长期负性情绪应激对D-gal大鼠认知功能及海马NR2B基因甲基化表达的影响

Effects of Long-term Negative Emotional Stress on Cognitive Function and Methylation of NR2B Gene in Hippocampus of D-gal Rats

目的探讨长期负性情绪应激对大鼠脑老化进程的影响机制。方法将雌雄各半的70只Wistar大鼠分别采用随机方法,分为空白对照组、D-gal脑老化模型组、应激脑老化组,每组14只且雌雄各半,其余大鼠做为攻击鼠参与负性情绪应激的诱导;采用颈后部皮下多点注射D-gal 0.1g/(kg·d)制备脑老化大鼠模型;采用不可预知性刺激,诱导应激脑老化组大鼠长期负性情绪应激;采用Morri′s水迷宫实验记录大鼠水下寻台时间(SPL),评价大鼠学习记忆功能;采用ELISA测定大鼠血浆促肾上腺皮质激素(ACTH)和皮质酮(CORT)含量;采集大鼠海马样本制备匀浆,采用甲基化特异性PCR法(MSP)检测海马N-甲基-D-天门冬氨酸受体-2B亚型(NR2B)基因调控区胞嘧啶-磷酸-鸟嘌呤位点(CpG)甲基化程度,采用ELISA法测定甲基化转移酶1(DNMT1)活性。结果与D-gal脑老化模型组比较,应激脑老化组大鼠的SPL明显延长,NR2B基因启动子区CpG甲基化程度及DNA DNMT1活性显著增高(P<0.05),血浆CORT、ACTH水平显著升高(P<0.05)。结论长期情绪调节不良能够加速机体大脑老化进程,其机制可能与慢性情绪应激引发机体特定基因甲基化程度增高有关。

Objective To study the mechanism of long-term negative emotional stress on the brain aging process in rats. Methods Totally 70 Wistar rats, half male and half female, were randomly divided into a blank control group, D-gal brain aging model group, and stress-induced brain aging group, 14 in half male and half female each group. As offense side, the remaining rats were involved in the induction of negative emotional stress. The D-gal brain aging model was prepared by subcutaneous injection of D-gal 0.1 g/(kg·d) into the posterior neck of a rat. Unpredictable stimulation was used to induce long-term negative emotional stress of rats in the stress-induced brain aging group. Morri′s water maze test was used to record the rats′ searching platform latency(SPL) and evaluate their learning and memory function. The contents of adrenocorticotropic hormone(ACTH) and corticosterone(CORT) in rat plasma were detected by ELISA. Rat hippocampal samples were collected to prepare homogenate, and methylation-specific PCR(MSP) was used to detect the degree of methylation of cytosine-phosphoric acid-guanin 2(CpG) in the region of NR2B gene regulation. The activity of DNA methyltransferase 1(DNMT1) was detected by ELISA. Results Compared with the D-gal brain aging model group, the SPL of was significantly prolonged, the degree of CpG methylation and DNA DNMT1 activity in the hippocampal NR2B gene regulation region were significantly elevated, and plasma CORT and ACTH levels significantly increased in the stress-induced brain aging group(P<0.05). Conclusions Long-term emotional maladjustment accelerated the brain aging process. And its mechanism might be related to increased methylation of specific genes in the body caused by chronic emotional stress.