中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (2): 184-191.doi: 10.12307/2022.923

• 骨组织构建 bone tissue construction • 上一篇    下一篇

补肾活血胶囊对激素性股骨头坏死大鼠骨破坏的修复

骆  帝1,2,梁学振1,2,阎博昭2,李嘉程1,许  波1,2,李  刚1,2   

  1. 1山东中医药大学附属医院,山东省济南市  250014;2山东中医药大学,山东省济南市  250355
  • 收稿日期:2021-07-22 接受日期:2021-09-11 出版日期:2023-01-18 发布日期:2022-06-18
  • 通讯作者: 李刚,博士,教授,主任医师,博士生导师,山东中医药大学附属医院,山东省济南市 250014;山东中医药大学,山东省济南市 250355
  • 作者简介:骆帝,1989年生,山东省菏泽市人,汉族,2020年山东中医药大学毕业,博士,博士后,主治医师,主要从事骨与关节疾病的研究。
  • 基金资助:
    国家自然科学基金资助项目(81774333),项目负责人:李刚;山东省自然科学基金联合专项基金项目(ZR2021LZY002),项目负责人:骆帝;山东省中医药科技发展计划项目(2019-0147),项目负责人:李刚;山东省医药卫生科技发展计划项目(202104070600),项目负责人:骆帝;济南市科技计划项目(201907103),项目负责人:李刚

Mechanism of Bushen Huoxue Capsule in repair of bone defects due to steroid-induced osteonecrosis of the femoral head in rats

Luo Di1, 2, Liang Xuezhen1, 2, Yan Bozhao2, Li Jiacheng1, Xu Bo1, 2, Li Gang1, 2   

  1. 1Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China; 2Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China
  • Received:2021-07-22 Accepted:2021-09-11 Online:2023-01-18 Published:2022-06-18
  • Contact: Li Gang, MD, Professor, Chief physician, Doctoral supervisor, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China; Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China
  • About author:Luo Di, MD, Attending physician, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China; Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81774333 (to LG); Joint Special Program of Shandong Provincial Natural Science Foundation, No. ZR2021LZY002 (to LD); Shandong Provincial Traditional Chinese Medicine Science and Technology Development Project, No. 2019-0147 (to LG); Shandong Provincial Medical and Health Scientific Development Plan, No. 202104070600 (to LD); Jinan Municipal Science and Technology Project, No. 201907103 (to LG)

摘要:

文题释义:
股骨头坏死:是指股骨头静脉淤滞、动脉血供受损或中断使骨细胞及骨髓成分部分死亡引起骨组织坏死及随后发生的修复,共同导致股骨头结构改变及塌陷,引起髋关节疼痛及功能障碍的疾病,可分为创伤性和非创伤性两大类,其中皮质类固醇类药物应用是造成非创伤性股骨头坏死的主要病因。
实时荧光定量聚合酶链式反应(Quantitative Real-time PCR,qPCR):是一种在DNA扩增反应中,以荧光化学物质测每次聚合酶链式反应(PCR)循环后产物总量的方法,可通过内参或者外参法对待测样品中的特定DNA序列进行定量分析的一种实验方法。

背景:课题组前期研究发现,糖皮质激素可抑制Hedgehog信号通路关键基因的表达,从而扰乱骨髓间充质干细胞成骨、成脂与成血管方向的分化,进而破坏骨内血供与骨组织结构,而补肾活血胶囊对此具有一定的治疗作用。
目的:观察补肾活血胶囊对激素诱导的股骨头坏死SD大鼠骨组织破坏的修复作用,进一步研究补肾活血胶囊对成骨与Hedgehog信号通路相关因子的影响机制。
方法:SD大鼠臀肌注射甲泼尼龙琥珀酸钠建立激素诱导相关的股骨头坏死大鼠模型;分别灌胃给予低、中、高不同剂量的补肾活血胶囊干预;并以臀肌注射生理盐水的大鼠为对照。给药8周后取材,通过剖检大体观察、组织病理学检查、qPCR与Western blot分析在不同层面综合评价补肾活血胶囊对激素性股骨头坏死大鼠骨组织修复的影响及其作用机制。
结果与结论:①大体观察:干预8周后,各组大鼠股骨头样本大体观无明显差异;②苏木精-伊红染色:与模型组比较,各剂量补肾活血胶囊组大鼠均显著提高了骨小梁面积、降低了空骨陷窝率;③免疫组化:与模型组比较,各剂量补肾活血胶囊组大鼠在不同程度上提高了碱性磷酸酶、核心结合因子α1、Ⅰ型胶原蛋白、骨桥蛋白的表达;④qPCR检测:与模型组比较,各剂量补肾活血胶囊组大鼠提升了成骨相关指标碱性磷酸酶、Ⅰ型胶原蛋白、核心结合因子α1与Hedgehog信号通路相关指标音猬因子、核转录因子1、核转录因子2的mRNA表达;⑤Western blot检测:与模型组比较,各剂量补肾活血胶囊组大鼠均在不同程度上提高了成骨相关指标碱性磷酸酶、核心结合因子α1、Ⅰ型胶原蛋白、骨桥蛋白与Hedgehog信号通路相关指标音猬因子、核转录因子2的蛋白表达;⑥研究初步证实了补肾活血胶囊对激素性股骨头坏死有一定的治疗作用,其作用机制与成骨相关的Hedgehog信号通路具有一定相关性。

https://orcid.org/0000-0002-3231-8879 (骆帝)

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: 股骨头坏死, 补肾活血胶囊, 甲泼尼龙琥珀酸钠, Hedgehog信号通路, 成骨

Abstract: BACKGROUND: Glucocorticoids can inhibit the expression of key genes in the Hedgehog signaling pathway, thereby disrupting the differentiation of bone marrow mesenchymal stem cells into osteogenesis, adipogenesis, and angiogenesis, thereby destroying the blood supply and bone tissue structure in the bone. We found that Bushen Huoxue Capsule has a certain therapeutic effect on these changes.
OBJECTIVE: To observe the effect of Bushen Huoxue Capsule on the repair and reconstruction of bone defect due to steroid-induced osteonecrosis of the femoral head in Sprague-Dawley rats, and to further study the mechanism of Bushen Huoxue Capsule on osteogenesis and hedgehog signaling pathway related factors.
METHODS: Animal model of steroid-induced osteonecrosis of the femoral head was established by injecting methylprednisolone sodium succinate into the gluteal muscle of Sprague-Dawley rats, and low-, middle- and high-dose Bushen Huoxue Capsule was given by gavage. Another rats given normal saline to the gluteal muscles were used as controls. After 8 weeks of injection, the effect of Bushen Huoxue Capsule on bone tissue repair in rats with steroid-induced osteonecrosis of the femoral head and its mechanism were comprehensively evaluated at different aspects through gross observation, histopathological examination, qPCR and western blot analysis. 
RESULTS AND CONCLUSION: Gross observation indicated no significant difference in the femoral head samples from different groups after 8 weeks of intervention. Hematoxylin-eosin staining revealed that Bushen Huoxue Capsule at different doses could significantly increase the trabecular area and decrease the empty bone lacuna ratio in rats. Immunohistochemical staining indicated that Bushen Huoxue Capsule at different doses could significantly upregulate the expression of alkaline phosphatase, core binding factor α1, type I collagen and osteopontin in rats. qPCR findings showed that compared with the control group, Bushen Huoxue Capsule at different doses could upregulate the mRNA expression of alkaline phosphatase, type I collagen, core binding factor α1 and Hedgehog signaling pathway-related indicators (sonic hedgehog, nuclear transcription factor 1, and nuclear transcription factor 2). Findings from western blot assay indicated that compared with the control group, Bushen Huoxue Capsule at different doses could upregulate the protein expression of alkaline phosphatase, type I collagen, core binding factor α1, osteopontin and Hedgehog signaling pathway-related indicators (sonic hedgehog and nuclear transcription factor 2). This study has preliminarily confirmed that Bushen Huoxue Capsule has a certain therapeutic effect on steroid-induced osteonecrosis of the femoral head, and its mechanism of action is related to the Hedgehog signaling pathway.

Key words: osteonecrosis of the femoral head, Bushen Huoxue Capsule, methylprednisolone sodium succinate, Hedgehog signaling pathway, osteogenesis

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