摘要
背景:SMAD家族成员4(SMAD4)能够促进骨质疏松大鼠骨重建,然而SMAD4是否干预骨质疏松大鼠铁代谢相关蛋白表达尚不清楚。目的:探究SMAD4过表达对骨质疏松大鼠铁代谢相关蛋白表达的影响。方法:大鼠随机分为假手术组、卵巢摘除组、转染对照组和SMAD4过表达组,后3组切除卵巢建立骨质疏松大鼠模型,假手术组仅切除脂肪组织。1周后行股骨髓腔腺病毒注射,SMAD4过表达组和转染对照组分别注射过表达SMAD4基因的腺病毒及对照空载病毒,注射1个月后进行指标检测。显微CT、苏木精-伊红染色、抗酒石酸酸性磷酸酶染色检测骨质疏松大鼠骨形成和骨吸收情况;ELISA检测血清铁蛋白、铁调素水平;免疫组化染色检测股骨组织碱性磷酸酶、骨钙素、核因子κB受体激活因子配体、抗酒石酸酸性磷酸酶水平;RT-qPCR检测股骨组织SMAD4、铁调素、二价金属离子转运体1、转铁蛋白受体1、膜铁转运蛋白1 mRNA水平;Western blot检测股骨组织SMAD4、碱性磷酸酶、骨钙素、骨保护素、核因子κB受体激活因子配体、抗酒石酸酸性磷酸酶、β-胶原降解产物、铁调素、二价金属离子转运体1、转铁蛋白受体1、膜铁转运蛋白1蛋白水平。结果与结论:①假手术组大鼠股骨组织骨小梁完整,几乎未见破骨细胞;卵巢摘除组、转染对照组骨小梁稀疏,存在大量破骨细胞;SMAD4过表达组大鼠骨小梁增多,破骨细胞减少;②与假手术组相比,卵巢摘除组大鼠股骨组织SMAD4、碱性磷酸酶、骨钙素、骨保护素蛋白水平、血清和股骨组织铁调素水平明显降低,股骨组织核因子κB受体激活因子配体、抗酒石酸酸性磷酸酶、β-胶原降解产物蛋白水平、二价金属离子转运体1、转铁蛋白受体1、膜铁转运蛋白1 mRNA和蛋白水平明显升高(P<0.05);③与转染对照组相比,SMAD4过表达组大鼠股骨组织SMAD4、碱性磷酸酶、骨钙素、骨保护素蛋白水平、血清和股骨组织铁调素水平明显升高,股骨组织核因子κB受体激活因子配体、抗酒石酸酸性磷酸酶、β-胶原降解产物蛋白水平、二价金属离子转运体1、转铁蛋白受体1、膜铁转运蛋白1 mRNA和蛋白水平明显降低(P<0.05);④提示过表达SMAD4通过干预铁代谢相关蛋白表达促进骨质疏松大鼠骨重建。
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.
作者
姚婷
官蓉威
高原
Yao Ting;Guan Rongwei;Gao Yuan(Department of Endocrinology,The Second Affiliated Hospital of Chengdu Medical College,Chengdu 610000,Sichuan Province,China;Department of Orthopedics,The Second Affiliated Hospital of Chengdu Medical College,Chengdu 610000,Sichuan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第23期3648-3653,共6页
Chinese Journal of Tissue Engineering Research
