恒古骨伤愈合剂对绝经后骨质疏松症模型大鼠炎症损伤及NF-κB/NFATc1信号通路的影响  

Effect of Osteoking on Inflammatory Injury and NF-κB/NFATc1 Signaling Pathway in Model Mice with Postmenopausal Osteoporosis

作　　者：余田甜 丰瑞兵 王晴 李朝文 陈家豪 蒋瑞轩 丁印帅 黄勇 胡昊 YU Tiantian;FENG Ruibing;WANG Qing;LI Chaowen;CHEN Jiahao;JIANG Ruixuan;DING Yinshuai;HUANG Yong;HU Hao(Hubei University of Chinese Medicine,Wuhan 430060,China;Hubei Provincial Hospital of Traditional Chinese Medicine Affiliated to Hubei University of Chinese Medicine,Wuhan 430074,China;School of Health Science,Wuhan Sports University,Wuhan 430079,China)

机构地区：[1]湖北中医药大学,武汉430060 [2]湖北中医药大学附属湖北省中医院,武汉430074 [3]武汉体育学院健康学院,武汉430079

出　　处：《中国实验方剂学杂志》2024年第10期62-69,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：2023年度湖北省自然科学基金面上项目(2023AFB945);湖北省自然科学基金创新发展联合重点项目(2023AFD126);湖北省中医药管理局2023—2024年度中医药面上项目(ZY2023M005,ZY2023M004);湖北省中医药管理局2023—2024年度中医药青年人才项目(ZY2023Q004);2022年度湖北省自然科学基金创新发展联合基金项目(2022CFD147)。

摘　　要：目的:探析恒古骨伤愈合剂作用绝经后骨质疏松症(PMOP)的可能机制。方法:将40只成年雌性大鼠随机分为假手术(Sham)组、骨质疏松模型(OVX)组、雌二醇(E2)干预组、恒古骨伤愈合剂(OK)干预组,每组10只。通过常规背部双切口取卵巢术完成建模,1周后给予相应药物处理。12周后分别检测大鼠体质量、子宫指数,分别通过;苏木素-伊红(HE)染色、抗酒石酸酸性磷酸酶(TRAP)染色明确骨组织病理结构变化情况、破骨细胞(OC)数目。微计算机断层扫描技术(Micro-CT)检测骨密度(BMD)、骨小梁数目(Tb.N)、骨小梁分离度(Tb.Sp)、骨体积分数(BV/TV)。三点弯曲实验检测股骨最大载荷。酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)及骨吸收标志物Ⅰ型胶原交联C-末端肽(CTX-1)的含量。蛋白免疫印迹法(Western blot)检测通路相关蛋白核转录因子-κB p65(NF-κB p65)、磷酸化核转录因子-p65(p-NF-κB p65)、核转录因子-κB抑制蛋白α(IκBα)、磷酸化核转录因子-κB抑制蛋白α(p-IκBα)、活化T细胞核因子(NFATc1)、原癌基因(c-Fos)蛋白表达水平。实时荧光定量聚合酶链式反应(Real-time PCR)测定OC相关特异性基因基质金属蛋白酶-9(MMP-9)、NFATc1、抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(CTSK)与c-Fos的mRNA表达水平。结果:与Sham组比较,OVX组子宫指数明显降低,体质量(BMI)显著升高,骨小梁结构彻底受损,OC数目提高,BMD、Tb.N、BV/TV、最大载荷下降,Tb.Sp上调,血清中TNF-α、CTX-1水平上调,c-Fos、p-NF-κB p65/NF-κB p65、NFATc1与p-IκBα/IκBα蛋白表达量增高,NFATc1、c-Fos、CTSK、TRAP、MMP-9 mRNA表达明显上调(P<0.05,P<0.01)。与OVX组比较,OK组、E2组大鼠体质量降低,子宫指数增高,骨小梁增多,OC数目下降,BMD、Tb.N、BV/TV、最大载荷增加,Tb.Sp下降,血清中TNF-α、CTX-1水平下降,c-Fos、p-NF-κB p65/NF-κB p65、NFATc1与p-IκBα/IκBα蛋白表达量减少,NFATc1、c-Fos、Objective:To explore the possible mechanism of Osteoking(OK)on postmenopausal osteoporosis(PMOP).Method:Forty adult female mice were randomly divided into a sham operation(Sham)group,osteoporosis model(OVX)group,estradiol intervention(E2)group,and OK group,with 10 mice in each group.The modeling was completed by conventional back double incision ovariectomy,and the corresponding drugs were given one week later.After 12 weeks,the body mass and uterine index of mice were measured,and the pathological changes of bone tissue and the number of osteoclasts(OCs)were determined by hematoxylin-eosin(HE)and tartrate-resistant acid phosphatase(TRAP)staining,respectively.Bone mineral density(BMD),trabecular number(Tb.N),trabecular separation(Tb.Sp),and bone volume fraction(BV/TV)were measured by microcomputed tomography(Micro-CT).The maximum load of the femur was detected by a three-point bending test.The contents of tumor necrosis factor-α(TNF-α)and bone resorption marker C-terminal telopeptide of type Ⅰ collagen(CTX-1)were measured by enzyme linked immunosorbent assay(ELISA).The protein expression levels of nuclear factor-kappa B p65(NF-κB p65),phosphorylated nuclear factor-kappa B p65(p-NF-κB p65),nuclear factor kappa B inhibitor alpha(IκBα),phosphorylated nuclear factor kappa B alpha(p-IκBα),nuclear factor of activated T cells 1(NFATc1),and proto-oncogene(c-Fos)were detected by Western blot.The mRNA expressions of OCs-related specific genes matrix metalloproteinase-9(MMP-9),NFATc1,TRAP,cathepsin K(CTSK),and c-Fos were detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Result:Compared with the Sham group,the uterine index decreased significantly in the OVX group,and the body mass(BMI)increased significantly.The structure of bone trabeculae was completely damaged,and the number of OCs increased.BMD,Tb.N,BV/TV,and maximum load decreased,while Tb.Sp was up-regulated.The levels of TNF-αand CTX-1 in serum were up-regulated.The protein expressions of c-Fos,p-NF-κB p65/NF-κB p65

关 键 词：恒古骨伤愈合剂 绝经后骨质疏松症 核转录因子-κB/活化T细胞核因子信号通路 炎症损伤 

分 类 号：R2-0[医药卫生—中医学] R33R289R816.8