moronic-acid and Inflammation

This study focuses on exploring the role and mechanism of moronic acid (MOA), a small triterpenoid molecule, against inflammatory bowel disease (IBD). Intestinal macrophages were cultured in vitro, and their M1 polarization was induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). After intervention with MOA, the proportion of M1 macrophages was detected, and the levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) were examined by ELISA. IFA staining was performed to determine the P50 and CD86 expressions, while DCFH-DA was used to determine the reactive oxygen species (ROS) level, as well as the p-P50 and NLRP3 protein levels. Additionally, we also used N-acetylcysteine, a ROS inhibitor, to further explore the association between MOA and ROS-NF-κB signaling. In murine experimentation, colitis was induced in mice with DSS. After MOA intervention, we assessed the mucosal barrier damage, tissue ROS, as well as protein and inflammatory cytokine levels. MOA could inhibit the M1 polarization of intestinal macrophages, suppress the expressions of inflammatory cytokines, and reduce the level of ROS-NF-κB-NLRP3 signaling. After inhibiting ROS through NAC treatment, the effect of MOA was evidently weakened. Clearly, MOA exerted its activity via ROS. In the murine model, MOA could lower the CD86 level in the intestinal tissues, inhibit the M1 polarization of macrophages, and reduce the tissue levels of inflammatory cytokines. This study finds that MOA can regulate ROS-NF-κB-NLRP3 signaling by inhibiting ROS, thereby suppressing the M1 polarization of intestinal macrophages, which plays a protective role in IBD.

Topics: Animals; Colitis; Cytokines; Inflammation; Inflammatory Bowel Diseases; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature