| In the development of central nervous system disease progression, neuron loss is one of the obvious pathological changes. Excitotoxicity, as one of the important mechanisms of neuronal damage, mainly induce apoptosis as the main mechanism of neuronal degeneration. Research have shown that reduction of the excitatory amino acid release and inhibition of glutamate receptor function can highlight effective against neuronal injury mediated by glutamate. Nonetheless, various adverse effects of glutamate receptor antagonist limited the clinical application, while the protective effects for the central nervous system was proved in the traditional Chinese medicine compound prescribe. Perivious experiments showed Shaoyao-gancao decoction (SGD) effective for spastic hemiplegia and ischemic brain injury, which can significantly improve the neurological dysfunction of stroke in rats, modulating neurotransmitter Glu, GABA and its receptor expressions. With the intervention of GABA receptor agonists and antagonists, the present study aimed to establish the NMDA-induced excitotoxic injury in PC 12 cell and explore the protective effects of peony Licorice compatibility, as well as the "Src-NR2-nNOS" pathway-based receptor-mediated signal interaction. This study attempted to clarify the synergies mechanism of the two amino acids in vitro model of excitotoxicity. and provide the experimental basis of SDG for the clinical prevention and treatment of the central nervous diseases and development of the active ingredients of Chinese medicine compound against excitotoxicity.ObjectiveTo observe the protective effects of SDG on PC12 cells and "Src-NR2-nNOS" signal pathway-based regulation of GABA-Glu balance.Methods1. Preparation and quality control of SGD2. To investigate the effect of SDG medicated serum on NMDA-induced PC 12 cells viability, apoptosis, and Ca2+ concentration.3. To detect the mRNA and protein phosphorylation expression of Src, NR2A, NR2B, nNOS in "Src-NR2-nNOS" signal pathway.Results1. Quality control of SGDFingerprint detected a total of ten characteristic peaks, where five peaks of peony, five peaks of licorice; Among SGD used in the experiment (TGP:Licorice=3:1), the paeoniflorin content of 19.13 mg/g, glycyrrhizin content of 13.39 mg/g, licorice ammonium content of 0.018 mg/g.2. Effect of SDG medicated serum on PC 12 cell viabilityNMDA significantly inhibited cell viability of PC 12 cells. Compared with the control group, the OD values of model group decreased remarkably (P<0.01), SGD 1:1 low-dose group can increase cell viability, middle dose group and high dose group compared with the model group was significantly increased (P<0.05, P<0.01), SGD 3:1 low dose group reduce cell viability, the middle and high dose group compared with the model group was significantly increased (P<0.05, P<0.01).GABAa agonist group (muscimol), GABAB receptor agonist group (Baclofen) cells OD values increased compared with model group increased; GABAA antagonist group (Bicuculline) increased compared with the model group, GABAB receptor antagonists(Saclofen) down-regulated the OD value.3. Effect of SDG medicated serum on PC 12 cell apoptosis3.1 Effect of SDG medicated serum on apoptosis rateNMD A significantly increased apoptosis of PC 12, the apoptosis rate of model group was significantly higher than the control group (P<0.05), SGD 1:1 group apoptosis rate compared with the model group decreased significantly (P<0.05), SDG 3:1 group compared with the model group decreased.The apoptosis rate of GABAa agonists group (muscimol), GABAB agonist group (Baclofen) compared with model group decreased significantly (P<0.05, P<0.01); GABAA receptor antagonist group (Bicuculline), the apoptosis rate GABAB antagonist group (Saclofen) compared with the model group increased significantly (P<0.05, P<0.01).3.2 Effect of SDG medicated serum on apoptosis protein Caspase-3 protein expressionNMDA remarkably increased the protein expression of Caspase-3 in model group (P<0.01), given SGD 1:1 and 3:1,Caspase-3 protein expression decreased significantly, compared with the model group (P<0.05).GABAA agonists group (muscimol) decreased significantly (P<0.05), GABAB agonist group (Baclofen) presented a downward trend; GABAA antagonist group (Bicuculline) down-regulated the protein expression of Caspase-3 group remarkably (P<0.05), GABAB antagonists group (Saclofen) declined compared with model group.4. Effect of SDG medicated serum on PC 12 cell Ca2+ concentrationCompared with the control group, NMDA significantly increased Ca2+ concentration (P<0.01), giving SGD 1:1,3:1 can inhibit cell Ca2+ concentration, compared with the model group was significantly decreased (P<0.05, P<0.01).GABAA agonists group (muscimol), GABAB agonist group (Baclofen) cells Ca2+ concentration in model group decreased significantly (P<0.05); compared with the model group, GABAA antagonist group (Bicuculline) Ca2+ concentration decreased, cells Ca2+ concentration decreased significantly compared with model group GABAB antagonist group (Saclofen) (P<0.01).5. Effect of SDG medicated serum on Src, NR2A, NR2B, nNOS mRNA expressionCompared with the control group, NMDA increased the mRNA expression of NR2A, nNOS significantly (P<0.01, P<0.05) and up-regulate the expression of the expression of NR2B, Src mRNA declined.Compared with the model group, SGD 1:1 group can down-regulate the expression of Src, NR2B mRNA. Significantly reduced NR2A, nNOS mRNA expression (P<0.01, P<0.05); SGD 3:1 group can down-regulate the expression of Src, NR2B, nNOS mRNA expressions. Significantly down-regulated the expressions of NR2A mRNA (P<0.01).Compared with model group, GABAA agonists group (Muscimol) NR2A, NR2B, nNOS mRNA expression significantly decreased (P<0.01, P<0.05, P<0.05), and mRNA expression of Src also declined; GABAB agonist group (Baclofen) NR2A, nNOS mRNA expression was significantly reduced(P<0.01, P<0.05), Src, NR2B of mRNA expression decreased.Compared with model group, GABAA antagonist group (Bicuculline) NR2A, NR2B, nNOS mRNA expression compared with model group decreased significantly (P<0.01, P<0.05, P<0.05), and Src mRNA expression was also decreased; NR2A, nNOS mRNA expression was significantly decreased (P<0.01, P<0.05), the mRNA expression of Src, NR2B declined in GABAB antagonist group(Saclofen).6. Effect of SDG medicated serum the p-Src (Tyr418) p-NR2A (Tyr1325) p-NR2B (Tyr1472) p-nNOS (Ser1417) protein expressionCompared with the control group, NMDA group significantly up-regulated p-NR2A, p-NR2B, p-nNOS protein expression (P<0.05, P<0.01, P<0.001), the expression of p-Src was increased.Compared with the model group, giving SGD 1:1 set lower p-NR2A p-NR2B p-nNOS expression and significantly reduced p-Src protein expression (P<0.05); giving SGD 3:1 set of down-regulated expression of p-Src. Significantly reduced p-NR2A, p-NR2B, p-nNOS protein expressions (P<0.05, P<0.01, P<0.01).Compared with model group, GABAA agonists group (Muscimol) p-Src, p-NR2A, p-nNOS protein expression decreased significantly (P<0.05, P<0.05, P<0.001), p-NR2B protein expression has increased; p-Src, p-NR2A, p-NR2B, p-nNOS protein expression decreased significantly (P<0.01, P<.01, P<0.05, P<0.01) in GABAN agonist group(Baclofen).Compared with model group, GABAA antagonist group (Bicuculline) significantly reduced protein expression of p-NR2B (P<0.01), protein expression of p-Src presented a downward trend, p-NR2A protein was significantly increased (P<0.01), the expression of p-nNOS was increased; in GABAN antagonist group (Saclofen), p-Src, p-nNOS decreased significantly (P<0.01), the expression of p-NR2B protein has declined,p-NR2A protein expression on the rise.Conclusion1. NMDA-induced excitotoxicity model was established on PC 12 cell, concentration at 200μmol/L. SDG medicated serum can increase cell viability, reduce the rate of apoptosis, and protein expression of apoptosis protein Caspase-3, reduce Ca2+ concentration. Thereby reducing the NMDA excitotoxicity.2. GABA agonists showed good antagonism of NMDA-induced excitatory injury, but did not rule out possible negative feedback regulation between extracellular GABA and NMDA. The over-excited GABA receptors may excit NMDA receptor at the same time, which increased excitotoxicity eventually; GABA antagonist can not significantly increased excitotoxicity.3. SDG presented Src inhibitor-like effects, mainly affecting NR2A receptor phosphorylation by the binding protein further weakening of nNOS synthesis and NO release.4. The experimental verified of "sour and sweet yin" features of SDG, regulated the brain and central inhibitive and excitatory neurotransmitter system, provided effective ideas for the development of NMDA inhibitors in traditional Chinese medicine. |
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