Network pharmacology-based investigation of the therapeutic efficacy and molecular targets of Xiangsha Liujunzi Decoction in thyroid cancer

ObjectiveTo explore the active components and potential molecular mechanisms of Xiangsha Liujunzi decoction (XSLJZ) in the treatment of thyroid cancer.MethodsActive components and corresponding targets of XSLJZ were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Thyroid cancer-related targets were collected from seven bioinformatics databases, including GeneCards, OMIM, DisGeNET, DrugBank, TCGA, GEO, and TTD. A protein-protein interaction (PPI) network and a multi-level “XSLJZ-medicinal materials-active components-potential targets-thyroid cancer” network were constructed to identify core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed, followed by molecular docking to assess binding affinities. In vitro, CCK-8, flow cytometry, colony formation, Transwell, and Western blotting assays were used to evaluate cell proliferation, death, colony formation, migration, and protein expression in 8505C and TPC-1 thyroid cancer cells. In vivo, a subcutaneous xenograft model was established in female BALB/c nude mice (6-8 weeks old, 15-16 g) by injecting 2×10⁶ 8505C cells. Mice were randomly divided into model control (vehicle), low-dose XSLJZ (2 mg/kg), and high-dose XSLJZ (4 mg/kg) groups (n=6 per group), receiving daily gavage for 24 days. Tumor volume and body weight were measured every four days. Histopathological changes were observed by HE and immunohistochemical staining.ResultsFive major active components of XSLJZ (isoliquiritigenin, naringenin, baicalein, nobiletin, and glycyrrhetinic acid) and 45 core targets for thyroid cancer were identified. XSLJZ and these five components significantly inhibited the proliferation of 8505C and TPC-1 cells without notable toxicity to normal thyroid cells, with isoliquiritigenin showing the most prominent effect. Flow cytometry revealed that XSLJZ (16 mg/mL) induced up to 84.91% death in TPC-1 cells. Molecular docking showed low binding energies between isoliquiritigenin and Akt1, Caspase-3, Caspase-9, and PARP (lowest: －9.9 kcal/mol with PARP). XSLJZ and isoliquiritigenin dose-dependently suppressed colony formation and migration, downregulated RAGE, p-PI3K, and p-Akt, upregulated Bax and mitochondrial cytochrome c, and activated Caspase-3/9-mediated apoptosis and GSDME-dependent pyroptosis via the AGE-RAGE→PI3K/Akt axis. In vivo, both low and high doses of XSLJZ significantly inhibited tumor growth, reduced Akt and Ki-67 expression in tumor tissues (all P<0.05), and showed no obvious toxicity to the heart, liver, or kidneys.ConclusionXSLJZ inhibits thyroid cancer cell proliferation and induces cell death through multi-component, multi-target synergistic regulation of the AGE-RAGE→PI3K/Akt signaling pathway.