The Effects Of Lipopolysaccharide On The Differentiation And Activity Of Mouse Osteoclasts In Vitro

Bone and joint infections promote osteoclast differentiation and activitation, which cause animbalance of bone resorption by osteoclasts and bone formation by osteoblasts, an important cause leadingto infective bone lysis and bone nonunion. Lipopolysaccharide (LPS), a component of the outer membranesof all gram-negative bacteria, was the first bacterial component shown to be capable of inducing osteoclastdifferentiation either directly or indirectly. Quercetin, as one of the most abundant flavonoids found inonions and other vegetables, has been highlighted as a bioactive substance with antioxidant activity andpotential clinical application in inflammatory diseases. Quercetin has been reported to prevent bone loss inovariectomy (OVx) animal models and to inhibit osteoclast formation in vitro. However, the effect ofquercetin on LPS-induced osteoclastogenesis has not yet been reported. In the present study, we aimed toinvestigate the effect of LPS-induced osteoclasts and to clarify the mechanism of bone formation ininflammatory diseases. The thesis consists of two parts.AIM OF THE STUDY: To investigate the effects of LPS on the differentiation and activity ofosteoclasts in vitro.METHODS:(1) A series concentrations of LPS (10,50,100,200,1000ng/ml) were used to inducemononuclear phagocyte line RAW264.7cells differentiation for4d. Osteoclast numbers were assessed byTRAP staining; the expressions of osteoclast-related genes including tartrate-resistant acid phosphatase(TRAP), matrix metalloproteinase-9(MMP-9) and cathepsin K (CK) were determined by real-timequantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-κB(RANK), tumor necrosis factor receptor-associated factor6(TRAF6) and cyclooxygenase-2(COX-2) weremeasured using Western blotting assays.(2) RAW264.7cells were treated with macrophage colony stimulating factor (M-CSF) andreceptor activator of NF-кB ligand (RANKL) for3d. Then a series concentrations of LPS (10,50,100,200,1000ng/ml) were added and cultured for another1d. The expressions of osteoclast-relatedgenes including TRAP, matrix metalloproteinase-9(MMP-9), receptor activator of NF-кB (RANK),cathepsin K and COX-2were measured by semiquantitative RT-PCR. RESULTS:(1) On differentiation day4, LPS directly promoted osteoclast differentiation ofRAW264.7cells; on differentiation day7bone slices resorption pits formatted. LPS at100ng/ml hassignificantly increased mRNA expression levels of TRAP, MMP-9and CK as well as up-regulated theprotein expression of RANK, TRAF6and COX-2than other treatment groups and non-treatment group(P<0.01).(2) LPS at100ng/ml has also significantly increased the expressions of osteoclast-related genesincluding TRAP, MMP-9, RANK, CK and COX-2.CONCLUSION:(1) LPS could stimulate preosteoclasts differentiation.(2) LPS promotes differentiated osteoclasts activity by up-regulated expression of osteoclast-relatedgenes.(3) LPS may play an important role in bone lysis in inflammatory diseases.