补肾益气方药左归丸和益气聪明汤对老年大鼠学习记忆能力及相关基因表达的影响
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摘要
目的:观察补肾方药(左归丸)、益气方药(益气聪明汤)及两方合用(合剂)对老年大鼠空间学习能力及学习记忆相关基因表达变化的影响,并探索相关表观遗传酶的变化。方法:以自然衰老(24月龄) SD雄性大鼠为动物模型,随机分为老年组,左归丸组(12. 12 g·kg~(-1)),益气聪明汤组(10. 18 g·kg~(-1)),合剂组(11. 15 g·kg~(-1))和老年皮质酮(glucocorticoid,GC)拮抗组(5×10-3g·kg~(-1)),另设青年组(同品系5月龄)。采用Morris水迷宫法观察各组大鼠空间记忆能力,激光共聚焦法观察各组大鼠大脑海马区乙酰化酶2(histone deacetylase 2,HDAC2)与甲基化Cp G岛结合蛋白2(methyl Cp G binding protein 2,MeCP2)的共定位情况,蛋白免疫印迹法(Western blot)检测海马组织HDAC2,组蛋白乙酰转移酶1(histone acetyltransferase 1,HAT1),糖皮质激素受体(glucocorticoid receptor,GR),突触蛋白I(synapsin,Syn-1)表达变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测HDAC2 mRNA的表达及各药物对上述指标异常变化的改善作用。结果:与青年组(正常组)比较,老年组(模型组)大鼠潜伏期显著延长(P <0. 05),穿越平台次数显著减少(P <0. 05);与老年组(模型组)比较,左归丸组、益气聪明汤组大鼠潜伏期显著缩短(P <0. 05),穿越平台次数有所增加。与青年组比较,老年组大鼠HDAC2蛋白与mRNA表达显著增多(P <0. 05,P <0. 01),且在细胞核内与MeCP2的共表达明显增多; HAT1,GR和Syn-I表达显著降低(P <0. 05,P <0. 01)。各给药组均能不同程度改善上述指标的异常变化,但益气方药以及两方合剂对HAT1无改善作用。结论:补肾方药左归丸、益气方药益气聪明汤可通过增加老年大鼠学习记忆相关蛋白GR,Syn-1与表观遗传修饰酶HAT1的表达,降低HDAC2的表达,并同时减少HDAC2蛋白与MeCP2的细胞核内共定位,以改善老年大鼠空间学习记忆能力。
Objective: To observe the effects of recipes for tonifying kidney and replenishing Qi,Zuoguiwan( ZG) and Yiqi Congming Tang( YQ) on memory capacity,expressions of learning and memory-related genes expression,and explore the changes in relevant epigenetic modification enzymes. Method: SD male rats with natural aging( 24 months old) were used as animal models and randomly divided into aged control group,aged ZG group( 12. 12 g·kg~(-1)),aged YQ group( 10. 18 g·kg~(-1)),aged compound group( 11. 15 g·kg~(-1)) and aged antagonist RU38486 group( 5 × 10-3 g·kg~(-1)). Another 5 months old male SD rats were included as the young control group. Morris water maze method was used to observe the spatial learning and memory ability of the rats.The co-localizations of histone deacetylase 2( HDAC2) and methyl CpG binding protein 2( Me CP2) in hippocampus of rats in each group were observed by laser confocal microscope. The changes in expressions of glucocorticoid receptor( GR),synapsin1( Syn-1),HDAC2,and histone acetyltransferase 1( HAT1) proteins in hippocampus of each group were detected by Western blot,and mRNA expression of HDAC2 was detected by Realtime fluorescence quantitative polymerase Chain reaction( Real-time PCR). At the same time,the effects of ZG,YQ and compound decoction in alleviating the above-mentioned abnormal changes were observed. Result:Compared with the young control group( control group),the latency of the aged control group was significantly prolonged( P < 0. 05),the number of crossing platforms was significantly reduced( P < 0. 05); compared with the aged control group( model group),the latencies of the ZG group and the YQ group were significantly shortened( P < 0. 05),and the numbers of crossing platforms were increased. Compared with the young control group,the protein and mRNA expressions of HDAC2 in the aged control group were significantly increased( P < 0. 05,P <0. 01),and the co-expression with MeCP2 in the nucleus was significantly increased; and the expressions of HAT1,GR and Syn-1 were significantly decreased( P < 0. 05,P < 0. 01). ZG,YQ and compound can all alleviate the abnormal changes of the above indicators to varying degrees,but the YQ and the compound group had no effect on HAT1. Conclusion: ZG group,YQ group,and compound group can improve the spatial learning and memory abilities of aged rats by increasing the expression of learning-memory-associated protein GR and epigenetic modification enzyme HAT1,and reducing the expression of HDAC2 and the co-localization of HDAC2 protein and MeCP2 in the nucleus.
Objective: To observe the effects of recipes for tonifying kidney and replenishing Qi,Zuoguiwan( ZG) and Yiqi Congming Tang( YQ) on memory capacity,expressions of learning and memory-related genes expression,and explore the changes in relevant epigenetic modification enzymes. Method: SD male rats with natural aging( 24 months old) were used as animal models and randomly divided into aged control group,aged ZG group( 12. 12 g·kg~(-1)),aged YQ group( 10. 18 g·kg~(-1)),aged compound group( 11. 15 g·kg~(-1)) and aged antagonist RU38486 group( 5 × 10-3 g·kg~(-1)). Another 5 months old male SD rats were included as the young control group. Morris water maze method was used to observe the spatial learning and memory ability of the rats.The co-localizations of histone deacetylase 2( HDAC2) and methyl CpG binding protein 2( Me CP2) in hippocampus of rats in each group were observed by laser confocal microscope. The changes in expressions of glucocorticoid receptor( GR),synapsin1( Syn-1),HDAC2,and histone acetyltransferase 1( HAT1) proteins in hippocampus of each group were detected by Western blot,and mRNA expression of HDAC2 was detected by Realtime fluorescence quantitative polymerase Chain reaction( Real-time PCR). At the same time,the effects of ZG,YQ and compound decoction in alleviating the above-mentioned abnormal changes were observed. Result:Compared with the young control group( control group),the latency of the aged control group was significantly prolonged( P < 0. 05),the number of crossing platforms was significantly reduced( P < 0. 05); compared with the aged control group( model group),the latencies of the ZG group and the YQ group were significantly shortened( P < 0. 05),and the numbers of crossing platforms were increased. Compared with the young control group,the protein and mRNA expressions of HDAC2 in the aged control group were significantly increased( P < 0. 05,P <0. 01),and the co-expression with MeCP2 in the nucleus was significantly increased; and the expressions of HAT1,GR and Syn-1 were significantly decreased( P < 0. 05,P < 0. 01). ZG,YQ and compound can all alleviate the abnormal changes of the above indicators to varying degrees,but the YQ and the compound group had no effect on HAT1. Conclusion: ZG group,YQ group,and compound group can improve the spatial learning and memory abilities of aged rats by increasing the expression of learning-memory-associated protein GR and epigenetic modification enzyme HAT1,and reducing the expression of HDAC2 and the co-localization of HDAC2 protein and MeCP2 in the nucleus.
引文
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[14]朱金华,徐义勇,万红娇,等.温胆汤对精神分裂症模型鼠海马组织PI3K,Akt和GSK3的影响[J].中国实验方剂学杂志,2019,25(1):101-106.
[15]黄倩倩,赵永烈,高俊峰,等.地黄饮子对AD大鼠中枢神经线粒体结构及功能的保护作用[J].中国实验方剂学杂志,2018,24(21):99-104.
[16]尹超,游秋云,张美娅,等.生慧汤对慢性睡眠剥夺小鼠学习记忆及海马IL-6,TNF-α,COX-2基因表达的影响[J].中国实验方剂学杂志,2019,25(1):96-100.
[17]Kutlu M G,Gould T J.Effects of drugs of abuse on hippocampal plasticity and hippocampus-dependent learning and memory:contributions to development and maintenance of addiction[J].Learn Memory,2016,23(10):515-533.
[18]Tabrizian K,Hashemzaei M,Nasiri A A,et al.Interactive involvement of hippocampal c AMP/PKA and cyclooxygenase-2 signaling pathways in spatial learning in the Morris water maze[J].Folia Neuropathol,2018,56(1):58-66.
[19]Madalena K M,Lerch J K.The effect of glucocorticoid and glucocorticoid receptor interactions on brain,spinal cord,and glial cell plasticity[J].Neural Plast,2017,doi:10.1155/2017/8640970.
[20]Tesic V,Perovic M,Lazic D,et al.Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat[J].J Steroid Biochem,2015,149:43-52.
[21]Daskalakis N P,De Kloet E R,Yehuda R,et al.Early life stress effects on glucocorticoid-BDNF interplay in the hippocampus[J].Front Mol Neurosci,2015,doi:10.3389/fnmol.2015.00068.
[22]Marte A,Messa M,Benfenati F,et al.Synapsins are downstream players of the BDNF-mediated axonal growth[J].Mol Neurobiol,2017,54(1):484-494.
[23]Kim S,Kaang B K.Epigenetic regulation and chromatin remodeling in learning and memory[J].Exp Mol Med,2017,49(1):e281.
[24]Duke C G,Kennedy A J,Gavin C F,et al.Experiencedependent epigenomic reorganization in the hippocampus[J].Learn Memory,2017,24(7):278-288.
[25]Schmauss C.The roles of class I histone deacetylases(HDACs)in memory,learning,and executive cognitive functions:a review[J].Neurosci Biobehav R,2017,83:63-71.
[26]Singh P,Srivas S,Thakur M K.Epigenetic regulation of memory-therapeutic potential for disorders[J].Curr Neuropharmacol,2017,15(999):1208-1221.
[27]Uchida S,Shumyatsky G P.Epigenetic regulation of Fgf1transcription by CRTC1 and memory enhancement[J].Brain Res Bull,2018,141:3-12.
[2]Sandi C.Glucocorticoids act on glutamatergic pathways to affect memory processes[J].Trends Neurosci,2011,34(4):165-176.
[3]金国琴,赵伟康,徐品初,等.补肾药延缓老年大鼠下丘脑-垂体-甲状腺轴的功能退化[J].标记免疫分析与临床,1998,5(1):32-35.
[4]康湘萍,金国琴.左归丸及右归丸对老年大鼠学习记忆相关信号分子表达的影响[J].中华中医药杂志,2012,27(8):2056-2059.
[5]Singh P,Srivas S,Thakur M K.Epigenetic regulation of memory-therapeutic potential for disorders[J].Curr Neuropharmacol,2017,15(8):1208-1221.
[6]张子洋,常富业.《普济方》论治健忘方药探析[J].中华中医药学刊,2014,32(10):2320-2322.
[7]孙丽娥,金国琴.微小RNA在衰老及衰老性疾病发生发展中的调控作用[J].生命的化学,2018,38(2):200-206.
[8]叶丹,朱建贵.基于数据挖掘技术的朱建贵从脾肾治老年病经验研究[J].中国医药导刊,2009,11(1):1-3.
[9]康湘萍,金国琴,梁超,等.补肾方药对老年大鼠BDNF介导的信号通路关键分子表达的影响[J].中华中医药杂志,2013,28(5):1309-1312.
[10]尹芳,郑静,康湘萍,等.老年大鼠学习记忆相关基因egr1表达变化的机制及补肾益气方药的调整作用[J].中国老年学杂志,2017,37(19):4689-4691.
[11]邱宏,金国琴,金如锋,等.水迷宫重复测量数据的方差分析及其在SPSS中的实现[J].中西医结合学报,2007,5(1):101-105.
[12]JanoutováJ,erO,Hosák L,et al.Is mild cognitive impairment a precursor of Alzheimer's disease?[J].Cent Eur J Publ Heal,2015,23(4):365-367.
[13]刘聪,李宁,敬舒,等.五味子-淫羊藿混合提取物对D-半乳糖致脑衰老小鼠学习记忆能力的改善作用[J].中国实验方剂学杂志,2017,23(21):147-152.
[14]朱金华,徐义勇,万红娇,等.温胆汤对精神分裂症模型鼠海马组织PI3K,Akt和GSK3的影响[J].中国实验方剂学杂志,2019,25(1):101-106.
[15]黄倩倩,赵永烈,高俊峰,等.地黄饮子对AD大鼠中枢神经线粒体结构及功能的保护作用[J].中国实验方剂学杂志,2018,24(21):99-104.
[16]尹超,游秋云,张美娅,等.生慧汤对慢性睡眠剥夺小鼠学习记忆及海马IL-6,TNF-α,COX-2基因表达的影响[J].中国实验方剂学杂志,2019,25(1):96-100.
[17]Kutlu M G,Gould T J.Effects of drugs of abuse on hippocampal plasticity and hippocampus-dependent learning and memory:contributions to development and maintenance of addiction[J].Learn Memory,2016,23(10):515-533.
[18]Tabrizian K,Hashemzaei M,Nasiri A A,et al.Interactive involvement of hippocampal c AMP/PKA and cyclooxygenase-2 signaling pathways in spatial learning in the Morris water maze[J].Folia Neuropathol,2018,56(1):58-66.
[19]Madalena K M,Lerch J K.The effect of glucocorticoid and glucocorticoid receptor interactions on brain,spinal cord,and glial cell plasticity[J].Neural Plast,2017,doi:10.1155/2017/8640970.
[20]Tesic V,Perovic M,Lazic D,et al.Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat[J].J Steroid Biochem,2015,149:43-52.
[21]Daskalakis N P,De Kloet E R,Yehuda R,et al.Early life stress effects on glucocorticoid-BDNF interplay in the hippocampus[J].Front Mol Neurosci,2015,doi:10.3389/fnmol.2015.00068.
[22]Marte A,Messa M,Benfenati F,et al.Synapsins are downstream players of the BDNF-mediated axonal growth[J].Mol Neurobiol,2017,54(1):484-494.
[23]Kim S,Kaang B K.Epigenetic regulation and chromatin remodeling in learning and memory[J].Exp Mol Med,2017,49(1):e281.
[24]Duke C G,Kennedy A J,Gavin C F,et al.Experiencedependent epigenomic reorganization in the hippocampus[J].Learn Memory,2017,24(7):278-288.
[25]Schmauss C.The roles of class I histone deacetylases(HDACs)in memory,learning,and executive cognitive functions:a review[J].Neurosci Biobehav R,2017,83:63-71.
[26]Singh P,Srivas S,Thakur M K.Epigenetic regulation of memory-therapeutic potential for disorders[J].Curr Neuropharmacol,2017,15(999):1208-1221.
[27]Uchida S,Shumyatsky G P.Epigenetic regulation of Fgf1transcription by CRTC1 and memory enhancement[J].Brain Res Bull,2018,141:3-12.