The mechanism of Shancigu and its monomer in the development of colorectal cancer based on network pharmacology

Abstract

Shancigu has traditionally been used for clearing heat, detoxification, resolving phlegm, and dissipating masses. However, its potential mechanisms in colorectal cancer (CRC) remain unclear. This study aimed to explore the molecular mechanisms of Shancigu and its active compound in CRC. The active ingredients of Shancigu and their predicted targets were identified, and differentially expressed genes (DEGs) associated with CRC metastasis and invasion were screened. Intersection genes were obtained and used to construct a protein-protein interaction (PPI) network. Core genes were identified, and their prognostic significance was analyzed. Molecular docking was performed between key survival-related genes and Shancigu compounds. Further in vitro, organoid, and in vivo experiments were conducted to investigate the regulatory effects of Stigmasterol, a major active component. A total of 18 active ingredients and 366 potential targets of Shancigu were identified. From 19,331 DEGs, 365 intersection genes and 18 core genes were screened. Among them, AKT1, AR, FN1, HRAS, ITGB1, and JUN showed significant prognostic relevance in CRC. Molecular docking revealed that Stigmasterol strongly binds to ITGB1 and JUN. In cellular experiments, Stigmasterol inhibited viability, proliferation, migration, and invasion, induced apoptosis, and downregulated JUN and ITGB1 expressions in HCT116 and Caco-2 cells. In CRC organoids, Stigmasterol reduced organoid viability and ATP activity. Animal studies demonstrated that both Shancigu and Stigmasterol reduced tumor weight and volume and inhibited Ki67, ITGB1, and JUN expression. Stigmasterol may suppress CRC proliferation and invasion by targeting the key genes JUN and ITGB1, providing insights into the potential therapeutic mechanisms of Shancigu against CRC.

Keywords: Colorectal cancer; ITGB1; JUN; Network pharmacology; Shancigu; Stigmasterol.

Fig. 1

Shancigu-active ingredients-target network. Blue represents Shancigu, purple represents active ingredients, and green represents targets.

Fig. 2

Differentially expressed genes related to metastasis and invasion of CRC. A Volcano map of genes associated with metastasis and invasion of CRC. Red indicates upregulated genes, while blue indicates downregulated genes. B Clustering heat map of genes associated with CRC metastasis and invasion.

Fig. 3

Intersection of gene associated with CRC metastasis and invasion and Shancigu target and functional analysis. A Intersection genes between CRC metastasis and invasion and the relevant action targets of the active ingredients of Shancigu. B–D DO, GO, and KEGG enrichment analysis of intersection genes.

Fig. 4

Construction of intersection gene protein interaction network and analysis of core genes. A Intersection gene protein interaction network. B–D. Degree, stress and betweenness algorithms were selected to analyze intersection gene protein interaction network. E Intersection genes of three algorithms (degree, stress and betweenness). F Survival analysis of AKT1. G Survival analysis of AR. H Survival analysis of FN1. I Survival analysis of HRAS. J Survival analysis of ITGB1. K Survival analysis of JUN.

Fig. 5

Molecular docking of significant survival related genes with monomer of Shancigu. A Heat map of molecular docking score. B Molecular docking of AKT1 with compound MOL007991. C Molecular docking of AR with compound MOL007991. D Molecular docking of FN1 with compound MOL007990. E Molecular docking of HRAS with compound MOL000358. F Molecular docking of ITGB1 with compound MOL000449. G Molecular docking of JUN with compound MOL000449.

Fig. 6

Stigmasterol regulated CRC cell viability, proliferation, apoptosis, migration, and invasion. A CCK-8 assay detection of HCT116 and Caco-2 cell viability. B Colony formation assay assessed the long-term proliferative capacity of HCT116 and Caco-2 cells following Stigmasterol treatment. C Flow cytometry determination of HCT116 and Caco-2 cell apoptosis. D Transwell assays detection of HCT116 and Caco-2 cell migration and invasion abilities. E Western blot analysis of Stigmasterol-binding proteins ITGB1 and JUN expressions. Data represent the mean ± SD of three independent biological replicates. *P < 0.05, **P < 0001, ***P < 0.001.

Fig. 7

Stigmasterol regulated the viability of organoids in CRC. A Changes in morphology and viability of CRC organoids after Stigmasterol concentration gradients (0, 1, 10, 30, 100, and 500 µM) treatment. B ATP activity detection of CRC organoid. Data represent the mean ± SD of three independent biological replicates. *P < 0.05, ***P < 0.001.

Fig. 8

Shancigu and its monomer Stigmasterol regulated the development of CRC in animal experiment. A Tumor images. B Tumor weight. C H&E staining of tumor histopathology and inflammatory cell infiltration. D IHC staining of the changes of tumor proliferation factor Ki67. E Western blot analysis of Stigmasterol-binding proteins ITGB1 and JUN expressions. All statistical data represent the mean ± SD from three independent biological experiments (n = 6 per group in each replicate). *P < 0.05, **P < 0.01, ***P < 0.001.