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中国临床药理学与治疗学 ›› 2025, Vol. 30 ›› Issue (1): 20-31.doi: 10.12092/j.issn.1009-2501.2025.01.003

• 基础研究 • 上一篇    下一篇

松脂醇二葡萄糖苷促骨质疏松性骨折愈合过程中血管生成的作用机制研究

王洁,田硕,李沂霖,魏君,马毓,刘艳秋   

  1. 山东中医药大学中医学院,济南  250355,山东
  • 收稿日期:2024-01-24 修回日期:2024-03-22 出版日期:2025-01-26 发布日期:2025-01-02
  • 通讯作者: 刘艳秋,女,博士,教授,博士生导师,研究方向:从事肾藏象理论的方药基础研究。 E-mail:qiusi1979@163.com
  • 作者简介:王洁,女,硕士研究生,研究方向:从事肾藏象理论的方药基础研究。 E-mail:wang062323@163.com
  • 基金资助:
    国家自然科学基金项目(82074134)

Study on the mechanism of pinoresinol diglucoside on angiogenesis during osteoporotic fracture healing

WANG Jie, TIAN Shuo, LI Yilin, WEI Jun, MA Yu, LIU Yanqiu   

  1. School of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong, China
  • Received:2024-01-24 Revised:2024-03-22 Online:2025-01-26 Published:2025-01-02

摘要:

目的:通过体内和体外实验探讨松脂醇二葡萄糖苷(PDG)促骨质疏松性骨折愈合过程中血管生成的调控机制。方法:将50只雌性C57BL/6J小鼠,随机分为5组:正常组、模型组、PDG 0.005、0.015 g/kg组、甲状旁腺激素1-34(PTH1-34)4×10-5 g/kg组。建立去卵巢联合骨折的骨质疏松性骨折模型,PDG给药组隔天灌胃给药8周,PTH1-34组隔天给药皮下注射PTH1-34 8周。Micro-CT扫描、免疫荧光和免疫组化染色检测相关参数及蛋白表达情况。培养人脐静脉血管内皮细胞(HUVECs),设置正常组、PDG 1、10、100 μmol/L组、PTH1-34 1 ng/mL组,CCK-8实验、划痕实验、管形成实验、免疫荧光染色以及Western blot实验检测相关参数及蛋白表达情况。结果:体内实验发现,与正常组比较,模型组小鼠骨折部位生成少量骨痂,股骨非骨痂部位骨密度(BMD)减少(P<0.01),骨体积分数(BV/TV)、骨小梁厚度(Tb. Th)、骨小梁数量(Tb. N)均降低(P<0.01),骨小梁分离度(Tb. Sp)升高(P<0.01),骨痂部位血管内皮标志分子(CD31)阳性表达减少(P<0.01);与模型组小鼠比较,PDG 0.005 g/kg组小鼠骨折部位骨痂体积增大,BMD、BV/TV指数均升高(P<0.05),PDG 0.015 g/kg组小鼠骨折部位骨痂体积进一步增大,BMD、BV/TV、Tb. Th、Tb. N指数均升高,Tb. Sp指数降低(P<0.01),PDG给药组骨痂部位CD31阳性表达均增加(P<0.01),PDG 0.015 g/kg组血管内皮生长因子(VEGF-A)(P<0.01)、Yes关联蛋白1(YAP1)(P<0.01)、PDZ结合域的转录共刺激因子(TAZ)(P<0.05)和TEA转录因子2(TEAD2)(P<0.01)蛋白表达增加。细胞实验发现,与正常组相比,PDG给药组可不同程度促进HUVECs的增殖、迁移和管形成(P<0.05),降低E-钙黏附蛋白(E-cadherin)蛋白表达(P<0.01),增加VEGF-A、TEAD2、TAZ、YAP1蛋白表达(P<0.05)。结论:PDG可能通过调控Hippo信号通路促进骨血管生成,从而加速骨质疏松性骨折愈合。

关键词: 松脂醇二葡萄糖苷, 骨质疏松性骨折, 骨生成, 血管生成, 血管内皮细胞

Abstract:

AIM: To explore the regulatory mechanism of pinoresinol diglucoside (PDG) on angiogenesis during osteoporotic fracture healing in vivo and in vitro. METHODS: Fifty male C57BL/6J mice were randomly divided into five groups: normal group, model group, PDG 0.005, 0.015 g/kg groups, and parathyroid hormone 1-34 (PTH1-34) 4×10-5 g/kg group. The osteoporotic fracture model of ovariectomized combined with femoral fracture was established, the PDG group was given intragastric administration every other day and the PTH1-34 group was given subcutaneous injection of PTH1-34 every other day for 8 weeks. Micro-CT scanning, immunofluorescence and immunohistochemical staining were used to detect the related parameters and protein expressions. Human umbilical vein endothelial cells (HUVECs) were cultured, normal group, PDG 1, 10, 100 μmol/L groups and PTH1-34 1 ng/mL group were set up. CCK-8 assay, scratch experiment, tubule formation experiment, immunofluorescence and Western blot were used to detect the related parameters and protein expressions. RESULTS: In vivo experiments found, compared with the normal control group, a small amount of bone callus volume of fracture site were increased in the model control group, while BMD of non-callus site of femur, trabecular bone fraction (BV/TV), trabecular thickness (Tb. Th) and trabecular number (Tb. N) were decreased (P<0.01), and trabecular separation (Tb. Sp) was increased (P<0.01). The positive expression of vascular endothelial marker vascular endothelial markers (CD31) was decreased (P<0.01). Compared with mice in the model control group, bone callus volume, index of BMD and BV/TV were increased in the PDG 0.005 g/kg group (P<0.05), index of BMD, BV/TV, Tb. Th, Tb. N were increased, and index of Tb. Sp was decreased in the PDG 0.015 g/kg group (P<0.05), the positive expression of CD31 was increased in the PDG administration groups (P<0.01), the protein expressions of vascular endothelial growth factor (VEGF-A) (P<0.01), Yes-associated protein 1 (YAP1) (P<0.01), PDZ-binding motif (TAZ) (P<0.05) and TEA domain transcription factor 2 (TEAD2) (P<0.01) were increased in callus in the PDG 0.015 g/kg groups. Cell experiments found, compared with the normal control group, PDG groups promoted the proliferation, migration and tubule formation activity of HUVECs to varying degrees (P<0.05), at the same time, the expression of endothelial cadherin (E-cadherin) was decreased (P<0.01), and VEGF-A, TEAD2, TAZ and YAP1 protein expression were increased (P<0.05). CONCLUSION: PDG may accelerate osteoporotic fracture healing by promoting bone angiogenesis through regulating Hippo signal pathway.

Key words: pinoresinol diglucoside, osteoporotic fracture, osteogenesis, angiogenesis, vascular endothelial cells

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