neoglycyrol and Inflammation

Glycyrol is a natural compound extracted from Glycyrrhiza uralensis, first reported by us to be a new immunosuppressant. Here, we demonstrate its beneficial effect in collagen-induced arthritis (CIA) in mice, a model for rheumatoid arthritis (RA) in man, and we document the underlying mechanisms. Peroral administration of glycyrol significantly reduced clinical scores, alleviated cartilage and bone erosion and reduced levels of serum inflammatory cytokines. Glycyrol also decreased delayed-type hypersensitivity, improved carbon clearance and reduced acetic acid-induced capillary permeability. Furthermore, glycyrol decreased NF-κB and NFAT transcriptional activities and inhibited IL-2 expression. The therapeutic effect of glycyrol was associated with down-regulation of both autoimmune and inflammatory reactions. In addition, we demonstrated that glycyrol has minimal acute toxicity in mice. Therefore, we propose that glycyrol may hold promise for future treatment of RA.

Topics: Animals; Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Experimental; Capillary Permeability; Cartilage, Articular; Cytokines; Down-Regulation; Drug Evaluation, Preclinical; Flavonoids; Hypersensitivity, Delayed; Immunosuppressive Agents; Inflammation; Interleukin-2; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; NF-kappa B; NFATC Transcription Factors; Phagocytosis; Phytotherapy; Transcription, Genetic

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

The anti-inflammatory effects of glycyrol, a benzofuran coumarin isolated from Glycyrrhizae Radix, were studied. Glycyrol of 5, 25 and 50 microM dose-dependently inhibited nitric oxide (NO) production by down-regulating inducible nitric oxide synthase (iNOS), and alleviated cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages, in both the mRNA and the protein. Furthermore, glycyrol dose-dependently decreased the mRNA of the pro-inflammatory cytokines IL-1beta and IL-6. LPS-induced NF-kappaB activation was prevented in RAW264.7 macrophages by inhibition of I-kappaBalpha phosphorylation. In addition, administration of glycyrol (30 and 100 mg/kg, i.p) reduced the thickness of carrageenan-induced mouse-paw edema swelling. Taken together, our results indicate that glycyrol is an important anti-inflammatory constituent of Glycyrrhizae Radix, and that its anti-inflammatory effect is attributed to the inhibition I-kappaBalpha phosphorylation.

Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cell Line; Cell Survival; Cyclooxygenase 2; Edema; Flavonoids; Glycyrrhiza uralensis; I-kappa B Kinase; Inflammation; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages; Male; Mice; NF-kappaB-Inducing Kinase; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Serine-Threonine Kinases