Toshiko Murakami

989 Roles of Cyclooxygenase-1/Prostaglandins and Their Receptors in Modulating Gastric Mucosal Integrity Under Stress Conditions

Background: Ethyl pyruvate exerts its anti-inflammatory effects by inhibiting the secretion of high-mobility group box-1 (HMGB1). HMGB1 was initially reported to be a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein. HMGB1 release from damaged cells activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 4, which leads to tissue injuries. Aim: To determine whether ethyl pyruvate diminishes the inflammatory response in the gastric damage induced by nonsteroidal anti-inflammatory drug (NSAIDs) Methods: Indomethacin (30 mg/kg body weight) was orally administered to C57BL/6J mice and TLR4 knockout mice. Then, human recombinant HMGB1 (rHMGB1), ethyl pyruvate, or vehicle was administered to mice intraperitoneally. The gastric tissues were removed at 6 h after the indomethacin treatment. Gastric damage was assessed by measuring the bleeding areas. The mRNA expression of tumor necrosis factor-α (TNF-α), TLR4, and HMGB1 were assessed by real-time reverse transcription-polymerase chain reaction. Localization of TLR4 and HMGB1 in the gastric tissue was determined by immunohistochemical examination. Additionally, RGM1 cells (rat gastric mucosal cell line) were treated with indomethacin (1 mM) or vehicle. RGM1 supernatants were harvested at 1 h, 3 h, and 6 h after the indomethacin treatment, and the concentration of HMGB1 was determined via ELISA. Results: HMGB1 was detected mainly in the nuclei of the gastric epithelial cells and inflammatory cells. Endothelial cells and inflammatory cells such as macrophages showed immunoreactivity for TLR4 proteins. Macroscopic gastric damage was accompanied by increased expression of TNF-α mRNA. Administration of rHMGB1 significantly aggravated small intestinal damage (1.9-fold increase) and increased TNF-α expression by 1.6-fold. In contrast, ethyl pyruvate decreased damage by 73% and lowered the increased TNF-α expression by 46%. Deficiency of TLR4 markedly inhibited the expression of TNF-α mRNA and prevented increase in bleeding areas in gastric tissues, whereas ethyl pyruvate failed to inhibit gastric damage and expression of TNFα mRNA in TLR4 knockout mice. In the in vitro study, indomethacin significantly increased the release of HMGB1 from RGM1 cells (3.4-fold increase). Conclusion: These results suggest that release of HMGB1 from gastric epithelial cells may trigger inflammatory responses and aggravate the damage in NSAIDs-induced gastric damage, and ethyl pyruvate ameliorates NSAIDs-induced gastric damage via inhibition of the HMGB1/TLR4 signaling pathway.