Preclinical Development of FA5, a Novel AMP-Activated Protein Kinase (AMPK) Activator as an Innovative Drug for the Management of Bowel Inflammation

Abstract

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.

Keywords: AMPK; DNBS colitis; immune system; inflammatory bowel diseases; oxidative stress.

Figure 1

2-DE coupled with WB was employed to detect specific phosphorylated proteins. Proteins from C2C12 cells were firstly separated according to pI on Immobiline Dry-Strips (18 cm, non-linear gradient pH 3–10) and then according to molecular weight using 11% polyacrylamide gels. Subsequently, proteins were transferred onto nitrocellulose membranes. Immediately after WB, membranes were stained with RuBP. Thereafter, membranes were incubated with the anti-phosphothreonine antibody. (A) A representative nitrocellulose image with a 2-DE protein map of C2C12 cells (control). Proteins were detected by RuBP staining. (B) The same membrane with detection of the immunoreactive spots. (C) A representative nitrocellulose membrane with detection of the immunoreactive spots of C2C12 cells treated with FA5. Spots differentially phosphorylated are highlighted.

Figure 2

Body (A) and spleen weight (B) estimated in rats with DNBS-induced colitis treated with FA5 (0.5, 1, 3, 10, 30 mg/kg/day) acadesine (10 mg/kg/day) or dexamethasone (0.1 mg/kg/day). Each column represents the mean ± standard error of the mean (SEM) (n = 8). *, p < 0.05, significant difference versus control group; ^a, p < 0.05, significant difference versus DNBS group.

Figure 3

Colonic length (A) and macroscopic damage score (B) evaluated in rats with colitis, either in the absence or in the presence of FA5 (0.5, 1, 3, 10, 30 mg/kg/day), acadesine (10 mg/kg/day), or dexamethasone (0.1 mg/kg/day). Each column represents the mean ± standard error of the mean (SEM) (n = 8). *, p < 0.05, significant difference versus control group; ^a, p < 0.05, significant difference versus DNBS group.

Figure 4

Microscopic damage score estimated for colon in rats under normal conditions or with DNBS-induced colitis, either alone or after treatment with FA5 (0.5–30 mg/kg/day), acadesine (mg/kg/day), or dexamethasone (1 mg/kg). Each column represents the mean ± standard error of the mean (SEM) (n = 8). * p < 0.05, significant difference vs. control group; ^a p < 0.05, significant difference vs. DNBS group.

Figure 5

Representative blots and densitometric analysis of the expression of phospho-AMPK and AMPK in colonic tissues from control rats and animals treated with DNBS alone or in combination with FA-5 (0.5, 1, 3, 10, 30 mg/kg/day). Each column represents the mean ± standard error of the mean (SEM) (n = 6). One-way ANOVA followed by Tukey’s post hoc test results: * p < 0.05, significant difference vs. the control group; ^a p < 0.05, significant difference vs. the DNBS group.

Figure 6

Representative blots (A) and densitometric analysis of the expression of ZO-1 (B), occludin (C), and claudin-1 (D) in colonic tissues from control rats and animals treated with DNBS alone or in combination with FA-5 (0.5, 1, 3, 10, 30 mg/kg/day). Each column represents the mean ± standard error of the mean (SEM) (n = 6). One-way ANOVA followed by Tukey’s post hoc test results: * p < 0.05, significant difference vs. the control group; ^a p < 0.05, significant difference vs. the DNBS group.

Figure 7

TNF (A), IL-10 (B), and MDA (C) levels in colonic tissues from control rats or in animals treated with DNBS either alone or in combination with FA5 (0.5, 1, 3, 10, 30 mg/kg/day), acadesine (10 mg/kg/day), or dexamethasone (0.1 mg/kg/day). Each column represents the mean ± standard error of the mean (SEM) (n = 8). *, p < 0.05, significant difference versus control group; ^a, p < 0.05, significant difference versus DNBS group.