摘要
目的:探讨丹参多酚酸酯(Sal)对骨质疏松症大鼠破骨细胞分化和骨吸收的影响,阐明其作用机制。方法:体内实验,采用去势法建立骨质疏松症大鼠模型,30只雌性大鼠随机分为假手术组、模型组和Sal组(建模4周后隔天腹腔注射40 mg·kg^(-1) Sal,共4周)。采用HE染色法观察各组大鼠股骨组织病理形态表现,双能X射线骨密度仪检测各组大鼠股骨骨密度(BMD),三点弯曲试验检测各组大鼠股骨最大载荷。体外实验,采用30μg·L^(-1)巨噬细胞集落刺激因子(M-CSF)和100μg·L^(-1)核因子κB(NF-κB)配体(RANKL)诱导骨髓巨噬细胞(BMMs)分化,细胞分为对照组、RANKL组、RANKL+Sal组(10 mg·L^(-1)Sal)、RANKL+Sal+空载体(vector)组(10 mg·L^(-1) Sal和2 mg·L^(-1) vector)、RANKL+Sal+LV-FKBP1A组[10 mg·L^(-1) Sal和2 mg·L^(-1)慢病毒介导的FKBP1A过表达载体(LV-FKBP1A)]、RANKL+Sal+LV-FKBP1A+QNZ组(10 mg·L^(-1) Sal、2 mg·L^(-1) LV-FKBP1A和10 nmol·L-1NF-κB抑制剂QNZ),MTT法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,Co-IP实验验证SMAD2和FKBP1A的相互作用,Westernblotting法检测各组大鼠骨组织和各组细胞中SMAD家族蛋白2(SMAD2)、FK506结合蛋白1A(FKBP1A)和磷酸化核因子-κB P65(p-P65)蛋白表达水平,酶联免疫吸附试验(ELISA)检测各组大鼠血清和各组细胞中抗酒石酸酸性磷酸酶5b(TRAP5b)水平,实时荧光定量PCR(RT-qPCR)法检测各组大鼠骨组织和各组细胞中组织蛋白酶K(CTSK)和降钙素受体(CTR)mRNA表达水平。结果:体内实验,与模型组比较,Sal组大鼠骨小梁数量明显增加,大鼠股骨BMD和最大负载明显升高(P<0.01),血清中TRAP5b水平明显降低(P<0.01),骨组织中SMAD2及FKBP1A蛋白表达水平和CTSK及CTRmRNA表达水平明显降低(P<0.01)。Co-IP实验结果证实SMAD2与FKBP1A蛋白存在相互作用。体外实验,与RANKL组比较,RANKL+Sal组细胞增殖活性明显降低(P<0.01),细胞凋亡率明显升高(P<0.01),细胞中TRAP5b水平,SMAD2、FKBP1A及p-P65蛋白表达水平和CTSK及CTRmRNA表达水平明显降低(P<0.01);与RANKL+Sal+vector组比较,RANKL+Sal+LV-FKBP1A组细胞凋亡率明显降低(P<0.01),细胞中TRAP5b水平,SMAD2、FKBP1A及p-P65蛋白表达水平和CTSK及CTRmRNA表达水平明显升高(P<0.01);与RANKL+Sal+LVFKBP1A组比较,RANKL+Sal+LV-FKBP1A+QNZ组细胞凋亡率明显升高(P<0.01),细胞中TRAP5b水平,SMAD2、FKBP1A及p-P65蛋白表达水平和CTSK及CTRmRNA表达水平明显降低(P<0.01)。结论:Sal通过调控SMAD2和FKBP1A蛋白的相互作用,抑制NF-κB活化,缓解大鼠骨质疏松症。
Objective:To investigate the effects of salvianolate(Sal)on the osteoclast differentiation and bone resorption in the osteoporotic rats,and to clarify its mechanism.Methods:Castration was used to establish the rat models of osteoporosis.A total of 30 rats were randomly divided into sham operation group,model group and Sal group(given with 40 mg·kg^(-1) Sal by intraperitoneal injection once two days 4 weeks after modeling for 4 weeks).HE staining method was used to observe the pathomorphology of femur tissue of the rats in various groups;dual-energy X-ray bone densitometer was used to detect the femur bone mineral density(BMD)of the rats in various groups;three-point bending test was used to detect the maximum loads of femur of the rats in various groups.In in vitro experiment,30μg·L^(-1) macrophage colony stimulating factor(M-CSF)and 100μg·L^(-1) nuclear factor kappa B(NF-κB)ligand(RANKL)were used to induce the differentiation of bone marrow macrophages(BMMs).The cells were divided into control group,RANKL group,RANKL+Sal group(10 mg·L^(-1) Sal),RANKL+Sal+vector group(10 mg·L^(-1) Sal and 2 mg·L^(-1) empty vector),RANKL+Sal+LV-FKBP1A group[10 mg·L^(-1) Sal and 2 mg·L^(-1) lentivirus-mediated FKBP1A overexpression vector(LV-FKBP1A)],RANKL+Sal+LV-FKBP1A+QNZ group(10 mg·L^(-1) Sal,2 mg·L^(-1)LV-FKBP1A and 10 nmol·L-1 NF-κB inhibitor QNZ).MTT assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the apoptotic rates of cells in various groups;Co-IP experiment was used to verify the interaction between SMAD2 and FKBP1A;Western blotting method was used to detect the expression levels of SMAD family member 2(SMAD2),FK506-binding protein 1A(FKBP1A)and phosphorylated NF-κB P65(p-P65)proteins in rat bone tissue and cells in various groups.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tartrate-resistant acid phosphatase 5b(TRAP5b)in serum of the rats and cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression levels of cathepsin K(CTSK)and calcitonin receptor(CTR)mRNA in bone tissue of the rats and cells in various groups.Results:The in vivo experimental results showed that compared with model group,the number of bone trabeculae in Sal group,the BMD and the maximum load of femur of the rats were significantly increased(P<0.01),and the level of TRAP5b in serum was decreased(P<0.01),the expression levels of SMAD2 and FKBP1A proteins and CTSK and CTR mRNA in bone tissue were significantly reduced(P<0.01).The Co-IP experiment results confirmed that SMAD2 interacted with FKBP1A protein.The in vitro experimental results showed that compared with RANKL group,the proliferation activity of cells in RANKL+Sal group was significantly decreased(P<0.01),the apoptotic rate was significantly increased(P<0.01),and the TRAP5b level and the expression levels of SMAD2,FKBP1A and p-P65 proteins and CTSK and CTR mRNA were significantly reduced(P<0.01).Compared with RANKL+Sal+vector group,the apoptotic rate in RANKL+Sal+LV-FKBP1A group was significantly reduced(P<0.01),and the TRAP5b level and the expression levels of SMAD2,FKBP1A and p-P65 proteins and CTSK and CTR mRNA in the cells were significantly increased(P<0.01).Compared with RANKL+Sal+LV-FKBP1A group,the apoptotic rate in RANKL+Sal+LV-FKBP1A+QNZ group was significantly increased(P<0.01),and the TRAP5b level and the expression levels of SMAD2,FKBP1A and p-P65 proteins and CTSK and CTR mRNA in the cells were significantly reduced(P<0.01).Conclusion:Sal inhibits the activation of NF-κB by regulating the interaction between SMAD2 and FKBP1A protein,and alleviates the osteoporosis in the rats.
作者
马运锋
韩小飞
MA Yunfeng;HAN Xiaofei(Department of Osteopathy,Second Affiliated Hospital,Henan University of Traditional Chinese Medicine,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,Henan;Department of Rheumatology,Second Affiliated Hospital,Henan University of Traditional Chinese Medicine,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,Henan)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2022年第1期111-121,共11页
Journal of Jilin University：Medicine Edition
基金
河南省卫健委中医药科学研究专项项目(2019ZY2149,2021ZY3013)。
作者简介
通信作者:马运锋(1983-),男,河南省长葛市人,住院医师,医学硕士,主要从事中医药治疗骨关节疾病方面的研究(E-mail:weiminwanghb@sina.com)。
