The Therapeutic Effect of Heme Oxygenase-1 in the Indomethacin-Induced Intestinal Injury in Mice

Abstract

Background: Previously, we have reported that inflammatory mediators such as lipopolysaccharide promote indomethacin (Indo)-induced small intestinal damage via Toll-like receptor (TLR) 4 (Watanabe T et al, GUT 2008 ). High-mobility group box-1 (HMGB1), originally identified as a DNA binding protein, also has potent proinflammatory properties as an alarmin. HMGB1 exerts proinflammatory effect via the receptor for advanced glycation endproducts (RAGE) and Toll-like receptors (TLRs). AIM: To investigate the role of HMGB1 and the receptor responsible for HMGB1 in Indo-induced small intestinal damage.Methods: Indo (10 mg/kg body weight) was administered orally to C57BL/6J mice, and the small intestines were removed at 3, 6, 12, and 24 h after the administration of Indo.The mRNA expression of tumor necrosis factor-α (TNF-α), keratinocyte chemoattractant (KC), monocyte chemotactic protein-1 (MCP1), TLR4, RAGE, and HMGB1 were assessed by real-time reverse transcription-polymerase chain reaction. Localization of TLR4, RAGE, and HMGB1 in the small intestine was determined by immunohistochemical examination. To clarify the role of HMGB1 in Indo-induced small intestinal damage, mouse recombinant HMGB1 (rHMGB1), anti-HMGB1 neutralizing antibodies, ethyl pyruvate, which inhibits secretion of HMGB1, or nonspecific chicken IgY were intraperitoneally administered to the mice after administration of Indo and the small intestinal damage was evaluated. To investigate the involvement of RAGE and TLR4 in HMGB-1-mediated Indo-induced small intestinal damage, RAGE knockout (KO) or TLR4 KO or wild-type mice were intraperitoneally administered rHMGB1 accompanied by Indo administration and the damage was evaluated. Results: Indo-induced small intestinal damage occurred 3 h after the administration of Indo and the damage was accompanied by an increase in the expression of TNF-α and KCmRNAs in the small intestine. HMGB-1, TLR4, and RAGE mRNAs were constitutively expressed. Immunoreactivity for HMGB1 was mainly observed in the nuclei of the intestinal epithelial cells and inflammatory cells. Immunoreactivity for RAGE and TLR4 was mainly observed in macrophage cells. Inhibition of HMGB1 by neutralizing antibodies and ethyl pyruvate reduced the small intestinal damage, accompanied by reduction in the expression of TNF-α, KC, and MCP1 mRNAs, while rHMGB1 further increased the Indo-induced small intestinal damage. The Indo-induced small intestinal damage observed in RAGE KO mice was almost of the same level as that observed in wild-type mice, while gene disruption of TLR4 resulted in decrease in Indo-induced small intestinal damage. Conclusion: These results suggest that HMGB1 acts as a factor promoting the Indo-induced small intestinal damage by upregulating the proinflammatory cytokines through TLR4.