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目的观察银杏叶提取物(GBE)对 N-甲基-D-天冬氨酸(NMDA)受体过度激活导致海马神经元和脑损伤作用的影响并探讨其可能机制。方法用锥虫蓝染色和乳酸脱氢酶(LDH)测定等方法评估 GBE 对海马神经元兴奋毒性损伤的影响,并运用全细胞膜片钳记录观察 GBE 对大鼠海马急性分离神经元 NMDA 受体激活电流的调制作用;以 Longa 评分、红四氮唑、神经元核蛋白和微管相关蛋白-2免疫组化染色等方法评估 GBE 对大鼠局灶性脑缺血损伤的保护作用。以上 GBE 干预作用均与 NMDA 受体特异性拮抗剂 MK-801作比较。结果 150μg/ml GBE 预处理可有效地保护暴露于 NMDA+甘氨酸的海马神经元,使细胞生存率提高到85.2%±5.2%,LDH 漏出减少到87 U/L±8U/L,但此效应弱于 MK-801(P<0.05);预加0.1 mg/ml GBE 可明显抑制由 NMDA 和甘氨酸共同作用能引起的内向电流(I_(NMDA)),抑制率为40%±17%,50 μmol/L MK-801的抑制率为78%±18%,两组差异有统计学意义(P<0.01);用标准细胞外液进行冲洗后,GBE 组 I_(NMDA)可恢复至91%±8%,而MK-801组不能(P<0.05)。GBE 预处理可有效缓解大鼠局灶性脑缺血损伤,其保护作用弱于 MK-801(P<0.05),但没有 MK-801所引起大鼠严重的行为学毒性反应。结论 GBE 预处理能对抗NMDA 受体过度激活所致海马神经元毒性损伤和大鼠局灶性脑缺血损伤,可能与通过拮抗 NMDA 受体有关,此保护作用及其与 NMDA 受体结合力均弱于 MK-801,无行为学毒性反应,使抗兴奋毒性临床干预成为可能.
Objective To observe the effect of Ginkgo biloba extract (GBE) on NMDA receptor over-activation leading to hippocampal neurons and brain injury and explore its possible mechanism. Methods The effect of GBE on excitotoxicity of hippocampal neurons was evaluated by trypan blue staining and lactate dehydrogenase (LDH) assay. Whole cell patch clamp recordings were used to observe the effect of GBE on the activation of NMDA receptors in hippocampal neurons. The current modulation effect; Longa score, red tetrazolium, neuronal nuclear protein and microtubule-associated protein-2 immunohistochemical staining and other methods to evaluate the protective effect of GBE on focal cerebral ischemia in rats. The above GBE interventions were compared with the NMDA receptor-specific antagonist MK-801. Results 150μg/ml GBE pretreatment effectively protected hippocampal neurons exposed to NMDA+glycine, improved the cell survival rate to 85.2%±5.2%, and the LDH leakage decreased to 87 U/L±8U/L, but this effect was weaker than MK-801 (P<0.05); Pretreatment with 0.1 mg/ml GBE could significantly inhibit the inward current (I_(NMDA)) induced by the combined action of NMDA and glycine, and the inhibition rate was 40%±17%, 50 μmol/L. The inhibition rate of MK-801 was 78%±18%, and the difference between the two groups was statistically significant (P<0.01). After flushing with standard extracellular fluid, the I_(NMDA) of GBE group could recover to 91%±8%. The MK-801 group could not (P<0.05). GBE pretreatment effectively relieves focal cerebral ischemic injury in rats, and its protective effect is weaker than that of MK-801 (P<0.05), but there is no severe behavioral toxicity reaction caused by MK-801 in rats. Conclusion GBE pretreatment can counteract the damage of hippocampal neurons induced by excessive activation of NMDA receptors and focal cerebral ischemic injury in rats. It may be related to the antagonism of NMDA receptors. This protective effect and its binding to NMDA receptors are both. Being weaker than MK-801, there is no behavioral toxicity reaction, making anti-excitotoxic clinical intervention possible.