The Role Of LPS, PGE₂ In Bone Regeneration

Background:The process of bone remodeling is complicated and many cells and cytokines inculding osteoblasts, osteoclasts participate in the process. The central of which is a dynamic balance precess result from the coupled activity of these two cell populations—the osteoblasts which mediate the bone formation process and the osteoclasts which mediate the bone resorbing process. However, the osteoporosis due to the bone resorbing rate exceed the bone formation rate and the non-union of bone owing to the stopping of the bone formation process are always the medical problems. Inflammation is a likely feature of bone repair processes, and several cytokines such as prostaglandins, leukotrienes, cytokines, and growth factors have been reported to be involved in bone metabolism. LPS (lipopolysaccharide, LPS) and PGE2 (prostaglandin E2, PGE2) are both important proinflammatory cytokines in bone regeneration.The mechanism of LPS on bone remodelling, however, is unclear as it has a biphasic response on cells and organs. PGE2 is the most potent stimulator during bone regeneration. The mechanism of PGE2 on bone formation has not been clarified. The activation of the fast response transcription factor nuclear factorκB (NF-κB),which exists normally as an complex composed of p65 and p50 had been reported to be involved in the regulation of the LPS-induced production of PGE2. The inhibition of NF-κB activity could block estrogen-deficiency induced bone loss. In this study, we focus on the relationship of LPS,PGE2 and NF-κB in bone regeneration in vitro and in vivo.Objective:To investigate the role of LPS and PGE2 in the bone regeneration process and the mechanism of signal conduction. To find the central factor for the therapy of nonunion or delayed union fractures providing a laboratory evidence.Methods:1 MC3T3-E1 osteoblasts were challenged with 500 ng/mL LPS,500 ng/mL LPS plus 50 nmol/L sc791 (selective COX-2 inhibitor), and 500 ng/mL LPS plus 5μmol/L BAY 11-7082(NF-κB inhibitor) respectively for Flow cytometry analysis,RT-PCR,western blot analysis, emsa and Immunofluorescence Analysis.2 Mouse MC3T3-E1 osteoblasts were challenged with 10μmol/L PGE2,5μmol/L BAY 11-7082 for 30 min,2 h and 4 h respectively. MTT method for Assessment of cell viability, cell differentiation was analyzed using ALP activity. Western blot analysis for NF-κB,BMP-7 and Id2 protein expression. Flow cytometry analysis for cell cycle and apoptosis rate. EMSA and Immunofluorescence analysis for NF-κB translocation and activation.3 Rats were randomly divided into three groups, including the controls, the radius fracture group-1 locally injected with normal saline and the radius fracture group-2 locally injected with 50 umol/L BAY 11-7082. The callus tissue were harvested at 3 d,7 d,14 d and 28 d after being fracture for Western blot analysis, ALP assessment, PGE2 production assay and histological observation.4 The cells were collected from the control group and the PGE2 treated group.The cell suspension with TRIZOL were immediately transmited to the "ShangHai biology gene chip company" for RAN extracted,measurement and hybridization with gene chip. By adopting software Feature extraction to analyze acquired genes of differential expression. Ratio≥2 or≤0.5, p Value Log Ratio<0.01 means the difference is significant. Results:1 500 ng/mL LPS could induce osteoblasts proliferation and increase the expression of NF-kB p65 in the nucleus and the specific NF-kB DNA binding activity, but had no influence on COX-2 expression at the same time. The cellular DPI value and NF-kB p65 protein expression had significant increase (P<0.01) at 6 h and 12 h after LPS treatment comparing to the control group. Whereas COX-2 expression had no significant difference (P>0.05) compared with untreated cells. NF-kB mRNA had no significant difference (P>0.05) but the protein expression had significant increase (P<0.01) comparing to the control group in the presence of LPS. BAY 11-7082 could inhibit the cell proliferation produced by 500 ng/mL LPS whereas sc791 could not do it. The results showed that low concentrations of LPS is beneficial for MC3T3-E1 osteoblast proliferation. LPS can induce proliferation of osteoblasts through NF-κB pathway and its induction is not correlated to COX-2.2 10μmol/L PGE2 exposure obviously caused inhibition of cell proliferation (P<0.01) and significant increasing the activity of ALP (P<0.01) in MC3T3-E1 cells at the time course of 2 h and 4 h. Preincubation with 5μmol/L NF-κB inhibitor can inhibit the PGE2-medicated ALP activity. Our data implied that PGE2 can increase ALP production through NF-κB activity. Western blot analysis indicated that the expression of NF-κB/p65 and BMP-7 protein in the stimulated cells were significant higher (P<0.01) than that of the controls after 10μmol/L PGE2 treated,whereas the Id2 expression was decreased (P<0.01) under the same conditions. NF-κB inhibitor BAY 11-7082 can inhibit the inductuion of PGE2. This means that PGE2 can inhibit the expresson of Id2 factors and increase the expression of NFKB/p65 and BMP-7 through NF-κB pathway. BAY 11-7082 plus PGE2 can induce osteoblast apoptosis but PGE2 alone can not do it.3 A significant increase of PGE2 production (P<0.01) was gained in the fracture groups compared with the controls after being fractured for 3 d and 7 d, while the production of PGE2 was substantially inhibited by the injection of specific inhibitors of NF-κB. In the same time, the ALP activity of callus was also increased and can be inhibited by the injection of NF-κB inhibitor BAY 11-7082. Just as the in vitro data, accompanying with the inhibition of NF-κB activation, the expression of BMP-7 was decreased, while the expression of Id2 was increased. These results implied that PGE2 production can be blocked through inhibition of the NF-κB pathway in vivo. Histological analysis results suggested that local injection of NF-κB inhibitor BAY 11-7082 at the fracture sites could result in delaying the union of fractures.4 Analysis on hybridezed chip sifts suggested that 276 significant up-regulated genes and 168 significant down-regulated genes after PGE2 treatment.The up-regulated genes induced by PGE2 related to inflammation and bone regeneration are:monocyte to macrophage differentiation-associated (MMD) gene. nuclear receptor subfamily 4, group A, member 2 gene, NR4A2, which is connected tightly with the p65-p50 heterodimer or homodimer of NF-κB. catenin (cadherin-associated protein). osteoblast specific factor (POSTN). bone morphogenetic protein 7 (osteogenic protein 1), BMP-7. The down-regulated genes induced by PGE2 related to inflammation and bone regeneration are:Id1, Id2 and Id3.Conclusion:1 Low concentration of lipopolysaccharide can induce directly proliferation of MC3T3-E1 osteoblasts mediated by COX-2-independent NF-κB pathway, and These studies provide insight into a potential mechanism by which NF-κB signal pathway has a central role in the initial phase of inflammation of the bone regeneratrion.2 PGE2 can induce osteoblast differentiation and promote bone formation by increasing BMP-7 protein expression and decreasing Id2 protein expression through NF-κB signal pathway. BAY 11-7082 can inhibit the osteoblast differentiation induced by PGE2 and induce the cell apoptosis with the role of PGE2. NF-κB can regulate the PGE2 production in vivo. BAY11-7082 can inhibit the PGE2 production, decrease the ALP activity and finally result in delaying the union of fractures. Cooperation between PGE2 production and NF-κB activation is the most primary step during the bone remodeling in vitro and in vivo.3 The up-regulated genes of promoting bone formation and nuclear receptor and the down-regulated genes of Id family found in expression of osteoblasts gene profile induced by PGE2 support our study results. Therefore, NF-κB could be taken as an important targeted gene in the bone healing process_and has a potential therapy role for nonunion or delayed union fractures.