三氧预处理保护大鼠脑缺血/再灌注损伤及其对谷氨酸受体的影响

Effects of trioxygen preconditioning on cerebral ischemia/reperfusion injury and glutamate receptor in rats

目的:研究三氧预处理对大鼠脑缺血/再灌注(I/R)损伤的作用。方法:选择24只清洁级雄性SD大鼠,按随机数字表法分为假手术组(Sham组)、脑I/R损伤组(I/R组)和三氧预处理组(Ozone组),每组8只。各组大鼠均常规喂养,Ozone组给予腹腔注射80 mg/L三氧水(剂量为0.01 mL/g)预处理,Sham组和I/R组分别输注等体积0.9%生理盐水;每日1次,共注射5 d。之后I/R组与Ozone组采用线栓法制备大鼠脑I/R模型,Sham组只分离不结扎动脉。缺血2 h后,测定大鼠神经功能缺损评分(NDS);再灌注24 h后,测定改良神经功能缺损评分(mNSS);并麻醉大鼠取脑组织,采用2,3,5-三苯基氯化四氮唑(TTC)染色观察大鼠的脑梗死情况,计算脑梗死体积占比;另外采用蛋白质免疫印迹试验(Western blotting)测定脑缺血半暗带区代谢型谷氨酸受体5(mGluR5)和离子型谷氨酸α-氨基-3-羟基-5甲基-4异恶唑丙酸受体(AMPAR)亚基GluA2的蛋白表达。结果:与Sham组相比,I/R组大鼠的NDS评分、mNSS评分和脑梗死体积占比均明显升高〔NDS评分(分):2.63±0.52比0,mNSS评分(分):9.63±1.19比1.13±0.64,脑梗死体积占比:(41.25±2.93)%比0%,均P<0.05〕,脑缺血半暗带区mGluR5和GluA2的蛋白表达水平均明显降低mGluR5蛋白(mGluR5/β-actin):0.44±0.14比1.00±0.10,GluA2蛋白(GluA2/β-actin):0.23±0.08比1.00±0.25,均P<0.05;与I/R组相比,Ozone组大鼠mNSS评分和脑梗死体积占比均明显降低〔mNSS评分(分):7.00±1.20比9.63±1.19,脑梗死体积占比:(27.23±6.21)%比(41.25±2.93)%,均P<0.05〕,脑缺血半暗带区mGluR5和GluA2的蛋白表达水平均明显升高mGluR5蛋白(mGluR5/β-actin):0.81±0.10比0.44±0.14,GluA2蛋白(GluA2/β-actin):0.76±0.13比0.23±0.08,均P<0.05。结论:三氧预处理可减轻大鼠脑I/R损伤,其机制可能与脑缺血半暗带区GluR5和GluA2蛋白表达上调有关。

Objective To study the effects of trioxygen pretreatment on cerebral ischemia/reperfusion(I/R)injury in rats.Methods A total of 24 clean grade male Sprague-Dawley(SD)rats were randomly divided into Sham group,brain I/R group(I/R group)and Ozone pretreatment group(Ozone group),with 8 rats in each group.The animals were routinely fed,and the operation was performed 5 days after the intervention of Ozone group by intraperitoneal injection of trioxygen water(concentration 80 mg/L,0.01 mL/g),and the Sham group and I/R group were injected with equal volume normal saline.The Sham group only separated the arteries without ligation,and the I/R group and Ozone group established the rat cerebral I/R model.Neurological deficit score(NDS)was performed 2 hours after ischemia and modified neurological deficit score(mNSS)was performed 24 hours after reperfusion.Brain tissue was collected after anesthesia.Cerebral infarction was observed by 2,3,5-triphenyltetrazolium chloride(TTC)staining and the percentage of cerebral infarction volume was calculated.Protein expression of metabolic glutamate receptor 5(mGluR5)and ionic glutamateα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor(AMPAR)subunit GluA2 in cerebral ischemic penumbra was determined by Western blotting.Results Compared with the Sham group,NDS score,mNSS score and percentage of cerebral infarction volume in I/R group were increased[NDS score:2.63±0.52 vs.0,mNSS score:9.63±1.19 vs.1.13±0.64,cerebral infarction volume:(41.25±2.93)%vs.0%,all P<0.05],and expressions of mGluR5 and GluA2 in penumbra area of cerebral ischemia were decreased[mGluR5 protein(mGluR5/β-actin):0.44±0.14 vs.1.00±0.10,GluA2 protein(GluA2/β-actin):0.23±0.08 vs.1.00±0.25,both P<0.05].Compared with the I/R group,mNSS score and percentage of cerebral infarction volume in the Ozone group were decreased[mNSS score:7.00±1.20 vs.9.63±1.19,cerebral infarction volume:(27.23±6.21)%vs.(41.25±2.93)%,both P<0.05],and mGluR5 and GluA2 expressions in the penumbra of cerebral ischemia were up-regulated[mGluR5 protein(mGluR5/β-actin):0.81±0.10 vs.0.44±0.14,GluA2 protein(GluA2/β-actin):0.76±0.13 vs.0.23±0.08,both P<0.05].Conclusion Trioxygen preconditioning can alleviate cerebral I/R injury in rats,and its mechanism may be related to the upregulation of GluR5 and GluA2 in the ischemic penumbra.