非诺贝特干预超氧化物歧化酶2转基因C57BL/6J小鼠的神经保护机制

Neuroprotective mechanism by which fenofibrate regulates superoxide dismutase 2 expression in transgenic C57BL/6J mice

背景:氧化损伤被认为是脑缺血再灌注损伤的重要因素之一,超氧化物歧化酶2是关键的线粒体抗氧化分子,非诺贝特可通过激活的PPARα调节超氧化物歧化酶2的表达。目的:验证非诺贝特治疗脑缺血再灌注损伤的机制是依赖超氧化物歧化酶2的表达。方法:用TALENs系统构建超氧化物歧化酶2转基因C57BL/6J小鼠,通过PCR和DNA测序技术鉴定转基因小鼠进行基因分型,免疫印迹法检测超氧化物歧化酶2蛋白在转基因小鼠体内表达情况。将野生型和超氧化物歧化酶2转基因小鼠随机分为4组,野生型对照组(6只)、野生型非诺贝特组(6只)、转基因对照组(超氧化物歧化酶2转基因型)(5只)、转基因非诺贝特组(5只)。采用线栓法制备大脑中动脉栓塞小鼠模型,90 min后拔出栓线,使脑血流再灌注30 min。使用脑血流监测仪监测局部脑血流;取脑组织切片,使用TTC染色分析各组脑梗死情况。结果与结论:①经PCR和DNA测序分析,成功构建超氧化物歧化酶2^(+/+)转基因小鼠9只。②缺血再灌注后,野生型非诺贝特组较野生型对照组的脑血流部分恢复、脑梗死体积明显缩小(P<0.001);转基因非诺贝特组与转基因对照组在脑血流与脑梗死体积方面无显著差异;转基因对照组在脑血流及脑梗死改善方面优于野生型对照组(P<0.001);野生型非诺贝特组与转基因对照组和转基因非诺贝特组在脑血流、脑梗死体积上均无显著差异。③结果说明,超氧化物歧化酶2的表达是非诺贝特治疗脑缺血再灌注损伤的机制之一。

BACKGROUND:Oxidative injury is considered to be one of the important factors of cerebral ischemia-reperfusion injury.Superoxide dismutase 2(SOD2)is a key mitochondrial antioxidant molecule,and fenofibrate can regulate the expression of SOD2 by activating peroxisome proliferator-activated receptorα.OBJECTIVE:To explore whether the mechanism of fenofibrate in the treatment of cerebral ischemia-reperfusion injury depends on the expression of SOD2.METHODS:The TALENs system was used to construct SOD2 transgenic mice.The transgenic mice were genotyped by PCR and DNA sequencing techniques.The expression of SOD2 protein in transgenic mice was detected by western blot assay.Wild-type and SOD2 transgenic mice were randomly divided into four groups:wild-type control group(n=6),wild-type fenofibrate group(n=6),SOD2 transgenic control group(n=5)and SOD2 transgenic fenofibrate group(n=5).A mouse model of middle cerebral artery occlusion was prepared using the suture-occlusion method.After 90 minutes of ischemia,the thread was removed to reperfuse cerebral blood flow for 30 minutes.A cerebral blood flow monitor was used to monitor local cerebral blood flow.Brain tissue slices were taken for 2,3,5-triphenyltetrazolium chloride staining to analyze the situation of cerebral infarction in each group.RESULTS AND CONCLUSION:After PCR and DNA sequencing analysis,nine SOD2+/+transgenic mice were successfully constructed.After cerebral ischemia-reperfusion,the wild-type fenofibrate group showed partial recovery of cerebral blood flow and significantly reduced cerebral infarction volume compared with the wild-type control group(P<0.001).There was no significant difference in cerebral blood flow and cerebral infarction volume between the SOD2 transgenic fenofibrate group and the SOD2 transgenic control group.The SOD2 transgenic control was superior to the wild-type control group in terms of improving cerebral blood flow and cerebral infarction(P<0.001).There were also no significant differences in cerebral blood flow and cerebral infarction volume between the wild-type fenofibrate group and the SOD2 transgenic control group and between the wild-type fenofibrate group and the SOD2 transgenic fenofibrate group.To conclude,the expression of SOD2 is one of the mechanisms of fenofibrate in the treatment of cerebral ischemia-reperfusion injury.