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
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摘要
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.
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.
引文
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[11]徐昌君,王鹏飞,刘杨,等.黄芪甲苷对特发性肺纤维化自噬活性作用PI3K/Akt/m TOR信号调控的影响[J].中国实验方剂学杂志,2017,23(18):75-82.
[12]Golob A L,Laya M B.Osteoporosis:screening,prevention,and management[J].Med Clin Nam,2015,99(3):587-606.
[13]Drake M T,Clarke B L,Lewiecki E M.The pathophysiology and treatment of osteoporosis[J].Clin Ther,2015,37(8):1837-1850.
[14]LI C W,LIANG B,SHI X L,et al.Opg/Rankl mRNA dynamic expression in the bone tissue of ovariectomized rats with osteoporosis[J].Genet Mol Res,2015,14(3):9215.
[15]GONG S,HAN X,LI X,et al.Development of a highthroughput screening strategy for upregulators of the OPG/RANKL ratio with the potential for antiosteoporosis effects[J].J Biomol Screen,2016,21(7):738-742.
[16]ZHOU Q,LI Z,ZHANG D,et al.Oxidative stress-related biomarkers in postmenopausal osteoporosis:a systematic review and Meta-analyses[J].Dis Markers,2016(3):1-12.
[17]Mai F T,El-Serafi A T,Omar H A.Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo:impact on oxidative stress and RANKL/OPG signals[J].Toxicol Appl Pharm,2017,324(4):26-35.
[18]DI L F,Giorgio M,Ferdinandy P,et al.New aspects of p66Shc in ischemia reperfusion injury and cardiovascular diseases[J].Brit J Pharmacol,2016,174(12):1690-1703.
[19]Wolski H,Drwska-Matelska N,Seremak-Mrozikiewicz A,et al.The role of Wnt/β-catenin pathway and LRP5protein in metabolism of bone tissue and osteoporosis etiology[J].Gineklo Pol,2015,86(4):311-314.
[20]CHEN X,LI L,GUO J,et al.Treadmill running exercise prevents senile osteoporosis and upregulates the Wnt signaling pathway in SAMP6 mice[J].Oncotarget,2016,44(7):71072-71086.
[21]ZHAO X L,CHEN J J,ZHANG G N,et al.Small molecule T63 suppresses osteoporosis by modulating osteoblast differentiation via BMP and WNT signaling pathways[J].Sci Rep,2017,7(1):10397.
[22]LIU X Y,WANG Q X,LU J,et al.Protective effect of Li Cl-activated Wnt/β-catenin signaling pathway on glucocorticoid-induced osteoporosis[J].AJSMMU,2017,38(2):201-205.
[2]YANG Y J,LIAO C.Regulation mechanisum of Fox O/Wnt pathway in osteoporosis mediated by oxidative stress[J].CPB,2013,29(1):27-30.
[3]孙振双.温肾固疏方调控Fox O/Wnt信号通路抗氧化应激防治绝经后骨质疏松的机制研究[D].南京:南京中医药大学,2017.
[4]Canalis E.Wnt signalling in osteoporosis:mechanisms and novel therapeutic approaches[J].Nat Rev Endocrinol,2013,9(10):575-583.
[5]曹玉冰.黄芪甲苷的药理作用及其机制的研究进展[J].现代药物与临床,2017,32(5):954-960.
[6]梅周芳,施天昀,都勇,等.黄芪甲苷研究进展[J].临床医药文献电子杂志,2017,4(3):586-587.
[7]陈倩,季旭明,阚东方,等.黄芪甲苷对复合水饮内停大鼠心脏功能的影响[J].中国实验方剂学杂志,2017,23(14):100-105.
[8]靳晓飞,高维娟.黄芪甲苷生物活性及神经保护作用研究进展[J].河北中医药学报,2017,32(3):54-57.
[9]黄海涛,梁单,金昱.黄芪甲苷拮抗过氧化氢引起的小鼠成骨细胞凋亡作用[J].延边大学医学学报,2012,35(2):86-91.
[10]祁珊珊,王永吉,辛薇.SD大鼠绝经后骨质疏松疾病动物模型的构建[J].临床与实验病理学杂志,2016,32(1):49-52.
[11]徐昌君,王鹏飞,刘杨,等.黄芪甲苷对特发性肺纤维化自噬活性作用PI3K/Akt/m TOR信号调控的影响[J].中国实验方剂学杂志,2017,23(18):75-82.
[12]Golob A L,Laya M B.Osteoporosis:screening,prevention,and management[J].Med Clin Nam,2015,99(3):587-606.
[13]Drake M T,Clarke B L,Lewiecki E M.The pathophysiology and treatment of osteoporosis[J].Clin Ther,2015,37(8):1837-1850.
[14]LI C W,LIANG B,SHI X L,et al.Opg/Rankl mRNA dynamic expression in the bone tissue of ovariectomized rats with osteoporosis[J].Genet Mol Res,2015,14(3):9215.
[15]GONG S,HAN X,LI X,et al.Development of a highthroughput screening strategy for upregulators of the OPG/RANKL ratio with the potential for antiosteoporosis effects[J].J Biomol Screen,2016,21(7):738-742.
[16]ZHOU Q,LI Z,ZHANG D,et al.Oxidative stress-related biomarkers in postmenopausal osteoporosis:a systematic review and Meta-analyses[J].Dis Markers,2016(3):1-12.
[17]Mai F T,El-Serafi A T,Omar H A.Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo:impact on oxidative stress and RANKL/OPG signals[J].Toxicol Appl Pharm,2017,324(4):26-35.
[18]DI L F,Giorgio M,Ferdinandy P,et al.New aspects of p66Shc in ischemia reperfusion injury and cardiovascular diseases[J].Brit J Pharmacol,2016,174(12):1690-1703.
[19]Wolski H,Drwska-Matelska N,Seremak-Mrozikiewicz A,et al.The role of Wnt/β-catenin pathway and LRP5protein in metabolism of bone tissue and osteoporosis etiology[J].Gineklo Pol,2015,86(4):311-314.
[20]CHEN X,LI L,GUO J,et al.Treadmill running exercise prevents senile osteoporosis and upregulates the Wnt signaling pathway in SAMP6 mice[J].Oncotarget,2016,44(7):71072-71086.
[21]ZHAO X L,CHEN J J,ZHANG G N,et al.Small molecule T63 suppresses osteoporosis by modulating osteoblast differentiation via BMP and WNT signaling pathways[J].Sci Rep,2017,7(1):10397.
[22]LIU X Y,WANG Q X,LU J,et al.Protective effect of Li Cl-activated Wnt/β-catenin signaling pathway on glucocorticoid-induced osteoporosis[J].AJSMMU,2017,38(2):201-205.