管花肉苁蓉醇提取物对缺氧/缺糖再灌注所致神经细胞损伤的保护作用及机制研究
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摘要
探究管花肉苁蓉醇提取物(CTEE)对缺氧/缺糖再灌注(OGD/R)所致的PC12细胞损伤的抑制作用及潜在机制。实验以OGD/R诱导PC12细胞作为损伤模型,通过MTT法检测细胞存活率、通过AO/EB与Hoechst 33258染色法考察细胞凋亡、通过JC-1染色法考察线粒体膜电位变化、通过MitoSOX染色法考察线粒体氧化应激反应,通过Western blot法考察凋亡相关蛋白(PARP,cleaved PARP,caspase-3,cleavd caspase-3,Bax,Bcl-2)的表达,研究了CTEE(12. 5,25,50 mg·L-1)对神经细胞的保护作用。结果显示,CTEE可以有效保护OGD/R诱导的PC12细胞损伤,并提高PC12细胞存活率,AO/EB与Hoechst 33258染色法显示CTEE可以有效抑制细胞凋亡。此外,JC-1与MitoSOX染色法显示CTEE可显著降低细胞中线粒体的氧化应激及线粒体膜电位的失调,且呈剂量依赖性。同时,CTEE可明显抑制线粒体凋亡信号通路中关键蛋白caspase-3和PARP的激活,且明显抑制Bax的升高和Bcl-2的下调。以上结果表明,管花肉苁蓉醇提取物对OGD/R所致的PC12细胞损伤具有较好的保护作用,其潜在机制可能是通过抑制线粒体氧化应激及相关凋亡信号通路实现的。
To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. Results showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.
To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. Results showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.
引文
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[2]杨薇,黎明全,李杨,等.缺血性中风病急性期中西药群组模块探索[J].中国中药杂志,2018,43(3):618.
[3] Bernardi P,Scorrano L,Colonna R,et al. Mitochondria and cell death. Mechanistic aspects and methodological issues[J]. Eur J Biochem,1999,264(3):687.
[4]张静,赵建军,杨伟,等.基于中医证候特征的缺血性中风复发风险的横断面分析[J].中国实验方剂学杂志,2018,24(12):194.
[5] Soustiel J F,Zaaroor M. Mitochondrial targeting for development of novel drug strategies in brain injury[J].Cent Nerv Syst Agents Med Chem,2012,12(2):131.
[6]孙丽新,严明,江振洲,等.银杏内酯对PC12细胞缺糖损伤凋亡相关基因的影响[J].中国中药杂志,2007,32(6):532.
[7]汤小蕾,孙平飞.管花肉苁蓉的研究进展[J].中医药导报,2009,15(5):101.
[8] Song D,Cao Z,Liu Z,et al. Cistanche deserticola polysaccharide attenuates osteoclastogenesis and bone resorption via inhibiting RANKL signaling and reactive oxygen species production[J].J Cell Physiol,2018,233(12):9674.
[9]刘文静,刘瑶,宋青青,等.利用1H-NMR比较管花肉苁蓉野生品和栽培品的化学成分组[J].中国中药杂志,2018,43(17):3506.
[10]安军明,柯铁军.苁蓉总苷胶囊治疗血管性痴呆99例[J].陕西中医学院学报,2011,34(2):36.
[11] Farajdokht F,Mohaddes G,Karimi-Sales E,et al. Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection[J]. Brain Res,2018,1692:100.
[12]杜肖,路畅,贺晓丽,等.小续命汤有效成分组对脑缺血/再灌注大鼠恢复早期脑线粒体的保护作用研究[J].中国中药杂志,2017,42(11):2139.
[13] Liu Y,Wang H,Yang M,et al. Cistanche deserticola polysaccharides protects PC12 cells against OGD/RP-induced injury[J]. Biomed Pharmacother,2018,99:671.
[14] Li M,Xu T,Zhou F,et al. Neuroprotective effects of four phenylethanoid glycosides on H2O2-induced apoptosis on PC12 cells via the Nrf2/ARE pathway[J]. Int J Mol Sci,2018,19(4):1135.
[15] Chen M,Wang X,Hu B,et al. Protective effects of echinacoside against anoxia/reperfusion injury in H9c2 cells via up-regulating p-AKT and SLC8A3[J]. Biomed Pharmacother,2018,104:52.
[16] Fu Z,Fan X,Wang X,et al. Cistanches Herba:an overview of its chemistry,pharmacology and pharmacokinetics property[J].J Ethnopharmacol,2018,219:233.
[17]骆新,李建英,许永华,等.肉苁蓉苯乙醇苷对大鼠高原脑水肿的防治作用[J].中南药学,2014,12(8):747.
[18]魏丽丽,陈虹,姜勇,等.高效液相法测定松果菊苷在脑缺血大鼠血浆和脑组织中的浓度[J].中国药理学通报,2011,27(5):737.
[19]陶义存,董翔,许永华,等.肉苁蓉苯乙醇总苷对高原脑水肿大鼠水通道蛋白4的影响[J].中国现代中药,2015,17(4):302.