The Synergistic Effects of 5-Aminosalicylic Acid and Vorinostat in the Treatment of Ulcerative Colitis

Abstract

Background: The drug 5-aminosalicylic acid (5-ASA) is the first-line therapy for the treatment of patients with mild-to-moderate ulcerative colitis (UC). However, in some cases, 5-ASA cannot achieve the desired therapeutic effects. Therefore, patients have to undergo therapies that include corticosteroids, monoclonal antibodies or immunosuppressants, which are expensive and may be accompanied by significant side effects. Synergistic drug combinations can achieve greater therapeutic effects than individual drugs while contributing to combating drug resistance and lessening toxic side effects. Thus, in this study, we sought to identify synergistic drugs that can act synergistically with 5-ASA. Methods: We started our study with protein-metabolite analysis based on peroxisome proliferator-activated receptor gamma (PPARG), the therapeutic target of 5-ASA, to identify more additional potential drug targets. Then, we further evaluated the possibility of their synergy with PPARG by integrating Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis, pathway-pathway interaction analysis, and semantic similarity analysis. Finally, we validated the synergistic effects with in vitro and in vivo experiments. Results: The combination of 5-ASA and vorinostat (SAHA) showed lower toxicity and mRNA expression of p65 in human colonic epithelial cell lines (Caco-2 and HCT-116), and more efficiently alleviated the symptoms of dextran sulfate sodium (DSS)-induced colitis than treatment with 5-ASA and SAHA alone. Conclusion: SAHA can exert effective synergistic effects with 5-ASA in the treatment of UC. One possible mechanism of synergism may be synergistic inhibition of the nuclear factor kappa B (NF-kB) signaling pathway. Moreover, the metabolite-butyric acid may be involved.

Keywords: 5-ASA; SAHA; butyric acid; protein-metabolite interactions; synergistic effects; ulcerative colitis.

FIGURE 1

Protein-metabolite network construction. A full network based on the primary protein, PPARG (orange node), associated metabolites (blue node), and related druggable proteins (green node). We also show PPARG-metabolite interactions (brown-yellow edges), and metabolite-protein interactions (light gray edges).

FIGURE 2

Measurement of the possibility of synergy. (A) KEGG pathway enrichment analysis on druggable target proteins. (B) Sankey plots of the pathways enriched for the druggable genes. (C) The top 10 candidate target proteins with semantic similarity scores and their UniProt IDs.

FIGURE 3

Validation of the prediction results in vivo. (A,C) CCK eight analysis was performed to evaluate the toxicity of 5-ASA (30 mM) and SAHA (5 µM) alone or in combination in Caco-2 cells or HCT-116 cells after 24 and 48 h of treatment; (B,D) The p65 mRNA levels were examined in Caco-2 cells and HCT-116 cells treated with 5-ASA and SAHA in . The data shown are the mean ± SD and represent three independent experiments, p values were calculated using an unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

FIGURE 4

In vitro validation of the synergetic effects of 5-ASA and SAHA on DSS-induced colitis mice. B57BL/6 mice were administered 2.5% DSS in drinking water for 7 days, followed by 3 days of water (n = 5 per group). (A) Body weight, (B) DAI score, (C) colon length, (D) HE staining (200 × magnification) and histological scores of colitis mice treated with 5-ASA (100 mg/kg/day) and SAHA (200 mg/kg/day) in combination or individually. (E) Immunohistochemistry of p65 and (F) the p65, (G) IL-6, IL-1β, and TNF-α mRNA levels in colonic tissues. The data shown are the mean ± SD and represent three independent experiments, p values were calculated using unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

FIGURE 5

PPARG and HDACs can interact with butyric acid. (A) Network of interaction among PPARG, HDACs and the metabolite-butyric acid. (B) 2D and (C) 3D versions of the molecular docking of butyric acid to PPARG, HDAC2, HDAC3, and HDAC4.

FIGURE 6

PPARG and HDACs can be regulated by butyrate. (A) PPARG, HDAC2, HDAC3 and HDAC4 mRNA levels were determined in Caco-2 cells treated with butyrate (5 mM) for 24 h; B57BL/6 mice were administered 2.5% DSS in drinking water for 7 days, followed by 3 days of water, in the presence or absence of butyrate (200 mM) (n = 5 per group). (B) Body weight, (C) DAI score, (D) colon length, (E) HE staining (200 × magnification) and histological scores or vechale. (F) PPARG, HDAC2, HDAC3, and HDAC4 mRNA levels in colonic epithelial cells. The data shown are the mean ± SD and represent three independent experiments, p values were calculated using unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.