Uncovering the Effects and Molecular Mechanisms of Shaoyao Decoction Against Colorectal Cancer Using Network Pharmacology Analysis Coupled With Experimental Validation and Gut Microbiota Analysis

BACKGROUND: Chronic gut inflammation and dysbiosis contribute significantly to colorectal cancer (CRC) development. Shaoyao decoction (SYD) is a well-established Chinese medicine prescription. Besides ameliorating CRC via anti-inflammatory effects, SYD modulates gut microbiota (GM) to improve inflammatory responses in ulcerative colitis (UC). However, whether and how SYD suppresses CRC by regulating GM remains largely unknown. METHODS: SD rats were orally administered SYD for 7 days to obtain medicated serum. We utilized liquid chromatography-mass spectrometry (LC-MS) analysis, GeneCards, DisGeNET, and SwissTargetPrediction databases to analyze blank and SYD-medicated rat serum, comparing the findings with those of SYD aqueous extract in previous studies to identify SYD circulating compounds/components with predictable target genes. Using network pharmacology, the potential active compounds and corresponding hub genes associated with modulating GM to suppress CRC were selected for molecular docking. In vivo experiments, a CRC transplantation tumor model was established in BALB/c mice using CT26 cells, with SYD gavage for 14 days. To investigate the mechanism of SYD-regulated GM against CRC, HE and IHC staining, Western blotting, and 16S rRNA sequencing were employed. RESULTS: LC-MS identified 26 SYD compounds with computationally predicted target genes. Network pharmacology prioritized 13 compounds targeting 8 inflammation/immunity-related genes (IL-17/TNF pathways), validated by molecular docking. In vivo experiments, SYD dose-dependently suppressed tumor growth (p < 0.05, medium/high doses), as confirmed by HE staining and IHC analysis of Ki-67. Notably, SYD potentially delayed CRC liver metastasis and alleviated hepatic injury in tumor-bearing mice. Western blotting demonstrated SYD's inhibition of the IL-17/TNF/NF-κB axis, aligning with computational predictions. 16S rRNA sequencing revealed SYD-enriched Akkermansia and GM structural shifts, mechanistically linking microbiota remodeling to anti-tumor efficacy. CONCLUSIONS: SYD combats CRC via dual modulation of IL-17/TNF/NF-κB signaling and GM ecosystems (e.g., Akkermansia enrichment). This microbiota-immune crosstalk positions SYD as a potential adjunct to conventional therapies, particularly for CRC patients with dysbiosis.