Mechanisms of Xiaozheng decoction for anti-bladder cancer effects via affecting the GSK3β/β-catenin signaling pathways: a network pharmacology-directed experimental investigation

PURPOSE: The combination of Xiaozheng decoction with postoperative intravesical instillation has been shown to improve the prognosis of bladder cancer patients and prevent recurrence. However, the mechanisms underlying the efficacy of this herbal formula remain largely unclear. This research aims to identify the important components of Xiaozheng decoction and explore their anti-bladder cancer effect and mechanism using network pharmacology-based experiments. METHODS: The chemical ingredients of each herb in the Xiaozheng decoction were collected from the Traditional Chinese Medicine (TCM) database. Network pharmacology was employed to predict the target proteins and pathways of action. Disease databases were utilized to identify target genes associated with bladder cancer. A Protein–Protein Interaction (PPI) network was constructed to illustrate the interaction with intersected target proteins. Key targets were identified using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. A compound-target-pathway network was established after molecular docking predictions. In vitro experiments with bladder cancer cell lines were conducted using core chemical components confirmed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) to verify the conclusions of network pharmacology. RESULTS: 45 active compounds were extracted, and their relationships with Traditional Chinese Medicines (TCMs) and protein targets were presented, comprising 7 herbs, 45 active compounds, and 557 protein targets. The intersection between potential TCM target genes and bladder cancer-related genes yielded 322 genes. GO and KEGG analyses indicated that these targets may be involved in numerous cancer-related pathways. Molecular docking results showed that candidate compounds except mandenol could form stable conformations with the receptor. In vitro experiments on three bladder cancer cell lines demonstrated that quercetin and two other impressive new compounds, bisdemethoxycurcumin (BDMC) and kumatakenin, significantly promoted cancer cell apoptosis through the B-cell lymphoma 2/Bcl-2-associated X (Bcl-2/BAX) pathway and inhibited proliferation and migration through the glycogen synthase kinase 3 beta (GSK3β)/β-catenin pathway. CONCLUSION: By employing network pharmacology and conducting in vitro experiments, the mechanism of Xiaozheng decoction’s effect against bladder cancer was tentatively elucidated, and its main active ingredients and targets were identified, providing a scientific basis for future research.