七氟醚对SD大鼠海马生物钟基因表达的影响

The effect of sevoflurane on the expression of circadian genes in SD rat hippocampus

目的七氟醚麻醉会导致学习记忆损伤，但其分子机制并不清楚。前期研究中，笔者利用基因芯片对SD大鼠海马的基因表达谱进行了分析，表明七氟醚能够改变海马内417个基因的表达，其中生物钟基因也有明显差异改变。本研究进一步系统调查了七氟醚对SD大鼠生物钟基因表达的影响，以期为七氟醚致学习记忆损伤研究提供理论基础。方法1．5％～4．5％七氟醚麻醉SD大鼠4h，54只完全随机分为样本组和对照组（每组6只），样本和对照分别在麻醉苏醒期0、4h、2d和10d处死并分离海马组织，提取总RNA。然后，采用实时定量聚合酶链反应（real-time quantative polymerase chain reaction, RT-qPCR）方法进行生物钟基因定量检测和分析。结果表明被调查的6个生物钟基因包括时钟周期基因2（period2，Per2）、白蛋白启动子D位点结合蛋白（the D site albumin promoter binding protein, Dbp）、活性调节细胞骨架蛋白（activity-regulated cytoskeletal protein, Arc）、早期生长反应蛋白1（early growth response protein 1, Egrl）、早期生长应答基因2（early growth response2，Krox20）和神经生长因子诱导蛋白B（nerve growth factor induced protein I-B, NGFI-B）。其中上调基因有Krox20，下调基因有Arc，Dbp和Per2；只有Egr随着苏醒时间增加先上调后下调表达。相比之下，Krox20生物钟基因受七氟醚影响最大。从剂量效应和时间相来看，只有Egr和Per有明显的剂量依赖性，其他4个基因则无此关系。此外，6个生物钟基因均有明显时间依赖关系，随着麻醉后苏醒时间增加，其表达水平逐渐趋予正常。结论七氟醚对海马生物钟基因表达有较大影响，这种改变会持续较长时间（至少10d），这可能与七氟醚引起的学习记忆损伤有关。

Objective Recent studies have shown that general anesthesia impairs memory function. Increasing evidences suggest that sevoflurane impairs memory processes due to gene expression changes in hippocampus. In our previous study, we found that sevoflurane changed the expression of 417 kinds of genes by microarray. The purpose of the present research is to study the time- course and the dose-effect of sevofurane on the expression of 6 kinds of circadian-related genes in hippocampus of SD rats. Methods 54 six-weeks-old male SD rats were anesthetized for 4 h with 1.5%-4.5% sevoflurane （n=6） and then were sacrificed 48 h later. Hippocampus were obtained at 0, 4 h, 2 d and 10 d after awakening. RNAs were extracted from the hippocampus by Trizol Extraction Method RNAs were transcribed to cDNA using M-MLV. RT-qPCR was carried out by SYBR Green method. The data were analyzed using 12.0 SPSS software. Results The resuhs indicated that among six circadian genes （ie, Krox20, Per2, Dbp, Arc, NGFI and Egrl ）, Krox20 and NGFI were up-regulated, Arc, Dhp and Per2 were down-regulated, and only Egr was initially up- regulated and then down-regulated after awakening. Especially, Krox20 change reached 67.0 fold at 0 h awakening after anesthesia. Comparatively, 2.5% sevoflurane had the most significant influence on the expression of Arc,Dbp,Krox20 and NGFI-B. As a whole, only Egr and Per2 had dose-dependent relationship, the other four genes didn＇t show obvious dose-dependent relationship. However, all six genes showed significant time-course relationship. Their expression tended to return to the normal level on the 10th day after anesthesia. Conclusion Sevoflurane has an obvious effect on the expression of circadian genes in rat hippocampus. These long-term changes （at least 10 d） may be related to memory impairment or other neural disorders.