6-cyano-7-nitroquinoxaline-2-3-dione and Colitis--Ulcerative

Our previous study has found that aureusidin can inhibit inflammation by targeting myeloid differentiation 2 (MD2) protein. Structural optimization of aureusidin gave rise to a derivative named CNQX. LPS was used to induce inflammation in intestinal macrophages; flow cytometry, PI staining and Hoechst 33342 staining were used to detect the apoptotic level of macrophages; enzyme-linked immunosorbent assay (ELISA) was utilized to detect the expression level of inflammatory factors (including IL-1β, IL-18 and TNF-α); immunofluorescence staining was used to investigate the expression of MD2; Western blot was employed to measure the protein level of TLR4, MD2, MyD88 and p-P65. As a result, CNQX with IC50 of 2.5 μM can significantly inhibit the inflammatory damage of macrophages, decrease apoptotic level, reduce the expression level of inflammatory factors and simultaneously decrease the expression level of TLR4, MD2, MyD88 as well as p-P65. Caco-2 cell line was used to simulate the intestinal mucosal barrier in vitro, LPS was employed to induce cell injury in Caco-2 (to up-regulate barrier permeability), and CNQX with IC50 of 2.5 μl was used for intervention. Flow cytometry was used to detect the apoptotic level of Caco-2 cells, trans-epithelial electric resistance (TEER) was measured, FITC-D was used to detect the permeability of the intestinal mucosa, and Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin-1, MyD88, TLR4 and MD2). As a result, CNQX decreased the apoptotic level of Caco-2 cells, increased TEER value, decreased the expression levels of MyD88, TLR4 and MD2, and increased the protein levels of tight junction proteins (including occludin and claudin-1). C57BL/6 wild-type mice were treated with drinking water containing Dextran sulphate sodium (DSS) to establish murine chronic colitis model. After CQNX intervention, we detected the bodyweight, DAI score and H&E tissue staining to evaluate the life status and pathological changes. Immunohistochemistry (IHC) staining was used to detect the expression of MD2 protein, tight junction protein (including occludin and claudin-1). Transmission electron microscopy and FITC-D were used to detect intestinal mucosal permeability. Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin-1, MyD88, TLR4 and MD2) in the intestinal mucosa tissue. Consequently, CNQX can inhibit the intestinal inflammatory r

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anti-Inflammatory Agents; Caco-2 Cells; Colitis, Ulcerative; Humans; Intestinal Mucosa; Lymphocyte Antigen 96; Macrophages; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Toll-Like Receptor 4; Transcription Factor RelA