Ya-Han-Lu-Duan decoction reshapes tumor microenvironment to treat colitis-associated colorectal cancer by regulating arachidonic acid metabolism

Figure 1 Ya-Han-Lu-Duan treatment alleviates pathology in colitis-associated colorectal cancer mice. A: Establishment of the colitis-associated colorectal cancer mouse model and timeline of drug administration; B: Survival curves of mice in each group; C: Weekly body weight changes in each group of mice; D: Colon lengths in each group of mice; E: Tumor counts in each group of mice; F: Tumor burden in each group of mice; G: Representative hematoxylin and eosin staining of colon tissue sections from each group of mice. n = 20 in control group, n = 12 in colitis-associated colorectal cancer group, n = 18 in sulfasalazine group, n = 13 in low-dose Ya-Han-Lu-Duan (YHLD) group, n = 15 in medium-dose YHLD group, n = 18 in high-dose YHLD group for panel (B-G). Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01. CAC: Colitis-associated colorectal cancer; AOM: Azoxymethane; DSS: Dextran sulfate sodium; CON: Control; SASP: Sulfasalazine; YHLD-H: High-dose Ya-Han-Lu-Duan; YHLD-L: Low-dose Ya-Han-Lu-Duan; YHLD-M: Medium-dose Ya-Han-Lu-Duan; HE: Hematoxylin and eosin.

Figure 2 UPLC-Q-TOF-MS analysis of Ya-Han-Lu-Duan extraction and plasma samples. A: Total ion chromatogram in ESI⁺ mode; B: Total ion chromatogram in ESI^- mode. YHLD: Ya-Han-Lu-Duan.

Figure 3 Effects of Ya-Han-Lu-Duan on transcriptomics of colon tissue in colitis-associated colorectal cancer mice. A: Differentially expressed genes between colitis-associated colorectal cancer vs control and high-dose Ya-Han-Lu-Duan vs colitis-associated colorectal cancer; B: KEGG pathway enrichment analysis of differentially expressed genes. n = 6 for A and B. CAC: Colitis-associated colorectal cancer; CON: Control; YHLD-H: High-dose Ya-Han-Lu-Duan; TNF: Tumor necrosis factor; ECM: Extracellular matrix.

Figure 4 Effects of Ya-Han-Lu-Duan on secretion and activation of myeloid-derived suppressor cells-associated and cancer-associated fibroblasts-related factors. A: Heatmap of genes related to the cytokine-cytokine receptor interaction and extracellular matrix–receptor interaction pathways; B: Enzyme-linked immunosorbent assay detection of levels of CXCL15, CXCL2, interleukin (IL)-1β, tumor necrosis factor, SPP1, IL-11, CSF3, and IL-23 in colon tissue; C: Western blot detection of COL9A3 and BMP7 expression levels in colon tissue; D: Immunofluorescence observation of FAP and CD11b⁺Gr-1⁺ expression. n = 6 for A and D, n = 20 in control group, n = 12 in colitis-associated colorectal cancer group, n = 18 in high-dose Ya-Han-Lu-Duan group for B; n = 3 for C. CAC: Colitis-associated colorectal cancer; CON: Control; YHLD-H: High-dose Ya-Han-Lu-Duan.

Figure 5 Effects of Ya-Han-Lu-Duan on metabolomics of colon tissue in colitis-associated colorectal cancer mice. A: Principal component analysis of metabolic profiles of control (CON), colitis-associated colorectal cancer (CAC), and high-dose Ya-Han-Lu-Duan (YHLD-H) groups; B: PLSDA validation between CAC vs CON and YHLD-H vs CAC; C: KEGG pathway enrichment analysis of differential metabolites between CAC vs CON and YHLD-H vs CAC. n = 6 for A-C. PCA: Principal component analysis; CAC: Colitis-associated colorectal cancer; CON: Control; YHLD-H: High-dose Ya-Han-Lu-Duan.

Figure 6 Ya-Han-Lu-Duan regulates arachidonic acid metabolism. A: Heatmap of differentially expressed genes involved in arachidonic acid (AA) metabolism; B: Heatmap of differential metabolites involved in AA metabolism; C: Schematic diagram of AA metabolism. n = 6 for A and B. AA: Arachidonic acid; CAC: Colitis-associated colorectal cancer; CON: Control; YHLD-H: High-dose Ya-Han-Lu-Duan.

Figure 7 Ya-Han-Lu-Duan regulates PTGS2 and ALOX12 in arachidonic acid metabolism. A: Western blot detection of PTGS2 and ALOX12 expression levels in colon tissue; B: Molecular docking results of the main components of Ya-Han-Lu-Duan (including Pentahydroxybufostane, Clematoside B, Psammosilenin A and Dulcoside A) with PTGS2 and ALOX12. n = 3 for A. CAC: Colitis-associated colorectal cancer; CON: Control; YHLD-H: High-dose Ya-Han-Lu-Duan.

Figure 8 Ya-Han-Lu-Duan regulates arachidonic acid metabolism by downregulating expression of PTGS2 and ALOX12, synergistically reducing myeloid-derived suppressor cells infiltration and cancer-associated fibroblast activation, improving the tumor microenvironment, and alleviating colitis-associated colorectal cancer. PGE2: Prostaglandin E2; PGG2: Prostaglandin G2; AA: Arachidonic acid; MDSC: Myeloid-derived suppressor cell; TNF: Tumor necrosis factor; IL: Interleukin; CAF: Cancer-associated fibroblast.