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
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.
