From Bmp/smad Signaling Pathways To Explore Epimedium Glycoside Of The Molecular Mechanisms Of Resistance To Ankylosing Spondylitis Ossification

Ankylosing spondylitis is a chronic inflammatory disease of axial joints and the pathogenesis is still unknown.Ankylosing spondylitis is one of the difficult medical problems, western medicine is still a lack of effective treatment.Traditional Chinese medicine has certain characteristics and advantages in the treatment of AS.Our preliminary experiments have shown:the BMP/Smad signaling pathway was activated in AS fibroblasts.With the interference of Bushenhuoxue Decoction, the BMP/Smad transductive signal of AS fibroblasts was restrained.And the expression of Cbfa-1was inhibited.Inhibiting the activation of BMP/Smad signal transduction pathway in AS fibroblast was one of the molecular mechanisms of Bushenhuoxue Decoction delaying ossification occurring in AS patients.Therefore, we speculate whether icariin have the same effect. This research using AS fibroblast system in vitro as the object, and taking the technique of western blotting, aimed to study the effect of Icariine on BMP/Smad signaling pathway on AS fibroblasts, and to dig out the molecular mechanisms of Icariine in anti-ossification of AS.1Experiment One:Objective:Eestablish a biological character stationary fibroblast line.Immunohistochemistry was used to identify the source of cells.To provide reliable material basis for the next experiment.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence and those AS patients.Identify the source of the cells with the method of Immunohistochemistry.Results:We estbalish a biological character stationary fibroblast line.And the Immunohistochemistry result was vimentin positive and ceratin negative.Conclusion:The result of the Immunohistochemistry shown the cells wewe originated from mesoderm.2Experiment TwoObjective:Expressions of Smadl,pSmad1, Smad4and cbfa-1in fibroblast from capsula articularis coxae of AS patients were compared with those from normal patients, and found out whether BMP/Smad pathway was activated in AS fibroblast. in order to testify that activation of BMP/Smad signal transduction pathway could induce AS fibroblast differentiating into osteoblast. Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence and those AS patients.Expressions of Smad1,pSmad1, Smad4and cbfa-1in normal fibroblast and AS fibroblast were dectected by western blotting.Results:Expressions of Smadl,pSmadl, Smad4and cbfa-1improved, comparing with those from control group (P<0.05).Conclusion:The elevated expression of Smad4,Smadl,pSmadl and cbfa-1reveal the BMP/Smad signal transduction pathway was activated in AS fibroblast.3Experiment ThreeObjective:Provide a theoretical basis for the next experiment by determining the effect of Icariine on AS fibroblast proliferation.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from AS patients. Fibroblast proliferation was detected by methyl thiazolyl tetrazolium (MTT) after different concentrations of Icariine (25,50,100200ug/ml) stimulating the cells for24h,48h and72h.Results:Icariine had obvious effect on AS fibroblast proliferation with the concentrations of100ug/ml (P<0.05).The concentrations of25ug/ml and50ug/ml had no obvious effect on AS fibroblast proliferation.(P>0.05).Conclusion:Icariine had obvious effect on AS fibroblast proliferation with the concentrations of100ug/ml.4Experiment FourObjective:Testify Icariine inhibited AS fibroblast differentiating into osteoblast through inhibiting BMP/Smad pathway from activating, and constraining the expression of Smad1,pSmad1, Smad4and cbfa-1, so that inhibiting ossification occurring was achieved.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from AS patients.Expressions of Smad1,pSmad1, Smad4and cbfa-1were dectected by western blotting.Results:The expression of Smad4,Smad1,pSmad1and cbfa-1were constrained after AS fibroblast was stimulated by Icariine,with the concentrations of100ug/ml. Conclusion:With the interference of Icariine, the expression of Smad1,pSmad1, Smad4and cbfa-1was constrained, and the BMP/Smad signal transduction pathway was restrained, so the differentiation of fibroblast into osteoblast was inhibited.Conclusion:Inhibiting the activation of BMP/Smad signal transduction pathway in AS fibroblast by Icariine was one of the molecular mechanisms of delaying ossification occurring in AS patients.