Identification of the effect and mechanism of Yiyi Fuzi Baijiang powder against colorectal cancer using network pharmacology and experimental validation

Abstract

Background: Yiyi Fuzi Baijiang powder (YFBP) is a traditional Chinese medicine used to treat colorectal cancer, although its bioactivity and mechanisms of action have not been studied in depth yet. The study intended to identify the potential targets and signaling pathways affected by YFBP during the treatment of colorectal cancer through pharmacological network analysis and to further analyze its chemical compositions and molecular mechanisms of action. Methods: The Traditional Chinese Medicine Systems Pharmacology (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), HitPredict (HIT), and Search Tool for Interactions of Chemicals (STITCH) databases were used to screen the bioactive components and promising targets of YFBP. Targets related to colorectal cancer were retrieved from the GeneCards and Gene Ontology databases. Cytoscape software was used to construct the "herb-active ingredient-target" network. The STRING database was used to construct and analyze protein-protein interactions (PPIs). Afterward, the R packages clusterProfiler and Cytoscape Hub plug-in were used to perform Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of target genes. The results of the network pharmacological analysis were also experimentally validated. Results: In total, 33 active components and 128 target genes were screened. Among them, 46 target genes were considered potential therapeutic targets that crossed the CRC target genes. The network pharmacology analysis showed that the active components of YFBP were correlated positively with CRC inflammatory target genes such as TLR4, TNF, and IL-6. The inflammation-related signaling pathways affected by the active components included the TNF-α, interleukin-17, and toll-like receptor signaling pathways. The active ingredients of YFBP, such as luteolin, β-sitosterol, myristic acid, and vanillin, may exert anti-tumor effects by downregulating SMOX expression via anti-inflammatory signaling and regulation of the TLR4/NF-κB signaling pathway. Conclusion: In the present study, the potential active components, potential targets, and key biological pathways involved in the YFBP treatment of CRC were determined, providing a theoretical foundation for further anti-tumor research.

Keywords: SMOX; Yiyi Fuzi Baijiang powder; colorectal cancer; inflammatory; network pharmacology.

FIGURE 1

Working flow chart of SMOX inhibitor prediction in CRC.

FIGURE 2

Chemical structure of LUT (A), SIT (B), MYA (C), and VAN (D).

FIGURE 3

Potential target genes and the PPI network map of YFBP against CRC. (A) Venn diagram of potential target genes of YFBP against CRC. (B) PPI network map of 46 target genes.

FIGURE 4

YFBP-CRC potential target gene network. The size of each node and the color depth in the network represent the size of its degree.

FIGURE 5

GO and KEGG pathway enrichment analyses. (A) Top 15 significantly rich GO analyses for the biological function of potential target genes of YFBP in CRC. (B) KEGG analysis of the first 15 significantly abundant YFBP potential target gene signaling pathways in CRC. (C) Top 15 vital signaling pathways’ core network. (D) Correlation between the core network and the top 15 significantly abundant gene biological function catalogs. (E) Correlation between the core network and the top 15 vital signaling pathways.

FIGURE 6

Colorectal cancer signaling pathway of YFBP potential target genes on CRC. The red represents YFBP target genes in the network.

FIGURE 7

Correlation between core target genes, the top 15 biological function catalogs, and the top 15 signaling pathways. The size of each node and the color depth in the network represent the size of its degree. Each node is interconnected by the gray connection line.

FIGURE 8

HPLC measurements of LUT (A), SIT (B), MYA (C), and VAN (D) concentrations in YFBP. Red represents the standard, and blue represents the sample.

FIGURE 9

YFBP active ingredients inhibit proliferation, migration, and invasion of colorectal cancer cells and promote apoptosis. (A–D) Viability assay of LUT, SIT, MYA, or VAN. (E ,F) Cell scratch assay of LUT, SIT, MYA, or VAN. (G ,H) Cell invasion assay of LUT, SIT, MYA, or VAN. (I,J) Cell apoptosis assay of LUT, SIT, MYA, or VAN. Quantitative results are shown as the mean ± SEM from three independent experiments. ^* p < 0.05, ^** p < 0.01 vs. the CTR group.

FIGURE 10

YFBP active ingredients inhibit in vivo HT-29 cell xenograft tumor growth. (A) Pictures of tumors taken after final treatment. (B) Tumor weight measured after final treatment. (C) Tumor size recorded during treatment. (E) HE staining of tumor. Scale bar = 20 μm. The green arrow indicates tumor cell necrosis, the blue arrow indicates nuclear fragmentation, the yellow arrow indicates cytoplasmic vacuolization, and the red arrow indicates neutrophil infiltration. (D ,F) Immunohistochemistry for SMOX. Scale bar = 50 μm. Quantitative results are shown as the mean ± SEM from three independent experiments. ^* p < 0.05, ^** p < 0.01 vs. the CTR group.

FIGURE 11

Effect of YFBP active ingredients on SMOX production and the expression of SMOX. (A) Level of SMOX was measured using ELISA kits. (B) mRNA expression of SMOX in the tumor tissue was measured by qRT-PCR. (C) Protein expression of SMOX in the tumor tissue was measured by Western blotting. Quantitative results are shown as the mean ± SEM from three independent experiments. ^** p < 0.01 vs. the CTR group.

FIGURE 12

Effect of YFBP active ingredients on the expression of the inflammatory mediator and the regulation of inflammatory signaling. (A–E) mRNA expression of TNFα, IL-1β, IL-6 TLR4, and NF-κB p65 in the tumor tissue was measured by qRT-PCR. (F) Protein expression of TLR4 in the tumor tissue was measured by Western blotting. (G) Activation of transcription factor NF-κB p65 which binds to SMOX was measured by ChIP. Quantitative results are shown as the mean ± SEM from three independent experiments. ^* p < 0.05, ^** p < 0.01 vs. the CTR group.