Rapamycin Prevents Heterotopic Ossification By Blocking The MTOR Pathway And Oxidative Stress

? BACKGROUND ? Rapamycin(RAPA)is a potent immunosuppressant that possesses activities of anti-fungal,tumor suppression and inhibition of m TOR.Heterotopic ossification(HO)is abnormal bona fida bone formation outside of the normal skeletal system(such as muscle,tendon,articular capsule and other soft tissues).HO can be broadly divided into hereditary and acquired HO.Hereditary HO is of low incidence but with severe symptoms and high mortality.For example,Fibrodysplasia Ossificans Progressiva(FOP)is caused by gain-of-function mutations of a BMP type I receptor ACVR1 and Progressive Osseous Heteroplasia(POH)is caused by inactivating mutation of GNAS gene encoding G protein.Acquired HO generally showed milder symptoms with lower mortality but higher incidence.The severity of symptoms is often closely associated with the intensity and the location of trauma.For FOP,potential drugs that could directly target the pathogenic factors are emerging.In contrast,specific drug that could target traumatic HO is still unavailable,largely due to our very limited understanding of the traumatic HO.In fact,all currently available treatments for acquired HO,such as surgery,pulse low intensity electromagnetic field and NSAIDS are non-specific treatments.In this study,we focused on studying the underlying mechanism of HO by the loss-of-function of m TOR pathway through specific inhibitor,Rapamycin(RAPA),which is a potent immunosuppressant.RAPA has been clinically used in the treatment of many diseases,such as systemic lupus erythematosus,stroke and lung cancer.In addition,RAPA also regulates the autophagy of cell,and inhibits the possible immune rejection of allograft,but our interest is that whether RAPA suppresses HO via cross-talking with other signaling pathways.?METHODS?1.Selecting and allocating sex,age and weight matched adult NSE-BMP4 mice into experimental and control groups(5/group).Injury was induced by cardiotoxin(CTX,100ng/100? l)injection in left legs by intramuscular injection.1,2 and 4 weeks after injury,the injured tissues were harvested and fixed as the early,middle and late stages of HO,respectively.2.RAPA(100? g/100? l)was intraperitoneally injected every other day for 1,2,4 weeks.The control group was given solvent with the same dosage and methods.3.After the end of the RAPA administration,X-ray images were taken under anesthetic(Bruker Small Animal Imaging Instrument)to compare the HO in the experimental and the control groups.4.The number and type of WBC were measured(Japan Sysmex XS-800 i five classification blood cell analyzer)in the clinical laboratory of the First Affiliated Hospital of Anhui Medical University.5.Frozen sections from above-mentioned tissues were stained with selected antibodies,according to standard procedure.6.Detecting the expression of target proteins,such as Hif1 a and P-m TOR in the injured tissue via Western Blot,according to standard procedure.?RESULTS?1.X-ray imaging showed that the experimental group treated with RAPA significantly inhibited the HO,in a dose dependent manner.2.RAPA could reduce the expression of P-m TOR in the injury sites.3.RAPA limited HO through inhibiting Hif1 a.4.Oxidative stress contributed to fibroproliferation in the early stage of HO.5.Rapamycin decreased vascularization/angiogenesis in HO.?CONCLUSIONS?Oxidative stress is associated with abnormal immune response(AIR)in the early stage of HO.Rapamycin could reverse the hypoxic condition,inhibit m TOR and decrease HO formation.