右美托咪定后处理对大鼠局灶性脑缺血-再灌注损伤的保护作用及机制研究

The Protective Effect and Mechanism of Dexmedetomidine after Treatment in Rats with Focal Cerebral Ischemia-reperfusion Injury

目的:研究右美托咪定(Dexmedetomidine)后处理对大鼠局灶性脑缺血-再灌注损伤的保护作用及机制。方法:将健康的120只Sprague-Dawley(SD)大鼠随机分为4组各30只,分别为假手术组(S组)、脑缺血再灌注组(Mod组)、右美托咪定预处理组(Dex1组,于MCAO模型制作前30min时经腹部注射浓度为100μg/mL的50μg/kg Dex)和右美托咪定后处理组(Dex2组,于恢复再灌注后即刻经腹腔注射浓度为100μg/mL的50μg/kg Dex)。除假手术组外,其余各组完成缺血再灌注24h后,比较各组大鼠间神经功能(bederson)评分,应用TTC染色法测定脑梗死梗死灶体积、再灌注计算梗死灶百分比;并分别应用酶联免疫吸附实验、黄嘌呤氧化酶法、硫代巴比妥酸法测定大鼠神经元特异性烯醇化酶(NSE)、超氧化物歧化酶(SOD)及丙二醛(MDA)水平,分别应用RT-PCR法及Western blot法检测各组大鼠环磷酸腺苷应答元件结合蛋白(CREB)、bcl-2、脑源性神经生长因子(BNDF)mRNA及蛋白表达量,并应用免疫酶标记法(SP)进行免疫组织化学染色。结果:Mod组与Dex1组大鼠bederson评分对比无差异,但Mod组及Dex1组大鼠bederson评分均显著高于Dex2组;同时,经TTC染色;Dex1组、Mod组大鼠梗死灶体积、梗死灶与全脑体积百分比显著高于Dex2组;Dex2组大鼠NSE、MDA水平显著较Dex1组低,SOD水平显著较Dex1组高;经RT-PCR及Westernblot检测、免疫组化分析均可见Dex2组大鼠CREM、bcl-2、BNDF mRNA及蛋白表达量、阳性表达率较Mod组、Dex1组高,且组间变化趋势为Dex2组﹥Dex1组﹥Mod组,上述对比差异均有统计学意义(P﹤0.05)。结论:右美托咪定对大鼠缺血再灌注损伤有一定的保护作用,而右美托咪定后处理能增强大鼠局灶性脑缺血再灌注抗氧化酶活性,抑制氧化应激反应,并能通过对环磷酸腺苷应答元件结合蛋白(CREB)的调控改善bcl-2、BDNF表达,脑保护作用更显著。

Objective： To study the protective effect and mechanism of dexmedetomidine（ Dex） after treatment in rats with focal cerebral ischemia-reperfusion injury. Methods： A total of 120 healthy Sprague-Dawley（ SD） rats were randomly divided into the sham operation group（ S group）,ischemia reperfusion group（ Mod group）,dexmedetomidine pretreatment group（ Dex1 group,treated with abdominal injection of50μg/kg Dex of 100μg/m L at 30 min before making MCAO models） and dexmedetomidine after treatment group（ Dex2 group,treated with intraperitoneal injection of 50μg/kg Dex of 100μg/m L immediately after reperfusion）,with 30 rats in each group. Except S group,24 h after ischemia reperfusion,the neurological function（ bederson） score was compared between the other groups,the volume of cerebral infarction was determined by TTC staining method,the percentage of infarct was calculated,and levels of neuron specific enolase（ NSE）,superoxide dismutase（ SOD） and malondialdehyde（ MDA） were determined by enzyme-linked immunosorbent assay,xanthine oxidase method and thiobarbituric acid method. The expression quantities of response element binding protein（ CREB）,Bcl-2 and brain-derived neurotrophic factor（ BNDF） mRNA and protein in all groups were detected by RT-PCR method and Western blot method. The immunoenzyme labeling assay（ SP） was used for immunohistochemical staining. Results： There was no significant difference of bederson score between Mod group and Dex1 group,but bederson scores of Mod group and Dex1 group were significantly higher than those of Dex2 group. TTC staining showed that the infarct volume and the percentage of infarct volume in total brain volume in Dex1 group and Mod group were significantly higher than those in Dex2 group. Levels of NSE and MDA in Dex2 group were significantly lower than those in Dex1 group while the SOD level was significantly higher than that in Dex1 group. RT-PCR,Westernblot and immunohistochemical analysis showed that expression quantities and positive expression rates of CREB,Bcl-2 and BDNF mRNA and protein in Dex2 group were higher than those in Mod group and Dex1 group,showing Dex2 group 〉Dex1 group〉 Mod group（ P〈0. 05）. Conclusion： Dexmedetomidine has certain protective effect in rats with ischemia reperfusion injury. Dexmedetomidine after treatment can enhance the activities of autioxidant enzymes and inhibit oxidative stress reactions in rats with focal cerebral ischemia reperfusion. It also can improve the expression of Bcl-2 and BDNF through regulating response element binding protein（ CREB） of cyclic adenosine monophosphate. The protective effect on brain is significant.