运动预适应介导氧化应激调控PI3K/Akt和Nrf2/HO-1通路对大鼠认知和学习障碍的影响

Effects of Exercise Preconditioning Mediating Oxidative Stress to Regulate PI3K/Akt and Nrf2/HO-1 Pathways to Alleviate Cognitive and Learning Disorders in Rats

目的:探究运动预适应调控PI3K/Akt、Nrf2/HO-1信号通路和氧化应激减轻低氧暴露大鼠认知、学习障碍的基本机制。方法:将60只6周龄SPF级雄性SD大鼠,按照随机数字表法分为对照组、运动组、暴露组和运动+暴露组,每组15只。对照组和暴露组不进行运动预适应干预,运动组与运动+暴露组接受运动预处理干预(跑台训练,2次/d,6 d/周,共训练4周);运动预适应结束次日,暴露组和运动+暴露组大鼠接受7 d低氧暴露。7 d低氧暴露后,采用旷场实验及Morris水迷宫实验评估大鼠认知、学习能力;采用酶联免疫吸附测定法(ELISA)检测大鼠血清SOD、GSH活性,MDA含量和IL-1β、IL-6、TNF-α水平;通过尼氏染色观察海马CA1区尼氏小体变化;RT-PCR检测PI3K、Nox1、Duox2、Akt mRNA相对表达水平;采用Western blot法检测海马组织Nox1、Duox2、GCLM蛋白及PI3K/Akt、Nrf2/HO-1通路蛋白相对表达量。结果:与对照组和运动组比较,暴露组3 d逃避潜伏期上升(P<0.05),穿越平台次数和平台所在象限停留时间明显降低(P<0.05);血清MDA含量,IL-1β、IL-6、TNF-α水平均明显上升(P<0.05),SOD、GSH活性明显下降(P<0.05);CA1区尼氏小体数量下降(P<0.05);PI3K、Nox1、Duox2 mRNA相对表达水平上升(P<0.05),同时Akt mRNA相对表达水平下降(P<0.05);海马组织Nox1、Duox2、GCLM蛋白相对表达量显著上升(P<0.05),p-PI3K/PI3K、p-Akt/Akt、p-GSK-3β/GSK-3β比值和Nrf2、HO-1蛋白相对表达量明显下调(P<0.05)。与暴露组比较,运动+暴露组中心区域活动时间、3 d逃避潜伏期降低(P<0.05),穿越平台次数和平台所在象限停留时间上升(P<0.05);血清MDA含量,IL-1β、IL-6、TNF-α水平均明显下降(P<0.05),SOD、GSH活性明显上升(P<0.05);CA1区神经元损伤减轻,尼氏小体数量上升;海马组织PI3K、Nox1、Duox2 mRNA相对表达水平下降(P<0.05),Akt mRNA相对表达水平上升(P<0.05);海马组织Nox1、Duox2、GCLM蛋白相对表达量明显降低(P<0.05),p-PI3K/PI3K、p-Akt/Akt、p-GSK-3β/GSK-3β比值和Nrf2、HO-1蛋白相对表达量明显上升(P<0.05)。结论:运动预适应可能通过激活PI3K/Akt和Nrf2/HO-1信号通路参与调节脑组织氧化应激并减轻损伤程度,进而改善低氧暴露大鼠神经行为功能。

Objective:To explore the basic mechanism of exercise preconditioning regulating PI3K/Akt and Nrf2/HO-1 signa-ling pathways and oxidative stress to alleviate cognitive and learning disabilities in rats with hypoxic exposure.Methods:A total of sixty 6-week-old SPF male SD rats were divided into control group,exercise group,exposure group and exercise+exposure group according to random number table method,with 15 rats in each group.The control and exposure groups received no exercise preconditioning intervention,while the exercise and exercise+exposure groups received exercise preconditioning intervention(treadmill training,twice a day,6 days a week,a total of 4 weeks of training).The day after exercise preconditioning,rats in the exposure and exercise+exposure groups received hypoxia exposure for 7 days.After 7 days of hypoxia exposure,the cognition and learning ability of rats were evaluated by open field test and Morris water maze test.SOD and GSH activities,MDA content and IL-1β,IL-6 and TNF-αlevels in serum of rats were detected by enzyme-linked immunosorbent assay(ELISA).The changes of Nissl corpuscles in CA1 region of hippocampus were observed by Nissl staining.mRNA expression levels of PI3K,Nox1,Duox2 and Akt were detected by RT-PCR.The relative expression levels of Nox1,Duox2,GCLM and PI3K/Akt and Nrf2/HO-1 pathway proteins in hippocampus were detected by Western blot.Results:Compared with the control group and the exercise group,the 3 d escape latency of the exposed group increased(P<0.05).The times of the platform crossings and the residence time in platform significantly decreased(P<0.05).Serum MDA content,IL-1β,IL-6 and TNF-αlevels significantly increased(P<0.05),while SOD and GSH activities signi-ficantly decreased(P<0.05).The number of Nissl bodies in CA1 region decreased(P<0.05).The mRNA expression levels of PI3K,Nox1 and Duox2 increased(P<0.05),while the mRNA expression level of Akt decreased(P<0.05).The relative expressions of Nox1,Duox2 and GCLM proteins in hippocampus significantly increased(P<0.05),while the ratios of p-PI3K/PI3K,p-Akt/Akt,p-GSK-3β/GSK-3βand the relative expressions of Nrf2 and HO-1 proteins significantly decreased(P<0.05).Compared with the exposure group,the movement time in the central area,the 3 d escape latency decreased(P<0.05),the number of platform crossings and the platform quadrant residence time increased(P<0.05)in the exercise+exposure group.Serum MDA content,IL-1β,IL-6 and TNF-αlevels significantly decreased(P<0.05),while SOD and GSH activities significantly increased(P<0.05).The damage of neurons in CA1 region was reduced and the number of Nissl corpuscles increased.The mRNA expression levels of PI3K,Nox1 and Duox2 in hippocampus tissues decreased(P<0.05),while the mRNA expression level of Akt increased(P<0.05).The relative expression levels of Nox1,Duox2 and GCLM proteins in hippocampus significantly decreased(P<0.05),while the ratio of p-PI3K/PI3K,p-Akt/Akt,p-GSK-3β/GSK-3βand the relative expression levels of Nrf2 and HO-1 proteins significantly increased(P<0.05).Conclusion:Exercise preconditioning may be involved in the regulation of oxidative stress in brain tissue by activating PI3K/Akt and Nrf2/HO-1 signaling pathways,and thereby improves the neurobehavioral functions of hypoxia exposed rats.