逍遥散对LPS所致海马神经元细胞损伤的保护作用及机制研究
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摘要
该文探讨逍遥散抗抑郁作用发挥与干预海马神经生长的关系。采用脂多糖(LPS)诱导的海马神经元细胞炎症损伤模型,给予逍遥散含药血清(终浓度4%,8%)干预,采用免疫荧光双标法检测细胞增殖活力,ELISA法检测模型细胞上清BD-NF,β-NGF水平,PCR法检测细胞裂解液中BDNF,NGF,Trk B,Trk A,CREB mRNA表达,Western blot法检测细胞裂解液中Trk B,CREB,p-CREB,SYP蛋白表达水平,揭示逍遥散的保护作用及作用机制。结果表明,8%逍遥散含药血清能明显提高损伤海马神经元细胞Brdu/Neu N的比值(P<0. 01),促进细胞增殖。4%,8%逍遥散含药血清明显上调模型细胞上清BDNF,β-NGF水平(P<0. 05或P<0. 01),上调模型细胞BDNF,NGF,Trk B,Trk A,CREB mRNA及Trk B,CREB,p-CREB,SYP蛋白的表达水平(P<0. 05或P<0. 01)。综上,可认为逍遥散对LPS诱导的海马神经元细胞损伤有一定保护作用,其保护作用的发挥与促进海马神经生长相关,是其抗抑郁作用机制之一。
To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and β-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and β-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.
To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and β-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and β-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.
引文
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[12]袁海霞,倪新强,吴正治,等.基于“肾脑相关”研究熟地黄对ADHD模型SHR大鼠前额叶、纹状体BDNF/TrkB及NRG-3的调控作用[J].中国中药杂志,2018,43(17):3539.
[13]赖仙辉,黄周青.芦丁对大鼠心脏缺血再灌注损伤的影响[J].中国现代应用药学,2017,34(8):1109.
[14] Matosin N,Fernandez-Enright F,Lum J S,et al. Molecular evi-dence of synaptic pathology in the CA1 region in schizophrenia[J]. NPJ Schizophr,2016,2:16022.
[2]王学,叶晓琳,刘小波,等.逍遥散抗抑郁作用的研究现状[J].中国实验方剂学杂志,2018,24(16):212.
[3] Tang M M,Lin W J,Pan Y Q,et al. Hippocampal neurogenesisdysfunction linked to depressive-like behaviors in a neuroinflam-mation induced model of depression.[J]. Physiol Behav,2016,161:166.
[4] Peng Z W,Xue Y Y,Wang H N,et al. Sertraline promotes hip-pocampus-derived neural stem cells differentiating into neuronsbut not glia and attenuates LPS-induced cellular damage[J].Prog Neuropsychopharmacol Biol Psychiatry,2012,36(1):183.
[5] Ma V D K,Groenendaal F,Kavelaars A,et al. Neuroprotectiveproperties and mechanisms of erythropoietin in in vitro and in vivoexperimental models for hypoxia/ischemia[J]. Brain Res Rev,2008,59(1):22.
[6] Diniz B S,Teixeira A L,Miranda A S,et al. Circulating Glial-derived neurotrophic factor is reduced in late-life depression[J].J Psychiatr Res,2012,46(1):135.
[7] Hashimoto K,Shimizu E,Iyo M. Critical role of brain-derivedneurotrophic factor in mood disorders[J]. Brain Res Rev,2004,45(2):104.
[8] Teche S P,Nuernberg G L,Sordi A O,et al. Measurementmethods of BDNF levels in major depression:a qualitative sys-tematic review of clinical trials[J]. Psychiat Quart,2013,84(4):485.
[9] Taliaz D,Stall N,Dar D E,et al. Knockdown of brain-derivedneurotrophic factor in specific brain sites precipitates behaviorsassociated with depression and reduces neurogenesis[J]. MolPsychiatr,2010,15(1):80.
[10]严灿,刘银伟,吴丽丽,等.加味四逆散调控抑郁症大鼠海马BDNF、NR1表达及促进海马DG区神经再生的研究[J].中国药理学通报,2016,32(4):569.
[11]龚胜兰,富文俊,黄彬青,等.抑郁症海马神经可塑性损伤机制探讨及丹栀逍遥散调控作用的研究进展[J].辽宁中医杂志,2016,43(3):663.
[12]袁海霞,倪新强,吴正治,等.基于“肾脑相关”研究熟地黄对ADHD模型SHR大鼠前额叶、纹状体BDNF/TrkB及NRG-3的调控作用[J].中国中药杂志,2018,43(17):3539.
[13]赖仙辉,黄周青.芦丁对大鼠心脏缺血再灌注损伤的影响[J].中国现代应用药学,2017,34(8):1109.
[14] Matosin N,Fernandez-Enright F,Lum J S,et al. Molecular evi-dence of synaptic pathology in the CA1 region in schizophrenia[J]. NPJ Schizophr,2016,2:16022.