醒脑静注射液组分促进缺氧复氧胶质细胞清除胞外谷氨酸提高缺氧神经元活力的研究
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摘要
[目的]研究醒脑静注射液(XNJI)8种成分促进缺氧复氧(Hypo/Reox)星形胶质细胞对胞外高浓度谷氨酸的清除,及其条件培养液对缺氧后神经元活力的影响。[方法]体外培养大鼠星形胶质细胞,胶质纤维酸性蛋白(GFAP)免疫荧光鉴定。采用环磷酸腺苷(d BcAMP)和Hypo/Reox诱导胶质细胞活化并进行造模,Cell Counting Kit-8(CCK-8)测定细胞活力;谷氨酸试剂盒检测细胞培养液中谷氨酸浓度,并利用此条件培养液培养缺氧神经元,CCK-8法测神经元活力。[结果]胶质细胞体外培养成功,GFAP染色阳性。dBcAMP 0.062 5 mmol/L协同缺氧4 h复氧3 h(Hypo 4 h/Reox 3 h)处理后胶质细胞对胞外高浓度谷氨酸的清除能力显著降低。XNJI组分β-榄香烯、吉马酮、龙脑、冰片能显著提高受损胶质细胞对胞外谷氨酸的清除能力;XNJI组分吉马酮、龙脑、冰片处理的胶质细胞条件培养液能显著提高缺氧神经元的活力。[结论] XNJI组分能促进缺氧复氧胶质细胞对谷氨酸的清除并提高神经元活力。
[Objective] To study the effects of eight active components of Xingnaojing injection on glutamate transport in hypoxia/reoxygenation(Hypo/Reox) astrocytes in vitro and astrocyte-conditioned medium on the viability of Hypo/Reox neurons. [Methods]Astrocytes were cultured in vitro and identified by GFAP. Dibutyryl cyclic adenosine monophosphate(dBcAMP) and Hypo/Reox were used to induce astrocytes activation,cell viability was determined by CCK-8. Extracellular glutamate concentrations were measured by the Glutamate Kit. Neurons were cultured with astrocytes conditioned medium,and neuronal cell viability was determined by CCK-8.[Results] Astrocytes express the marker protein GFAP. dBcAMP 0.0625 mmol/L synergistic hypoxia 4 h plus reoxygenation 3 h(Hypo 4 h/Reox 3 h) can significantly reduce the ability of astrocytes to clear extracellular glutamate. The effective components of β-elemene,genistein,camphol and borneol can significantly increase the clearance ability of astrocytes in Hypo 4 h/Reox 3 h. At the same time,they also significantly improve neuronal viability cultured with astrocytes-conditioned medium after Hypo 4 h/Reox 3 h. [Conclusion] XNJI components promote glutamate clearance in hypoxia/reoxygenation astrocytes and improve cell viability in hypoxia neurons.
[Objective] To study the effects of eight active components of Xingnaojing injection on glutamate transport in hypoxia/reoxygenation(Hypo/Reox) astrocytes in vitro and astrocyte-conditioned medium on the viability of Hypo/Reox neurons. [Methods]Astrocytes were cultured in vitro and identified by GFAP. Dibutyryl cyclic adenosine monophosphate(dBcAMP) and Hypo/Reox were used to induce astrocytes activation,cell viability was determined by CCK-8. Extracellular glutamate concentrations were measured by the Glutamate Kit. Neurons were cultured with astrocytes conditioned medium,and neuronal cell viability was determined by CCK-8.[Results] Astrocytes express the marker protein GFAP. dBcAMP 0.0625 mmol/L synergistic hypoxia 4 h plus reoxygenation 3 h(Hypo 4 h/Reox 3 h) can significantly reduce the ability of astrocytes to clear extracellular glutamate. The effective components of β-elemene,genistein,camphol and borneol can significantly increase the clearance ability of astrocytes in Hypo 4 h/Reox 3 h. At the same time,they also significantly improve neuronal viability cultured with astrocytes-conditioned medium after Hypo 4 h/Reox 3 h. [Conclusion] XNJI components promote glutamate clearance in hypoxia/reoxygenation astrocytes and improve cell viability in hypoxia neurons.
引文
[1]廉全荣,董华丽.醒脑静注射液治疗急性脑梗死临床观察[J].中国中医急症,2010,19(3):430-431.
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[3]李红琴.醒脑静择时治疗大面积脑梗死的临床观察[D].武汉:湖北中医药大学,2011.
[4]李春艳,刘晓明,高善语,等.醒脑静对急性脑梗死的血管炎性反应干预[J].菏泽医学专科学校学报,2014,26(4):77-78,93.
[5]张国妮,徐耀琳.醒脑静注射液对急性脑出血患者神经功能恢复的影响[J].中国民间疗法,2017,25(1):53-54.
[6]韩斌.醒脑静对OGD损伤后血管内皮细胞生存活性的影响及炎性反应抑制机制的体外研究[D].长春:吉林大学,2013.
[7]宿宝贵,郑发武,吕来清,等.电针对脑梗塞大鼠缺血半影区星形胶质细胞形态的影响[J].中国病理生理杂志,2007,23(10):1964-1967.
[8]张敏,李文斌,王彦华.胶质细胞谷氨酸转运体及其在脑缺血和脑缺血预适应中的变化[J].生命科学,2010,23(5):431-436.
[9]邱永明.缺血后处理对全脑缺血大鼠海马GLT-1和GS表达的影响及意义[D].上海:上海交通大学,2011.
[10]张欢欢,刘寒,何林,等.星形胶质细胞对脑缺血后神经元保护机制的研究进展[J].山东医药,2017,57(36):97-99.
[11]Haghighat N,Mc Candless DW,Geraminegad P.Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP[J].Neurochemical Research,2000,25(2):277-284.
[12]陈宇亮.星形胶质细胞中腺苷和谷氨酸代谢在中枢神经系统氧中毒中的作用研究[D].上海:第二军医大学,2014.
[13]Gouix E,Buisson A,Nieoullon A,et al.Oxygen glucose deprivation-induced astrocyte dysfunction provokes neuronal death through oxidative stress[J].Pharmacology Research,2014(87):8-17.
[14]杨晓运,李智,秦绿叶,等.星形胶质细胞和神经元之间谷氨酸-谷氨酰胺的代谢偶联[J].生理科学进展,2003,34(4):350-352.
[15]康文博,张赛,梁海乾.星形胶质细胞转分化为神经元的研究进展[J].天津医药,2015,56(6):694-697.
[16]闫荣,罗晓光,张尧,等.星形胶质细胞条件培养液诱导骨髓基质细胞向神经元样细胞分化的神经营养机制探讨[J].中国医科大学学报,2013,63(8):714-721.
[17]Kleinkauf-Rocha J,Bobermin LD,Machado Pde M,et al.Lipoic acid increases glutamate uptake,glutamine synthetase activity and glutathione content in C6 astrocyte cell line[J].International Journal of Developmental Neuroscience,2013,31(3):165-170.
[18]Ueki T,Kawakami Z,Kanno H,et al.Yokukansan,a traditional japanese medicine,enhances the glutamate transporter GLT-1 function in cultured rat cortical astrocytes[J].Evidence-Based Complementary and Alternative Medicine:e CAM,2018(2018):6804017.
[2]孙蓉媚.醒脑静注射液治疗脑梗塞132例---附虚实两证疗效分析[J].浙江中医杂志,2002,47(9):43.
[3]李红琴.醒脑静择时治疗大面积脑梗死的临床观察[D].武汉:湖北中医药大学,2011.
[4]李春艳,刘晓明,高善语,等.醒脑静对急性脑梗死的血管炎性反应干预[J].菏泽医学专科学校学报,2014,26(4):77-78,93.
[5]张国妮,徐耀琳.醒脑静注射液对急性脑出血患者神经功能恢复的影响[J].中国民间疗法,2017,25(1):53-54.
[6]韩斌.醒脑静对OGD损伤后血管内皮细胞生存活性的影响及炎性反应抑制机制的体外研究[D].长春:吉林大学,2013.
[7]宿宝贵,郑发武,吕来清,等.电针对脑梗塞大鼠缺血半影区星形胶质细胞形态的影响[J].中国病理生理杂志,2007,23(10):1964-1967.
[8]张敏,李文斌,王彦华.胶质细胞谷氨酸转运体及其在脑缺血和脑缺血预适应中的变化[J].生命科学,2010,23(5):431-436.
[9]邱永明.缺血后处理对全脑缺血大鼠海马GLT-1和GS表达的影响及意义[D].上海:上海交通大学,2011.
[10]张欢欢,刘寒,何林,等.星形胶质细胞对脑缺血后神经元保护机制的研究进展[J].山东医药,2017,57(36):97-99.
[11]Haghighat N,Mc Candless DW,Geraminegad P.Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP[J].Neurochemical Research,2000,25(2):277-284.
[12]陈宇亮.星形胶质细胞中腺苷和谷氨酸代谢在中枢神经系统氧中毒中的作用研究[D].上海:第二军医大学,2014.
[13]Gouix E,Buisson A,Nieoullon A,et al.Oxygen glucose deprivation-induced astrocyte dysfunction provokes neuronal death through oxidative stress[J].Pharmacology Research,2014(87):8-17.
[14]杨晓运,李智,秦绿叶,等.星形胶质细胞和神经元之间谷氨酸-谷氨酰胺的代谢偶联[J].生理科学进展,2003,34(4):350-352.
[15]康文博,张赛,梁海乾.星形胶质细胞转分化为神经元的研究进展[J].天津医药,2015,56(6):694-697.
[16]闫荣,罗晓光,张尧,等.星形胶质细胞条件培养液诱导骨髓基质细胞向神经元样细胞分化的神经营养机制探讨[J].中国医科大学学报,2013,63(8):714-721.
[17]Kleinkauf-Rocha J,Bobermin LD,Machado Pde M,et al.Lipoic acid increases glutamate uptake,glutamine synthetase activity and glutathione content in C6 astrocyte cell line[J].International Journal of Developmental Neuroscience,2013,31(3):165-170.
[18]Ueki T,Kawakami Z,Kanno H,et al.Yokukansan,a traditional japanese medicine,enhances the glutamate transporter GLT-1 function in cultured rat cortical astrocytes[J].Evidence-Based Complementary and Alternative Medicine:e CAM,2018(2018):6804017.