脑缺血再灌注损伤大鼠Beclin-1与Bcl-xL、Caspase-2的表达及Cystatin C干预作用的影响

陈 萌， 梁秀军， 王爱乐， 常晓栋， 杨嘉磊

脑缺血再灌注损伤大鼠Beclin-1与Bcl-xL、Caspase-2的表达及Cystatin C干预作用的影响

陈 萌1， 梁秀军2， 王爱乐3， 常晓栋4， 杨嘉磊4

目的探讨大鼠脑缺血再灌注后自噬蛋白Beclin-1与凋亡蛋白Bcl-xL、Caspase-2表达的变化以及Cystatin C对其的干预作用。方法Sprague-Dawley大鼠雄性60只随机分成假手术组(sham)、模型组(model)、Cystatin C低(low)、中(middle)、高(high)浓度组，每组12只。用线栓法制备大鼠右侧局灶性脑缺血2 h再灌注24 h模型。各组大鼠进行神经损伤严重程度评分(mNSS)，Western Blot检测损伤区脑皮质组织中凋亡相关蛋白Bcl-xL、Caspase-2的表达；免疫荧光法检测Beclin-1蛋白表达的平均荧光强度；TUNEL法观察神经细胞的凋亡指数(AI)。结果与模型组相比，Cystatin C低、中浓度组中Bcl-xL的表达较模型组明显升高(P<0.05)，Caspase-2的表达降低(P<0.05)；而Cystatin C高浓度组Bcl-xL表达明显降低，Caspase-2的表达则显著上升(P<0.05)；Cystatin C低、中、高浓度组Beclin-1的表达逐渐升高(P<0.05)。结论应用低、中浓度的Cystatin C干预脑缺血再灌注损伤大鼠后，自噬的增强能够促进Bcl-xL的表达，抑制Caspase-2的表达；而高浓度的Cystatin C使Caspase-2的表达增强，Bcl-xL的表达降低。

脑缺血再灌注； 自噬； Caspase-2； Bcl-xL； Beclin-1

Abstract：ObjectiveTo investigate the expressions of autophagy related proteins Beclin-1 and apoptosis related proteins Bcl-xL、Caspase-2 after focal cerebral ischemia-reperfusion with Cystatin C pretreatment in rats.Methods60 male SD rats were randomly divided into sham operation group，the model group and Cystatin C pretreatment group(Cystatin C 2 mg/l，5 mg/l，10 mg/l，respectively)，12 rats in each group. The model of right middle cerebral artery occlusion(MCAO) was established by thread embolism. After ischemia for 2 h and reperfusion 24 h，the Modified neurological severity scores (mNSS) were tested. All rats were killed at 24 h after MCAO. The brains were removed and using Western Blot method to detect apoptosis related proteins Bcl-xL and Caspase-2，located in the center area of the cerebral cortex tissue injury；Beclin-1 protein expression was detected by immunofluorescence method，and TUNEL method was used to evaluate the apoptosis of neuron.ResultsCompared with model group，the expression of Bcl-xL had different degree of increase in the Cys low and Cys middle group(P<0.05)，the expression of Caspase-2 decreased(P<0.05)；While the expression of Caspase-2 in the Cys high group was higher than the Cys low and Cys middle group(P<0.05)，the Bcl-xL was obviously lesser(P<0.05)；the expression of Beclin-1 protein in the each concentration Cystatin C group increased gradually(P<0.05).ConclusionLow and middle concentration of Cystatin C intervention ischemia reperfusion injury in rats，the activity of autophagy increased could promote the pression of Bcl-xL，inhibit the expression of Caspase-2.While high concentration of Cystatin C (reach a certain degree of autophagy flow) could inhibit the expression of Bcl-xL，promote the expression of Caspase-2.

Keywords： Cerebral ischemia reperfusion； Autophagy； Beclin-1； Bcl-xL； Caspase-2

脑卒中(Stroke)又叫脑血管意外，是一种突然起病的脑血液循环障碍性疾病，临床上分为缺血性脑卒中和出血性脑卒中。在国内的脑血管疾病患者中，缺血性脑卒中患者可达66.4%[1]。脑缺血后继发性脑损伤将严重影响患者预后并导致病情加重，其主要参与原因包括炎症反应、细胞凋亡、兴奋性氨基酸毒性、自噬及钙超载等等。自噬在缺血再灌注损伤时是一把双刃剑，当细胞启动适度的自噬来清除受损细胞器时，对细胞主要起保护作用，但当不完全或过度自噬发生时可能导致细胞死亡[2]。半胱氨酸蛋白酶抑制剂C(Cystatin C，Cys C)已证实可以诱导细胞自噬，从而保护小鼠皮质神经元[3]。本实验预先给予大鼠不同浓度的Cystatin C进行处理，然后建立局灶性脑缺血再灌注损伤模型，研究其对大鼠脑缺血再灌注损伤后神经细胞自噬蛋白Beclin-1和凋亡蛋白Bcl-xL、Caspase-2表达的影响。……