中药健脾益肺方对EAAT2表达的影响
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摘要
目的探索健脾益肺方(JPYF)对谷氨酸转运体2(EAAT2)表达的影响及可能机制。方法取新生24 h内的SD幼鼠,无菌条件下取出幼鼠大脑皮层星形胶质细胞,分离、提纯、原代培养以进行后期实验;调整适宜的细胞浓度分5组种板,分别为A:谷氨酸组(250μmo L/L),B-D:JPYF方(0.25、0.5、1 g/kg)+谷氨酸组,E:正常组,以JPYF方(终浓度为0.25、0.5、1 g/kg)作用B-D组24 h后,谷氨酸250μmo L/L作用A-D组4 h诱导建立体外培养原代星形胶质细胞损伤模型;采用噻唑蓝(MTT)法测定各组星形胶质细胞存活率,紫外比色法检测培养基上清谷氨酸浓度,细胞免疫荧光法(immunofluorescence)、Western blot检测星形胶质细胞EAAT2的表达量,IF法检测原代脊髓神经元细胞存活情况。结果 JPYF方可显著保护星形胶质细胞(P<0.01),提高谷氨酸转运蛋白的表达(P<0.05,P<0.01),降低细胞外液谷氨酸浓度(P<0.05,P<0.01),减少神经元损害(P<0.05,P<0.01),在0.25～1g/L的剂量上呈现一定的量效关系。结论 JPYF方可减少谷氨酸对原代脊髓运动神经元细胞的损伤,其机制可能是JPYF方提高EAAT2的表达,降低细胞外液谷氨酸浓度,从而起到保护神经元的作用。
Objective To explore the effect of Jianpi Yifei prescription(JPYF) on the expression of glutamate transporter 2(EAAT2) and the possible mechanism behind this.Methods Neonatal Sprague-Dawley rats within 24 h of birth were used and and the astrocytes of cerebral cortex were isolated under aseptic conditions,then purified,and cultured for later experiments.The appropriate cell concentration was adjusted in plates for five groups:A,glutamic acid group(250μmo L/L),B-D,JPYF prescription(final concentrations 0.25,0.5,and 1 g/kg) + glutamic acid group;and E,normal group,After the B-D groups had been treated with JPYF prescription(final concentrations of 0.25,0.5,and 1 g/kg) for24 h,glutamate at 250 μmol/L was applied to the A-D groups for 4 h to induce an in vitro primary astrocyte injury model.Cell viability was determined by MTT assay.Determination of glutamic acid concentration in culture medium was performed by ultraviolet colorimetry.The expression of glutamate transporter protein in astrocytes was detected by immunofluorescence and western blotting.The injury of spinal cord neurons was detected by Immunofluorescence.Results JPYF Fang significantly protected the astrocytes(P< 0.01),improved the expression of glutamate transporters(P< 0.05,P< 0.01),reduced glutamate concentrations(P< 0.05,P< 0.01),protectd neurons(P< 0.05,P< 0.01),and showed a certain dose-related effect within the range of 0.25-1 g.L-1.Conclusions Within a certain dose range,JPYF Fang can reduce the damage of glutamate to neuronal cells,the mechanism of which may involve improving the expression of glutamate transporter and decreasing the concentration of glutamate in extracellular fluid,so as to protect neurons.
Objective To explore the effect of Jianpi Yifei prescription(JPYF) on the expression of glutamate transporter 2(EAAT2) and the possible mechanism behind this.Methods Neonatal Sprague-Dawley rats within 24 h of birth were used and and the astrocytes of cerebral cortex were isolated under aseptic conditions,then purified,and cultured for later experiments.The appropriate cell concentration was adjusted in plates for five groups:A,glutamic acid group(250μmo L/L),B-D,JPYF prescription(final concentrations 0.25,0.5,and 1 g/kg) + glutamic acid group;and E,normal group,After the B-D groups had been treated with JPYF prescription(final concentrations of 0.25,0.5,and 1 g/kg) for24 h,glutamate at 250 μmol/L was applied to the A-D groups for 4 h to induce an in vitro primary astrocyte injury model.Cell viability was determined by MTT assay.Determination of glutamic acid concentration in culture medium was performed by ultraviolet colorimetry.The expression of glutamate transporter protein in astrocytes was detected by immunofluorescence and western blotting.The injury of spinal cord neurons was detected by Immunofluorescence.Results JPYF Fang significantly protected the astrocytes(P< 0.01),improved the expression of glutamate transporters(P< 0.05,P< 0.01),reduced glutamate concentrations(P< 0.05,P< 0.01),protectd neurons(P< 0.05,P< 0.01),and showed a certain dose-related effect within the range of 0.25-1 g.L-1.Conclusions Within a certain dose range,JPYF Fang can reduce the damage of glutamate to neuronal cells,the mechanism of which may involve improving the expression of glutamate transporter and decreasing the concentration of glutamate in extracellular fluid,so as to protect neurons.
引文
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[18]Chen Benkler,Yael Barhum,Tali Ben-Zur,et al.Multifactorial gene therapy enhancing the glutamate uptake system and reducing oxidative stress delays symptom onset and prolongs survival in the SOD1-G93A ALS mouse model[J]Mol Neurosci,2019,58(1):46-58.
[19]Clements JD,Lester RA,Tong G,et al.The time course of glutamate in the synaptic cleft[J].Science,1992,258(5087):1498-1501.
[2]Hertz L.Intercellular metabolic compartmentation in the brain:past,present and future[J]Neurochem Int,2004,45(2-3):285-296.
[3]Hubbard JA,Binder DK.Targeting glutamate transporter-1 in neurological diseases[J]Oncotarget,2017,8(14):22311-22312.
[4]郑瑜,任展能,王安琦,等.肺脾论治对肌萎缩侧索硬化运动和生活质量的疗效观察[J]时珍国医国药,2016,27(02):391-392.
[5]郑瑜,周炜,杨碧莹,等.肺脾论治对肌萎缩侧索硬化肌力及呼吸功能的影响[J]辽宁中医杂志,2016,43(08):1676-1677.
[6]王安琦,李秀兰,任展能,等.健脾益肺法治疗肌萎缩侧索硬化的疗效研究[J]世界中医药杂志,2017,12(6):1364-1367.
[7]崔梅,陆征宇,董强.黄芪对谷氨酸损伤海马神经元的保护作用[J]中西医结合心脑血管病杂志,2014,12(6):734-736.
[8]朱兆洪.黄芪益母草注射液对脑缺血再灌注大鼠NMDA受体的影响[J]浙江中医学院学报,2005,29(6):47-9.
[9]张敏,徐丽,刘黎星,等.人参皂苷RS2对脑缺血再灌注大鼠学习记忆及海马神经元Glu和NMDA受体亚单位表达的影响[J]齐鲁医学杂志,2006,21(4):286-9.
[10]Andersena PM,Borasiob GD,Dengler R,et al.EFNS task force on management of amyotrophic lateral sclerosis:guidelines for diagnosing and clinical care of patients and relatives[J]Eur JNeurol,2005,12(12):921-938.
[11]曾含漪,徐仁伵.肌萎缩侧索硬化运动神经元选择性损伤的可能发病机制[J].中国老年学杂志,2013,33(4):972-975.
[12]Heath PR,Shaw PJ.Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis[J].Muscle Nerve,2002.26(4):438-458.
[13]Sheldon AL,Robinson MB.The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention[J]Neurochem Int,2007,51(6-7):333-355.
[14]Kong Q,Chang LC,Takahashi K,et al.Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection[J]J Clin Invest,2014.124(3):1255-1267.
[15]Rothstein JD,Martin LJ,Kuncl RW.Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis[J]N Engl Med,1992,326(22),1464-1468.
[16]Rothstein JD,Van Kammen M,Levey AI,et al.Selective loss of glial glutamate transporter GLT-1 inamyotrophic lateral sclerosis[J]Ann Neurol,1995,38(1),73-84.
[17]Rothstein JD,Dykes-Hoberg MP,ardo CA.et al.Knockout of glutamate transporters reveals amajor role for astroglial transport in excitotoxicity and clearance of glutamate[J]Neuron,1996,16(3),675-686.
[18]Chen Benkler,Yael Barhum,Tali Ben-Zur,et al.Multifactorial gene therapy enhancing the glutamate uptake system and reducing oxidative stress delays symptom onset and prolongs survival in the SOD1-G93A ALS mouse model[J]Mol Neurosci,2019,58(1):46-58.
[19]Clements JD,Lester RA,Tong G,et al.The time course of glutamate in the synaptic cleft[J].Science,1992,258(5087):1498-1501.