Overexpression of SMAD4 interferes with the expression of iron metabolism related proteins in osteoporotic rats / 中国组织工程研究

ABSTRACT

BACKGROUND:SMAD family member 4(SMAD4)can promote bone remodeling in osteoporotic rats,but it is unclear whether SMAD4 interferes with the expression of iron metabolism related proteins in osteoporotic rats. OBJECTIVE:To explore the effect of SMAD4 overexpression on the expression of iron metabolism related proteins in osteoporotic rats. METHODS:Rats were randomized into sham group,ovariectomy group,transfection control group and SMAD4 overexpression group.Animal models of osteoporosis were established in the latter three groups by ovariectomy,and only adipose tissue was removed in the sham group.One week later,adenovirus was injected into the femoral bone marrow cavity.SMAD4 overexpression group and transfection control group were injected with adenovirus overexpressing SMAD4 gene and control empty virus,respectively.Index detection was performed at 1 month after injection.Micro-CT,hematoxylin-eosin staining and tartrate resistant acid phosphatase staining were used to detect bone formation and bone resorption in osteoporotic rats.ELISA was used to detect serum ferritin and hepcidin levels.Immunohistochemical staining was used to detect alkaline phosphatase,osteocalcin,receptor activator for nuclear factor-κB ligand and tartrate resistant acid phosphatase levels in femoral tissue.RT-qPCR was used to detect SMAD4,hepcidin,divalent metal transporter 1,transferrin receptor1 and ferroportin1 mRNA levels in femoral tissue.Western blot was used to detect SMAD4,alkaline phosphatase,osteocalcin,osteoprotegerin,receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps,hepcidin,divalent metal transporter 1,transferrin receptor 1,and ferroportin 1 protein levels. RESULTS AND CONCLUSION:In the sham group,bone trabeculae in femur tissue were intact,and almost no osteoclasts were found.In the ovariectomy and transfection control groups,the bone trabeculae were sparse and a large number of osteoclasts were present.In the SMAD4 overexpression group,the number of bone trabeculae was increased and the number of osteoclasts was decreased.Compared with the sham group,the ovariectomy group showed a significant reduction in the protein expression of SMAD4,alkaline phosphatase,osteocalcin,and osteoprotegerin in femoral tissue and hepcidin levels in serum and femoral tissue,while receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps protein levels,divalent metal transporter 1,transferrin receptor1,ferroportin1 mRNA and protein levels were significantly increased(P＜0.05).Compared with the transfection control group,the SMAD4 overexpression showed a significant increase in SMAD4,alkaline phosphatase,osteocalcin,and osteoprotegerin protein levels in femoral tissue and hepcidin levels in serum and femoral tissue,while the expressions of receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps protein levels,divalent metal transporter 1,transferrin receptor1,and ferroportin 1 at mRNA and protein levels were significantly decreased(P＜0.05).To conclude,overexpression of SMAD4 promotes bone remodeling in osteoporotic rats by interfering with the expression of iron metabolism related proteins.