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目的在前期研究已经明确新银杏叶提取物(nGBE)具有抗谷氨酸兴奋毒性作用的基础上,通过干预谷氨酸受体的磷酸化及糖基化作用进一步探讨nGBE预处理给药抗兴奋毒性神经损害的机制。方法利用新生Wistar大鼠原代培养的海马神经元谷氨酸毒性模型,采用锥虫蓝活细胞拒染及培养液乳酸脱氢酶(LDH)测定的方法,比较nGBE单独应用、各种磷酸化及糖基化抑制剂单独应用以及将nGBE与各抑制剂联合应用对兴奋毒性神经损害的影响。结果磷脂依赖性蛋白激酶(PKC)抑制剂白屈菜赤碱(chelerythrine)和蛋白合成抑制剂环己米特(放线菌酮,cicloheximide)可不同程度地对抗兴奋毒性作用,与nGBE联合应用呈现出协同保护效应;而糖基化抑制剂衣霉素(tunicamycin)和酪氨酸激酶(PTK)抑制剂染料木黄酮(金雀异黄素,genistein)对细胞的存活率及LDH释放量无影响,nGBE与其共同作用也没有显示出协同保护或损害作用。结论 nGBE预处理给药优于急性与急救给药的机制可能是通过抑制谷氨酸受体的磷酸化作用从而改变谷氨酸受体的功能而实现的。
OBJECTIVE: In the previous study, the effect of nGBE on glutamate-induced excitotoxicity was clarified, and the anti-excitement of nGBE pretreatment was further investigated by intervention of glutamate receptor phosphorylation and glycosylation Mechanism of toxic nerve damage. Methods The neonatal Wistar rat primary cultured hippocampal neurons glutamate toxicity model was used to detect the inhibitory activity of trypan blue in vitro and culture medium lactate dehydrogenase (LDH), compared with nGBE alone, a variety of phosphorylation and Glycosylation inhibitors alone and the combination of nGBE and inhibitors on excitotoxic neurotoxicity. Results Chelerythrine, a phospholipid-dependent protein kinase (PKC) inhibitor, and cicloheximide, a protein synthesis inhibitor, were able to counteract the excitotoxicity in varying degrees and were shown to be associated with nGBE And synergistic protective effects were observed. However, the glycosylation inhibitor tunicamycin and tyrosine kinase (PTK) inhibitor genistein had no effect on cell viability and LDH release , nGBE and its synergy did not show synergistic protection or damage. Conclusion The mechanism of nGBE pretreatment administration superior to acute and first aid administration may be achieved by inhibiting glutamate receptor phosphorylation and thereby changing glutamate receptor function.