Trioxolone Methyl, a Novel Cyano Enone-Bearing 18βH-Glycyrrhetinic Acid Derivative, Ameliorates Dextran Sulphate Sodium-Induced Colitis in Mice

Abstract

Semi-synthetic triterpenoids, bearing cyano enone functionality in ring A, are considered to be novel promising therapeutic agents with complex inhibitory effects on tissue damage, inflammation and tumor growth. Previously, we showed that the cyano enone-containing 18βH-glycyrrhetinic acid derivative soloxolone methyl (SM) effectively suppressed the inflammatory response of macrophages in vitro and the development of influenza A-induced pneumonia and phlogogen-stimulated paw edema in vivo. In this work, we reported the synthesis of a novel 18βH-glycyrrhetinic acid derivative trioxolone methyl (TM), bearing a 2-cyano-3-oxo-1(2)-en moiety in ring A and a 12,19-dioxo-9(11),13(18)-dien moiety in rings C, D, and E. TM exhibited a high inhibitory effect on nitric oxide (II) production by lipopolysaccharide-stimulated J774 macrophages in vitro and dextran sulfate sodium (DSS)-induced colitis in mice, displaying higher anti-inflammatory activity in comparison with SM. TM effectively suppressed the DSS-induced epithelial damage and inflammatory infiltration of colon tissue, the hyperproduction of colonic neutral mucin and TNFα and increased glutathione synthesis. Our in silico analysis showed that Akt1, STAT3 and dopamine receptor D2 can be considered as mediators of the anti-colitic activity of TM. Our findings provided valuable information for a better understanding of the anti-inflammatory activity of cyano enone-bearing triterpenoids and revealed TM as a promising anti-inflammatory candidate.

Keywords: 18βH-glycyrrhetinic acid; CDDO-Me; colitis; cyano enone; inflammation; molecular docking; molecular targets; soloxolone methyl.

Figure 1

The chemical structures of cyano enone-bearing semi-synthetic triterpenoids.

Scheme 1

The synthesis of trioxolone methyl (TM) [methyl 2-cyano-3,12,19-trioxo-olean-1(2),9(11),13(18)-trien-30-oate].

Figure 2

TM effectively inhibited the production of on nitric oxide (II) (NO) by lipopolysaccharide (LPS)-challenged J774 macrophages. (A) The viability of J774 cells incubated with or without (control) soloxolone methyl (SM) and trioxolone methyl (TM) for 24 h. (B) The effect of triterpenoids on NO production by LPS-activated J774 cells. J774 cells were co-treated by LPS (10 µg/mL) and TM or SM (0.05–1 µM) for 24 h followed by the measurement of the nitrite concentration in a culture medium using the Griess reaction. The results are expressed as the means ± SD of three (A) and two (B) experiments performed in tetra- (A) and pentaplicate (B).

Figure 3

TM displayed pronounced anti-colitic activity. (A) The experimental scheme. Colitis was induced in C57Bl/6 mice by the administration of 2.5% dextran sulfate sodium (DSS) in drinking water for 7 days. SM and TM (5 mg/kg) in sesame oil were administered via the gastric gavage simultaneously with DSS and within 3 days after the withdrawal of DSS (10 days in total). Sulfasalazine (SLZ; 100 mg/kg) was used as a reference drug. (B) TM suppressed the colon shortening. Colon length was measured between the ileo–cecal junction and the proximal rectum. (C) The relative body weight (% of initial body weight) and disease activity index (DAI) showing the level of blood in the stool of the mice. The data are presented as the means ± SD (n = 7 per group).

Figure 4

TM inhibited DSS-induced epithelial damage, inflammatory infiltration and mucin hyperproduction in colon tissue and could target thrombin. (A) The effect of triterpenoids on the epithelial damage and the inflammatory infiltration in colitis mice. Black arrows indicate the ulcerative foci. Hematoxylin and eosin staining, magnification ×100. (B) The effects of SM and TM on the colitis severity were quantified by the histological scoring system. The data are presented as the means ± SD (n = 7 per group). (C) The effect of SM and TM on the mucin production of goblet cells of epithelial crypts in colitis mice. Periodic Acid-Schiff (PAS) staining, magnification ×100.

Figure 5

TM inhibited the production of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and enhanced the antioxidant defense via glutathione (GSH) in DSS-induced colitis mice. (A) The effect of SM and TM on TNFα production in colitis mice. Immunohistochemical staining with anti-TNFα monoclonal antibodies, magnification ×400. (B) The effect of SM and TM on the GSH production in colitis mice. The data are presented as the means ± SD (n = 7 per group).

Figure 6

AKT serine/threonine kinase 1 (Akt1), tyrosine protein kinase Src, signal transducer and activator of transcription 3 (STAT3) and dopamine receptor D2 (DRD2) are probable primary targets of TM. (A) Venn diagram of the genes that were differentially expressed in the DSS-inflamed colon tissues in comparison with the healthy control (fold change > 1.5, p < 0.05) identified by the re-analysis of the Gene Expression Omnibus (GEO) datasets. (B) The protein–protein interaction network containing the colitis-associated differentially expressed genes (DEGs) and primary targets of TM, predicted by the Polypharmacology browser 2.0 tool (PB2) and the STITCH database, was reconstructed using the STRING database (confidence score > 0.7) in Cytoscape. The top 5 interconnected probable targets of SM are highlighted in yellow. (C) The binding energies of TM and the known inhibitors with the top 5 probable targets of TM that are the most associated with the colitis regulome. The binding energies were calculated by Autodock Vina. (D) The mode of binding of TM and SM to Akt1, Src, STAT3, and DRD2. Stereo presentation of the docked poses of TM and SM in the mentioned proteins, superimposed on the corresponding inhibitor-bound structures, created by BIOVIA Discovery Studio. Two-dimensional representations of the docked poses of TM and SM in Akt1, Src, STAT3, and DRD2, depicted by LigPlot+. The green lines and comb represent the hydrogen bonds and nonbonding contacts, respectively. Common amino acid residues, interacting with both TM/SM and the corresponding inhibitors, are highlighted in red circles.

Figure 7

TM can target thrombin. (A) A stereo presentation of the docked poses of TM and SM in thrombin superimposed on the thrombin inhibitor-bound structure. (B) A 2D representation of the docked poses of TM and SM in thrombin. Common residues, interacting with both the inhibitor and TM, are highlighted in red circles. The comb and green dashed lines represent nonbonding contacts and hydrogen bonds, respectively.