电针预处理对大鼠脑缺血再灌注时皮质Ubc9表达的影响

Effect of electroacupuncture preconditioning on expression of cortical Ubc9 during cerebral ischemia-reperfusion in rats

目的评价电针预处理对大鼠脑缺血再灌注时皮质Ubc9表达的影响。方法清洁级健康雄性SD大鼠80只,8~12周龄,体重200~250 g,按照随机数字表法分为4组(n=20):假手术组(S组)、脑缺血再灌注组(I/R组)、电针预处理组(E组)和假电针组(SE组)。S组仅暴露颈部血管,不插入线栓阻闭;其余3组采用线栓法制备大鼠脑缺血再灌注损伤模型,栓塞2 h后拔出线栓恢复灌注。E组和SE组造模前5 d行电针刺激,E组选取百会穴(大鼠头顶部两耳连线中点)电针刺激,参数为2/12 Hz疏密波,强度1 mA,30 min/d,连续5 d,最后1次电针刺激24 h后造模,SE组选取刺激点为百会穴旁开1 cm,其余操作参数同E组。于再灌注24和48 h时行神经功能缺陷评分,随后处死大鼠取大脑皮质缺血半暗带组织,TUNEL法检测细胞凋亡情况并计算凋亡率,Western blot法检测Ubc9和结合状态小泛素样修饰蛋白(SUMO)2/3的表达。结果与S组比较,其余3组再灌注24和48 h时神经功能缺陷评分和皮质细胞凋亡率升高,Ubc9和结合状态SUMO2/3表达上调(P<0.05);与I/R组和SE组比较,E组再灌注24和48 h时神经功能缺陷评分和皮质细胞凋亡率降低,Ubc9和结合状态SUMO2/3表达上调(P<0.05)。结论电针预处理减轻大鼠脑缺血再灌注损伤的机制与进一步上调Ubc9表达,从而增强SUMO2/3化修饰有关。

Objective To evaluate the effect of electroacupuncture(EA)preconditioning on expression of cortical Ubc9 during cerebral ischemia-reperfusion(I/R)in rats.Methods A total of 80 healthy clean-grade male Sprague-Dawley rats,aged 8-12 weeks,weighing 200-250 g,were divided into 4 groups(n=20 each)using a random number table method:sham operation group(group S),group I/R,EA preconditioning group(group E)and sham EA group(group SE).Blood vessels were only exposed,without occlusion in group S.In the other three groups,the cerebral I/R model was established by middle cerebral artery occlusion using suture-occluded method,and the suture was removed after 2-h occlusion to restore the perfusion in anesthetized rats.EA was performed at 5 days before establishing the model in group E and group SE.Baihui acupoints were stimulated with an electric stimulator(2/12 Hz disperse-dense waves,intensity 1 mA)for 30 min once a day for 5 consecutive days,and the model was established at 24 h after the last stimulation.EA was performed at the points 1 cm lateral to the acupoints of Baihui,and the other operating parameters were the same as those previously described in group E.Neurological deficit scores(NDSs)were evaluated at 24 and 48 h of reperfusion.Then the rats were sacrificed,and tissues in the ischemic penumbra of cerebral cortex were obtained for determination of cell apoptosis(by TUNEL)and expression of Ubc9 and conjugated small ubiquitin-like modifier(SUMO)2/3(by Western blot).The apoptosis rate was calculated.Results Compared with group S,NDSs at 24 and 48 h of reperfusion and apoptosis rate were significantly increased,and the expression of Ubc9 and conjugated SUMO2/3 was up-regulated in the other three groups(P<0.05).Compared with group I/R and group SE,NDSs at 24 and 48 h of reperfusion and apoptosis rate were significantly decreased,and the expression of Ubc9 and conjugated SUMO2/3 was up-regulated in group E(P<0.05).Conclusion The mechanism by which EA preconditioning reduces cerebral I/R injury is related to up-regulating Ubc9 expression and thus enhancing SUMO2/3ylation in rats.