Novel FXR agonist nelumal A suppresses colitis and inflammation-related colorectal carcinogenesis

Abstract

FXR is a member of the nuclear receptor superfamily and bile acids are endogenous ligands of FXR. FXR activation has recently been reported to inhibit intestinal inflammation and tumour development. This study aimed to investigate whether the novel FXR agonist nelumal A, the active compound of the plant Ligularia nelumbifolia, can prevent colitis and colorectal carcinogenesis. In a mouse colitis model, dextran sodium sulfate-induced colonic mucosal ulcer and the inflammation grade in the colon significantly reduced in mice fed diets containing nelumal A. In an azoxymethane/dextran sodium sulfate-induced mouse inflammation-related colorectal carcinogenesis model, the mice showed decreased incidence of colonic mucosal ulcers and adenocarcinomas in nelumal A-treated group. Administration of nelumal A also induced tight junctions, antioxidant enzymes, and FXR target gene expression in the intestine, while it decreased the gene expression of bile acid synthesis in the liver. These findings suggest that nelumal A effectively attenuates colonic inflammation and suppresses colitis-related carcinogenesis, presumably through reduction of bile acid synthesis and oxidative damage. This agent may be potentially useful for treatment of inflammatory bowel diseases as well as their related colorectal cancer chemoprevention.

Figure 1

Effects of nelumal A on expression levels of TNF-α and anti-oxidant enzymes in the colon of colitis model mice. The mRNA expression levels of Tnfa, Catalase, and Gpx1 in the colonic mucosa were measured by quantitative real-time reverse transcription PCR with specific primers. Parallel amplification of 18S was used as the internal control. Each column represents the mean ± SD. Asterisk indicates statistically significant differences compared to DSS-treated group; P < 0.05. Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test. n = 5. Nel-A, nelumal A.

Figure 2

Effects of nelumal A on cellular proliferation and apoptosis in colorectal tumour tissues, and on inflammatory cytokines and anti-oxidant enzymes in colonic mucosa of AOM/DSS-treated mice. (a) Sections of the colon were stained with anti-PCNA or anti-cleaved caspase-3 antibodies. Representative photographs from each group are shown in the left panels. The positive cell indices, which were determined by counting positive cells, are shown in the right panels. (b) The mRNA expression levels of F4/80, Mcp1, Tnfa, Catalase, Gpx1, and Sod1 in the colonic mucosa were measured by quantitative real-time reverse transcription PCR with specific primers. Parallel amplification of 18S was used as the internal control. Scale bars, 100 μm. Each column represents the mean ± SD of triplicate assays. Asterisk indicates statistically significant differences compared to AOM/DSS group; P < 0.05. Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test. n = 6. Ctrl, control. Nel-A, nelumal A.

Figure 3

Effects of nelumal A on cell proliferation and barrier function in the colon of carcinogenesis model mice. The mRNA expression levels of Cox2, Cyclind1, Inos, Pcna (a), Muc2, and Tjp1 (b) in the colonic mucosa were measured by quantitative real-time reverse transcription PCR with specific primers. Parallel amplification of 18S was used as the internal control. Each column represents the mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test. n = 6. (c) Immunohistochemical analyses for MUC2 and ZO1, with enlarged pictures of the section from enclosed areas with square, in the colon of the experimental mice. Bars, 100 μm. (d) Data for quantitative analysis of immunohistochemistry were expressed as box-and-whisker plots with median values and 10, 25, 75, and 90 percentiles. Asterisk indicates statistically significant differences compared to AOM/DSS-treated control group; P < 0.05. Ctrl, control. Nel-A, nelumal A.

Figure 4

Effects of nelumal A on FXR signals in the ileum and FGF pathways in the liver in the colon of carcinogenesis model mice. (a) The mRNA expression levels of Fgf15, Fxr, and Shp in the ileum were measured by quantitative real-time reverse transcription PCR with specific primers. Parallel amplification of 18S was used as the internal control. Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test. n = 6. (b) Total proteins were extracted from the ileum samples of experimental mice, and protein levels of FGF15, FXR, and SHP were examined by western blot analysis using specific antibodies. GAPDH served as the loading control. (c) The bar graphs show the mean intensities of each protein. Statistical analyses were performed using Kruskal–Wallis test and following Steel–Dwass test. n = 4. (d) The mRNA expression levels of Cyp7a1 and Fgfr4 in the liver were measured by quantitative real-time reverse transcription as described above. Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test. n = 6. Each column represents the mean ± SD. Asterisk indicates statistically significant differences compared to AOM/DSS-treated control group; P < 0.05. Ctrl, control. Nel-A, nelumal A.