[Network pharmacology-based investigation of the therapeutic efficacy and molecular targets of Xiangsha Liujunzi decoction in thyroid carcinoma].

Abstract

Objectives: To explore the active components and potential molecular mechanisms of Chinese medicine Xiangsha Liujunzi decoction (XSLJZ) in the treatment of thyroid carcinoma.

Methods: Active 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, Online Mendelian Inheritance in Man Database (OMIM), DISGENET, DrugBank, The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Therapeutic Target Database (TTD). A protein-protein interaction (PPI) network and a multi-level "XSLJZ-medicinal materials-active components-potential targets-thyroid carcinoma" 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, cell counting kit-8 (CCK-8), flow cytometry, colony formation, Transwell, and Western blotting assays were used to evaluate cell viability, death, proliferation ability, migration, and protein expression in 8505C and TPC1 thyroid carcinoma 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 (200 mg/kg), and high-dose XSLJZ (400 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 hematoxylin and eosin staining and immunohistochemical staining.

Results: Five major active components of XSLJZ (isoliquiritigenin, naringenin, baicalein, nobiletin, and glycyrrhetinic acid) and 45 core targets for thyroid carcinoma were identified. XSLJZ and these five components significantly inhibited the proliferation of 8505C and TPC1 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±0.49)% death in TPC1 cells. Molecular docking showed low binding energies between isoliquiritigenin and protein kinase B 1 (Akt1), cysteine aspartic acid specific protease (Caspase)-3, Caspase-9, and poly ADP-ribose polymerase (PARP) (lowest －9.9 kcal/mol with PARP). XSLJZ and isoliquiritigenin dose-dependently suppressed colony formation and migration, downregu-lated receptor for advanced glycation end product (RAGE), phosphorylated phosphoino-sitide 3-kinase (PI3K), and phosphorylated Akt, upregulated B-cell lymphoma protein 2 associated X protein (BAX) and mitochondrial cytochrome c, and activated Caspase-3/9-mediated apoptosis and gasdermin E (GSDME)-dependent pyroptosis via the advanced glycation end product (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 kidney.

Conclusions: XSLJZ inhibits thyroid carcinoma cell proliferation and induces cell death through multi-component, multi-target synergistic regulation of the AGE-RAGE→PI3K/Akt signaling pathway.