齐墩果酸局部给药对去卵巢小鼠骨折愈合的影响

Effect of local administration of oleanic acid on bone fracture healing in ovariectomized mice

目的探讨齐墩果酸局部给药对去卵巢小鼠骨折愈合的影响及作用机制。方法采用3月龄雌性C57BL/6小鼠45只,随机分为假手术组、模型组、齐墩果酸(oleanic acid, OA)组,每组15只。模型组和OA组通过双侧卵巢切除术(ovariectomy, OVX)建立小鼠骨质疏松症模型,假手术组仅切除卵巢周围脂肪组织。OVX术后3个月建立左侧胫骨中段骨折模型,同时OA组术中植入植入式渗透泵,局部缓释OA。术后14、28 d取材,观察骨痂组织的形态、骨小梁微结构、病理改变以及Notch信号通路靶基因和成骨相关基因的表达情况。结果左侧胫骨组织三维重建显示,骨折14 d时,假手术组骨折线模糊,骨折端有大量骨痂形成,模型组仍有部分骨折线,OA组骨折线模糊;28 d时,假手术组骨折已基本愈合,骨痂较少,模型组骨折部位仍有较大的骨痂,OA组骨折部位骨痂较少。与假手术组相比,术后14 d时模型组小鼠骨痂组织中Notch信号通路靶基因Hes1 mRNA表达水平升高(P<0.05),Runt相关转录因子2(runt-related transcription factor 2, Runx 2)、骨钙素(osteocalcin, OCN)和碱性磷酸酶(alkaline phosphatase, ALP)的mRNA表达水平降低(P<0.05);与模型组相比,OA组骨痂组织中Hes1 mRNA表达水平降低(P<0.05),Runx 2、OCN的mRNA表达水平升高(P<0.05);术后28 d时,各组Hes1 mRNA表达水平未见显著差异,OA组和模型组Runx 2、ALP和OCN的mRNA表达水平均低于假手术组(P<0.05),但OA组和模型组Runx 2、ALP的mRNA表达水平未见差异,而OA组OCN的mRNA表达水平高于模型组(P<0.05)。骨折14 d时,步态分析结果显示,OA组左后肢平均迈步时间比模型组小鼠减少(P<0.05)。结论局部缓释OA,可以下调骨折愈合中期Notch信号通路下游靶基因Hes1的表达,上调成骨相关基因Runx 2、OCN的表达,促进去卵巢小鼠的骨折愈合。

Objective To investigate the effect and mechanism of local administration of oleanic acid on bone fracture healing in ovariectomized mice. Methods A total of 45 female C57 BL/6 mice aged 3 months were randomly divided into sham group, model group and oleanic acid(OA) group, with 15 mice in each group. In the model group and OA group, osteoporosis model in mice was established by bilateral ovariectomy(OVX), while only parovarian adipose tissue was removed in the sham mice. Three months after OVX, a middle fracture model of the left tibia was established, and an implantable osmotic pump was implanted simultaneously during the operation, with local sustained-release of OA in OA group. At 14 and 28 days after the operation, the morphology of callus tissue, bone trabecular microstructure, pathological changes, and the expression levels of Notch signaling pathway target gene and osteogenic related genes were observed. Results The three-dimensional reconstruction of left tibia showed that the fracture lines of the sham group and OA group were fuzzy at 14 days after fracture, and a large number of callus were formed. The fracture line of model group was still clear. At 28 days after fracture, the callus of the sham group and OA group had almost finished remodeling, while there was still larger callus in the model group. The mRNA expression level of Notch signaling pathway target gene Hes1 and RUNt-related transcription factor 2(Runx 2), osteocalcin(OCN) and alkaline phosphatase(ALP) in callus of the model group were increased by 14 days after fracture compared with those of sham operation group(P<0.05). Compared with the model group, the expression level of Hes1 mRNA in callus of the OA group was decreased(P<0.05), while the expression levels of Runx 2 and OCN mRNA were increased(P<0.05). At 28 days after fracture, there was no significant difference in the mRNA expression levels of Hes1 among different groups, and the mRNA expression levels of Runx2, ALP and OCN in the OA group and the model group were lower than those in the sham operation group(P<0.05), but there was no significant difference in the mRNA expression levels of Runx 2 and ALP between the OA group and the model group, while the mRNA expression level of OCN in the OA group was higher than that in the model group(P<0.05). At 14 days after fracture, gait analysis result showed that the average walking time of left posterior limb in OA group was less than that in model group(P<0.05). Conclusion Local sustained-release OA down-regulated the expression of the target gene of Notch signaling pathway, and up-regulate the expressions of osteogenic related genes, so as to promote the fracture healing of ovariectomized mice.