Anti-inflammatory synergy effect of synthetic human peptides hBD-3 and LL-37 in a macrophage and gingival epithelial cell co-culture model activated by lipopolysaccharide from Porphyromonas gingivalis

Abstract

Periodontitis is a infection caused by certain microorganisms, such as Porphyromonas gingivalis which is one of the most important pathogens. The major virulence factors of this bacteria is the endodoxin lipopolysaccharide (LPS) that induces an inflammatory response in the individual and lead a exacerbated production of cytokines, which consequently leads to destruction of tooth supporting tissues, such as the periodontal ligament. So the mechanical treatment as a way to control dental plaque is well established as a preventive activity, and antibiotic drugs is used as adjuvant therapy. However, many cases the bacteria become resistant to antibiotics therapy. In this context, the use of antimicrobial peptides (AMPs) have demonstrated not only antimicrobial effects but also as modulators of inflammatory process in response to stimulation of Toll like receptors (TLR) by bacterial lipopolysaccharide leading to a decreased production of inflammatory cytokines has the advantage of broad-spectrum. Thus, the purpose of this study are to analyze the effect of anti-inflammatory peptides LL-37 (Catelicidin) and human beta-defensin-3 (hBD-3), as well as the synergistic effect of the combination of both, after stimulation with LPS from P. gingivalis and the molecular mechanisms involved in anti-inflammatory activity of the peptides in macrophage (U937 + PMA) and epithelial cell (OBA-09) co-culture model. Initially we will determine the cytotoxicity of peptides in co-culture model (OBA-9 and macrophages-U937) through the reduction assay Methyl-tetrazolium (MTT). The ability of peptides to regulated the production of cytokines and in the activation of intracellular signaling pathways involved in producing these inflammatory mediators used in vitro after stimulation with lipopolysaccharide (LPS) of P. gingivalis (agonist of TLR2) and Escherichia coli (agonist TLR4) will be determined by tests qPCR Array. Then the 6 cytokines that will be more express by qPCR Array will be quantify by ELISA. The 3 intracellular signaling pathways mediated by the Toll receptor more expressed by qPCR Array, will be assessed its phosphorylation by Western blot. Statistical analysis will be done using the SPSS software. (AU)