Genome-wide Transcriptome Analysis of Hippocampus, Frontal cortex and Pituitary gland in Rats Elucidated the Pathogenisis of Depressive Disorder Induced by Chronic Restraint

Objective: To elucidate the potential effects of stressful factors for depression on the hippocampal, frontal cortex and pituitary gland genome-wide transcriptomes at the molecular level, we evaluated the transcriptomic profiles of depression rats under chronic restraint stress (CRS). Methods: Forty-eight rats were randomly divided into control, model, fluoxetine and acupuncture groups. Experimental period was 28 days. Open-field test, Sucrose preference and body weight were investigated respectively at the experiment before and after the experiment. The experiment having been finished, solexa sequencing technology (Illumina Nextseq 500/151nt) was applied to investigate and verify the altered hippocampal, frontal cortex and pituitary gland genome-wide transcriptome analysis in rats of all groups. Results: Based on the data from RNA-seq analysis, it revealed that compared the expression of genes in the control group with model group in the respective of three brain tissues, 101, 134, 148 differential expression genes were found in the hippocampus, frontal cortex and pituitary gland, respectively; compared the expression of genes in the fluoxetine group with model group in the respective of three brain tissues, 41, 46, 87 differential expression genes were found in the hippocampus, frontal cortex and pituitary gland, respectively; compared the expression of genes in the acupuncture group with model group in the respective of three brain tissues, 107, 89, 179 differential expression genes were found in the hippocampus, frontal cortex and pituitary gland, respectively. Furthermore, we used the GO (Gene ontology) analysis and KEGG (Kyo-To conservation of Genes and Genomes) pathway analysis for these differentially expressed genes. We found that the results of these two analyses were consistent, both mainly related to monoamine neurotransmitters, inflammation and immune response in control group VS model group. It is noted that this phenomenon also appears in fluoxetine group compared with model group, acupuncture group VS model group. Importantly, the antidepressant effect of acupuncture was more extensive, which was more consistent with the pathogenesis of depression induced by CRS in rats. Conclusions: Our study represents the first detailed analysis of the hippocampal, frontal cortex and pituitary gland genome-wide transcriptomes in depression rats under CRS by RNA-seq technology. The results of this study revealed multiple DEGs and possible mechanisms specifying the function of hippocampal, frontal cortex and pituitary gland in depression rats induced by CRS. These results provide a basis for further investigation of the signaling mechanisms that affect central nervous system output related to stress-sensitive depressive disorder development. Forty-eight rats were randomly divided into control, model, fluoxetine and acupuncture groups. The rat model of depression was established by subjecting to chronic restraint stress (6h/day) for 28 days. Rats in acupuncture group were acupunctured at GV20 (Baihui) and GV29 (Yintang) one hour before the CRS procedures every day. Rats in fluoxetine group were administered with fluoxetine (10mg/kg) by gavage one hour before the CRS procedures every day. Open-field test, Sucrose preference and body weight were investigated respectively at the experiment before and after the experiment. The experiment having been finished, the hippocampal, frontal cortex and pituitary gland were harvested, and their RNA profiles were generated by deep sequencing, in triplicate, using Illumina Nextseq 500/151nt.研究目的：为从分子层面阐明抑郁相关应激因素对海马体、前额叶皮层及垂体全基因组转录组的潜在影响，本研究针对慢性束缚应激（chronic restraint stress, CRS）模型抑郁大鼠的转录组特征展开分析。 研究方法：将48只大鼠随机分为对照组、模型组、氟西汀组与针刺组，实验周期为28天。分别于实验前后开展旷场实验、蔗糖偏好实验与体重检测。实验结束后，采用Solexa测序技术（Illumina Nextseq 500/151nt）对各组大鼠海马体、前额叶皮层及垂体的全基因组转录组差异进行分析与验证。 研究结果：基于RNA测序（RNA-seq）数据分析结果显示：以三组脑组织分别进行组间比较，对照组与模型组相比，海马体、前额叶皮层及垂体中分别筛选出101、134、148个差异表达基因（differentially expressed genes, DEGs）；氟西汀组与模型组相比，上述脑组织中分别筛选出41、46、87个DEGs；针刺组与模型组相比，上述脑组织中分别筛选出107、89、179个DEGs。随后，对上述差异表达基因开展基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析。结果显示，对照组与模型组的富集分析结果主要集中于单胺类神经递质、炎症及免疫反应相关通路；氟西汀组与模型组、针刺组与模型组的比较亦呈现相似结果。值得注意的是，针刺的抗抑郁作用更为广泛，其调控通路与慢性束缚应激诱导的大鼠抑郁发病机制更为契合。 研究结论：本研究首次采用RNA测序技术，对慢性束缚应激模型抑郁大鼠的海马体、前额叶皮层及垂体全基因组转录组开展系统性详细分析。本研究筛选出多个差异表达基因，并阐明了慢性束缚应激诱导抑郁大鼠海马体、前额叶皮层及垂体功能异常的潜在分子机制。本研究结果为进一步探究影响应激敏感性抑郁发生发展的中枢神经系统信号通路机制提供了理论依据。 补充实验细节：将48只大鼠随机分为对照组、模型组、氟西汀组与针刺组；通过每日6小时慢性束缚应激连续28天构建大鼠抑郁模型。针刺组大鼠于每日束缚应激前1小时，于GV20（百会穴）与GV29（印堂穴）进行针刺干预；氟西汀组大鼠于每日束缚应激前1小时，以10mg/kg剂量灌胃给予氟西汀。分别于实验前后开展旷场实验、蔗糖偏好实验与体重检测。实验结束后，采集各组大鼠的海马体、前额叶皮层及垂体组织，采用Illumina Nextseq 500/151nt平台进行三次重复深度测序，获取其转录组图谱。