δ阿片受体激动剂DADLE对大鼠皮质神经元缺氧损伤的影响

Effects of δ-opioid receptor agonist DADLE on primary cultured rat＇s cortical neurons following hypoxia injury

目的探讨δ阿片受体激动剂DADLE对于体外培养的大鼠皮质神经元抗物理缺氧损伤是否具有保护作用。方法将体外培养8d的大鼠皮质神经元进行MAV2免疫荧光鉴定后，随机分为4组：缺氧组（n=6），缺氧＋DADLE预处理组（H=6），对照组（n=6），对照＋DADLE预处理组（n=6）。缺氧细胞置入含体积分数1％02，5％CO2及94％N2的缺氧箱内37℃培养72h。DADLE预处理组在将细胞置入孵箱之前加入DADLE（终浓度为10μmol／L）；对照组则将细胞置入含95％空气和5％CO2的恒温培养箱中培养。用hochest33258核染色法评价细胞凋亡情况，检测细胞乳酸脱氢酶LDH漏出量；检测各组细胞上清液葡萄糖水平，以评价其葡萄糖／能量代谢状况。应用单因素方差分析LDH漏出量及葡萄糖水平，X2检验分析神经元生存率。结果皮质神经元培养8d后，经MAP2免疫荧光鉴定为95％细胞为神经元。DADLE预处理缺氧细胞组细胞存活率为（71．88±1．77）％，高于未处理缺氧细胞组的（43．58±3．07）％，P〈0．05，DADLE预处理＋缺氧组的LDH活力单位为（3824．27±294．86），低于未处理缺氧组的（4516．59±605．02），P〈0．05。DADLE预处理＋缺氧组细胞上清液葡萄糖水平为（11．92±2．05）mmol／L，高于未处理缺氧组的（9.88±0．71）mmoL／L，P〈0．05。结论δ阿片受体激动剂DADLE对体外培养的大鼠皮质神经元抗缺氧损伤具有保护作用，可能作用机制为其降低缺氧皮质神经元的葡萄糖／能量代谢需求，减轻缺氧损伤时细胞的葡萄糖／能量代谢障碍。

Objective To study the protective effect of δ-opioid receptor agonist DADLE on hypoxia-indueed injury of rat cortical neurons. Method Cortical neurons cultured for 8 days were assessed by MAP2 immunofluorescence and randonly divided into bypoxia group （ n = 6）, and DADLE preconditioning plus hypoxia group（ n = 6）, normoxia group（ n = 6）, and DADLE preconditioning plus normoxia group（ n = 6）. The cells in hypoxia group were cultured in a hypoxic incubator containing 1% 02,5% CO2, and 94% N2 at 37℃. The cells in DADLE preconditioning groups were preconditioned with 10 μmol/L DADLE before culture. The ceils in nonuoxia group were cultured in an incubator containing 95 % air and 5 % CO2 at 37℃. The survival rate of neurons cultured for 72 hours was analyzed with hochest-33258 staining, and lactate dehydrogenase（LDH） release was detected. Glucose in the culture medium was detected to assess glucose/energy metabolism. The data of LDH re- lease and glucose level were analyzed by ANOVA, and cell survival rates were analyzed by Cbi-square Tests. Resuits When cortical neurons were cultured for 8 days, 95 % the cells were cortical neurons as assayed by MAtt2 i mmunofluorescence. Cell survival rate was significantly higher in DADLE preconditioning plus hypoxia group than in hypoxia group [（71.88 ± 1.77） % vs. （43.58 ± 3.07 ） % （ P 〈 0.05 ） ]. LDH release was significantly lower in DADLE preconditioning plus hypoxia group than in hypoxia group [（3824.27 ± 294.86） vs. （4516.59 ± 605.02）, P 〈 0.05 ]. Glucose level in the culture medium was significantly higher in DADLE preconditioning plus hypoxia group than in hypoxia group[ （ 11.92 ± 2.05） mmol/L vs. （9.88 ± 0.71 ） mmol/L, P 〈 0.051. Conclusions δ-opioid receptor agonist DADLE can protect rat cortical neurons from hypoxia injury, which might be the mechanism of reducing glucose/energy metabolism in cortical neurons during the period of hypoxia.