补骨脂提取物干预骨质疏松模型大鼠骨密度及骨生物力学的变化

Effects of psoralen corylifolia extract on bone mineral density and bone biomechanics in osteoporosis rats

背景:地塞米松作为一种糖皮质激素,长期应用会破坏成骨与破骨的动态平衡,降低骨密度,损伤骨生物力学,调控核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)/骨保护蛋白通路可能影响地塞米松诱导的骨质疏松症大鼠骨密度及骨生物力学。目的:探讨基于RANKL/骨保护蛋白通路研究补骨脂提取物对地塞米松诱导骨质疏松症大鼠骨密度及骨生物力学的影响。方法:SPF级Wistar大鼠肌肉注射地塞米松,建立骨质疏松大鼠模型。选择1×107 TU/mL浓度的慢病毒载体进行实验。将模型大鼠随机分为模型组、空载组(空慢病毒载体)、骨保护蛋白沉默组(含骨保护蛋白基因干扰片段的慢病毒载体)、补骨脂提取物组、补骨脂提取物+骨保护蛋白沉默组,每组12只,另取12只正常大鼠设为对照组。药物处理后,采用骨密度仪测定大鼠左侧股骨骨密度,采用力学实验测试机测定大鼠右侧股骨生物力学指标弹性模量、最大载荷、屈服载荷,测定大鼠股骨骨矿物盐含量,酶联免疫吸附法检测血清中RANKL、骨保护蛋白水平,蛋白免疫印迹法检测骨组织中RANKL、骨保护蛋白表达水平。实验方案经青海大学医学院动物实验伦理委员会批准(批准号为2017081501)结果与结论:①大鼠骨密度、弹性模量、最大载荷、屈服载荷、骨矿物盐含量、血清中骨保护蛋白水平、骨组织中骨保护蛋白表达:模型组较对照组降低,骨保护蛋白沉默组较模型组降低,补骨脂提取物组较模型组升高,补骨脂提取物+骨保护蛋白沉默组较骨保护蛋白沉默组升高,较补骨脂提取物组降低(均P<0.05);②大鼠血清中RANKL水平、骨组织中RANKL蛋白表达结果显示,模型组较对照组升高;骨保护蛋白沉默组较模型组升高,补骨脂提取物组较模型组降低,补骨脂提取物+骨保护蛋白沉默组较骨保护蛋白沉默组降低,较补骨脂提取物组升高(均P<0.05);③模型组与空载组两组间各指标比较无明显变化(P>0.05);④结果说明,补骨脂提取物可提高地塞米松诱导骨质疏松症大鼠的骨密度,改善其骨生物力学,可能是通过上调骨保护蛋白表达,下调RANKL表达实现的。

BACKGROUND:Long-term use of dexamethasone as a glucocorticoid will destroy the dynamic balance of osteogenesis and osteoclastation,reduce bone mineral density,damage bone biomechanics.A regulation of receptor activator of nuclear factor-κB ligand(RANKL)/osteoprotegerin(OPG)protein pathway may be an approach to improve bone mineral density and bone biomechanics in dexamethasone-induced osteoporosis rats.OBJECTIVE:To investigate the effects of psoralen corylifolia extract on bone mineral density and bone biomechanics in dexamethasone-induced osteoporosis rats based on RANKL/OPG pathway.METHODS:Osteoporosis models were established in Wistar rats,SPF grade,using intramuscular injection of dexamethasone.Lentiviral vectors containing OPG gene interference fragments at concentration of 1×107 TU/mL were selected for subsequent experiments.The model rats were randomly divided into model group,no-load group(lentivirus empty vector),OPG silencing group(lentivirus vector containing OPG gene interference fragment),psoralen corylifolia extract group,psoralen corylifolia extract+OPG silencing group,with 12 rats in each group.Another 12 rats were selected as the control group.After drug treatment,bone mineral density of the left femur in rats was measured by bone densitometer;the elastic modulus,maximum load and yield load of the right femur of rats were measured by mechanical testing machine;the mineral salt content in the femur of rats was determined;the levels of RANKL and OPG in the serum of rats were detected by ELISA kit;and the expression levels of RANKL and OPG in bone tissues of rats were detected by western blot.The study protocol was approved by the Animal Ethics Committee of Qinghai University Medical School with an approval No.2017081501.RESULTS AND CONCLUSION:(1)Bone mineral density,modulus of elasticity,maximum load,yield load,bone mineral salt content,serum OPG level and OPG protein expression in bone tissues were measured,which were lower in the model group than the control group,lower in the OPG silencing group than the model group,higher in the psoralea corylifolia extract group than the model group,and higher in the psoralen corylifolia extract+OPG silencing group than the OPG silencing group,but lower than the psoralea corylifolia extract group(all P<0.05).(2)The level of RANKL in serum and the expression of RANKL in bone tissue were measured,which were higher in the model group than the control group,higher in the OPG silencing group than the model group,lower in the psoralea corylifolia extract group than the model group,and lower in the psoralen corylifolia extract+OPG silencing group than the OPG silencing group,but higher than the psoralea corylifolia extract group(all P<0.05).(3)There was no significant difference in each index between model group and no-load group(P>0.05).(4)All these findings indicate that psoralea corylifolia extract can increase bone mineral density and improve bone biomechanics in dexamethasone-induced osteoporosis rats,possibly by up-regulating the expression of OPG and down-regulating the expression of RANKL.