Study On The Roles And Mechanisms Of SPTBN1 In The Preventing Of Primary Osteoporosis

Background:Senile and postmenopausal osteoporosis(primary osteoporosis)accounts for 85-90%of osteoporosis.But the specific mechanism is still unclear.Genome-wide association study(GWAS)are based on human genomics analysis and are tools for the study of the molecular genetics and pathogenesis of osteoporosis.Our team combined GWAS data and transcriptome data to screen out one member of the spectrin family--SPTBN1,which may be a key regulator of osteoporosis.SPTBN1 is an actin_linking scaffold protein,and has been confirmed to have key pathophysiological functions in cancer,heart,brain and so on.However,its role in osteoporosis,especially osteoblast,osteoclast,and bone vessels has not been reported.Therefore,by up-regulating or down-regulating the expression level of SPTB1 in mice,the role of SPTBN1 in primary osteoporosis and its regulatory mechanism are explored,providing a new target for its treatment.Objective:To investigate the role of SPTBN1 in primary osteoporosis and explore the mechanism.Materials and Methods:1.Expression level of SPTBN1 in primary osteoporosis.The patients were divided into the Senile group(16 months),the Young group(3 months),the OVX group(16 weeks after ovariectomy),and the Sham group.The bone microstructure was detected by microCT and SPTBN1 was detected by immunohistochemistry.2.Changes in bone microsfructure and blood vessels in primary osteoporosis mice after up-regulating or down-regulating SPTBN1.In the Senile group and the OVX group,the AAV virus(Senile-AAV group,OVX-AAV group)or SAM virus(Senile-SAM group,OVX-SAM group)were injected into the right femur,or negative control virus(Senile-Control group,OVX-Control group)was injected to the left femur.Analyzed bone microstructure by MicroCT scan,hematoxylin-eosin staining one month after injection,and analyzed morphometry of trabecular,changes in blood vessel volume fraction and number.ALP/Trap staining was performed,and SPTBN1,Runx2,Osx,and Ocn were detected by immunohistochemistry,and osteoclast and osteoblast numbers were analyzed.3.The effect of SPTBN1 on the proliferation and differentiation of osteoblasts.Transfected the preosteoblast cell line MC3T3 with LV virus(LV group)to down-regulate SPTBN1,or SAM virus(SAM group)to up-regulate SPTBN1,negative control(Negative group),and blank control group(Control group).CCK8 assay was used to detect the proliferative activity of SPTBN1 and immunoblotting was used to analyze Cycline E1 cells.Flow cytometry was used to detect SPTBN1 and immunoblotting was used to analyze caspase3.ALP staining and alizarin red staining were used to define the differentiation and mineralization of cells.5.Mechanism of how SPTBN1 preventing osteoporosis.After treatment as 2nd step,VEGF was detected by immunohistochemistry.After treatment as 4th step,the expression levels of SPTBN1,Smad3,STAT1 and TGF-β were detected by immunoblotting,and the cell supernatant was taken to detect the expression level of Cxcl9 by ELISA.Results:1.Compared to the Young group,the expression of SPTBN1 in the Senile group was lower.Compared to the Sham group,the expression of SPTBN1 in the OVX group was lower.2.Compared with the Senile-Control group,the Senile-AAV group had a lower bone density,a smaller number of trabecular bones,and wider gap.Compared with the OVX-Control group,the OVX-AAV group had a lower bone density,a smaller number of trabecular bones,and wider gap.Compared with that of the Senile-Control group,the expression of SPTBN1,Runx2,Osx,and Ocn in the Senile-AAV group was decreased,as well as the volume fraction of blood vessels in the bone,the number of blood vessels,and the expression of VEGF,while an increase in osteoclasts.Compared with that of the OVX-Control group,the expression of SPTBN1,Runx2,Osx,and Ocn in the OVX-AAV group was decreased,as well as the volume fraction of blood vessels in the bone,the number of blood vessels,and the expression of VEGF,while an increase in osteoclasts.3.Compared with the Senile-Control group,the Senile-SAM group had a higher bone density,a larger number of trabecular bones.Compared with the OVX-Control group,the OVX-SAM group had a higher bone density,a larger number of trabecular bones.Compared with that of the Senile-Control group,the expression of SPTBN1,Hunx2,Osx,and Ocn in the Senile-SAM group was increased,as well as the volume fraction of blood vessels in the bone,the number of blood vessels,and the expression of VEGF,while an decreased in osteoclasts.Compared with that of the OVX-Control group,the expression of SPTBN1,Runx2,Osx,and Ocn in the OVX-SAM group was increased,as well as the volume fraction of blood vessels in the bone,the number of blood vessels,and the expression of VEGF,while an decreased in osteoclasts.4.Compared with the Negative group and the Control group,the LV group proliferated slowly,the number of apoptosis increased,and the number of cells stained by ALP and alizarin red staining decreased.In contrast,the SAM group proliferated faster,apoptosis decreased,and the number of cells stained by ALP and alizarin red staining increased.5.The expression of p-Smad3/TGF-β and p-STAT1/Cxc19 was increased in LV group,and the expression of p-Smad3/TGF-β and p-STAT1/Cxcl9 were decreased in SAM group.Conclusion:SPTBN1 can inhibit senile and postmenopausal osteoporosis.SPTBN1 can promote osteoblast proliferation and differentiation and inhibit apoptosis.The above effects were partially achieved by regulating activities of p-Smad3/TGF-β,p-STAT1/Cxcl9.SPTBN1 was an important regulator of osteoporosis.