Genistein后处理通过激活eNOS保护缺血后海马CA1区神经元

Genistein postconditioning protects neurons against ischemic injury through eNOS activation in hippocampal CA1 region

目的研究Genistein后处理(GPC)对脑缺血再灌注后大鼠海马CA1区神经元的神经保护作用,及其对eNOS磷酸化水平的影响。方法建立大鼠4动脉结扎全脑缺血模型,实验动物随机分为假手术组、缺血再灌注(I/R)组、GPC组(尾静脉注射)、抑制剂L-NAME组(脑室注射)、溶剂对照组。采用Western blot法检测大鼠海马CA1区eNOS、p-eNOS的表达;NeuN染色和TUNEL法分别观察海马CA1区神经元的存活和凋亡样损伤。结果与I/R组相比,GPC后再灌注30 min和3 d p-eNOS水平显著升高(P<0.001),而eNOS蛋白表达在各个时间点没有明显变化。激光扫描共聚焦结果显示,GPC组与I/R组相比较,海马CA1区存活的神经元明显增加(213.0±21.0 vs 45.0±15.0,P<0.05),而凋亡样损伤的神经元显著减少(29.0±6.0 vs 192.0±31.0,P<0.05);NOS抑制剂L-NAME不但可显著减弱GPC诱导的神经保护作用(P<0.05),而且有效降低p-eNOS水平(1.149±0.218 vs 1.583±0.155,P<0.001)。结论 Genistein后处理可抑制大鼠海马CA1区神经元凋亡,其机制可能与p-eNOS表达上调有关。

Objective To investigate the neuroprotective role of Genistein postconditioning （GPC） against cerebral ischemic injury and its effects on the level of eNOS phosphorylation in hippocampal CA1 region of rats. Methods The rats were subjected to global cerebral isehemia by four-vessel occlusion and randomly divided into five groups, sham, isehemia/reperfusion （I/R）, GPC （intravenously through the tail vein）, L-NAME （an inhibitor of NOS, icv. ） and vehicle （ I/R ± DMSO, I/R ± Genistein ± NaC1） groups. The protein level of p-eNOS and eNOS was de- tected by Western blotting. Additionally, NeuN staining and TUNEL analysis was used to detect the survival neu- rons and apoptotic neurons of hippocampal CA1 region, respectively by Laser Scanning Confocal Microscope. Results Compared with I/R groups, in （;PC groups p-eNOS levels at 30 rain or 3 d of reperfusion significantly in-creased （P 〈 0. 001 ）, while eNOS protein expression in all time points had no obviously change. GPC groups compared with I/R groups, the number of surviving neurons of hippocampal CA1 region significantly increased （213.0 ±21.0 vs 45.0 ± 15.0, P 〈0. 05） and apoptotic neurons markedly reduced （29.0 ±6.0 vs 192. 0±31.0, P 〈 0.05 ）. Furthermore, L-NAME can not only significantly abate neuroprotection induced by genistein postcondi- tioning （P 〈 0. 05 ）, but also effectively reduce the level of p-eNOS （ 1. 149 ± 0. 218 vs 1. 583 ± 0. 155, P 〈 0. 001 ）. Conclusions GPC significantly prevents neuronal injury from global cerebral ischemia in the hippocampal CA1 region of rats. The mechanism might be explained by the increase of p-eNOS level.