Effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice

CHENG Shao; TAO Yuren; SHU Bing; WANG Jing; ZHAO Yongjian; ZHAO Shitian; ZHANG Yan; TANG Dezhi; ZHAO Dongfeng; SUN Yueli; SHI Qi; WANG Yongjun

doi:10.16306/j.1008-861x.2021.04.012

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Effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice

Experiment Research | 更新时间：2022-09-09

- Effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 35, Issue 4, Pages: 79-88(2021)
- 作者机构：
  
  1.上海中医药大学附属龙华医院（上海　200032）
  2.上海市中医药研究院脊柱病研究所（上海　200032）
  3.教育部筋骨理论与治法重点实验室（上海　200032）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2021.04.012
  CLC：
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CHENG Shao, TAO Yuren, SHU Bing, et al. Effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):79-88(2021)
DOI：

CHENG Shao, TAO Yuren, SHU Bing, et al. Effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):79-88(2021) DOI： 10.16306/j.1008-861x.2021.04.012.

摘要

目的：,2,观察化瘀补肾方对去卵巢小鼠骨折愈合及氧化应激的影响。,方法：,2,3月龄雌性C57BL/6小鼠随机分为假手术组、模型组、化瘀补肾方组，每组10只。模型组和化瘀补肾方组小鼠行双侧卵巢切除术（OVX），假手术组仅切除卵巢周围部分脂肪组织。术后3个月，各组小鼠均建立左侧胫骨骨折复合髓内固定模型。造模后，化瘀补肾方组小鼠灌胃给予7.33 g/kg化瘀补肾方药液，另两组小鼠灌胃给予等体积0.9%NaCl溶液，连续14 d。末次给药后，取血，分离胫骨组织。X线及Micro-CT扫描观察骨折愈合情况；阿尔新蓝/橙黄G染色后镜下观察骨痂组织形态学变化。试剂盒测定血清超氧化物歧化酶（SOD）、丙二醛（MDA）水平；免疫组化检测骨痂组织Ⅰ型胶原蛋白（CollagenⅠ）表达；免疫荧光检测骨痂组织Runt相关转录因子2（Runx2）、SOD表达；试剂盒检测骨痂组织MDA含量。TUNEL法检测骨痂组织细胞凋亡。,结果：,2,①胫骨X线及Micro-CT检测显示，模型组小鼠骨痂稀疏，骨折线清晰；化瘀补肾方组小鼠骨痂较致密，骨折线略模糊。②阿尔新蓝/橙黄G染色显示，模型组小鼠骨痂组织中软骨基质较多，骨小梁稀疏；化瘀补肾方组骨痂组织中软骨基质较少，骨小梁致密。③模型组骨痂组织Runx2、CollagenⅠ的表达较假手术组显著降低（,P,<,0.01），化瘀补肾方组骨痂组织Runx2、CollagenⅠ的表达较模型组明显升高（,P,<,0.05）。④化瘀补肾方组骨痂组织细胞凋亡较模型组明显减少（,P,<,0.01）。⑤与假手术组相比，模型组小鼠血清及骨痂组织SOD水平显著降低（,P,<,0.01），MDA含量显著升高（,P,<,0.01）；与模型组相比，化瘀补肾方组小鼠血清及骨痂组织SOD水平明显升高（,P,<,0.05），MDA含量明显下降（,P,<,0.01）。,结论：,2,化瘀补肾方能够促进去卵巢小鼠的骨折愈合，其作用机制可能与提高血清及局部骨痂组织的抗氧化酶水平、减轻氧化应激状态、抑制细胞凋亡有关。

Abstract

Objective:,2,To observe the effects of Huayu Bushen Formula on fracture healing and oxidative stress in ovariectomized mice.,Methods:,2,Three months old female C57BL/6 mice were randomly divided into the sham operation group, model group and Huayu Bushen Formula group, 10 mice in each group.Bilateral ovariectomy(OVX) was performed in the model group and Huayu Bushen Formula group, while only part of the adipose tissue around the ovary was removed in the sham operation group.Three months after operation, the model of left tibial fracture combined with intramedullary fixation was established in all groups.After modeling, mice in the Huayu Bushen Formula group were treated with decoction of Huayu Bushen Formula at dose of 7.33 g/kg by intragastric administration for 14 days, while mice in the sham operation group and model group were treated with 0.9% NaCl solution at equal volume.After last administration, blood was taken and tibia tissue was separated.The fracture healing was observed by X-ray and Micro-CT scanning.After alcian blue/orange G staining, the histomorphological changes of callus were observed under microscope.The serum levels of superoxide dismutase(SOD) and malondialdehyde(MDA) were measured by kits.The expression of CollagenⅠ in callus was detected by immunohistochemistry.The expressions of Runt-related transcription factor 2(Runx2) and SOD in callus were detected by immunofluorescence.The content of MDA in callus was detected by kit.The cell apoptosis in callus was detected by TUNEL assay.,Results:,2,①X-ray and Micro-CT examination of tibia showed that, the calluses were few and scattered and the fracture line was clear in the model group, while the calluses were dense and the fracture line was slightly blurred in the Huayu Bushen Formula group.②Alcian blue/orange G staining showed that, there were more cartilage matrix and less bone trabecula in callus of the model group, but less cartilage matrix and dense bone trabecular were observed in callus of the Huayu Bushen Formula group.③The expressions of Runx2 and CollagenⅠ in callus of the model group were significantly lower than those of the sham operation group(,P,<,0.01), and the expressions of Runx2 and CollagenⅠ in callus of the Huayu Bushen Formula group were significantly higher than those of the model group(,P,<,0.05) .④The cell apoptosis of callus tissue in the Huayu Bushen Formula group was significantly decreased compared with that in the model group(,P,<,0.01) .⑤Compared with the sham operation group, the SOD level in serum and callus tissue of the model group was significantly decreased(,P,<,0.01), and the MDA content was significantly increased(,P,<,0.01) .Compared with the model group, the SOD level in serum and callus tissue of the Huayu Bushen Formula group was significantly increased(,P,<,0.05), and the MDA content was significantly decreased(,P,<,0.01) .,Conclusion:,2,Huayu Bushen Formula can promote the fracture healing in ovariectomized mice, and its mechanisms may be related to increasing the levels of antioxidant enzymes in serum and local callus, alleviating oxidative stress and inhibiting cell apoptosis.

关键词

化瘀补肾方骨质疏松性骨折氧化应激小鼠

Keywords

Huayu Bushen Formulaosteoportic fractureoxidative stressmouse

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