Neutrophil Transmigration into the Joint of RA-Induced Mouse Is Markedly Blocked By EC-18, Via STAT3 Signaling

Neutrophil trafficking during the inflammatory response is effectively regulated by the enhanced IL-6 expression which orchestrates chemokine production and leukocyte apoptosis. Inappropriate regulation of leukocyte trafficking can lead to impaired neutrophil clearance and increased tissue damage from the accumulation of neutrophil-secreted proteases and reactive oxygen species at the site of inflammation. In collagen-induced arthritis (CIA) mouse model, arthritic symptoms were recapitulated with an increase of IL-6 level in the synovium, which was recuperated by the treatment of EC-18 to the level comparable with commercial therapeutics (such as Remicade or Methotrexate). When the tissues were stained with neutrophil-specific antibodies, EC-18 significantly reduced the infiltration of neutrophils into the synovium. EC-18, a monoacetyl-diglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), has been isolated from the antlers of Sika deer (Cervus nippon Temminck), which were known to have immunosuppressive and anti-arthritic activities. In this study, we have discovered that EC-18 regulates the activity of signal transducer and activator of transcription 3 (STAT3), which is a master regulator of IL-6 expression. EC-18 caused the decrease of IL-6 production in a macrophage cell line, RAW264.7, and RA-fibroblast-like synoviocyte (RA-FLS) via the regulation of STAT3 signaling without affecting NF-κB signaling, which is also a well-known regulator of IL-6 expression. The expression of RANKL, a key factor for osteoclast differentiation and activation, was also regulated by STAT3, and decreased by EC-18 treatment. In fact, EC-18 inhibited osteoclastogenesis of hematopoietic stem cells. Therefore, blocking STAT3 activity by EC-18 treatment was able to inhibit the inflammatory cytokine loop triggering RANKL expression, thereby inhibiting joint destruction. Cardinal features of rheumatoid arthritis (RA) are chronic synovial inflammation and joint damage caused by the activity of cytokines and proteins expressed from cells in synovial fluid. Interleukin (IL)-6, a multifunctional pro-inflammatory cytokine, plays a critical role in the pathogenesis of joint and systemic inflammation in RA. Therefore, EC-18 could be utilized as a potential therapeutic agent for the treatment of sustained inflammation and joint destruction.