缺血预处理通过减轻内质网应激保护全脑缺血大鼠

lschemic preconditioning in the protection of global cerebral ischemia in rats by reducing endoplasmic reticulum stress

目的探讨抑制内质网应激（endoplasmic reticulum stress，ERS）在缺血预处理诱导脑缺血耐受中的作用。方法120只成年雄性Sprague—Dawley大鼠，随机分为假手术组、全脑缺血组和缺血预处理组（缺血预处理3min，2d后全脑缺血15min），设1、3和7d共3个时间点。应用Sugawara法观察大鼠行为学变化，TUNEL染色观察皮质神经元凋亡情况，免疫荧光染色和蛋白质印迹分析检测ERS相关蛋白CHOP、GRP78和胱天蛋白酶-12表达情况。结果神经行为学评分显示，假手术组无神经功能缺损，全脑缺血组和缺血预处理组均出现明显的神经功能缺损，而且随着时间推移出现逐步改善，但全脑缺血组在模型制作后各时间点的神经功能评分均显著性低于缺血预处理组：1d时：（11．00±0．63）分对（14．33±0．33）分（t=21．74，P=0．001）；3d时：（12．17±0．31）分对（15．17±0．48）分（t=27．93，P=0．000）；7d时：（14．67±0．49）分对（16．33±0．33）分（t=7．81，P=0．020）。TUNEL染色显示，缺血后7d时，假手术组、全脑缺血组和缺血预处理组每娜奇TUNEL染色阳性细胞计数分别为（4．83±1．85）个、（395．67±43．43）个和（146．17±27．38）个（F=23．62，P=0．001），缺血预处理组显著性低于全脑缺血组（P=0．001）。免疫荧光染色显示，缺血后7d时，缺血预处理组CHOP[（26．50±3．89）个对（82．33±4．25）个；P=0．000]、GRP78[（15．00±2．02）对（35．67±2．99）个；P=0．000]和胱天蛋白酶一12[（22．33±2．76）个对（66．50±7．25）个；P=0．000]阳性细胞数量均显著性少于全脑缺血组。蛋白质印迹法显示，缺血后7d时，缺血预处理组CHOP[（1．22±0．38）对（3．22±0．51）；t=24．50，P=0．001]、GRP78[（1．78±0．45）对（3．16±0．76）；t=14．29，P=0．005]和胱天蛋白酶-12[（2．89±0．53）对（5．96±0．67）；t=77．73；P=0．000]表达显著性低于全脑缺血组。结论缺血预处理对第2次致死性缺血表现出神经保护作用，其机制可能与ERS减轻，ERS相关蛋白表达下调有关。

Objective To investigate the effect of inhibition of endoplasmic reticulum stress （ERS） in ischemic preconditioning-induced cerebral ischemia tolerance. Methods A total of 120 adult male Sprague- Dawley rats were randomly allocated into three groups： sham operation, global cerebral ischemic and ischemic preconditioning groups （ischemlc preconditioning for 3 minutes, and global cerebral ischemia for 15 minutes after 2 days）. Three time points （day 1, day 3 and day 7） were set. Sugawara method was used to observe the changes of neurological behavior in rats. TUNEL staining was used to observe the conditions of cortical neuronal apoptosis. Immunofluorescence staining and Western blot analysis were used to detect the expression levels of ERS-related protein CHOP, GRP78, and caspase-12. Results The neurological behavior score showed that the sham operation group did not have neurological deficits. Both the global cerebral ischemic group and the ischemic preconditioning group had obvious neurological deficits, and they improved gradually with the passage of time, but after modeling, the neurological scores at each time point in the global cerebral ischemic were significantly lower than those in the ischemic preconditioning group： at day 1 ： 11.00 ± 0. 63 vs. 14. 33 ± 0. 33 （t = 21.74, P = 0. 001）; at day 3：12.17±0.31 vs. 15.17±0.48 （t=27.93, P=0.000）; at day 7： 14.67±0.49vs. 16. 33 ±0. 33 （t =7. 81, P=0. 020）. TUNEL staining showed that at day 7 after ischemia, the positive cell count per mm2 in the sham operation, global cerebral ischemic and ischemic preconditioning groups were 4. 83 ± 1.85vs. 395.67± 43.43 and 146.17± 27.38 respectively （F= 23.62, P= 0.001）. The ischemic preconditioning group was significantly lower than that in the global cerebral ischemic group （P = 0. 001 ）. Immunofluorescence staining showed that at day 7 after ischemia, the numbers of positive cells of CHOP （26. 50±3.89vs. 82. 33±4.25, P=0.000）, GRP78 （15.00±2.02vs. 35.67±2.99; t=0.000）, and caspase-12 （22. 33 ± 2. 76 vs. 66. 50 ± 7. 25; P = 0. 000） in the ischemic preconditioning group were significantly less than those in the global cerebral ischemic group. Western blotting showed that at day 7 after ischemia, the expression levels of CHOP （1.22 ± 0. 38 vs. 3.22 ± 0. 51; t = 24. 50, P= 0. 001）, GRP78 （1.78±0.45vs. 3.16±0.76; t=14.29, P=0.005）, and caspase-12 （2. 89±0.53vs. 5.96±0.67; t= 77. 73; P=0. 000） in the ischemic preconditioning group were significantly lower than those in the global cerebral ischemic group. Conclusions Ischemic preconditioning demonstrated a neuroprotective effect for the second lethal ischemia, its mechanism may be associated with the relief of ERS and downregulation of ERS- related protein.