Toll-like receptor 2 mediates ischemia-reperfusion injury of the small intestine in adult mice.

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.

Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine.

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates innate and adaptive immunity. Recent studies have shown that the activation of TLR-dependent signaling pathways plays important roles in the pathogenesis of ischemia-reperfusion (I/R) injuries in many organs. All TLRs, except TLR3, use a common adaptor protein, MyD88, to transduce activation signals. We investigated the role of MyD88 in I/R injury of the small intestine. MyD88 and cyclooxygenase-2 (COX-2) knockout and wild-type mice were subjected to intestinal I/R injury. I/R-induced small intestinal injury was characterized by infiltration of inflammatory cells, disruption of the mucosal epithelium, destruction of villi, and increases in myeloperoxidase activity and mRNA levels of TNF-α and the IL-8 homolog KC. MyD88 deficiency worsened the severity of I/R injury, as assessed using the histological grading system, measuring luminal contents of hemoglobin (a marker of intestinal bleeding), and counting apoptotic epithelial cells, while it inhibited the increase in mRNA expression of TNF-α and KC. I/R significantly enhanced COX-2 expression and increased PGE(2) concentration in the small intestine of wild-type mice, which were markedly inhibited by MyD88 deficiency. COX-2 knockout mice were also highly susceptible to intestinal I/R injury. Exogenous PGE(2) reduced the severity of injury in both MyD88 and COX-2 knockout mice to the level of wild-type mice. These findings suggest that the MyD88 signaling pathway may inhibit I/R injury in the small intestine by inducing COX-2 expression.

Dual action of nitric oxide in the pathogenesis of ischemia/reperfusion-induced mucosal injury in mouse stomach.

AIM: We investigated the roles of NO/NOS isoforms in the pathogenesis of ischemia/reperfusion (I/R)-induced gastric injury in mouse stomachs. METHODS: Under urethane anesthesia, the celiac artery was clamped, and then reperfusion was established 30 min later by removal of the clamp. After a 60-min reperfusion, the stomach was examined for macroscopic lesions. RESULTS: Following I/R, hemorrhagic lesions were generated in the mucosa, although ischemia alone caused no visible damage. Prior administration of L-NAME (a nonselective NOS inhibitor) significantly aggravated these lesions, in a L-arginine-inhibitable manner. By contrast, the selective iNOS inhibitor 1400W significantly prevented the occurrence of I/R-induced gastric lesions. The mucosal MPO activity was increased after I/R, and this response was enhanced and attenuated by prior administration of L-NAME and 1400W, respectively. Interestingly, the later treatment with L-NAME, given 10 min before reperfusion, significantly reduced the severity of the I/R-induced gastric damage, in a L-arginine-dependent manner. The expression of iNOS mRNA was up-regulated in the stomach following I/R, with an increase of mucosal NO content, and the NO production was significantly inhibited by both L-NAME and 1400W. CONCLUSION: Endogenous NO plays a dual role in the pathogenesis of IR-induced gastric damage; NO/cNOS is protective while NO/iNOS is proulcerogenic during I/R.

Pathogenic importance of pepsin in ischemia/reperfusion-induced gastric injury.

We investigated the role of pepsin in the development of ischemia/reperfusion (I/R)-induced gastric lesions in rats. Under urethane anesthesia, the pylorus was ligated, the celiac artery was clamped, and 1 ml of HCl (50-150 mM) was instilled in the stomach. Then, reperfusion was established 15 min later by removing the clamp, and 2 h later the stomach was assessed for gross mucosal damage. Pepstatin (a specific pepsin inhibitor) or pepsin was given i.g. after the pylorus was ligated while cimetidine, omeprazole, or atropine was given s.c. 30 min before the ligation. I/R produced hemorrhagic gastric injury, with a concomitant increase in the amount of pepsin secreted, and the degree of both these responses was dependent on the concentration of HCl. The formation of lesions by IR in the presence of 100 mM HCl was significantly prevented by atropine or bilateral vagotomy, but neither omeprazole nor cimetidine had any effect. Intragastric administration of pepstatin dose-dependently reduced the severity of the I/R-induced gastric lesions, the effect being significant even at 0.1 mg/kg, while that of pepsin markedly aggravated these lesions. The increased pepsin output during I/R was associated with luminal acid loss and significantly inhibited by bilateral vagotomy or pretreatment with atropine but not cimetidine or omeprazole, while pepstatin significantly inhibited the pepsin activity. In conclusion, we suggest that pepsin plays a pivotal role in the pathogenesis of I/R-induced gastric lesions, and pepsin secretion is increased during I/R, the process being associated with acid back-diffusion and mediated through a vagal-cholinergic pathway.

Roles of cyclooxygenase-2 and prostacyclin/IP receptors in mucosal defense against ischemia/reperfusion injury in mouse stomach.

We examined the roles of cyclooxygenase (COX) isozymes, prostaglandins (PGs), and their receptors in the mucosal defense against ischemia/reperfusion (I/R)-induced gastric lesions in mice. Male C57BL/6 mice, including wild-type animals and those lacking prostaglandin E(2) (EP)1, EP3, or prostaglandin I(2) (IP) receptors, were used after 18 h of fasting. Under urethane anesthesia, the celiac artery was clamped (ischemia) for 30 min, and then reperfusion was achieved for 60 min through the removal of the clamp, and the stomach was examined for lesions. I/R produced hemorrhagic gastric lesions in wild-type mice. The severity of lesions was significantly increased by pretreatment with indomethacin (a nonselective COX inhibitor) and rofecoxib (a selective COX-2 inhibitor) but not 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; a selective COX-1 inhibitor). The expression of COX-2 mRNA was up-regulated in the stomach following I/R but not by sham operation or ischemia alone. The ulcerogenic response was markedly aggravated in IP receptor knockout mice but not those lacking EP1 or EP3 receptors. I/R increased the levels of 6-keto-PGF(1alpha) and PGE(2) in the stomach of wild-type mice, and this response was attenuated by indomethacin and rofecoxib but not SC-560. Pretreatment of wild-type mice with iloprost, a prostacyclin (PGI(2)) analog, significantly prevented the I/R-induced gastric lesions in the absence and presence of indomethacin or rofecoxib. PGE(2) also reduced the severity of I/R-induced gastric lesions, yet the effect was much less pronounced than that of iloprost. These results suggest that endogenous PGs derived from COX-2 play a crucial role in gastric mucosal defense during I/R, and this action is mainly mediated by PGI(2) through the activation of IP receptors.

Mechanisms underlying capsaicin-stimulated secretion in the stomach: comparison with mucosal acidification.

The effects of capsaicin and mucosal acidification on gastric HCO3(-) secretion were compared in wild-type and prostacyclin (PGI2) IP receptor or prostaglandin E receptor EP1 or EP3 knockout C57BL/6 mice as well as rats. Under urethane anesthesia, the stomach was mounted on an ex vivo chamber, perfused with saline, and the secretion of HCO3(-) was measured at pH 7.0 using the pH-stat method. Capsaicin or 200 mM HCl was applied to the chamber for 10 min. Capsaicin increased the secretion of HCO3(-) in rats and wild-type mice, the response at 0.3 mg/ml being equivalent to that induced by acidification. This effect of capsaicin in rats was abolished by ablation of capsaicin-sensitive afferent neurons and attenuated by indomethacin, N(G)-nitro-L-arginine methylester (L-NAME), and capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist] but not FR172357 [3-bromo-8-[2,6-dichloro-3-[N[(E)-4-(N,N-dimethylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-metylimidazo[1,2-a]pyridine; bradykinin B2 antagonist] or the EP1 antagonist. The acid-induced HCO3(-) secretion was attenuated by indomethacin, L-NAME, the EP1 antagonist, and sensory deafferentation, but not affected by capsazepine or FR172357. Prostaglandin E2 (PGE2), NOR-3 [(+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine] (NO donor), and bradykinin stimulated the secretion of HCO3(-), and the effect of bradykinin was blocked by indomethacin and L-NAME as well as FR172357. The stimulatory effect of capsaicin disappeared in IP (-/-) mice, whereas that of acidification disappeared in EP1 (-/-) mice. Intragastric application of capsaicin increased mucosal PGI2 but not PGE2 levels in the rat stomach. These results suggested that both capsaicin and acid increase gastric HCO3(-) secretion via a common pathway, involving PG and NO as well as capsaicin-sensitive afferent neurons, yet their responses differ concerning TRPV1 or prostanoid receptor dependence.