脑啡肽乙酸酯对大鼠急性全脑缺血－再灌注后海马区神经元保护的机制研究

Neuroprotective effect of DADLE on the neurons in rat brain hippocampus with acute global cerebral ischemia -reperfusion injury

目的：研究脑啡肽乙酸酯（ DADLE）是否通过调控胞外信号调控激酶（ ERK）信号转导通路而抑制大鼠急性全脑缺血－再灌注后海马区神经细胞凋亡。方法健康雌性SD大鼠50只（180～220 g）随机分为五组，建立大鼠急性全脑缺血－再灌注模型：假手术组（ Sham组，n＝10），只行手术分离血管，不予结扎血管及再灌注；缺血－再灌注组（I／R组，n＝10），缺血15 min，再灌注120 min；DADLE＋PD98059组（DA＋PD组，n＝10），缺血15 min后再灌注开始前静脉注射DADLE（3 mg／kg）及PD98059（0．3 mg／kg），然后行再灌注120 min； PD98059组（PD组，n＝10），缺血15 min后再灌注开始前静脉注射PD98059（0．3 mg／kg）；DADLE处理组（DA组，n＝10），缺血15 min后再灌注开始前静脉注射DADLE（3 mg／kg），然后行再灌注120 min。 HE染色观察各组海马CA1区神经元病理变化情况，TUNEL法检测大鼠海马凋亡细胞，Western blot检测脑组织磷酸化ERK1／2（P－ERK1／2）的表达状况。结果 HE染色显示，I／R组、DA＋PD组、PD组、DA组较Sham组正常神经元细胞计数显著减少（P＜0．01）；DA组神经元的损伤、脱失、细胞肿胀均较I／R组轻，正常神经元细胞计数较I／R组显著增多（P＜0．01）；PD组、DA＋PD组及I／R组正常神经元细胞计数组间两两比较差异无统计学意义。 TUNEL法检测显示，I／R组、DA＋PD组、PD组、DA组大鼠海马CA1区神经细胞凋亡指数均较Sham组显著升高（P＜0．01）；与I／R组比较，DA＋PD组及DA组凋亡指数显著下降（P＜0．01），PD组凋亡指数则升高（P＜0．05）；DA＋PD组、PD组及DA组组间两两比较差异有统计学意义（ P＜0．01）。Western blot检测显示，I／R组（0．69±0．07）、DA＋PD组（0．59±0．03）及DA组（0．83±0．06）的P－ERK1／2表达较Sham组（0．42±0．06）增多（P＜0．01），而PD组（0．41±0．05）则无显著变化；PD组对P－ERK1表达呈完全抑制，予DADLE处理后P－ERK1表达则显著增加；与I／R组比较， P－ERK1／2在DA组的表达显著增加（P＜0．01），而在DA＋PD组及PD组则显著降低（P＜0．01）；DA＋PD组、PD组及DA组组间两两比较差异有统计学意义（ P＜0．01）。结论 DADLE可通过激活ERK1／2信号转导通路特别是ERK1通路而发挥抑制急性全脑缺血－再灌注损伤诱导的神经细胞凋亡作用。

Objective To investigate whether DADLE could affect rat brain hippocampus neuronal apoptosis after acute global cerebral ischemia-reperfusion injury by regulating ERK signaling pathway.Methods Fifty female Sprague-Dawley rats （180 ~220 g） were randomly divided into 5 groups：sham group （Sham, n=10） exposed bilateral common artery but without occlusion;ischemia/reperfusion group （ I/R, n =10 ） was induced by bilateral common artery occlusion combined with hypotension for 15 min global ischemia followed by 120 min reperfusion; DADLE ＋PD98059 group （DA＋PD, n=10）, DADLE （3 mg/kg, iv） and PD98059 （0.3 mg/kg, iv） was applied through external jugular vein before reperfusion; PD98059 group （PD, n=10）, PD98059 （0.3 mg/kg, iv） were applied through external jugular vein before reperfusion; DADLE group： DADLE （3 mg/kg, iv） was applied through external jugular vein before reperfusion. Neuropathological changes in rat hippocampus CA1 area were observed by Hernatoxylin-Eosin （ H-E） staining.Apoptotic cells in rat hippocampus were detected by TUNEL method, and the expression of phosphorylated ERK （ P-ERK） in rat brain tissue was assessed by immunoblotting.Results H -E staining demonstrated that the normal neurons in hippocampal CA1 area were significantly decreased in I/R, DA＋PD, PD and DA group compared with that in Sham group （P〈0.01）;and in DA group when compared with I/R group the normal neurons was remarkably increased （P〈0.01） with less neuronal damage, demyelination, cell swelling.But there were no significant difference of normal neurons counting among the PD, DA＋PD and DADLE groups.TUNEL assay showed I/R group, DA ＋PD group, PD group, DA rat hippocampal CA1 neuronal apoptosis index was significantly higher than that in the Sham group （ P〈0.01）;compared with I/R group, apoptotic index was significantly decreased （P〈0.01） in DA＋PD and and DA group, but was increased （P〈0.05） in PD group;there were significant difference （P〈0.01） among DA ＋PD, PD and DA group.Western blotting showed that, compared with the sham group （0.42 ±0.06）, the P -ERK1/2 expression in I/R group （0.69 ±0.07）, DA ＋PD group （0.59 ±0.03） and the DA group （0.83 ±0.06） was increased （P〈0.01）, while the PD group （0.41 ±0.05） showed no significant change; in PD group the P -ERK1 expression was completely suppressed, but P -ERK1 expression was increased significantly after treated by DADLE; compared with I/R group, P-ERK1/2 in the DA group was significantly increased （ P〈0.01）, while in the DA＋PD group and the PD group was significantly decreased （ P 〈0.01 ）; the pairwise comparisons among DA ＋PD group, PD group and the DA group showed significant difference （ P 〈0.01 ） . Conclusion DADLE could inhibit acute global cerebral ischemia -reperfusion injury -induced neuronal apoptosis by activating ERK1/2 especially ERK1 signaling pathway.