黄芪甲苷通过调控FoxO3a/Wnt2/β-catenin通路抑制去卵巢大鼠骨质疏松的作用

Inhibitory Effect of Astragaloside on Osteoporosis in Ovariectomized Rats by Regulating FoxO3a/Wnt2/β-catenin Pathway

目的：通过调控转录因子叉头框蛋白O3a（FoxO3a）/分泌型糖蛋白2（Wnt2）/β-连环蛋白（β-catenin）通路评价黄芪甲苷对去卵巢大鼠骨质疏松的作用,并探讨其作用机制。方法：雌性SD大鼠随机均分6组,分别为假手术组,模型组,阳性药组（尼尔雌醇,1.5 mg·kg-1）,黄芪甲苷低、中、高剂量组（20,40,80 mg·kg-1）,每组8只。通过摘除大鼠双侧卵巢复制绝经后大鼠骨质疏松模型。术后6周,灌胃给药,每日1次,连续8周。酶联免疫吸附测定（ELISA）试剂盒检测大鼠骨钙素（bone gla protein,BGP）,降钙素（calcitonin,CT）,骨保护素（osteoprotegerin,OPG）,核转录因子-κB（NF-κB）受体活化因子配体（receptor activator for nuclear factor-κB ligand,RANKL）,丙二醛（malondialdehyde,MDA）,过氧化氢酶（catalase,CAT）,谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）和超氧化物歧化酶（superoxide dismutase,SOD）含量,双能X射线骨密度检测仪分别测定股骨和腰椎的骨密度值（bone mineral density,BMD）,采用三点弯曲法测量胫骨生物力学指标,蛋白免疫印迹法（Western blot）检测大鼠股骨组织中β-catenin,FoxO3a,Wnt2,p66（shc）,p-p66（shc）蛋白的表达。结果：与模型组比较,黄芪甲苷组CT和OPG水平显著升高,BGP和RANKL水平则显著降低（P〈0.05,P〈0.01）;黄芪甲苷显著升高腰椎、股骨的BMD和胫骨的最大载荷及最大应力（P〈0.05,P〈0.01）;同时,黄芪甲苷显著降低MDA水平（P〈0.05）,升高CAT,SOD和GSH-Px水平（P〈0.05,P〈0.01）。Western blot显示,黄芪甲苷显著降低p-p66（shc）/p66（shc）水平（P〈0.05）;升高β-catenin和Wnt2蛋白表达水平（P〈0.05,P〈0.01）,抑制FoxO3a蛋白表达水平（P〈0.01）。结论：黄芪甲苷能够有效缓解氧化应激介导的去卵巢大鼠的骨质疏松,该作用可能与其调节FoxO3a/Wnt2/β-catenin通路有关。

Objective： To investigate the effect of astragaloside on osteoporosis in ovariectomized rats and its mechanism by forkhead box O3a（FoxO3 a）/Wnt2/β-catenin signal pathway. Method： Female SD rats were randomly divided into 6 groups： shamed operation group,positive control group（Nilestriol,1. 5 mg·kg-1）,model group,low,middle and high-dose astragaloside groups（20,40,80 mg·kg-1）（n = 8）. The animal model of osteoporosis in postmenopausal rats was replicated through bilateral ovariectomy. After 6 weeks,the drugs were given by gavage once a day for 8 weeks. The serum contents of bone gla protein（BGP）, calcitonin（CT）,osteoprotegerin（OPG）,receptor activator for nuclear factor-κB ligand（RANKL）,malondialdehyde（MDA）,catalase（CAT）,glutathione peroxidase（GSH-Px） and superoxide dismutase（SOD） were detected by enzymelinked immunosorbent assay（ELISA） kits; bone mineral density（BMD） in lumbar spine and lumbar vertebra was tested by double energy X rays（DEXA）,and bone biomechanical properties were analyzed by three point bending test. The expressions of β-catenin,FoxO3 a,Wnt2,p66（shc）,p-p66（shc）were measured by Western blot.Result： Compared with the model group,the levels of CT and OPG were significantly decreased,while BGP and RANKL were significantly increased（P 〈 0. 05） in the astragaloside groups. astragaloside significantly elevated BMD in lumbar spine and lumbar vertebra（P 〈 0. 05）,and the maximum load and maximum stress of tibia（P〈0. 01）. Meanwhile,the content of MDA was significantly decreased（P 〈 0. 05）,whereas the levels of CAT,SOD and GSH-PX were increased. Western blot showed that astragalloside significantly reduced p-p66（shc）/p66（shc）（P〈0. 05）,inhibited the expression of FoxO3 a（P 〈 0. 01） and increased β-catenin and Wnt2（P 〈 0. 05,P 〈 0. 01）.Conclusion： Astragaloside can effectively relieve osteoporosis induced by oxidative stress in ovariectomized rats,which may be related to the regulation of FoxO3 a/Wnt2/β-catenin pathway.