补肾中药通过调控Notch1蛋白的表达治疗大鼠骨质疏松性骨折的作用研究

The effect of Chinese kidney-tonifying herbal medicine on osteoporotic fractures by regulating Notch1 protein in rats

目的探究补肾中药对大鼠骨质疏松骨折的治疗作用和机制。方法选取老年雌性大鼠60只,分为正常组、骨质疏松骨折模型组、高、中、低剂量补肾中药治疗组,各组均摘除卵巢,术后3个月形成骨质疏松模型。随后除正常组外,其余各组均建立股骨骨折模型。骨质疏松骨折模型建立后,每日给予正常组与模型组生理盐水进行灌胃,治疗组分别给予高、中、低剂量补肾中药灌胃12 w进行治疗。末次实验结束后麻醉大鼠,应用双能X射线检测大鼠股骨的骨密度。采取血清样品通过ELISA法测量碱性磷酸酶、骨钙素、TRAP破骨标志物等指标含量,对股骨样品进行病理学检查以及Western blot法检测Notch1蛋白的表达。结果病理组织学切片结果显示,高剂量治疗组在骨折端的成骨细胞数量较多,破骨细胞数量较少,提示补肾中药具有促进骨性愈合的效果。经补肾中药治疗后,高剂量治疗组的骨密度与模型组相比骨密度显著回升。研究发现,strACP经过补肾中药治疗后,治疗组中的碱性磷酸酶与血清抗酒石酸性磷酸酶的含量显著降低,骨钙素含量明显提升,上述指标在高剂量治疗组中与模型组相比差异均具有统计学意义(P<0.01),呈明显的剂量依赖性。另外,Western blot法检测结果也显示,Notch1蛋白的表达在模型组中受到显著抑制(与正常组相比,P<0.01),而补肾中药具有调节Notch1蛋白表达的作用,成明显剂量相关性,且高剂量治疗组的表达水平与正常组接近。结论补肾中药能够通过调节Notch1蛋白的表达来抑制血清中破骨标志物碱性磷酸酶和TRAP的水平,并提高骨钙素的含量来刺激成骨细胞生成、抑制破骨细胞分化,从而加速骨折的愈合,提高骨密度,对大鼠股骨骨质疏松骨折具有良好的治疗作用。

Objective To investigate the effect of compound Chinese kidney-tonifying herbal medicine on osteoporotic fractures in rats and its mechanism.Methods 60 elder male rats were divided into 5 groups：normal control group（NC）,osteoporotic fracture model control group（MC）,and low/middle/high dose of compound Chinese kidney-tonifying herbal medicine-treated groups（BS-L/M/H）.After removal of the ovaries for 3 months,the osteoporotic fracture model was established in each group except for NC group.Rats in NC group and MC group received normal saline after model establishment for 12 weeks,while rats in BS groups received low/middle/high dose of extracts of compound Chinese kidney-tonifying herbal medicine,respectively,during the experimental period.24 hours after the last treatment,rats were anaesthetized to take X-ray photos in order to measure bone mineral density（BMD）.Serum was collected to measure the content of alkaline phosphatase（AKP）,osteocalcin（OC） and serumtar trate-resistant acid phosphatase（strACP）.Femurs were collected.Histopathological observation and Western blotting analysis were used to detect the expression of Notch1 protein in bone.Results Histopathological observation showed that more osteoblasts and less osteoclasts in BS-H group than in MC group,suggesting that application of compound Chinese kidney-tonifying herbal medicines accelerated the healing of fractured bone.BMD in BS-H group was significantly higher than that MC group.Mechanism study showed that activities of AKP and strACP in the treatment groups decreased,but OC increased significantly,and there was statistical difference between BS-H group and MC group（P〈0.01）.The expression of Notch1 protein was significantly inhibited in MC group compared to NC group（P〈0.01）.Compound Chinese kidney-tonifying herbal medicine up-regulated Notch1 protein expression dose-dependently,and in BS-H group the expression was close to that in NC group.Conclusion Compound Chinese kidney-tonifying herbal medicine inhibits the activities of AKP and strACP by regulating the expression of Noth1 protein,and upregulates the growth of osteoblasts and downregulates the differentiation of osteoclasts by elevating the content of OC,thus helps the bone fracture healing and increases BMD,showing a good therapeutic effect on osteoporotic femoral fracture in rats.