葛根素对氧糖剥夺细胞模型CaM、CaMKⅡ、MECP2、BDNF及Akt表达的影响
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摘要
目的研究葛根素对氧糖剥夺(oxygen and glucose deprivation,OGD)血管性痴呆细胞模型细胞黏附分子(CaM)、钙调蛋白激酶Ⅱ(CaMKⅡ)、脑源性神经营养因子(BDNF)及Akt表达的影响。方法选取生长良好的PC12细胞传代、分化,行OGD准备血管性痴呆细胞模型,随机分为对照组、模型组及低、中、高剂量葛根素组。MTT法测定细胞存活率并确定合适的葛根素干预浓度及OGD处理时间;检测乳酸脱氢酶(LDH)释放量评定细胞损伤程度,鉴定细胞模型;Western blot检测CaM、CaMKⅡ、MECP2、BDNF及Akt蛋白的表达水平。结果 PC12细胞存活率随OGD时间延长而逐渐降低,呈时间依赖性;PC12细胞存活率随葛根素浓度增加而逐渐升高,呈浓度依赖性。葛根素有效干预浓度为0.1～10μmol/L;OGD最佳处理时间为6h。与对照组相比,模型组LDH释放量明显增高(P<0.05);葛根素干预组LDH释放量随葛根素浓度增加而减少(P<0.05)。模型组CaM蛋白表达明显升高,BDNF表达量明显减少(P<0.05),MECP2表达及CaMKⅡ、Akt蛋白磷酸化水平均未见明显变化(P>0.05)。葛根素干预可下调CaM蛋白水平,提高MECP2、BDNF的表达及CaMKⅡ磷酸化水平,中、高剂量葛根素组亦能升高Akt蛋白磷酸化水平(P<0.05)。结论葛根素可能通过提高Ca2+-CaM复合物介导CaMKⅡ自身磷酸化水平,诱导MECP2磷酸化,上调BDNF的表达,激活下游PI3K-Akt通路,抑制凋亡基因及蛋白表达,发挥神经保护作用。
Objective To observe the effects of puerarin on the expressions of CaM,CaMKⅡ,BDNF and Akt in vascular dementia cell models induced by oxygen and glucose deprivation(OGD).Methods The passaged welldifferentiated PC12 cells were randomly divided into control group,model group,and low-dose,medium-dose and high-dose intervention groups.Vascular dementia cell model was established by OGD.Suitable OGD time and concentration of puererin were obtained from the cell viability measured by MTT assay.The release of LDH was measured to assess the extent of cell damage and identify cell models.The expressions of CaM,CaMKⅡ,MECP2,BDNF and Akt were detected by Western blot.Results PC12 cells with OGD prolonged viability decreased in a time-dependent manner,with increased concentrations of puerarin increased in a concentration-dependent manner.Effective intervention of puerarin was 0.1-10μmol/L and optimal time of OGD was 6h.Compared with control group,the release of LDH in model group was significantly increased(P<0.05),while the release in puerarin group was decreased with the increase of puerarin concentration(P<0.05).In model group,the expression of CaM was significantly increased and that of BDNF was significantly decreased(P <0.05).However,the expression of MECP2 and the phosphorylation of CaMKⅡand Akt did not differ(P>0.05).Puerarin could down-regulate the level of CaM protein,increase the expressions of MECP2 and BDNF and the phosphorylation of CaMKⅡ,and also increase the phosphorylation of Akt in addition to the low-dose group(P<0.05).Conclusion The neuroprotective effect of puerarin may be related to the increase of the autophosphorylation of CaMKⅡ mediated by Ca2+-CaM complex,induce the phosphorylation of MECP2,up-regulate the expression of BDNF and activate the PI3K-Akt pathway to inhibit the expression of apoptotic genes and proteins.
Objective To observe the effects of puerarin on the expressions of CaM,CaMKⅡ,BDNF and Akt in vascular dementia cell models induced by oxygen and glucose deprivation(OGD).Methods The passaged welldifferentiated PC12 cells were randomly divided into control group,model group,and low-dose,medium-dose and high-dose intervention groups.Vascular dementia cell model was established by OGD.Suitable OGD time and concentration of puererin were obtained from the cell viability measured by MTT assay.The release of LDH was measured to assess the extent of cell damage and identify cell models.The expressions of CaM,CaMKⅡ,MECP2,BDNF and Akt were detected by Western blot.Results PC12 cells with OGD prolonged viability decreased in a time-dependent manner,with increased concentrations of puerarin increased in a concentration-dependent manner.Effective intervention of puerarin was 0.1-10μmol/L and optimal time of OGD was 6h.Compared with control group,the release of LDH in model group was significantly increased(P<0.05),while the release in puerarin group was decreased with the increase of puerarin concentration(P<0.05).In model group,the expression of CaM was significantly increased and that of BDNF was significantly decreased(P <0.05).However,the expression of MECP2 and the phosphorylation of CaMKⅡand Akt did not differ(P>0.05).Puerarin could down-regulate the level of CaM protein,increase the expressions of MECP2 and BDNF and the phosphorylation of CaMKⅡ,and also increase the phosphorylation of Akt in addition to the low-dose group(P<0.05).Conclusion The neuroprotective effect of puerarin may be related to the increase of the autophosphorylation of CaMKⅡ mediated by Ca2+-CaM complex,induce the phosphorylation of MECP2,up-regulate the expression of BDNF and activate the PI3K-Akt pathway to inhibit the expression of apoptotic genes and proteins.
引文
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[2]ZHOU YX,ZHANG H,PENG C.Puerarin:A review of pharmacological effects[J].Phytother Res,2014,28(7):961-975.
[3]PENG W,ZHANG Y,ZHENG M,et al.Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1and subsequent expression of inducible heat shock protein 70[J].Circ Res,2010,106(1):102-110.
[4]HUA Z,XIAO G,DONG Y,et al.PI3Kand MAPK pathways mediate the BDNF/TrkB-increased metastasis in neuroblastoma[J].Tumour Biol,2016,37(12):1-10.
[5]AMBIGAPATHY G,ZHENG Z,KEIFER J.Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning[J].Epigenetics,2015,10(10):981-993.
[6]张茹,郭荷娜,吴海琴,等.葛根素对大鼠局灶性脑缺血后海马CA1区NMDA受体的影响及其意义[J].四川大学学报(医学版),2011,42(1):52-55.
[7]ZHU X,WANG K,ZHANG K,et al.Puerarin protects human neuroblastoma SH-SY5Ycells against glutamate-induced oxidative stress and mitochondrial dysfunction[J].J Biochem Mol Toxicol,2016,30(1):22-28.
[8]王秀力,陈东,刘佳梅.NGF诱导PC12细胞向神经元的分化[J].吉林大学学报(医学版),2007,33(5):827-830.
[9]ZHAO J,LI L,LING C,et al.Marine compound xyloketal B protects PC12cells against OGD-induced cell damage[J].Brain Res,2009,1302:240-247.
[10]ZHANG Y,QIAO L,XU W,et al.Paeoniflorin attenuates cerebral ischemia-induced injury by regulating Ca2+/CaMKII/CREB signaling pathway[J].Molecules,2017,22(3):359.
[11]ZHU X,WANG K,ZHANG K,et al.Puerarin protects human neuroblastoma SH-SY5Ycells against glutamate-induced oxidative stress and mitochondrial dysfunction[J].J Biochem Mol Toxicol,2016,30(1):22-28.
[12]RADAK D,KATSIKI N,RESANOVIC I,et al.Apoptosis and acute brain ischemia in ischemic stroke[J].Curr Vasc Pharmacol,2016,15(2):115-122.
[13]齐中华,刘群.葛根素对实验性脑缺血GAD67mRNA表达变化的影响[J].中风与神经疾病杂志,2006,23(2):83-85.
[14]邱根全,石秦东,蔡云,等.益气活血开窍复方对缺血再灌注脑组织兴奋性氨基酸及超微结构的影响[J].西安交通大学学报(医学版),2002,23(5):489-492.
[15]CHABITZ WR,STEIGLEDER T,COOPER-KUHN CM,et al.Intravenous brain-derived neurotrophic factor enhances poststroke sensorimotor recovery and stimulates neurogenesis[J].Stroke,2007,38(7):2165-2172.
[16]ZHAO J,CHENG YY,FAN W,et al.Botanical drug puerarin coordinates with nerve growth factor in the regulation of neuronal survival and neuritogenesis via activating ERK1/2and PI3K/Akt signaling pathways in the neurite extension process[J].CNS Neurosci Ther,2015,21(1):61-70.
[17]ZHANG Q,HUANG WD,LV XY,et al.Puerarin protects differentiated PC12 cells from H(2)O(2)-induced apoptosis through the PI3K/Akt signalling pathway[J].Cell Biol Int,2012,36(5):419-426.