All experimental procedures were approved by the Animal Care Committee of the Osaka City University Graduate School of Medicine (Permit Number: 11006)
All experimental procedures were approved by the Animal Care Committee of the Osaka City University Graduate School of Medicine (Permit Number: 11006). to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1s effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and Metanicotine the expression of tumor necrosis factor (TNF) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNF expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNF expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNF, whereas ulcer healing in TLR2 knockout (KO) mice was comparable to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNF expression. Thus, we showed that HMGB1 is usually a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses. Introduction High-mobility group box protein 1 (HMGB1), a member of the high-mobility group protein superfamily, is usually a nuclear protein [1]. HMGB1 interacts with DNA as a chromatin-associated nonhistone protein to stabilize nucleosomes and to regulate the transcription Metanicotine of many genes in the nucleus [2]. When leaked from a cell during necrotic cell death [3] or actively secreted into the extracellular environment by monocytes and macrophages [3,4], HMGB1 acts as an alarmin with potent proinflammatory properties [5]. The best studied HMGB1 receptors are Toll-like receptor (TLR) 2 [6,7], TLR 4 [6-9], and receptor for advanced glycation end products (RAGE) MGF [6,8]. TLR2 and TLR4 are members of the TLR family, and they play a crucial role in innate immune responses to pathogen-associated molecular patterns and damage-associated molecular pattern molecules [10]. TLR2 primarily recognizes components of Metanicotine the gram-positive bacterial cell wall, and TLR4 primarily recognizes lipopolysaccharide, which is the major cell wall component of gram-negative bacteria. Triggering TLR2 and TLR4 signaling pathways leads to the activation of nuclear factor B (NF-B), through the accessory protein MyD88, and the subsequent regulation of immune and inflammatory genes, including inflammatory cytokines such as tumor necrosis factor (TNF), with the activation of mitogen-activated protein kinases [11-13]. Receptor for advanced glycation end products (RAGE) is usually a multi-ligand receptor that belongs to the immunoglobulin superfamily [14]. Other known RAGE ligands include amyloid [15] and S100 [16]. Multiple experiments have suggested that this ligand-RAGE conversation also activates NF-B and mitogen-activated protein kinases [17-20]. Many pathological conditions are related to the proinflammatory properties of HMGB1. Previous reports exhibited that HMGB1 plays a critical role in endotoxemia [21], acute pancreatitis [22], acute respiratory distress syndrome [23], some autoimmune diseases [24], cerebral ischemia injury [25], and ischemia-reperfusion (I-R) injuries of the liver [26], heart [27], and kidney [28]. With regard to the gastrointestinal tract, HMGB1 is usually a complicating factor in experimental colitis [29,30], and non-steroidal anti-inflammatory drug induced small intestinal injury [31]. At present, the role of HMGB1 in wound healing is usually unclear, although its ability to induce inflammation has been well documented, as described above. In the gastrointestinal field, no study has examined the role of HMGB1 in wound healing. The aim of this study was to investigate the role of HMGB1 in gastric ulcer healing. We investigated the role of HMGB1 in the healing process by using an established experimental chronic gastric ulcer model created in rodent by topical application of Metanicotine acetic acid from the gastric serosal side. The model closely mimics human peptic gastric ulcer in histology and morphology [32]. We also investigated whether HMGB1 affects ulcer healing through TLR2, TLR4, or RAGE. Materials and Metanicotine Methods Animals TLR2- and TLR4-knockout (KO) mice, which were originally generated by Dr. S. Akira (Osaka University, Osaka, Japan) and backcrossed 8 occasions onto a C57BL/6 background,.