丁公藤注射液促进骨质疏松性大鼠骨折愈合的作用机制研究

Study on the Mechanism of Injection Erycibe in Promoting Fracture Healing in Osteoporotic Rats

关键词:  丁公藤  骨质疏松  成骨分化  酪氨酸激酶1的非催化区  蛋白激酶B

Key words:  erycibe  osteoporosis  osteogenic differentiation  non-catalytic domain of tyrosine kinase 1  protein kinase B

摘要:

目的 研究丁公藤注射液(injection erycibe，IE)对骨质疏松性大鼠骨折愈合的促进作用及其作用机制。方法 通过卵巢切除术和骨折术建立骨质疏松性骨折大鼠模型，50只健康SD大鼠(♀)随机分为假手术组、模型组和1，2，4mL·kg^-1 IE组，检测大鼠股骨骨密度、生物力学指标和血清钙、碱性磷酸酶含量，HE染色观察大鼠骨骼病理组织学变化。对分离的骨髓间充质干细胞(mesenchymal stem cells，MSCs)进行药物干预，分为对照组、5，10，20mg·L^-1 IE组、诱导组(成骨诱导培养基诱导分化)、NC siRNA组、NCK1 siRNA组、IE+NC siRNA组和IE+NCK1 siRNA组，采用MTT检测MSCs增殖，RT-qPCR检测成骨分化相关基因ALP、Runx2和Osterix mRNA水平，Western blotting检测NCK1和p-AKT蛋白表达水平，茜素红染色观察MSCs钙结节数量。结果 与模型组比较，2，4mL·kg^-1 IE组大鼠股骨骨密度、生物力学指标、血清生化指标均明显升高(P<0.05)，且呈剂量依赖性，4mL·kg^-1 IE组效果最佳。与对照组比较，10，20mg·L^-1IE组MSCs增殖和成骨分化相关基因表达量明显升高(P<0.05或P<0.01)，NCK1和p-AKT蛋白表达水平明显升高(P<0.05)，MSCs内钙结节量明显增加，且呈剂量依赖性，20mg·L^-1 IE组效果最佳。与IE+NC siRNA组比较，IE+NCK1 siRNA组成骨分化相关基因表达量明显降低(P<0.01)，NCK1和p-AKT蛋白表达水平明显降低(P<0.01)。结论 IE通过促进NCK1蛋白表达，激活AKT信号通路，对骨质疏松性大鼠骨折愈合起到一定促进作用。

Abstract:

OBJECTIVE To study the promoting effect and mechanism of injection erycibe(IE) on fracture healing in osteoporotic rats. METHODS Castration and fracture surgery were used to establish a rat model of osteoporotic fracture. Fifty female healthy SD rats were randomly divided into sham operation group, model group and 1, 2, 4 mL·kg^-1 IE group. The rat femur bone mineral density, biomechanical indicators, serum calcium and alkaline phosphatase levels were detected. HE staining was used to observe the pathological changes of rat skeleton. The isolated bone marrow mesenchymal stem cells(MSCs) were intervened with drugs, which were divided into control group, 5, 10, 20 mg·L^-1IE group, induction group(using osteogenic induction medium), NC siRNA group, NCK1 siRNA group, IE+NC siRNA group and IE+NCK1 siRNA group. MTT method was used to detect the proliferation of MSCs. RT-qPCR was used to detect the mRNA levels of osteogenic differentiation related genes ALP, Runx2 and Osterix. Western blotting was used to detect the protein expression levels of NCK1 and p-AKT. Alizarin red staining was used to observe the number of calcium nodules in MSCs. RESULTS Compared with the model group, the bone mineral density, biomechanical indexes, and serum biochemical indexes of rats in 2, 4 mL·kg^-1 IE groups were significantly increased(P<0.05) in a dose-dependent manner, and 4 mL·kg^-1 IE group had the best effect. Compared with the control group, the proliferation of MSCs and the expression of osteogenic differentiation-related genes in 10, 20 mg·L^-1IE group were significantly increased(P<0.05 or P<0.01), and the expression levels of NCK1 and p-AKT proteins were significantly increased(P<0.05) and the amount of calcium nodules in MSCs was significantly increased in a dose-dependent manner. Moreover, the 20 mg·L^-1 IE group had the best effect. Compared with IE+NC siRNA group, the expression of bone differentiation-related genes ALP, Runx2 and Osterix mRNA were significantly decreased(P<0.01), the expression levels of NCK1 and p-AKT proteins were significantly decreased(P<0.01) in IE+NCK1 siRNA group. CONCLUSION EI can promote fracture healing in osteoporotic rats by promotes the expression of NCK1 protein and activates the AKT signaling pathway.