The Mechanism And Efficacy Of Irisin In The Mouse Model Of Osteogenesis Imperfecta

Background and ObjectiveOsteogenesis imperfecta?OI?is a systemic connective tissue disease characterized by increased bone fragility and disordered collagen metabolism.Most of the OI cases are caused by the collagen metabolism disorders due to mutations in one of the genes encoding collagen type??1 chain?Col1?1?and collagen type??2 chain?Col1?2?.OI is divided into five subtypes?Type 1-5?based on the characteristic symptoms.The clinical features of OI is characterized by low bone mass,recurrent fractures,bony deformities,dentinogenesis imperfect,joint laxity and short stature.Symptoms often appear in childhood and seriously affect the quality of life,bring heavy burden to the family and society.To date,there is no curative treatment for OI.Currently,the primary means for OI therapy are medical treatment,surgical operation,rehabilitation exercise,molecular and cellular therapy.Drug therapy is the main treatment,among which bisphosphonates?BPs?appear to be the most widely used one now.BPs is an inhibitor of bone resorption,which combines with hydroxyapatite in bone matrix and improves bone structure of OI by inhibiting the activity of osteoclasts.However,the medicine-using pattern of BP is complex and it has several dverse reactions such as acute phase response?APR?,atypical femoral fracture?AFF?,osteonecrosis of the jaw?ONJ?,delayed bone union,renal damage and hypocalcemia.Denosumab is a specific antibody targeting the receptor activator of nuclear factor-?B ligand?RANKL?,which can effectively improve the bone mineral density?BMD?of OI.However,the rebound vertebral fracture is can not be ignored when applied for children.Teripartide is the only synthesis drug of bone approved by FDA for the treatment of osteoporosis,which can effectively increase the BMD of patients with osteoporosis and reduce the risk of fracture.Nevertheless,it has little effect on patients with moderate or severe OI and may lead to osteosarcoma.Strontium ranelate have the dual regulation of bone metabolism with inhibiting osteoclasis and promoting osteogenesis,which can effectively reduce the fracture rate of OI mouse model.However,strontium ranelate is at risk of delisting due to the possibility of increasing cardiovascular accidents.Therefore,there is a strong incentive for the exploration of more effective and safe therapies to treat OI.Irisin is a kind of N-glycosylated myogenic protein hormone,which is secreted from skeletal muscle with the stimulus of exercise.Irisin can promote the differentiation of osteoblasts by activating Wnt/?-catenin signaling pathway,and activate p38 mitogen activated protein kinase?MAPK?and extracellular regulated protein kinases?ERK?for inducing the proliferation,differentiation and mineralization of osteoblasts.Irisin can also inhibit the formation of osteoclasts by inhibiting the RANKL of RAW264.7 osteoclast cell line.The results of animal experiments demonstrated that Irisin can increase the cortical BMD,enhance the bone strength and improve the bone microstructure of mice.Moreover,Irisin can also prevent and restore bone loss and muscle atrophy in hindlimb suspended mice.Given these observations,we speculate that Irisin can also effectively improve the clinical symptoms of OI.In this study,we select oim/oim mice?OI mouce model?as the objects of study.To explore the dual effects of Irisin on primary osteoblasts and osteoclasts using oim/oim mice?OI mouse model?in the context of collagen deficiency.Part ?:Effect of Irisin on osteoblasts and osteoclasts of OI mice model in vitro.Methods:?1?Primary osteoblasts and bone marrow monocytes?BMMs?of homozygous mice?oim/oim or wt/wt?were obtained for Inducing differentiation with different concentrations?10 and 100 ng/ml?of Irisin.Quantitative real-time PCR?q RT-PCR?and Western blotting were used to quantify the expression of osteoblast differentiation-related genes and osteoclast differentiation-related genes.ALP staining and alizarin red S?ARS?staining were analysised for observing the osteogenic differentiation and mineralization.Trap and immunofluorescence assay were used for evaluating osteoclast formation,and bone resorption assay were applied for assessing the resorption function of osteoclasts.Results:?1?q RT-PCR and Western blotting showed that the expression of osteoclast transcription regulatory factor?Runx2?and differentiation marker genes?ALP,OCN and col1?1?in oim/oim mice were signifcantly increased at m RNA and protein levels after stimulation with Irisin.Irisin also caused signifcant decreases in osteoclast differentiation related genes and transcription factors?Calcr,Trap,CTSK,c-fos and NFATc1?both at m RNA and protein levels.?2?ALP staining and ARS staining demonstrated that Irisin significantly increased osteoblast differentiation and mineralization of oim/oim mice in vitro.?3?Results of Trap staining,immunofluorescence assay and bone resorption assay indicated that Irisin signifcantly decreased the formation number of osteoclasts and inhibited the resorption activity of osteoclasts in oim/oim mice.Conclusions:Irisin can effectively improve the imbalance of bone metabolism in oim/oim mice?OI mouse model?.In the context of collagen deficiency,Irisin can also increased osteoblast differentiation and mineralization,and decreases in formation and resorption of the osteoclasts.Part ?:Therapeutic effect of Irisin on OI mouse modelMethods:?1?Three-week-old B6C3Fe-?/?-Coll?2^oim/+mice were randomly divided into four groups based on genotype?oim/oim or wt/wt?and treatment?Irisin or Veh?:Oim+Veh group,Oim+Irisin group group,Wt+Veh group and Wt+Irisin group.From week4,the experimental mice?oim/oim or wt/wt?were injected with 100?g/kg-body weight of Irisin or a comparable volume of physiological saline solution?Veh?via a tail,once a week for 8 weeks.?2?Each mouse weighed weekly and the length of isolated femur was measured to evaluate the growth and development of mice.?3?Oim/oim mice were X-rayed for fracture counting before and after treatment.?4?Serum of each mice was harvested as they were euthanized for assessing the activities of OCN,NTx and TRACP5b.?5?At the end of experiment,isolated left femur was scanned by micro-computed tomographic?Micro CT?for bone structure analysis.?6?Left tibias were obtained for dynamic bone histomorphometry.?7?Right tibias were stained with TRAP and HE solution for histomorphometric examination,and number of osteoclasts and osteoblasts were further counted.?8?Left femurs and femoral segments of fractured one were obtained for further biomechanical analysis.Results:?1?Irisin intervention had no significant effect the body weight and femur length of each genotype mice.?2?Irisin can significantly reduce the incidence of fracture in oim/oim mice.?3?Rresults of ELISA suggested that Irisin could increase the osteoblast-associated markers of OCN,and decrease the osteoclast-associated markers of NTx and TRACP5b.?4?Irisin improves bone microstructure of OI mouse model with both cortical and trabecular bone involved.?5?Irisin promotes bone mineral apposition via increasing BFR in OI mouse model.?6?Irisin induces osteogenesis and inhibits osteoclast formation in OI mouse model.?7?Irisin enhances the mechanical properties of bone in OI mouse model by improving structural properties and reducing bone brittleness.Conclusions:Irisin can positively improve bone mass,bone microstructure and bone strength by inhibiting bone absorption and improving bone formation,and reduce the fracture incidence in a growing mouse model of OI.SummaryThis is the first report of the therapeutic effect of Irisin in a growing B6C3Fe-?/?-Coll?2^oim/+mouse model of OI.In vitro and animal experiments,Irisin reduces fracture incidence by exerting a positive influence on BMD,bone microstructure and bone mechanical properties,which might involve the increases in osteoblast differentiation and mineralization and decreases in formation and resorption of the osteoclasts in vitro.