Pathophysiology of NSAID-Associated Intestinal Lesions in the Rat: Luminal Bacteria and Mucosal Inflammation as Targets for Prevention

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) can damage the small intestine, mainly through an involvement of enteric bacteria. This study examined the pathophysiology of NSAID-associated intestinal lesions in a rat model of diclofenac-enteropathy and evaluated the effect of rifaximin on small bowel damage. Enteropathy was induced in 40-week old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Rifaximin (delayed release formulation) was administered (50 mg/kg BID) 1 h before the NSAID. At the end of treatments, parameters dealing with ileal damage, inflammation, barrier integrity, microbiota composition, and TLR-NF-κB-inflammasome pathway were evaluated. In addition, the modulating effect of rifaximin on NLRP3 inflammasome was tested in an in vitro cell system. Diclofenac induced intestinal damage and inflammation, triggering an increase in tissue concentrations of tumor necrosis factor and interleukin-1β, higher expression of TLR-2 and TLR-4, MyD88, NF-κB and activation of caspase-1. In addition, the NSAID decreased ileal occludin expression and provoked a shift of bacterial phyla toward an increase in Proteobacteria and Bacteroidetes abundance. All these changes were counterbalanced by rifaximin co-administration. This drug was also capable of increasing the proportion of Lactobacilli, a genus depleted by the NSAID. In LPS-primed THP-1 cells stimulated by nigericin (a model to study the NLRP3 inflammasome), rifaximin reduced IL-1β production in a concentration-dependent fashion, this effect being associated with inhibition of the up-stream caspase-1 activation. In conclusion, diclofenac induced ileal mucosal lesions, driving inflammatory pathways and microbiota changes. In conclusion, rifaximin prevents diclofenac-induced enteropathy through both anti-bacterial and anti-inflammatory activities.

Keywords: enteroprotection; intestinal bleeding; intestinal damage; microbiota; non-steroidal anti-inflammatory drugs; rifaximin.

FIGURE 1

Histological analysis of damage in the ileum of rats treated with vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days. Representative pictures showing the microscopic appearance of ileal mucosa from control animals (A), as well as type 1 (B), type 2 (C) or type 3 (D) lesions, observed in animals treated with DIC. Effects of treatments on type 1 (E), type 2 (F), or type 3 (G) lesions. Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference vs. diclofenac alone.

FIGURE 2

Effects of vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days on blood hemoglobin (A) or fecal calprotectin levels (B). Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference versus diclofenac alone.

FIGURE 3

Effects of vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days on myeloperoxidase (MPO) (A), malondialdehyde (MDA) (B), tumor necrosis factor (TNF) (C), or interleukin-1β (IL-1β) (D) in the ileum. Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference versus diclofenac alone.

FIGURE 4

Western blot analysis of toll-like receptor-2 (TLR-2) (A), toll-like receptor-4 (TLR-4) (B), myeloid-differentiation primary response gene 88 (MyD88) (C), activated nuclear factor-κB (p65) (D), pro-caspase-1 (E), and caspase-1 (p20) (F) in the ileum of rats treated with vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days. Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference versus diclofenac alone. Note that the beta actin signals in (A,D) result from stripping and re-probing the same membrane, where TLR-2- and p65 proteins have been detected.

FIGURE 5

Western blot analysis of occludin in the ileum of rats treated with vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days. Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference versus diclofenac alone.

FIGURE 6

(A) Relative abundance of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria and (B) prevalence of Lactobacillus spp. in the ileum of rats treated with vehicle (Control), diclofenac (DIC, 4 mg/kg BID), diclofenac plus rifaximin (DIC+RIF, 50 mg/kg BID), or rifaximin (RIF) for 14 days. Each column represents the mean ± SEM obtained from 10 animals. ^∗p < 0.05, significant difference versus Control; ^ap < 0.05, significant difference versus diclofenac alone.

FIGURE 7

Lipopolysaccharide-primed THP-1 cells treated with rifaximin (RIF, 25, 50, or 100 μM) before the addition of nigericin (10 μM). (A) Effect of rifaximin (RIF) on interleukin-1β (IL-1β) release in cell supernatants. (B) Effect of rifaximin (RIF) on caspase-1 p20 expression in cell supernatants as well as on pro-caspase-1 expression in cell lysates. Each column represents the mean ± SEM value obtained from four separate experiments. ^∗p < 0.05, significant difference versus cells treated with nigericin alone.