中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (31): 5028-5034.doi: 10.12307/2023.513

• 骨科植入物相关基础实验 Basic experiments of orthopedic implant • 上一篇    下一篇

红茴香提取物干预骨质疏松性大鼠骨折后的血管生成

史桂荣1,任博文2,张仲博2,王莉莎2,张啟威2,史栋梁2   

  1. 1商丘医学高等专科学校临床医学院中医教研室,河南省商丘市  476000;2河南省中医院骨病一科,河南省郑州市  450000
  • 收稿日期:2022-04-14 接受日期:2022-08-08 出版日期:2023-11-08 发布日期:2023-01-31
  • 通讯作者: 史栋梁,硕士,副主任医师,河南省中医院骨病一科,河南省郑州市 450000
  • 作者简介:史桂荣,女,1970年生,河南省商丘市人,汉族,2006年河南中医学院毕业,硕士,副教授,主要从事骨与关节相关疾病的研究。

Effect of Illicium henryi extract on angiogenesis in rats with osteoporotic fractures

Shi Guirong1, Ren Bowen2, Zhang Zhongbo2, Wang Lisha2, Zhang Qiwei2, Shi Dongliang2   

  1. 1Department of Traditional Chinese Medicine, Clinical School of Shangqiu Medical College, Shangqiu 476000, Henan Province, China; 2First Department of Orthopedics, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou 450000, Henan Province, China
  • Received:2022-04-14 Accepted:2022-08-08 Online:2023-11-08 Published:2023-01-31
  • Contact: Shi Dongliang, Master, Associate chief physician, First Department of Orthopedics, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou 450000, Henan Province, China
  • About author:Shi Guirong, Master, Associate professor, Department of Traditional Chinese Medicine, Clinical School of Shangqiu Medical College, Shangqiu 476000, Henan Province, China

摘要:


文题释义:

红茴香提取物:是一种具有消炎镇痛功效的中药制剂,其注射液经化学成分鉴定分析发现主要是黄酮类化合物,含量最高的是槲皮素。槲皮素被报道是一种骨骼保护药物,具有促进血管生成的作用。
核因子E2相关因子1(NF-E2-related factor 2,NRF1):是CNC(Cap’n’collar)家族成员,NRF1表达水平上调可能与血管内皮生长因子诱导的血管生成有关。有报道称红茴香的乙醇提取物治疗小鼠肾损伤时可以提高小鼠肾脏中NRF2 mRNA的表达水平。

背景:红茴香提取物是一种具有消炎镇痛和活血化瘀等功效的中药制剂,其乙醇提取物对大鼠软组织损伤具有显著疗效,而红茴香是否对骨质疏松性骨折具有治疗效果尚未报道。
目的:探讨红茴香提取物对骨质疏松性骨折大鼠血管生成的保护作用及可能机制。
方法:①体内实验:应用去势法和闭合性骨折法建立骨质疏松性骨折大鼠模型。36只健康雌性SD大鼠随机选取30只分为假手术组、模型组和红茴香提取物组(建模后给予0.05 mL/kg红茴香提取物),每组10只,其余6只纳入空白对照组。采用双能X射线和三点弯曲力学试验法检测大鼠股骨骨密度值和股骨弹性段终点载荷;苏木精-伊红染色法测定骨痂组织血管数量和血管面积;ELISA法检测血清中血管内皮生长因子、碱性成纤细胞生长因子、血小板衍生生长因子和一氧化氮水平。②体外实验:对人微血管内皮细胞进行药物干预,分为对照组、红茴香提取物(50,100或200 mg/L)组、Vector+二甲基亚砜组、NRF1过表达组、LY294002(10 μmol/L)组和NRF1过表达+ LY294002组。MTT法检测人微血管内皮细胞增殖;Transwell法检测人微血管内皮细胞迁移率;血管生成实验检测人微血管内皮细胞血管生成;Western blot法检测人微血管内皮细胞中NRF1,PI3K,p-PI3K,AKT和p-AKT蛋白表达,ELISA法检测人微血管内皮细胞中血管内皮生长因子、碱性成纤细胞生长因子、血小板衍生生长因子和一氧化氮水平。

结果与结论:①红茴香提取物改善了骨质疏松大鼠股骨骨密度和最大负荷(P < 0.05),增加骨痂组织中血管数量、血管面积和血清中血管形成相关因子水平(P < 0.05)。②红茴香提取物能促进人微血管内皮细胞中NRF1、p-PI3K和p-AKT蛋白表达(P < 0.05),促进人微血管内皮细胞增殖、迁移率、血管生成和血管形成相关因子水平(P < 0.05),NRF1过表达可起到相同作用,但LY294002处理可逆转NRF1过表达的作用。③结果说明,红茴香提取物可能通过提高NRF1蛋白表达、激活PI3K/AKT信号通路,对骨质疏松性骨折大鼠血管生成起到一定的促进作用。 

https://orcid.org/0000-0003-1120-1417(史桂荣)

中国组织工程研究杂志出版内容重点:人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程

关键词: 红茴香提取物, 骨质疏松, 骨折, 人微血管内皮细胞, NRF1, PI3K/AKT通路

Abstract: BACKGROUND: The extract of Illicium henryi is a Chinese herbal preparation with anti-inflammatory and analgesic properties and invigorating blood circulation and stasis. Its ethanol extract has markedly curative effect on rat soft tissue injury. However, there is yet no report on whether Illicium henryi has therapeutic effect on osteoporotic fractures. 
OBJECTIVE: To investigate the protective effect of Illicium henryi extract on angiogenesis in osteoporotic fracture rats and to clarify its possible mechanism. 
METHODS: (1) In vivo experiments: A rat model of osteoporotic fracture was prepared using castration method and closed fracture method. A total of 36 healthy female Sprague-Dawley rats were selected, 30 of which were randomized into sham, model, and treatment groups, with 10 rabbits in each group, and the remaining 6 were included in the control group. Each rat in the treatment group was given 0.05 mL/kg Illicium henryi extract after modeling. The dual-energy X-ray and three-point bending mechanics methods were used to detect femoral bone mineral density and the end-point load of the femoral elastic segment. Hematoxylin-eosin staining was used to measure the number and area of blood vessels in bone calluses. ELISA was used to detect serum levels of vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth factor, and nitric oxide. (2) In vitro experiments: Human microvascular endothelial cells were given drug intervention. Treated cells were divided into control group, Illicium henryi extract (50, 100, or 200 mg/L) groups, Vector+dimethyl sulfoxide group, NRF1 overexpression group, LY294002 (10 μmol/L) treatment group, and NRF1 overexpression+LY294002 treatment group. MTT, Transwell, and angiogenesis assays were used to detect the proliferation, migration rate and angiogenesis of human microvascular endothelial cells, respectively. Western blot assay was used to detect the levels of NRF1, PI3K, p-PI3K, AKT, and p-AKT proteins in human microvascular endothelial cells. ELISA was used to detect the levels of vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth factor, and nitric oxide in human microvascular endothelial cells. 
RESULTS AND CONCLUSION: (1) Illicium henryi extract significantly improved the bone mineral density and maximum load of the femur in osteoporotic rats (P < 0.05), and increased the number and area of blood vessels in the callus tissue, and the serum levels of angiogenesis-related factors (P < 0.05). (2) Illicium henryi extract promoted the expression of NRF1, p-PI3K and p-AKT proteins in human microvascular endothelial cells (P < 0.05), and increased the proliferation, migration, angiogenesis, and angiogenesis-related factor expressions of human microvascular endothelial cells (P < 0.05). Overexpression of NRF1 had the same effects, but LY294002 treatment reversed the effects of NRF1 overexpression. (3) To conclude, Illicium henryi extract can promote angiogenesis in osteoporotic fracture rats by increasing the expression of NRF1 protein and activating the PI3K/AKT signaling pathway.  

Key words: Illicium henryi, osteoporosis, fracture, human microvascular endothelial cell, NRF1, the PI3K/AKT pathway

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