N-Palmitoyl-D-Glucosamine Inhibits TLR-4/NLRP3 and Improves DNBS-Induced Colon Inflammation through a PPAR-α-Dependent Mechanism

Abstract

Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy.

Keywords: IBD; NLRP3; PPARs; intestinal barrier; intestinal inflammation; micronized N-palmitoyl-D-glucosamine; toll-like receptors; ulcerative colitis.

Figure 1

Micronized PGA significantly improves colitis hallmarks in PPAR-α-dependent manner. The effects of oral administration of micronized PGA on (A) DAI score, (B,D) colonic length, and (C) spleen weight in DNBS-treated mice. Results are expressed as the mean ± SD of n = 5 experiments ° p < 0.05 vs. vehicle; °°°° p < 0.0001 vs. vehicle; * p < 0.05 vs. DNBS; *** p < 0.001 vs. DNBS; **** p < 0.0001 vs. DNBS. Peroxisome proliferator-activated receptors-α (PPAR-α); Disease Activity Index (DAI); 2,4-dinitrobenzene sulfonic acid (DNBS).

Figure 2

Micronized PGA prevents the loss of tight junction proteins ZO-1 and occludin, and colonic barrier disruption through PPAR-α involvement. (A) Representative images show double-label immunohistochemistry for occludin (green) and ZO-1 (red) in the colon with (B,C) relative quantification. Nuclei were also labeled by DAPI. (E) Representative images of H&E-stained on distal colon sections, and (D) relative histological damage score. Results are expressed as the mean ± SD of n = 5 experiments (25 slices for each animal). °°°° p < 0.0001 vs. vehicle; **** p < 0.0001 vs. DNBS. Scale bar = 100 μm; magnification 20×. Zonula occludens (ZO-1); peroxisome proliferator-activated receptors-α (PPAR-α); hematoxylin and eosin (H&E), 2,4-dinitrobenzene sulfonic acid (DNBS).

Figure 3

Micronized PGA decreases TLR-4/NLRP3/iNOS expression in mice colon and pro-inflammatory cytokines release in plasma samples throughout selective PPAR-α involvement in DNBS-treated mice. (A) Immunofluorescence images display TLR-4 (green), NLRP3 (magenta), and iNOS (red) staining, and the relative quantification for (B) TLR-4, (C) NLRP3, and (D) iNOS. (E) IL-1β and (F) PGE₂ release in mice plasma. Results are expressed as the mean ± SD of n = 5 experiments (25 slices for each animal). °°°° p < 0.0001 vs. vehicle; * p < 0.05 vs. DNBS; **** p < 0.0001 vs. DNBS. Scale bar = 100 μm; magnification 20× 40×. Toll-like receptor (TLR)-4; nucleotide-binding oligomerization domain leucine-rich repeat and pyrine domain-containing protein 3 (NLRP3); inducible nitric oxide synthase (iNOS); peroxisome proliferator-activated receptors-α (PPAR-α); interleukin-1β (IL-1β); prostaglandin E2 (PGE_2.), 2,4-dinitrobenzene sulfonic acid (DNBS).