Investigating the mechanism of Fuzheng Sanjie Formula in regulating osimertinib resistance in NSCLC via the CAFs-TIMP3-MMP9 axis: a study integrating network pharmacology, data mining.

BACKGROUND: Osimertinib resistance limits EGFR-mutant non-small cell lung cancer (NSCLC) treatment efficacy. Cancer-associated fibroblasts (CAFs), tissue inhibitor of metalloproteinases 3 (TIMP3), and matrix metalloproteinase 9 (MMP9) are involved in resistance. Fuzheng Sanjie Formula (FZSJF) is used clinically, but its mechanism is unclear. OBJECTIVE: To explore the mechanism of FZSJF in reversing osimertinib resistance via the CAFs-TIMP3-MMP9 axis using network pharmacology, in vitro, and in vivo studies. METHODS: The main components of FZSJF were analyzed. Network pharmacology and molecular docking were used to assess binding to MMP9/TIMP3. TCGA data were used for expression and prognostic analysis. CompuSyn was used to calculate the combination index (CI) to assess whether the combination of osimertinib and FZSJF was synergistic Osimertinib-resistant cells (H1975OR) and CAFs were established and treated with control, osimertinib, FZSJF-containing serum, or their combination. Proliferation, invasion, migration, and MMP9/TIMP3 expression were evaluated by CCK-8, Transwell, wound healing, colony formation, Western blot (WB), quantitative real-time PCR (qPCR), and immunofluorescence (IF). TIMP3-silencing CAFs were co-cultured with H1975N cells to assess invasion and MMP9 expression. Nude mouse xenografts (H1975N, H1975OR, H1975N+CAFs) were used to evaluate tumor growth and protein expression. RESULTS: MMP9 was identified as a key target. FZSJF components and osimertinib showed good binding to MMP9/TIMP3. TCGA revealed high MMP9 and low TIMP3 in lung cancer, with MMP9 linked to shorter overall survival (OS) and TIMP3 to longer OS. CI values confirmed synergy between osimertinib and FZSJF. H1975OR cells and CAFs showed upregulated MMP9 and downregulated TIMP3. FZSJF-containing serum inhibited the proliferation, invasion, and migration of H1975OR cells, upregulated TIMP3, and downregulated MMP9; when combined with osimertinib, these effects were enhanced. TIMP3-silencing CAFs promoted H1975N invasion and MMP9 expression. In vivo, H1975OR and H1975N+CAFs tumors grew faster with higher MMP9 and lower TIMP3. FZSJF alone or combined inhibited tumor growth, restored MMP9/TIMP3 balance, without affecting body weight. CONCLUSION: FZSJF reverses osimertinib resistance by upregulating TIMP3 and inhibiting CAFs-mediated MMP9 overexpression, restoring MMP9/TIMP3 balance and suppressing NSCLC invasion. This study provides experimental evidence for the efficacy of FZSJF in treating resistant NSCLC.