G-CSF Mediates The Staphylococcus Aureus Osteomyelitis-induced Bone Loss

Object:To investigate the association and mechanism of granulocyte colony-stimulating factor-mediated Staphylococcus aureus infection-induced bone loss.Methods:1?To establish and test the model of Staphylococcus aureus chronic osteomyelitis:white blood cell count(WBC count),bacterial culture,radiography,magnetic resonance imaging.2?Staphylococcus aureus infection induced bone loss caused by decreased bone formation capacity:(1)Micro-CT study of the parameters of bone tissue;(2)q-PCR test:osteogenic and osteoclastic related gene expression in bone tissue samples;(3)bone formation capacity test:immunohistochemical staining of osteocalcin(OCN);(4)bone destruction capacity test:tartrate-resistant acid phosphatase(TRAP)staining.3?G-CSF antibody can alleviate the bone loss caused by Staphylococcus aureus infection:(1)screen of bone loss related inflammatory factors by bioinformatics methods;(2)G-CSF in chronic osteomyelitis patients serum was measured using enzyme-linked immunosorbent assay(ELISA);(3)G-CSF in Mouse serum and bone marrow supernatant were measured using enzyme-linked immunosorbent assay;(4)mononuclear macrophage immunohistochemistry in bone tissue;(5)Micro-CT study of bone tissue in mouse after G-CSF antibody injection;(6)leukocyte counts in mouse after G-CSF antibody injection;(7)bone tissue from mouse after G-CSF was examined for the expression of OCN by immunohistochemistry.4?The mechanism of G-CSF-induced bone loss:(1)pre-osteoblast cells were isolated from the femur bone of newborn mice;(2)The expression of osteoblast specific genes in bone tissues after G-CSF treatment was measured by q-PCR;(3)Alizarin Red staining test;(4)Wnt/?-catenin pathway and BMP/Smdl pathway were measured by Western Blot.Result:1?(1)Our results showed that in the 1st,2nd and 3rd week,the WBC count was significantly higher than that in the control group(p<0.05).While in the 4th and 5th week,compared with the control group,the WBC count was decreased,but the difference was not statistically significant;(2)X-ray examination showed that high-density shadow was found in the ilium and coccyx,which was near the injection site of the tail vein;(3)the results of magnetic resonance imaging showed thatabnormal signals was found in the tibial medullary;(4)results of bacterial culture identification:Staphylococcus aureus.2?(1)MicroCT-related parameters of bone:BV/TV,Tb/N,Tb/Th and Con.D were significantly lower than that in the control group;Tb/Sp and Tb.Pf were significantly higher than that in the control group.(2)q-PCR results showed that the expression of OCN and RUNX2 decreased significantly,while the expression of RANKL had no obvious change.(3)The number of OCN positive osteoblasts in infected group was significantly lower than that in control group.(4)the number of TRAP positive osteoclasts was higher than that in the control group,but the difference was not statistically significant.3?G-CSF antibody can alleviate the bone loss caused by Staphylococcus aureus infection:(1)we found that granulocyte-colony stimulating factor(G-CSF)showed the biggest change in all the inflammatory factors;(2)The serum level of G-CSF in patients with Staphylococcus aureus osteomyelitis was significantly higher than that of healthy controls;(3)ELISA results of animal infection model confirmed that the expression of G-CSF increased significantly in serum and bone marrow,especially in bone marrow;(4)The result of immunohistochemistry using the F4/80 antibody found that the number of mononuclear macrophages in the marrow secreting G-CSF also increased significantly;(5)after neutralizing G-CSF in serum,the phenomenon of the bone mass loss can partially reversed in model of Staphylococcus aureus chronic osteomyelitis.4.(1)Q-PCR result:Compared with the control group,the expression of RUNX2,ALP and Osterix rose initially and then decreased with the increasing of G-CSF concentration.Low dose(10 ng/mL)of G-CSF could promote the expression of RUNX2,ALP and Osterix.Middle dose(50 ng/mL)of G-CSF also show a positive effect.While,high dose(100 ng/mL)of G-CSF significant reduce the expression of RUNX2,ALP and Osterix;(2)The results of Alizarin Red showed that:Compared with the control group,the number of mineralized nodules rose initially and then decreased with the increasing of G-CSF concentration.Low dose(10 ng/mL)of G-CSF could promote the formation of mineralization nodules./While,high dose(100 ng/mL)of G-CSF significant reduce the the formation of mineralization nodules;(3)G-CSF could inhibit the Wnt/?-catenin and BMP/Smdl signaling pathway,thereby inhibiting osteogenic differentiation of osteoblast cells.Conlusion:G-CSF may inhibit the osteoblastic differentiation of osteoblast cells by inhibiting the Wnt/?-catenin and BMP/Smdl signaling pathway,and ultimately lead to the result of bone loss caused by Staphylococcus aureus infection.