[Effect mechanism of NOD like receptor signaling pathway on intestinal mucosal barrier of rat during early phase of acute intra-abdominal infection].
OBJECTIVE
To initially investigate the expressing regularity and effect of enterocyte NOD like receptors on gut mucosal barrier during early phase of acute intra-abdominal infection.


METHODS
Sprague-Dawley (SD) rats were randomly allocated into control group (n=6) and experimental group (n=24). Acute intra-abdominal infection model was induced by cecal ligation and puncture (CLP). The level of NOD2 and NOD like receptor 3 (NLRP3) mRNA expression in gut mucosa was determined using fluorescent polymerase chain reaction (PCR); the expression of caspase-1 and tight junction protein was determined by Western blotting; the activity of nuclear factor-ΚB (NF-ΚB) was determined by electrophoretic mobility shift assay (EMSA); the level of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme linked immunosorbent assay (ELISA). The dead cell percentage of enterocyte was observed by terminal deoxynucleotidyl transferase mediated nick end labeling, and the gut mucosal permeability using an in situ loop preparation of gut with fluorescence isothiocyanate-conjugated dextran was determined.


RESULTS
NOD2 mRNA expression was quickly increased to a very high apex at 2 hours after operation, compared with the control group, the difference was statistically significant (75.50±13.03 vs. 1.00±0.00, P<0.01), and quickly descended at 6 hours, and then slowing descended. The expression of NLRP3 mRNA was decreased at 2 hours after the operation, then increased gradually, and peaked at 12 hours, which was significantly higher than that in control group (4.03±0.71 vs. 1.00±0.00,P<0.05). The level of caspase-1 was significantly higher than that in control group at 2 hours (3.56±0.14 vs. 2.10±0.11,P<0.01) and then gradually increased. The levels of Occludin, ZO-1 and Claudin-4 were obviously lowered than that in control group at 2-6 hours (2 hours Occludin: 7.24±1.13 vs. 12.72±1.34, 6 hours ZO-1: 0.47±0.09 vs. 1.57±0.17, 2 hours Claudin-4: 1.63±0.28 vs. 3.40±0.34, P<0.05 or P<0.01), and then all slowly decreased. The activity of NF-ΚB was quickly increased at 2 hours, obviously higher than that in control group (24.85±0.57 vs. 12.42±0.73, P<0.01), and then slowly decreased at a state of high expression. The expression of IL-6 in experimental group had a peak at 6 hours (compared with the control group, 3088.07±330.03 vs. 26.19±7.58,P<0.01), and then slowly decreased. The level of TNF-α was significantly higher than that in control group at 2 hours (110.75±19.18 vs. 7.86±3.58,P<0.01), and then gradually increased. The percentage of dead enterocyte was higher than that in control group with infection progress (0.12±0.02 vs. 0.03±0.01,P<0.05), and then gradually increased, so mucosal permeability was gradually increased too. Compared with the control group, the difference was statistically significant through 2 hours [glucosans: (35.75±4.66)% vs. (2.84±0.35)%, P<0.01]. The relevance analysis showed that NLRP3 have a little higher correlation with mucosal permeability and caspase-1 protein expression than other targets. Caspase-1 had a strong correlation with the percentage of dead cell, TNF-α and gut mucosal permeability. Gut mucosal permeability had highest correlation with the expression of caspase-1 protein.


CONCLUSIONS
The data of our study suggested that NOD2 and NLRP3 take role in early phase of intra-abdominal infection, the huge wave of the expression level of NOD2 hinted that it was feed backed by some accurate mechanism in case of its express was too strong or too weak. The correlation of NLRP3, caspase-1, and percentage of dead cell imply they maybe have some extent of causation, and the percentage of dead cell in gut mucosa was as important as tight junction protein in maintaining the function of intestinal mucosal barrier.
Department of General Surgery, Tianjin Tianhe Hospital, Tianjin 300050, China. Corresponding author: Wang Xi-mo, Department of General Surgery, Tianjin Union Medical Center, Colorectal and Anal Disease Institute, Tianjin 300121, China, Email: wangximo@126.com.
Yuan-ting
YT
Xiao
Guo-xun
GX
Li
Xi-mo
XM
Wang
D000818:Animals; D002352:Carrier Proteins; D020170:Caspase 1 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D059413:Intraabdominal Infections / Q000378:metabolism*; D008297:Male; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D053473:Nod2 Signaling Adaptor Protein / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015398:Signal Transduction
D002352:Carrier Proteins; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C512493:NOD2 protein, rat; C573346:Nlrp3 protein, rat; D053473:Nod2 Signaling Adaptor Protein; D018160:Receptors, Cytoplasmic and Nuclear; D020170:Caspase 1