葛根素对大鼠星形胶质细胞增殖、凋亡和β-catenin、cyclinD1、Bax蛋白表达的影响
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摘要
目的:探讨葛根素对H_2O_2诱导的大鼠星形胶质细胞(AST)增殖、凋亡的影响及作用机制。方法:采用MTT法检测100、200、400、600、800、1 000μmol/L H_2O_2对AST活力的影响;将AST分为空白对照组(细胞未经任何处理)、H_2O_2组(400μmol/L H_2O_2处理AST 24 h)和葛根素+H_2O_2组(1 mmol/L葛根素处理30 min后,400μmol/L H_2O_2处理24 h),应用MTT、Annexin V-FITC/PI双染法及Western blot分别检测细胞活力、凋亡率及β-catenin、cyclin D1和Bax蛋白的表达。结果:随着H_2O_2浓度增加,AST活力降低(P<0.05)。与空白对照组比较,H_2O_2组细胞活力明显降低,凋亡率升高,β-catenin和cyclin D1表达降低,Bax表达升高(P<0.05);与H_2O_2组比较,葛根素+H_2O_2组细胞活力升高,凋亡率降低,β-catenin和cyclin D1表达升高,Bax表达降低(P<0.05)。结论:葛根素可减轻H_2O_2诱导的大鼠AST氧化应激损伤,机制与激活Wnt/β-catenin信号通路有关。
Aim:To investigate the effects and mechanism of puerarin on proliferation and apoptosis of rat astrocytes induced by H_2O_2.Methods:The effects of different concentrations(100,200,400,600,800,1 000μmol/L)of H_2O_2 on the viability of astrocytes were detected by MTT assay.Rat astrocytes were divided into blank control group(untreated cells),H_2O_2 group(treated with 400μmol/L H_2O_2 for 24 hours),puerarin+H_2O_2 group(treated with 1 mmol/L puerarin for 30min and 400μmol/L H_2O_2 for 24 hours).MTT,flow cytometry and Western blot were used to detect cell viability,apoptosis and expressions ofβ-catenin,cyclin D1 and Bax proteins,respectively.Results:With the increase of H_2O_2 concentration,the activity of rat astrocytes decreased(P<0.05).Compared with the blank control group,the cell viability in the H_2O_2 group was significantly decreased,the apoptosis rate was significantly increased,the expressions ofβ-catenin and cyclin D1 were significantly decreased,and the expression of Bax was significantly increased(P<0.05).Compared with the H_2O_2 group,the cell viability in the puerarin+H_2O_2 group was significantly increased,the apoptosis rate was significantly decreased,the expressions ofβ-catenin and cyclin D1 were significantly increased,and the expression of Bax was significantly decreased(P<0.05).Conclusion:Puerarin can promote the proliferation and inhibit apoptosis of rat astrocytes induced by H_2O_2,and the mechanism is related to the activation of Wnt/β-catenin signaling pathway.
Aim:To investigate the effects and mechanism of puerarin on proliferation and apoptosis of rat astrocytes induced by H_2O_2.Methods:The effects of different concentrations(100,200,400,600,800,1 000μmol/L)of H_2O_2 on the viability of astrocytes were detected by MTT assay.Rat astrocytes were divided into blank control group(untreated cells),H_2O_2 group(treated with 400μmol/L H_2O_2 for 24 hours),puerarin+H_2O_2 group(treated with 1 mmol/L puerarin for 30min and 400μmol/L H_2O_2 for 24 hours).MTT,flow cytometry and Western blot were used to detect cell viability,apoptosis and expressions ofβ-catenin,cyclin D1 and Bax proteins,respectively.Results:With the increase of H_2O_2 concentration,the activity of rat astrocytes decreased(P<0.05).Compared with the blank control group,the cell viability in the H_2O_2 group was significantly decreased,the apoptosis rate was significantly increased,the expressions ofβ-catenin and cyclin D1 were significantly decreased,and the expression of Bax was significantly increased(P<0.05).Compared with the H_2O_2 group,the cell viability in the puerarin+H_2O_2 group was significantly increased,the apoptosis rate was significantly decreased,the expressions ofβ-catenin and cyclin D1 were significantly increased,and the expression of Bax was significantly decreased(P<0.05).Conclusion:Puerarin can promote the proliferation and inhibit apoptosis of rat astrocytes induced by H_2O_2,and the mechanism is related to the activation of Wnt/β-catenin signaling pathway.
引文
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[2]MAO XX,LIU J,CHEN C,et al.PCBP2 modulates neural apoptosis and astrocyte proliferation after spinal cord injury[J].Neurochem Res,2016,41(9):2401
[3]HAMDI Y,KADDOUR H,VAUDRY D,et al.The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a pka/mapk-dependent mechanism[J].PLo S One,2012,7(8):e42498
[4]MA YL,ZHANG LX,LIU GL,et al.N-myc downstreamregulated gene 2(ndrg2)is involved in ischemia-hypoxiainduced astrocyte apoptosis:a novel target for stroke therapy[J].Mol Neurobiol,2017,54(5):3286
[5]白群华,李文明,刘洪涛,等.葛根素对脂多糖诱导N9小胶质细胞激活的抑制作用[J].细胞与分子免疫学杂志,2010,26(3):227
[6]WANG N,ZHANG YM,WU L,et al.Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J].Neuropharmacology,2014,79(4):282
[7]袁芳,王忠诚,王天佑,等.葛根素对星形胶质细胞肿胀及大鼠脑水肿的对抗作用[J].首都医科大学学报,2001,22(3):206
[8]REDDY VP,ZHU XW,PERRY G,et al.Oxidative stress in diabetes and alzheimer's disease[J].J Alzheimers Dis,2009,16(4):763
[9]WANG M,LI YJ,DING Y,et al.Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury[J].Mol Neurobiol,2016,53(2):932
[10]LING YZ,LI XH,LI Y,et al.Protective effects of parecoxib on rat primary astrocytes from oxidative stress induced by hydrogen peroxide[J].J Zhejiang Univ Sci B,2016,17(9):692
[11]WANG YC,WU YT,HUANG HY,et al.Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury[J].Biomaterials,2008,29(34):4546
[12]孙文阁,李春鹏,张晔,等.脊髓半切损伤后髓鞘碱性蛋白及胶质纤维酸性蛋白的表达及意义[J].苏州大学学报(医学版),2005,25(6):947
[13]LIU CM,MA JQ,LIU SS,et al.Puerarin protects mouse liver against nickel-induced oxidative stress and inflammation associated with the TLR4/p38/CREB pathway[J].Chem Biol Interact,2016,243(8):29
[14]LIU Y,TANG Q,SHAO SY,et al.Lyophilized powder of catalpol and puerarin protected cerebral vessels from ischemia by its anti-apoptosis on endothelial cells[J].Int J Biol Sci,2017,13(3):327
[15]ZHU GQ,WANG XC,WU SB,et al.Neuroprotective effects of puerarin on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced parkinson's disease model in mice[J].Phytother Res,2014,28(2):179
[16]王艳竹.体外缺氧/复氧对星形胶质细胞AQP4表达的影响及葛根素干预的实验研究[D].长春:吉林大学,2005.
[17]MOHAMMED MK,SHAO C,WANG J,et al.Wnt/β-catenin signaling plays an ever-expanding role in stem cell selfrenewal,tumorigenesis and cancer chemoresistance[J].Genes Dis,2016,3(1):11
[18]GAO K,SHEN ZL,YUAN YJ,et al.Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/beta-catenin signaling pathway after spinal cord injury[J].J Neurochem,2016,138(1):139
[19]WANG L,ZHAO Y,WU Q,et al.Therapeutic effects ofβ-elemene via attenuation of the Wnt/β-catenin signaling pathway in cervical cancer cells[J].Mol Med Rep,2018,17(3):4299
[20]赵明明,黄艳群,李莲,等.Wnt/β-catenin信号通路激活剂对过氧化氢诱导的星形胶质细胞凋亡的影响[J].临床和实验医学杂志,2018,17(9):917
[21]TEIXEIRA A,CHAVEROT N,STROSBERG AD,et al.Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1[J].J Neurochem,2000,74(3):1034
[22]CHEN XY,LU M,HE XJ,et al.TRPC3/6/7 Knockdown protects the brain from cerebral ischemia injury via astrocyte apoptosis inhibition and effects on nf-db translocation[J].Mol Neurobiol,2017,54(10):7555