Effect Of Porphyromonas Gingivalis Lipopolysaccharide On The Expression Of EphA2in RAW264.7Cell

Background:Chronic periodontitis is a chronic inflammatory disease, which ischaracterized by the destruction of periodontal connective tissue andalveolar bone loss. Pg in dental plaque biofilm is one of the majorperiodontal pathogenic bacteria. Pg produces large amounts of virulencefactors, such as bacterial endotoxin, trypsin-like protease. The endotoxin,also called lipopolysaccharide (LPS), is a cell wall component inGram-negative bacteria, is a key factor caused by the resorption ofalveolar bone. It can cause bone resorption by promoting thedifferentiation of osteoclasts, but on the other hand, it also inhibitsosteoblast differentiation and bone formation. Bone remodeling is anorderly process coupling bone resorption and bone formation, where adynamic equilibrium is maintained for the environment within the bonehomeostasis, and lasts a lifetime. Recent studies show ephrinA2/EphA2bidirectional signal transduction pathway regulates bone homeostasis,particularly important role in the reconstruction of bone resorption phase.However, whether Pg-LPS activates the process of bone resorptionthough EphA2/ephrinA2signaling pathway has not been reported.Basedon the above background research, we propose a hypothesis, whether Pg-LPS involved in alveolar bone loss through EphA2/ephrinA2bidirectional signaling pathway? In this study, we use Pg-LPS co-culturedwith murine macrophages RAW264.7to focus on the effect of Pg-LPS onthe expression of EphA2in osteoclasts, and try to investigate the functionof EphA2signaling molecules in Pg-LPS-mediated inflammatory boneresorption.Methods:RAW264.7cell was co-cultured with10g/ml of Pg-LPS at1,3and5d. EphA2and the osteoclast-related genes (MMP9, c-fos,ACP5,CtsK and NFATc1) were detected by RT-PCR. EphA2protein wasdetected by Weston blotting. Tartrate-resistant acid phosphatase(TRAP)staining was used to detected osteoclast differentiation.Results:1. RT-PCR result showed that: After RAW264.7cell was co-culturedwith Pg-LPS for0.5h、1h and2h, EphA2gene expression in theexperimental group was significantly higher than control group.2. RT-PCR results showed that: After RAW264.7cell wasco-cultured with Pg-LPS, at3d and5d, EphA2gene expression in theexperimental group was significantly higher than control group (P<0.01),EphA2gene expression is more obvious at3d than5d. But there was nosignificant difference at1day between the two groups. Meanwhile, theexpression of osteoclast-related genes ACP5, MMP9, CtsK, c-fos andNFATc1was also significantly upregulated (P <0.05), but the expressionof CtsK at1d and c-fos at5d remained unchanged.3. Weston blotting result showed that:After RAW264.7cell was co-cultured with Pg-LPS, at1d, EphA2expression in theexperimental group was significantly higher than control group. However,at3d and5d, there were no significant differences.4. The TRAP staining result showed that: After RAW264.7cell wasco-cultured with Pg-LPS for5d, compared with the control, the numberof TRAP-positive multinucleated cells (containing≥3nuclei) wassignificantly increased in the experimental groups.Conclusions:1. In the absence of osteoclast-induced conditions, Pg-LPS canpromote RAW264.7cells expressing EphA2gene.2. In osteoclast-induced conditions, Pg-LPS can promote RAW264.7cell differentiate into osteoclast in the middle and later stages ofosteoclast differentiation, but has no obvious effect in the early stage.3. Pg-LPS possibly can promote RAW264.7cells into osteoclastdifferentiation through EphA2/ephrinA2signaling pathways.