Bacterial signaling through Nod2/GSK3b protects the small intestine against ischemia‐reperfusion induced injury in mice

Abstract

Ischemia/reperfusion (I/R) injury represents an important mucosal injury and is one of the leading causes of death in critically ill patients.AimTo investigate the impact of bacteria/Nod2 signaling in I/R‐mediated injury.MethodsThe human colonic HCT‐116 (expressing endogenous Nod2) cells and rat IEC‐6 cells infected with Ad5Nod2 viral vector were used to study muramyl dipeptide (MDP; 50 ug/mL) signaling. GSK3β (Ser9), histone 3 (Ser10), β‐catenin phosphorylation levels were determined by Western blot or immunohistochemistry analysis. Germ‐free (GF), conventionalized (CNV), Nod2 knock‐out (Nod2−/−) and wild‐type (WT) mice (C57BL/6; 6–8 mice/group) were subjected to intestinal I/R by partial occlusion of the 2 distal branches of the superior mesenteric artery for 60 min, followed by 4 h reperfusion. Proliferation was determined using Ki67 IHC staining.ResultsGSK3β phosphorylation and nuclear accumulation of β‐catenin is induced within 60 min in MDP‐stimulated HCT‐116 cells and Ad5Nod2‐infected IEC‐6 cells. I/R‐induced ileal injury worsen in GF mice compared to CNV mice (1.5±0.22 vs 0.7±0.20, p<0.05) which associated with shorten crypt/villin length (11.9±0.19 μm vs 18.1±0.21 μm, p<0.01). Importantly, I/R‐induced ileal injury is more pronounced in Nod2−/− mice compared to WT mice (1.9±0.23 vs 0.9±0.29, p<0.05), with concomitant shorten crypt/villi length (11.8±0.17 μm vs 18.0±0.16 μm, p<0.05) and decreased IL‐6 mRNA accumulation (18.5±5.7 vs. 54.7±9.4). This increased injury response was accompanied by reduced Ki67 staining, GSK3β and H3 phosphorylation.ConclusionBacteria/Nod2 signaling to GSK3β/β‐catenin pathway represents an important host response to the microbiota and may protect the small intestine against I/R‐induced injury.