Objective:To explore the neuroprotective effects of the Shaoyao Gancao decoction(SGD)against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulatingγ-aminobutyric acid(...Objective:To explore the neuroprotective effects of the Shaoyao Gancao decoction(SGD)against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulatingγ-aminobutyric acid(GABA)-glutamate(Glu)homeostasis.Methods: N-Methyl-d-aspartic acid(NMDA)was used to establish a PC12 cell excitability injury model.To investigate the neuroprotective effect of SGD,a cell counting kit-8(CCK-8)assay was used to determine PC12 cell viability,Annexin V/Propidium Iodide(Annexin V/PI)double staining was used to determine PC12 cell apoptosis,and Ca^(2+)concentration was observed using laser confocal microscopy.GABA receptor agonists and antagonists were used to analyze the neuroprotective interactions betweenγ-aminobutyric acid(GABA)and NMDA receptors.Additionally,molecular biology techniques were used to determine mRNA and protein expression in the Src-NR2-nNOS pathway.We analyzed the correlations between the regulatory sites of GABA and NMDA interactions,excitatory neurotoxicity,and brain damage at the molecular level.Results: NMDA excitotoxic injury manifested as a significant decrease in cell activity,increased apoptosis and caspase-3 protein expression,and a significant increase in intracellular Ca^(2+)concentration.Administration of SGD,a GABAA receptor agonist(muscimol),or a GABAB receptor agonist(baclofen)decreased intracellular Ca^(2+)concentrations,attenuated apoptosis,and reversed NMDA-induced upregulation of caspase-3,Src,NMDAR2A,NMDAR2B,and nNOS.Unexpectedly,a GABA_(A)receptor antagonist(bicuculline)and a GABA_(B)receptor antagonist(saclofen)failed to significantly increase excitatory neurotoxicity.Conclusions: Taken together,these results not only provide an experimental basis for SGD administration in the clinical treatment of central nervous system injury diseases,but also suggest that the Src-NR2A-nNOS pathway may be a valuable target in excitotoxicity treatment.展开更多
目的 :了解 n NOS m RNA和 i NOSm RNA在局灶性脑梗死表达中的时程变化与细胞定位。方法 :鼠局灶性脑缺血 /再灌注模型 ;用原位杂交技术进行缺血 /再灌注后 1,3,5天 n NOS m RNA、i NOSm RNA表达的细胞定位并用点杂交方法测量不同时间...目的 :了解 n NOS m RNA和 i NOSm RNA在局灶性脑梗死表达中的时程变化与细胞定位。方法 :鼠局灶性脑缺血 /再灌注模型 ;用原位杂交技术进行缺血 /再灌注后 1,3,5天 n NOS m RNA、i NOSm RNA表达的细胞定位并用点杂交方法测量不同时间段梗死半球与非梗死半球内 n NOS m RNA、i NOSm RNA水平。结果 :正常鼠 n NOSm RNA以神经元表达为主 ,i NOSm RNA无表达 ,梗死后 i NOSm RNA表达以小胶质细胞为主 ,梗死区未见 n NOSm RNA;n NOSm RNA表达从 4h就开始下降 ,1～ 3天最低 ,5天左右又上升 ,i NOSm RNA在缺血再灌注后 12 h开始表达 ,1天左右升至高峰 ,3天左右缓慢下降。结论 :n NOSm RNA在缺血损伤后有一短暂表达增高 ,而在缺血损伤 4h后就开始缓慢下降 ;而 i NOSm RNA在缺血 /再灌注后 12 h开始表达 ,高峰在 1天左右 ,3天左右开始下降 ,其细胞定位以小胶质细胞为主。展开更多
基金supported by the National Natural Science Foundation of China(82074036).
文摘Objective:To explore the neuroprotective effects of the Shaoyao Gancao decoction(SGD)against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulatingγ-aminobutyric acid(GABA)-glutamate(Glu)homeostasis.Methods: N-Methyl-d-aspartic acid(NMDA)was used to establish a PC12 cell excitability injury model.To investigate the neuroprotective effect of SGD,a cell counting kit-8(CCK-8)assay was used to determine PC12 cell viability,Annexin V/Propidium Iodide(Annexin V/PI)double staining was used to determine PC12 cell apoptosis,and Ca^(2+)concentration was observed using laser confocal microscopy.GABA receptor agonists and antagonists were used to analyze the neuroprotective interactions betweenγ-aminobutyric acid(GABA)and NMDA receptors.Additionally,molecular biology techniques were used to determine mRNA and protein expression in the Src-NR2-nNOS pathway.We analyzed the correlations between the regulatory sites of GABA and NMDA interactions,excitatory neurotoxicity,and brain damage at the molecular level.Results: NMDA excitotoxic injury manifested as a significant decrease in cell activity,increased apoptosis and caspase-3 protein expression,and a significant increase in intracellular Ca^(2+)concentration.Administration of SGD,a GABAA receptor agonist(muscimol),or a GABAB receptor agonist(baclofen)decreased intracellular Ca^(2+)concentrations,attenuated apoptosis,and reversed NMDA-induced upregulation of caspase-3,Src,NMDAR2A,NMDAR2B,and nNOS.Unexpectedly,a GABA_(A)receptor antagonist(bicuculline)and a GABA_(B)receptor antagonist(saclofen)failed to significantly increase excitatory neurotoxicity.Conclusions: Taken together,these results not only provide an experimental basis for SGD administration in the clinical treatment of central nervous system injury diseases,but also suggest that the Src-NR2A-nNOS pathway may be a valuable target in excitotoxicity treatment.
文摘目的 :了解 n NOS m RNA和 i NOSm RNA在局灶性脑梗死表达中的时程变化与细胞定位。方法 :鼠局灶性脑缺血 /再灌注模型 ;用原位杂交技术进行缺血 /再灌注后 1,3,5天 n NOS m RNA、i NOSm RNA表达的细胞定位并用点杂交方法测量不同时间段梗死半球与非梗死半球内 n NOS m RNA、i NOSm RNA水平。结果 :正常鼠 n NOSm RNA以神经元表达为主 ,i NOSm RNA无表达 ,梗死后 i NOSm RNA表达以小胶质细胞为主 ,梗死区未见 n NOSm RNA;n NOSm RNA表达从 4h就开始下降 ,1～ 3天最低 ,5天左右又上升 ,i NOSm RNA在缺血再灌注后 12 h开始表达 ,1天左右升至高峰 ,3天左右缓慢下降。结论 :n NOSm RNA在缺血损伤后有一短暂表达增高 ,而在缺血损伤 4h后就开始缓慢下降 ;而 i NOSm RNA在缺血 /再灌注后 12 h开始表达 ,高峰在 1天左右 ,3天左右开始下降 ,其细胞定位以小胶质细胞为主。
