补肾健脾方增加成肌分化表达促进去睾丸小鼠骨质疏松性骨折愈合的实验研究

The Study of Efficacy of the Nourishing Kidney and Invigorating Spleen Formula in Improving Osteoporotic Fracture Repair by Promoting Myogenic Differentiation

目的:研究补肾健脾方对去睾丸小鼠骨质疏松性骨折愈合的疗效,探讨“先后天之本”“肾脾相关”“骨肉不相亲”等中医理论在骨质疏松性骨折防治中的应用。方法:按照体重并采用随机方法将30只雄性C57BL/6小鼠分为对照组和补肾健脾方组,每组各15只;补肾健脾方组给予补肾健脾方灌胃,对照组给予生理盐水灌胃。两组制备去睾丸小鼠骨质疏松性骨折模型,于骨折术后第2天分别给予相应药物灌胃治疗,连续灌胃28 d,隔天1次,治疗结束后处死小鼠并取材,进行影像学观察、病理组织学染色以及逆转录聚合酶链式反应(RT-PCR)、蛋白质免疫印迹试验(Western Blot)方法检测MyoD1、MEF2C、Myf5及Myogenin的表达量。结果:微型CT结果显示,补肾健脾方可促进骨痂形成及加速骨折愈合;比目鱼肌苏木精-伊红(HE)染色结果显示,与对照组比较,补肾健脾方组肌纤维排列紧密,肌细胞较多。RT-PCR和Western Blot结果发现,与对照组比较,补肾健脾方组胫骨后肌MyoD1、MEF2C、Myf5及Myogenin等成肌分化基因和蛋白表达量均较高,且差异都有统计学意义(P<0.05)。结论:补肾健脾方可促进去睾丸小鼠骨质疏松性骨折的愈合,其机制可能与增加胫骨附着肌内MyoD1、MEF2C、Myf5及Myogenin的表达有关。

Objective:To examine the ability of the nourishing kidney and invigorating spleen formula to promote osteoporotic fracture healing,and to discuss the traditional Chinese medicine(TCM)theories of the relationship between spleen and kidney,the origin of the innateness and acquirement,along with disharmony between bone and muscle in the application of the osteoporotic fracture prevention.Methods:30 male C57BL/6 mice were divided into control group and nourishing kidney and invigorating spleen formula group,with 15 mice in each group.The nourishing kidney and invigorating spleen formula group was treated with the nourishing kidney and invigorating spleen formula,and control group was treated with normal saline by gastric perfusion.The osteoporotic fracture model after castration were prepared in two groups.On the second day after fracture operation,each group was given corresponding drugs by gavage for 28 d,once every other day,and after the treatment,the mics were killed to take the samples for imaging observation,histopathological staining and reverse transcription-polymerase chain reaction(RT-PCR)and Western Blot methods to detect the expression of MyoD1,MEF2C,Myf5 and Myogenin.Results:The nourishing kidney and invigorating spleen formula had an effective effect in stimulating the formation of callus and improving the quality of fracture healing according to Micro-CT.The hematoxylin-eosin(HE)staining result of solus muscle showed that treatment of nourishing kidney and invigorating spleen formula could result in tighter arrangement of muscle fibers and more muscle cells than the control group.Western Blot and RT-PCR results showed that,compared with control group,the specific myogenic transcription factors of posterior tibial muscle,such as MyoD1,MEF2C,Myf5 and Myogenin,and protein expression in the nourishing kidney and invigorating spleen formula group were higher,and the differences were statistically significant.Conclusion:The nourishing kidney and invigorating spleen formula enhanced the repair of osteoporotic fracture,and its mechanism may be related to increasing the expression of MyoD1,MEF2C,Myf5 and Myogenin in tibial attachment muscle.