Integrated Network Pharmacology, Molecular Docking and Experimental Validation Reveal That Quercetin Suppresses Clear...

Background: Dodder, the dried mature seed of Cuscuta chinensis Lam. (CCL), has demonstrated anti-tumor activity, but its molecular and immunological mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear. Objective: To identify potential targets and elucidate the immune mechanisms by which CCL exerts therapeutic effects against ccRCC. Methods: A network pharmacology approach was employed to predict CCL's bioactive components and their putative targets in ccRCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to explore relevant pathways. Molecular docking validated the binding of key compounds to hub proteins. In vitro assays-including cell viability, colony formation, invasion, and apoptosis measurements-assessed the effects of quercetin, a principal CCL constituent, on 786-O renal carcinoma cells. Flow cytometry was performed to determine the percentage of CD163⁺ cells. An in vivo xenograft model evaluated CCL's anti-tumor efficacy. Western blotting, flow cytometry, and multiplex immunohistochemistry (mIHC) examined the modulation of signaling pathways and immune cell markers. Results: Network pharmacology identified IL-6, EGFR, TLR4, MMP9, CD44, and IFN-γ as core targets of CCL in ccRCC. Enrichment analyses implicated immune regulation, inflammation modulation, and PI3K/AKT signaling inhibition. Molecular docking revealed strong quercetin-MMP9 binding affinity. Immuno-correlation analyses indicated that high MMP9 levels positively correlated with macrophage infiltration and M2 polarization, suggesting a role in tumor immune escape. Quercetin significantly reduced the viability of 786-O cells in a dose-dependent manner, showing approximately 45% inhibition at 80 μM (p < 0.01). In addition, quercetin decreased MMP9 expression and reduced the proportion of CD163-positive macrophages. These effects were reversed by FSL-1 TFA (Toll-like receptor 2/6 agonist), which is the agonist of MMP-9. In the xenograft model, tumor volume in the quercetin-treated group was reduced by approximately 50% compared with the control group. Conclusions: CCL, particularly its active component quercetin, may inhibit ccRCC progression via inhibiting MMP9-mediated M2 macrophage polarization.