An Aqueous Extract of Freeze-dried Protaetia brevitarsis Larvae Enhances Immunostimulatory Activity in RAW 264.7 Macrophages by Activating the NF-κB Signaling Pathway

White-spotted flower chafer (Protaetia brevitarsis) larvae are a potential nutritional supplement and have been used in traditional Asian dietary medicine. In this study, we found that an aqueous extract of freeze-dried P. brevitarsis larvae (AEPB) promotes immunostimulation in RAW 264.7 macrophages. No significant cytotoxicity was observed below 800 mu g/mL AEPB. Moreover, AEPB treatment enhanced the production of nitric oxide (NO), prostaglandin E2 (PGE(2)), interleukin (IL)-6, and IL-12 through the upregulation of their regulatory genes. AEPB also promoted the nuclear translocation of nuclear factor-kappa B (NF-kappa B), and pyrrolidine dithiocarbamate, an inhibitor of NF-kappa B activation, remarkably prevented the expression of AEPB-induced inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6, and IL-12, indicating that AEPB promotes the production of immunostimulants such as NO and PGE2 and pro-inflammatory cytokines such as IL-6 and IL-12 in RAW 264.7 macrophages by activating the NF-kappa B signaling pathway. Moreover, AEPB upregulated the extracellular expression of Toll-like receptor 4 (TLR4) and subsequently increased myeloid differentiation primary response 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4) expression, which indicates that AEPB activated the NF-kappa B signaling pathway through the TLR4-mediated MyD88 and IRAK4 axis. Collectively, this study provides evidence that AEPB is a promising nutritional supplement for stimulating macrophage-mediated immune responses.