Lipoxin A4 Suppresses Osteoclastogenesis In RAW264.7 Cells And Prevents Ovariectomy-Induced Bone Loss

Objective1.To study the influence of LXA4 on the differentiation of RAW264.7cell to osteoclast and the function of mature osteoclast.2.To investigate the molecular mechanism of the effection of LXA4 on the ditterentiation and function of osteoclast in RAW264.7 cell.3.To observe the change if the microstructure of bone and other serological indicators in ovariectomized mice models treated with LXA4.Methods1.CCK-8 was used to test the influence of LXA4 and Boc-2 on the proliferation of RAW264.7 cell in certain concentration.Expression of the LXA4receptor FPR2/ALX in RAW264.7 and mature osteoclast was measured,then the influence of the receptor antagonist on their expression were furthe observed.RANKL was used to induce the differentiation of RAW264.7 to osteoclast and TRAP staining was performed to detect the influence of LXA4 and its antagonist on osteoclast formation and activity of TRAP enzyme.Osseous lamella resorption test was proceeded to observe the influence of LXA4 on the resorption ability of mature osteoclast.2.Real time quantitative PCR and Western Blot were used to detect the influence of LXA4 on the expression of marker genes and relevant signals in the process of inducing RAW264.7 to osteoclast differentiation with RANKL.The effect of LXA4 on NF-κB and AP-1 was detected by Electrophoretic Mobility Shift Assay.3.Use 12 weeks old female C57/BL6 mice to establish osteoporosis animal models by ovariectomy.Experimental group was intraperitoneal injected with LXA4(10ug/kg/d).6 weeks later,all the mice were sacrificed and the femurs were dissected for Micro-CT scanning and TRAP staining.Inflammatory factors level in serum were also examined.Results1.Differentiation of RAW264.7 to osteoclast and function of mature osteoclast could be significantly inhibited by LXA4,while this effect could be blocked by the Boc-2 which was a specific antagonist of LXA4 receptor FPR2/ALX.2.LXA4 could inhibit the expression of specific genes of osteoclast including TRAP,MMP-9,Cathepsin K,RANK.Transcription factors related to osteoclast namely c-Fos and NFATcl could also be inhibited by LXA4.Meanwhile,activation of p-38,ERK and JNK in MAPKs and Akt pathway were also down-regulated,inhibiting the activation of NF-kB and AP1 induced by RANKL and the formation of ROS.3.Bone loss in ovariectomize mice were obviously inhibited by LXA4.Ration between RANK and osteoprotegerin as well as inflammatory factors level were also reduced.ConclusionLXA4 can inhibit the RANK-RANKL pathway and down-regulation of its downstream signal transduction pathways and related genes,protein expression.In this way,LXA4 can inhibiting osteoclast differentiation and activation,as well as improve bone micro structure in the ovariectomized osteoporotic mice animal model.Therefor,LXA4 may providing a novel solution for the treatment of osteoclast relevant deseases...