Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

Figure 1 Analysis of pharmacodynamic material basis of Calculus bovis. A: Positive and negative ion chromatograms of Calculus bovis (CB) extract; B: Structural diagrams of 22 components of CB; C: Ion chromatogram of the standardized substances of CB extract; D: Ion chromatograms of CB-enriched serum and blank serum.

Figure 2 In vivo anti-tumor activity of Calculus bovis. A: Pictures of tumors in each group after treatment; B: Tumor size changes in each group during treatment; C: Tumor weight of each group during treatment; D: Changes in weight of mice in each group during treatment; E: Hematoxylin-eosin staining results of tumor tissue sections in each group after treatment. Control: Model group. CB: Calculus bovis; L-CB: Low-dose CB group; M-CB: Medium-dose CB group; H-CB: High-dose CB group; Differences were assessed using one-way ANOVA and multiple comparisons were determined using Tukey's test. ^aP < 0.01 vs control, ^bP < 0.05 vs L-CB, ^cP < 0.01 vs L-CB.

Figure 3 Cyberpharmacological study on anti-liver cancer mechanism of active components of Calculus bovis based on UPLC-Q-TOF-MS analysis. A: Intersection plot of potential targets of Calculus bovis (CB) and liver cancer targets; B: Component-target-disease interaction network of CB against liver cancer. The purple quadrilaterals represent the active components of CB; C: Protein-protein interaction network; D: Gene Ontology function analysis of CB against liver cancer; E: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of CB against liver cancer; F: Minimum binding energy of the active components of CB with core target proteins; G: Molecular docking diagram of some active components.

Figure 4 Differentially expressed genes between Calculus bovis-treated group and control group (q < 0. 05 and log2FlodChange > 0) after transcriptome sequences were analyzed by bioinformatics. A: Differential gene volcano plot between the Calculus bovis (CB)-treated and control groups; B: Histogram of Gene Ontology enrichment results of differentially expressed mRNAs in the CB-treated and control groups (top 10); C: Bubble plots of Kyoto Encyclopedia of Genes and Genomes enrichment results of differentially expressed mRNAs in the CB-treated and control groups (top 20); D: Heatmap of selected genes involved in the Wnt pathway.

Figure 5 Calculus bovis-enriched serum inhibits polarization of M2 tumor-associated macrophages. A: Cytotoxicity of Calculus bovis-enriched serum (CBS) on THP-1 cells by Cell Counting Kit-8 assay. The blank group served as a control; B: CD206 quantification in THP-1 cells after IL-4 and IL-13 induction by flow cytometry; C: The mRNA expression levels of CD206 after intervention of M2 tumor-associated macrophages (M2-TAMs) with different concentrations of blank serum and CBS; D: Quantification of CD206 in M2-TAMs after 10% (100 mL/L) CBS intervention by flow cytometry; E: The mRNA expression levels of CCL22, TGF-β2, IL-10, and Arg-1 after 10% (100 mL/L) CBS intervention in M2-TAMs. ^dP < 0.05, ^eP < 0.01. BS: Blank serum; CBS: CB-enriched serum.

Figure 6 Calculus bovis-enriched serum inhibits polarization of M2 tumor-associated macrophages through the Wnt pathway and suppresses malignant behaviors of liver cancer cells. A: Inhibition rate of HepG2 proliferation by M2 + Calculus bovis-enriched serum (CBS) conditioned medium; B: Effect of M2 + CBS conditioned medium on HepG2 apoptosis detected by flow cytometry; C: Inhibitory effect of M2 + CBS conditioned medium on migration of HepG2 cells by cell scratch assay; D: Inhibitory effect of M2 + CBS conditioned medium on migration of HepG2 cells by Transwell assay. M2 + CBS conditioned medium: Supernatants of differentiated M2 macrophages cultured in DMEM medium enriched with CBS and penicillin-streptomycin, and the conditional media were collected after culture. ^dP < 0.05, ^eP < 0.01.

Figure 7 Calculus bovis-enriched serum inhibits polarization of M2 tumor-associated macrophages through the Wnt pathway to suppress against liver cancer. A: Effect of Calculus bovis-enriched serum (CBS) on Wnt5B, β-catenin, and Axin2 mRNA levels in THP-1 cells detected by real-time reverse transcriptase-polymerase chain reaction; B: Effect of CBS on Wnt5B, β-catenin, and Axin2 protein levels in THP-1 cells detected by Western blot analysis; C: Effect of the Wnt pathway agonist SKL2001 on β-catenin protein expression; D: Changes in CD206 detected by flow cytometry in THP-1 cells after CBS combined with SKL2001 intervention for polarization. ^eP < 0.01.

Figure 8 In vivo experimental validation of regulation of M2 macrophage polarization by Calculus bovis through the Wnt pathway. A: Quantification of mRNA expression of Wnt5B, β-catenin, and Axin2 by real-time reverse transcriptase-polymerase chain reaction; B: Quantification of protein expression of Wnt5B, β-catenin, and Axin2 by Western blot analysis; C: Ratio of cells expressing CD206 molecules in tumor tissues analyzed by flow cytometry. ^eP < 0.01.

Figure 9 Schematic diagram of anti-liver cancer mechanism of Calculus bovis. Calculus bovis exerts its anti-liver cancer effect by inhibiting the Wnt/β-catenin pathway and suppressing the polarization of M2 tumor-associated macrophage. M2-TAMs: M2 tumor-associated macrophages; TME: Tumor microenvironment.