Research On Changes Of Bone Metabolism And Its Regulation Mechanism In Ankylosing Spondylitis

BackgroundAnkylosing spondylitis(AS)is a disease characterized by inflammation of the sacroiliac joint attachment.Its main pathological changes are inflammation and ossification.Osteoporosis(OP)is a common pathological change in AS.Although there are many studies on bone metabolism of AS,its mechanism has not been clarified,and there is no medicine to cure the disease.ObjectiveTo study the changes of bone metabolism indexes in model mice of ankylosing spondylitis;To investigate the effects of GSK3β,Wnt signaling pathway and NF-κB signaling pathway on bone metabolism in AS model mice;To research preliminarily on the effect of metformin and bone metabolism in AS model mice.Methods1.BLAB/c mice were induced by proteoglycan to establish animal model.The experimental animals were divided into four groups:AS group,OP group,AS&OP group and control group.Bone mineral density(BMD)and bone biomechanical properties of mice were measured.Serum concentrations of CA,P,ALP and Trap in mice were measured.2.Serum concentration of bone metabolic indexes and signaling pathway protein in all mice were determined by ELISA:PINP,β-CTX,25(OH)D,OPG,RANKL and β-Catenin,protein expression of OPG,RANKL and β-Catenin was determined by Western blot.C2C12 cells were induced with BMP2.Next,IWP-2 was used.Protein expressions of Wnt,β-catenin,ALP,OCN and RUNX2 were measured.3.Western-blot assay was used to detect Wnt and GSK-3β protein in AS group.RAW264.7 cells were induced by GSK-3β,then the SB216763 inhibitor was used.the protein expression of p65 and RANKL in RAW264.7 cells was determined.4.As model mice were given metformin by gavage.The peripheral arthritis score of mice was measured.The pathological characteristics and immunohistochemical expression of bone and joint specimens in each group mice were observed under microscope.Results1.Bone mineral density,maximum load and flexural elastic modulus in AS&OP group model mice were the lowest,and the concentrations of serum Ca,ALP and trap changed most significantly.The difference was statistically significant compared with the control group.2.The ELISA assay showed that the serum concentrations of PINP,25(OH)D and OPG in AS&OP model mice decreased significantly.However,the serum concentrations of β-CTX and RANKL in AS&OP model mice were significantly up-regulated.The difference was statistically significant compared with the control group.The serum concentration of β-catenin was up-regulated in AS group and down-regulated in OP group and AS&OP group.Western-blot assay showed that the expression of OPG was significantly down-regulated and the expression of RANKL protein were significantly up-regulated in AS&OP group.The expression ofβ-catenin protein was up-regulated in AS group,while in OP group and AS&OP group the expression of β-catenin protein was down regulated.BMP2 was used to induce C2C12 cells.The expression of Wnt and β-catenin protein was significantly up-regulated.After adding iwp-2,expressions of ALP,OCN and Runx-2 were significantly down regulated.3.Western-blot assay showed the expression of GSK3β and Wnt protein was up-regulated in AS model mice.GSK3 β was used to induce RAW264.7 cells.the expression of RANKL and p65 protein was significantly up-regulated.After adding sb216763,the expression of RANKL and p65 protein was down regulated.4.The peripheral arthritis score of AS mice reached the peak at the 12th week.Pathological sections assay under microscope showed degeneration and destruction of articular cartilage were found in AS model mice,and a large number of vacuole like cells were found in the medullary cavity.In metformin group,the structure of medullary cavity was complete,and the cells in medullary cavity were arranged orderly.The articular periosteum was smooth and flat.In the sulfasalazine group,osteophytes were formed,degeneration and destruction of articular cartilage were also found.Immunohistochemical assay showed that expression of BMP2 was strongly positive in metformin group,while expressions of Runx2 and Wnt3a were positive.In sulfasalazine group,expressions of BMP2,Runx2 and RANKL were positive.RANKL expression was strongly positive in AS group.ConclusionBone metabolism in AS model mice is out of balance.Wnt/β-catenin signaling pathway has a positive regulatory role in osteogenic metabolism,and NF-κB signaling pathway has a negative regulatory role in osteoclast metabolism..GSK3 βnegatively regulates Wnt/β-catenin and positively regulates NF-κB,which leads to imbalance of bone metabolism in AS model mice.After AS mice was treated with metformin,Wnt signaling pathway was activated,NF-κB signaling pathway was inhibited.Osteogenic metabolism enhanced significantly,and osteoclastic metabolism weakened.The curative effect of metformin was better than sulfasalazine.