选择性脑低温对大鼠脑缺血再灌注时皮质小泛素样修饰蛋白化的影响

Effect of selective brain hypothermia on SUMOylation of cerebral cortex during cerebral ischemiareperfusion in rats

目的评价选择性脑低温对大鼠脑缺血再灌注时皮质小泛素样修饰蛋白(SUMO)化的影响。方法清洁级健康雄性sD大鼠120只,8周龄,体重200~250g,采用随机数字表法分为4组(n=30):假手术组(S组)、缺血再灌注组(I/R组)、选择性脑低温组(H组)和37℃生理盐水组(N组)。采用线栓法阻断大鼠右侧大脑中动脉,缺血2h后I/R组拔除线栓恢复灌注,H组拔除线栓即刻由右侧颈内动脉以100ml·kg^-1·h^-1速率输注10~13℃生理盐水20min,N组以同样速率输注37℃生理盐水20min。于再灌注后6、24和48h时进行神经功能缺陷评分,采用TUNEL法检测缺血侧皮质细胞凋亡率,Western blot法测定缺血侧皮质SUMO特异性蛋白酶3(SENP3)、结合状态SUM02/3表达水平。结果与S组比较,其余3组大鼠各时点神经功能缺陷评分和细胞凋亡率升高,结合状态SUM02/3表达上调,I/R组和N组SENP3表达上调,H组SENP3表达下调(P<0.01);与I/R组比较,H组神经功能缺陷评分和细胞凋亡率降低,结合状态SUM02/3表达上调,SENP3表达下调(P<0.05)。结论选择性脑低温减轻大鼠脑缺血再灌注损伤的机制与其增强皮质SUMO化有关。

Objective To evaluate the effect of selective brain hypothermia on small ubiquitin-like modifier (SUMO)ylation of cerebral cortex during cerebral ischemia-reperfusion (I/R)in rats.Methods A total of 120healthy clean-grade male Sprague-Dawley rats,aged 8weeks,weighing 200-250g,were divided into 4groups (n=30each)by a random number table method:sham operation group (group S), group I/R,selective brain hypothermia group (group H)and 37℃ normal saline group (group N).Rats were anesthetized with 10%chloral hydrate 3ml/kg.Cerebral ischemia was induced by middle cerebral artery occlusion using a nylon thread with rounded tip inserted into the right internal carotid artery and advanced cranially until resistance was met.The right middle cerebral artery was occluded for 2h followed by reperfusion in group I/R.10-13℃ normal saline was infused at the rate of 100ml ·kg^-1·h^-1for 20min starting from the time point immediately after removing the nylon thread in group H.37℃ normal saline was infused instead at the same rate for 20rain in N group.The neurological deficit were assessed and scored at 6,24and 48h after reperfusion.The cerebral cortex on ischemic side was then removed for determination of cell apoptosis (by TUNEL)and expression of SUMO sepcific proteases 3(SENP3)and SUMO2/3-conjugated protein (by Western blot).The apoptotic rate was calculated.Results Compared with group S, the neurological deficit score and apoptosis rate were significantly increased,and the expression of SUMO2/3-conjugated protein was up-regulated at each time point after reperfusion in the other three groups (P<0.05),and the expression of SENP3was significantly up-regulated in I/R and N groups and down-reg-ulated in group H (P<0.01).Compared with group I/R,the neurological deficit score and apoptosis rate were significantly decreased,the expression of SUMO2/3-conjugated protein was up-regulated,and the expression of SENP3was down-regulated in group H (P<0.05).Conclusion The mechanism by which selective brain hypothermia reduces cerebral I/R injury is related to enhancing SUMOylation of cerebral cortex in rats.