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The effect of Lipoxin A4 on the interaction between macrophage and osteoblast: possible role in the treatment of aseptic loosening.

Li G, Wu P, Xu Y, Yu Y, Sun L, Zhu L, Ye D - BMC Musculoskelet Disord (2009)

Bottom Line: Exogenous 0-100 nM LXA4 presented an inhibitory effect on both generation of above cytokines and PMMA stimulated calvarial bone resorption with a dose-dependent manner.LXA4 in supernatant from neither rest macrophages nor macrophages cultured alone exposing to PMMA was detectable.In the present study, we demonstrated that LXA4 had a favorable inhibitory effect on PMMA-induced inflammation in a macrophage and OB co-culture system.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Department of surgery, Liyuan Hospital, Huazhong University of Science and Technology, Wuhan, PR China. lg30003000@yahoo.com.cn

ABSTRACT

Background: Aseptic loosening (AL) is the main problem of total joints replacement (TJR) by the implantation of permanently prosthetic components. In vitro and in vivo studies have clearly demonstrated that wear debris and its byproducts could trigger inflammation in the peri-implant tissue. Lipoxins (LXs) are endogenous eicosanoids synthesized locally from arachidonate acid (AA) at sites of inflammation and mediate pro-resolving activity. A number of studies have demonstrated the effect of LXA4 to counteract inflammation in different cell and animal models, but till now, no relative report about the role of LXs in progress or prevention of AL.

Methods: Murine RAW264.7 macrophage cell line and MC3T3-E1 osteoblasts (OB) cell line were purchased. Co-cultured model of these two cell lines was established. To explore the effect of exogenous Lipoxin A4 (LXA4) on polymethylmethacrylate (PMMA) induced inflammation, pro-inflammatory cytokines including TNF-alpha, IL-1beta, PGE2 and GM-CSF were measured by ELISA kits and bone resorption was quantified by measuring calcium release from 5-day-old mice calvaria in vitro. To determine further the endogenous effect of LXA4, cells were co-cultured and with or without 15-lipoxygease (15-LO) blocking by 15-LO siRNA. Both real-time PCR and western blotting were applied to confirm the inhibitory efficiency of 15-LO by siRNA.

Results: 0.1 mg/ml, 0.5 mg/ml and 1.0 mg/ml PMMA showed a time-dependent manner to trigger production of all the pro-inflammatory cytokines studied. Exogenous 0-100 nM LXA4 presented an inhibitory effect on both generation of above cytokines and PMMA stimulated calvarial bone resorption with a dose-dependent manner. LXA4 in supernatant from neither rest macrophages nor macrophages cultured alone exposing to PMMA was detectable. In co-cultured cells challenged by PMMA, LXA4 was increased significantly, while, this enhance could be partly inhibited by 15-LO siRNA. When LXA4 generation was blocked with 15-LO siRNA, the PMMA induced pro-inflammatory cytokines were elevated and bone resorption was accelerated.

Conclusion: In the present study, we demonstrated that LXA4 had a favorable inhibitory effect on PMMA-induced inflammation in a macrophage and OB co-culture system.

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Related in: MedlinePlus

Effect of PMMA on endogenous LXA4 production. A, LXA4 content in culture media was monitored with ELISA kit. **, P < 0.01 compared to macrophages+PMMA; #, P < 0.05 compared with macrophage+OB+PMMA. B, Inhibition of 15-LO siRNA on 15-LO mRNA expression measured by RT-QPCR. Results were normalized to GAPDH and expressed as fold induction over cells co-cultured with OB without 15-LO siRNA. C, Inhibition of 15-LO siRNA on 15-LO protein expression measured by western blotting. GAPDH was applied as internal control.
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Figure 4: Effect of PMMA on endogenous LXA4 production. A, LXA4 content in culture media was monitored with ELISA kit. **, P < 0.01 compared to macrophages+PMMA; #, P < 0.05 compared with macrophage+OB+PMMA. B, Inhibition of 15-LO siRNA on 15-LO mRNA expression measured by RT-QPCR. Results were normalized to GAPDH and expressed as fold induction over cells co-cultured with OB without 15-LO siRNA. C, Inhibition of 15-LO siRNA on 15-LO protein expression measured by western blotting. GAPDH was applied as internal control.

Mentions: Above data showed an important anti-inflammatory role of exogenous LXA4 on the PMMA induced inflammation in cultured macrophages. To determine whether endogenous LXA4 formation also played a role in the resolution of peri-implant inflammation induced by wear debris, we next determined weather PMMA could change the production of LXA4 in RAW 264.7 cells. RAW 264.7 macrophages were cultured alone or co-cultured with MC3T3-E1 OB cell line and treated with 1.0 mg/ml PMMA for 48 hours. It was found that, in supernatant from macrophages cultured alone, LXA4 could be detected in neither control cells nor cells treated with PMMA (Fig. 4.). In supernatant from co-cultured cells exposed to PMMA, LXA4 was increased significantly, while, this enhance could be partly inhibited by 15-LO siRNA, which could block the expression of 15-LO, a key enzyme to LXs production in macrophages[31]. The block of 15-LO expression by 15-LO siRNA in macrophages were confirmed by RT-QPCR and western blotting (Fig. 4B and 4C).


The effect of Lipoxin A4 on the interaction between macrophage and osteoblast: possible role in the treatment of aseptic loosening.

Li G, Wu P, Xu Y, Yu Y, Sun L, Zhu L, Ye D - BMC Musculoskelet Disord (2009)

Effect of PMMA on endogenous LXA4 production. A, LXA4 content in culture media was monitored with ELISA kit. **, P < 0.01 compared to macrophages+PMMA; #, P < 0.05 compared with macrophage+OB+PMMA. B, Inhibition of 15-LO siRNA on 15-LO mRNA expression measured by RT-QPCR. Results were normalized to GAPDH and expressed as fold induction over cells co-cultured with OB without 15-LO siRNA. C, Inhibition of 15-LO siRNA on 15-LO protein expression measured by western blotting. GAPDH was applied as internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698870&req=5

Figure 4: Effect of PMMA on endogenous LXA4 production. A, LXA4 content in culture media was monitored with ELISA kit. **, P < 0.01 compared to macrophages+PMMA; #, P < 0.05 compared with macrophage+OB+PMMA. B, Inhibition of 15-LO siRNA on 15-LO mRNA expression measured by RT-QPCR. Results were normalized to GAPDH and expressed as fold induction over cells co-cultured with OB without 15-LO siRNA. C, Inhibition of 15-LO siRNA on 15-LO protein expression measured by western blotting. GAPDH was applied as internal control.
Mentions: Above data showed an important anti-inflammatory role of exogenous LXA4 on the PMMA induced inflammation in cultured macrophages. To determine whether endogenous LXA4 formation also played a role in the resolution of peri-implant inflammation induced by wear debris, we next determined weather PMMA could change the production of LXA4 in RAW 264.7 cells. RAW 264.7 macrophages were cultured alone or co-cultured with MC3T3-E1 OB cell line and treated with 1.0 mg/ml PMMA for 48 hours. It was found that, in supernatant from macrophages cultured alone, LXA4 could be detected in neither control cells nor cells treated with PMMA (Fig. 4.). In supernatant from co-cultured cells exposed to PMMA, LXA4 was increased significantly, while, this enhance could be partly inhibited by 15-LO siRNA, which could block the expression of 15-LO, a key enzyme to LXs production in macrophages[31]. The block of 15-LO expression by 15-LO siRNA in macrophages were confirmed by RT-QPCR and western blotting (Fig. 4B and 4C).

Bottom Line: Exogenous 0-100 nM LXA4 presented an inhibitory effect on both generation of above cytokines and PMMA stimulated calvarial bone resorption with a dose-dependent manner.LXA4 in supernatant from neither rest macrophages nor macrophages cultured alone exposing to PMMA was detectable.In the present study, we demonstrated that LXA4 had a favorable inhibitory effect on PMMA-induced inflammation in a macrophage and OB co-culture system.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Department of surgery, Liyuan Hospital, Huazhong University of Science and Technology, Wuhan, PR China. lg30003000@yahoo.com.cn

ABSTRACT

Background: Aseptic loosening (AL) is the main problem of total joints replacement (TJR) by the implantation of permanently prosthetic components. In vitro and in vivo studies have clearly demonstrated that wear debris and its byproducts could trigger inflammation in the peri-implant tissue. Lipoxins (LXs) are endogenous eicosanoids synthesized locally from arachidonate acid (AA) at sites of inflammation and mediate pro-resolving activity. A number of studies have demonstrated the effect of LXA4 to counteract inflammation in different cell and animal models, but till now, no relative report about the role of LXs in progress or prevention of AL.

Methods: Murine RAW264.7 macrophage cell line and MC3T3-E1 osteoblasts (OB) cell line were purchased. Co-cultured model of these two cell lines was established. To explore the effect of exogenous Lipoxin A4 (LXA4) on polymethylmethacrylate (PMMA) induced inflammation, pro-inflammatory cytokines including TNF-alpha, IL-1beta, PGE2 and GM-CSF were measured by ELISA kits and bone resorption was quantified by measuring calcium release from 5-day-old mice calvaria in vitro. To determine further the endogenous effect of LXA4, cells were co-cultured and with or without 15-lipoxygease (15-LO) blocking by 15-LO siRNA. Both real-time PCR and western blotting were applied to confirm the inhibitory efficiency of 15-LO by siRNA.

Results: 0.1 mg/ml, 0.5 mg/ml and 1.0 mg/ml PMMA showed a time-dependent manner to trigger production of all the pro-inflammatory cytokines studied. Exogenous 0-100 nM LXA4 presented an inhibitory effect on both generation of above cytokines and PMMA stimulated calvarial bone resorption with a dose-dependent manner. LXA4 in supernatant from neither rest macrophages nor macrophages cultured alone exposing to PMMA was detectable. In co-cultured cells challenged by PMMA, LXA4 was increased significantly, while, this enhance could be partly inhibited by 15-LO siRNA. When LXA4 generation was blocked with 15-LO siRNA, the PMMA induced pro-inflammatory cytokines were elevated and bone resorption was accelerated.

Conclusion: In the present study, we demonstrated that LXA4 had a favorable inhibitory effect on PMMA-induced inflammation in a macrophage and OB co-culture system.

Show MeSH
Related in: MedlinePlus