activity of 18β-glycyrrhetinic acid and its targeting of KEAP1/NRF2 proteins.

Conclusion: HGXJ formula and its blood-entry bioactive compounds exerted antitumor effects on NSCLC via suppressing the KEAP1-NRF2 antioxidant axis and inducing ROS-dependent cell death. These findings provide robust evidence supporting the scientific basis for the application of HGXJ formula in advanced NSCLC treatment.