非诺贝特对大鼠局灶性脑再灌注损伤的脑保护作用
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摘要
非诺贝特的脑缺血保护机制
目的：脑缺血可引起脑组织水肿，神经细胞坏死、凋亡，导致脑功能障碍。MAPK(促有丝分裂蛋白激酶)家族可分为以下三组：ERK、JNK、(C-Jun氨基末端激酶)以及P38MAPK(胞外信号调节酶)。有研究表明，MAPK在脑缺血再灌注损伤后被激活，这些激酶信号通路被认为是造成脑缺血再灌注后致脑损伤的重要分子成份。另外，目前国内外研究表明非诺贝特具有脑保护作用，其保护机制是否与调节JNK，ERK信号通路有关尚不清楚。本实验研究非诺贝特对大鼠局灶性脑缺血再灌注损伤的影响，并评价JNK，C-Jun氨基未端激酶和胞外信号调节酶(ERK)在非诺贝特产生的神经保护中起的作用。
方法：采用大脑中动脉线拴法(MACO)建立大鼠局灶性脑缺血再灌注模型，60只成年SD大鼠，体重220克～250克，被随机分为三组：假手术组(n=20)，脑缺血再灌注组(IR组，n=20)；非诺贝特预治序组(n=20)。动物模型制备：采用线栓法，大鼠用10％水合氯醛腹腔注射麻醉，用特制的尼龙线阻断大脑中动脉血流，2小时后抽出栓线，形成再灌注，假手术组仅暴露颈总动脉及分叉处，不插入阻断大脑中动脉。各组动物术中均用加热板使其肛温保持在37℃左右，术后单笼饲养。假手术组再灌注6小时后处死，IR组再灌注22小时后处死。非诺贝特预治疗组在模型制作前14天用非诺贝特喂养。
本实验(1)测定梗死面积：各组大鼠处死后断头取脑，立即做冠状切片，放入2％TTC磷酸盐缓冲液中染色，各个脑片摄像，用HPIAS-1000图像分析软件计算出梗死面积。(2)NDS神经功能缺损评分(3)脑组织含水量测定：再灌注22小时后，根据Gotoh'sformala方法：脑组织含水量(％)=(湿重-干重)／湿重×100％。(4)脑组织病理观察：光镜下观察所有切片，HE染色，存J E M—1200E电子显微镜下观察。(5)TUNEL检测碉亡细胞数：玻片脱腊、乙醇水化，滴加TUNEL染色缓冲液，DAB显色后，在光镜44倍下选择具有代表性的视野，计数1mm~2阳性细胞数。(6)免疫组织化学法和免疫印迹法检测ERK和JNK的活性，免疫印迹法检测Bcl-2和Bax的表达。取出全脑，分离出背侧海马约100毫克，匀浆后超声粉碎，离心，转膜，用过氧化物酶标记的二抗孵育，X光片上的显色条带进行光密度扫描半定最分析。
结果：同假手术组比较，IR组脑梗死体积，神经功能评分，脑组织含水量，调亡细胞数有显著性差异。非诺贝特组与IR组比较显著降低了上述指标，在分子水平上，显著增加了JNK和ERK的表达活性，同时增加了Bax和Bcl-2基因的表达。非诺贝特下调JNK和Bax蛋白的活性，上调ERK和Bcl-2蛋白的活性表达。
结论：上述结果表明非诺贝特对大鼠局灶性脑缺血再灌注损伤具有保护作用，这可能与非诺贝特对ERK和JNK活性的不同调节有关。实验结果可能对脑缺血再灌注损伤脑保护治疗方面提供了一条新途径。
Objective Improving the ability of the brain to tolerate ischemic injury has important implications. We investigated the effect of Fenofibrate on the focal cerebral ischemia-reperfusion(IR)injury in the rat and evaluated the role of C-Jun NH2-terminal kinase(JNK)and extracelluar-regulated protein kinases(ERK)in Fenofibrate-induced neuroprotection. Methods 60 adult Sprague-Dawley rats were randomized into three groups: sham group(n=20); IRgroup(n=20); Fenoflbrate-pretreated group(n=20). The rats were subjected to middle cerebral artery occlusion with intralimunal filament occlusion, and killed at 6h and 22h after IR injury. Fenoflbrate was administered 14 d before IR injury. Neuroprotective effect of Fenoflbrate on IR was evaluated in terms of neural function(neurological deficit score [NDS])and morphology(HEstaining, EM, TTC staining, cerebral water content, and TUNEL assay)In addition, immunohistochemistry and immunoblotting were used to detect JNK and ERK expression and immunoblotting was used to detect Bcl-2 and Bax expression. Results Compared with the sham group, the cerebral infarct volume ratio, NDS, the cerebral water content, necrosis, and apoptotic cell death were evident in IR-treated rats. Fenofibrate significantly decreased all of above parameters compared with the IR group. At a molecular basis, IR significantly increased JNK and ERK immunoreactivity and expression while increased Bax and Bcl-2 expression. Fenofibrate decreased JNK and Bax but increased ERK and Bcl-2 expression. Conclusions These findings suggest that preconditioning with Fenofibrate have neuroprotective effect against IR injury in the rat, and that this may be associated with differentially regulation of ERK and JNK expression. Our observation may provide a new avenue for therapy to prevent brain damage in IR injury.
目的：脑缺血可引起脑组织水肿，神经细胞坏死、凋亡，导致脑功能障碍。MAPK(促有丝分裂蛋白激酶)家族可分为以下三组：ERK、JNK、(C-Jun氨基末端激酶)以及P38MAPK(胞外信号调节酶)。有研究表明，MAPK在脑缺血再灌注损伤后被激活，这些激酶信号通路被认为是造成脑缺血再灌注后致脑损伤的重要分子成份。另外，目前国内外研究表明非诺贝特具有脑保护作用，其保护机制是否与调节JNK，ERK信号通路有关尚不清楚。本实验研究非诺贝特对大鼠局灶性脑缺血再灌注损伤的影响，并评价JNK，C-Jun氨基未端激酶和胞外信号调节酶(ERK)在非诺贝特产生的神经保护中起的作用。
方法：采用大脑中动脉线拴法(MACO)建立大鼠局灶性脑缺血再灌注模型，60只成年SD大鼠，体重220克～250克，被随机分为三组：假手术组(n=20)，脑缺血再灌注组(IR组，n=20)；非诺贝特预治序组(n=20)。动物模型制备：采用线栓法，大鼠用10％水合氯醛腹腔注射麻醉，用特制的尼龙线阻断大脑中动脉血流，2小时后抽出栓线，形成再灌注，假手术组仅暴露颈总动脉及分叉处，不插入阻断大脑中动脉。各组动物术中均用加热板使其肛温保持在37℃左右，术后单笼饲养。假手术组再灌注6小时后处死，IR组再灌注22小时后处死。非诺贝特预治疗组在模型制作前14天用非诺贝特喂养。
本实验(1)测定梗死面积：各组大鼠处死后断头取脑，立即做冠状切片，放入2％TTC磷酸盐缓冲液中染色，各个脑片摄像，用HPIAS-1000图像分析软件计算出梗死面积。(2)NDS神经功能缺损评分(3)脑组织含水量测定：再灌注22小时后，根据Gotoh'sformala方法：脑组织含水量(％)=(湿重-干重)／湿重×100％。(4)脑组织病理观察：光镜下观察所有切片，HE染色，存J E M—1200E电子显微镜下观察。(5)TUNEL检测碉亡细胞数：玻片脱腊、乙醇水化，滴加TUNEL染色缓冲液，DAB显色后，在光镜44倍下选择具有代表性的视野，计数1mm~2阳性细胞数。(6)免疫组织化学法和免疫印迹法检测ERK和JNK的活性，免疫印迹法检测Bcl-2和Bax的表达。取出全脑，分离出背侧海马约100毫克，匀浆后超声粉碎，离心，转膜，用过氧化物酶标记的二抗孵育，X光片上的显色条带进行光密度扫描半定最分析。
结果：同假手术组比较，IR组脑梗死体积，神经功能评分，脑组织含水量，调亡细胞数有显著性差异。非诺贝特组与IR组比较显著降低了上述指标，在分子水平上，显著增加了JNK和ERK的表达活性，同时增加了Bax和Bcl-2基因的表达。非诺贝特下调JNK和Bax蛋白的活性，上调ERK和Bcl-2蛋白的活性表达。
结论：上述结果表明非诺贝特对大鼠局灶性脑缺血再灌注损伤具有保护作用，这可能与非诺贝特对ERK和JNK活性的不同调节有关。实验结果可能对脑缺血再灌注损伤脑保护治疗方面提供了一条新途径。
Objective Improving the ability of the brain to tolerate ischemic injury has important implications. We investigated the effect of Fenofibrate on the focal cerebral ischemia-reperfusion(IR)injury in the rat and evaluated the role of C-Jun NH2-terminal kinase(JNK)and extracelluar-regulated protein kinases(ERK)in Fenofibrate-induced neuroprotection. Methods 60 adult Sprague-Dawley rats were randomized into three groups: sham group(n=20); IRgroup(n=20); Fenoflbrate-pretreated group(n=20). The rats were subjected to middle cerebral artery occlusion with intralimunal filament occlusion, and killed at 6h and 22h after IR injury. Fenoflbrate was administered 14 d before IR injury. Neuroprotective effect of Fenoflbrate on IR was evaluated in terms of neural function(neurological deficit score [NDS])and morphology(HEstaining, EM, TTC staining, cerebral water content, and TUNEL assay)In addition, immunohistochemistry and immunoblotting were used to detect JNK and ERK expression and immunoblotting was used to detect Bcl-2 and Bax expression. Results Compared with the sham group, the cerebral infarct volume ratio, NDS, the cerebral water content, necrosis, and apoptotic cell death were evident in IR-treated rats. Fenofibrate significantly decreased all of above parameters compared with the IR group. At a molecular basis, IR significantly increased JNK and ERK immunoreactivity and expression while increased Bax and Bcl-2 expression. Fenofibrate decreased JNK and Bax but increased ERK and Bcl-2 expression. Conclusions These findings suggest that preconditioning with Fenofibrate have neuroprotective effect against IR injury in the rat, and that this may be associated with differentially regulation of ERK and JNK expression. Our observation may provide a new avenue for therapy to prevent brain damage in IR injury.
引文
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