矾酸钠和染料木黄酮对脑缺血／再灌注诱导海马神经元损伤的作用及分子机制

Effect and mechanism of sodium vanadate and genistein on neuronal injury induced by cerebral ischemia/repervusion

目的研究矾酸钠和染料木黄酮对脑缺血／再灌注后海马神经元损伤的作用及其分子机制。方法采用蒙古沙土鼠双侧颈总动脉结扎诱导的短暂(15min)前脑缺血模型，用底物γ^-32P掺入法测定酪氨酸蛋白激酶(PIK)的活性，免疫印迹法分析NMDA受体2B亚基(NR2B)的酪氨酸磷酸化变化；采用体外孵育的大鼠海马脑片模拟缺血模型，测定海马神经元细胞乳酸脱氢酶(lactate dehydrogenase，LDH)的漏出量作为神经元损伤的指标。结果沙土鼠缺血／再灌6h，海马突触体总PIK活性显著升高，缺血前20min腹腔注射染料木黄酮对PIK总活性有明显的降低作用，而矾酸钠对其却无明显作用；短暂前脑缺血／再灌注6h后，NR2B的酪氨酸磷酸化显著增加，矾酸钠与染料木黄酮可分别增加、减少这种磷酸化的增加，而对NR2B的蛋白表达无明显作用；大鼠海马脑片“缺血”30min／再灌1h，细胞LDH的漏出量明显增加，“缺血”前15min，在海马脑片孵育液中加入矾酸钠与染料木黄酮可分别增加、减少LDH的漏出量。结论PIK和酪氨酸蛋白磷酸酶(PTP)共同参与缺血性脑损伤的调节，而PIK起着更为重要的作用。

Objective To study the effects and molecular mechanisms of vanadate and genistein on the injury of hippocampal neuron induced by ischemia/reperfusion (I/R). Methods Transient (15 min) cerebral ischemia was induced by the method of bilateral carotid artery occlusion in mongolian gerbils. Total PTK activity was measured by [γ- 32 P] incorporation. Tyrosine phosphorylation of NMDA receptor subunit 2B (NR2B) was analyzed by immunoblot assay. The extent of hippocampal injury induced by I/R was monitored using the release of cytosolic enzyme lactate dehydrogenase (LDH). Results Transient cerebral ischemia followed by reperfusion for 6 h caused a significant increase in total PTK activity. Administration of genistein 20 min before ischemia caused a pronounced decrease in total PTK activity, but the administration of vanadate had no effect. Transient cerebral ischemia followed by 6-h reperfusion caused a significant increase in tyrosine phosphorylation of NR2B. This increase of tyrosine phosphorylation was further elevated by administrating vanadate, but decreased remarkably by genistein; whereas the expression of NR2B protein was unaffected by vanadate or genistein. In addition, the measurement of LDH efflux, as an indicator of hippocampus injury, indicated that vanadate aggravated the cerebral injury induced by brain I/R, while genistein could protect the cerebral injury. Conclusion PTK and PTP are commonly involved in the ischemic injury, while PTK plays a more important role than PTP.