奥氮平诱导体重增加小鼠的全脑转录组学分析

doi:10.12300/j.issn.1674-5817.2023.006

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (3): 262-270.DOI: 10.12300/j.issn.1674-5817.2023.006

奥氮平诱导体重增加小鼠的全脑转录组学分析

张渊¹()(), 李晗²()(), 张成芳¹()()

^1.同济大学附属精神卫生中心(上海市浦东新区精神卫生中心), 同济大学精神疾病临床研究中心, 上海 200124
^2.上海交通大学医学院附属精神卫生中心, 上海市重性精神病重点实验室, 上海 201108

收稿日期:2023-02-01 修回日期:2023-04-11 出版日期:2023-06-25 发布日期:2023-07-18
通讯作者: 张成芳（1987—），女，博士，主治医师，研究方向：抗精神病药物代谢不良反应的机制和临床防治。E-mail：callup1987@126.com。ORCID： 0000-0002-5977-0312
作者简介:张渊（1988—），女，硕士，主治医师，研究方向：抗精神病药物相关动物模型。E-mail：450718469@qq.com。ORCID： 0000-0001-6662-4738；
李晗（1984—），男，硕士，助理研究员，研究方向：精神疾病模型。E-mail：erjunda140@126.com。ORCID: 0000-0003-3903-5254
基金资助:
上海市浦东新区优秀青年医学人才培养计划“基于全脑转录组测序筛选奥氮平调控小鼠中枢摄食网络的关键基因”(PWRq2020-19);上海市浦东新区精神卫生中心院级科研面上项目“稳定期精神分裂症患者外周血氧化应激与认知功能相关性分析”(PDJWM-202002)

Whole-brain Transcriptomic Analysis of Weight Gain Mice induced by Olanzapine

Yuan ZHANG¹()(), Han LI²()(), Chengfang ZHANG¹()()

^1.Clinical Research Center for Mental Disorders, Shanghai Pudong New Area Mental Health Center, School of Medicine, Tongji University, Shanghai 200124, China
^2.Shanghai Mental Health Center, Shanghai Jiao Tong University of Medicine, Shanghai Key Laboratory of Psychotic Disorders, Shanghai 201108, China

Received:2023-02-01 Revised:2023-04-11 Published:2023-06-25 Online:2023-07-18
Contact: ZHANG Chengfang (ORCID: 0000-0002-5977-0312), E-mail: callup1987@126.com

摘要/Abstract

摘要：

目的分析奥氮平给药组与对照组的小鼠脑组织的转录组测序结果，筛选出差异表达基因，探索非典型抗精神病药奥氮平导致体重增加的潜在作用靶点。方法 20只雌性C57BL/6小鼠被随机分为对照组和奥氮平给药组，分别给予生理盐水和奥氮平溶液灌胃，8周后留取全脑组织进行转录组测序（transcriptome sequencing，RNA-Seq）。通过对测序结果进行基因本体论（gene ontology，GO）功能注释分析和基因组百科全书（Kyoto encyclopedia of genes and gnomes，KEGG）途径富集分析以及蛋白互作（protein-protein interaction，PPI）网络分析等，寻找奥氮平诱导体重增加的可能靶点，并通过实时荧光定量PCR法进行验证。结果对照组和奥氮平给药组的差异表达基因共591个，其中上调基因251个，下调基因340个。GO分析显示差异基因广泛参与转录过程，其中消化系统的调节和冷诱导的产热相关基因表达属于显著富集项目。KEGG分析显示差异基因广泛参与神经活性物质与受体之间的相互作用，差异基因显著富集在催产素信号、脂肪的消化吸收以及胆固醇代谢通路。实时荧光定量PCR结果表明，富集在摄食调控、胃运动、产热、脂肪代谢等过程的基因（Oxt、Trpv1、Adipoq、Phox2b、Abcg5、Mogat2、Dbh、Plac8、Neurog1）以及PPI网络中的枢纽基因（Fos、Dusp1、Egr2）的表达改变与RNA-Seq趋势一致。结论奥氮平给药导致小鼠中枢的摄食调控、胃肠运动、产热等生理过程发生改变，这些改变可能参与了奥氮平诱导的体重增加。

关键词: 奥氮平, 体重增加, 全脑, 转录组测序, 小鼠

Abstract:

Objective The transcriptome sequencing results of brain tissues of olanzapine-treated mice were analyzed to screen out differentially-expressed genes and explore potential targets of atypical antipsychotics leading to body weight gain. Methods Twenty female C57BL/6 mice were randomly divided into control group (Ctrl) and Olanzapine administration group (Olz), which were given saline and Olanzapine solution by gavage, respectively. The whole brain tissues were collected 8 weeks later for Transcriptome sequencing (RNA-Seq). The possible targets of olanzapine-induced body weight gain were identified by the Gene Ontology (GO) functional annotation analysis, the Kyoto Encyclopedia of Genes and Gnomes (KEGG) pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. Differential expression levels of mRNAs were further verified by real-time quantitative fluorescence PCR (RT-qPCR). Results Compared with Ctrl group, 591 differentially expressed genes were screened in Olz group, including 251 up-regulated genes and 340 down-regulated genes. GO analysis showed that differential genes were widely involved in transcriptional process, among which the expression of genes related to the regulation of digestive system and cold-induced thermogenesis were significantly enriched. KEGG analysis showed that differential genes were widely involved in the interaction between neuroactive ligands and receptors, and the differential genes were significantly enriched in oxytocin signaling, fat digestion and absorption, and cholesterol metabolism pathways. RT-qPCR were performed to verify the expression levels of genes enriched in feeding regulation, gastric kinesis, thermogenesis, fat metabolism and other processes (Oxt, Trpv1, Adipoq, Phox2b, Abcg5, Mogat2, Dbh, Plac8 and Neurog1) as well as hub genes in PPI network (Fos, Dusp1 and Egr2), and the results were consistent with the trend of RNA-Seq. Conclusion Olanzapine administration resulted in changes in central feeding regulation, gastrointestinal motility, thermogenesis and other physiological processes in mice, which might be involved in body weight gain induced by olanzapine.

Key words: Olanzapine, Body weight gain, Whole brain, RNA-Seq, Mice

中图分类号:

R-332

张渊, 李晗, 张成芳. 奥氮平诱导体重增加小鼠的全脑转录组学分析[J]. 实验动物与比较医学, 2023, 43(3): 262-270.

Yuan ZHANG, Han LI, Chengfang ZHANG. Whole-brain Transcriptomic Analysis of Weight Gain Mice induced by Olanzapine[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 262-270.

图/表 6

表1 差异基因的实时荧光定量PCR引物序列

Table 1 Primer sequences of differential genes used in real-time quantitative fluorescence PCR

基因名称	引物序列（5'→3'）
β-Actin	F: GTGCTATGTTGCTCTAGACTTCG
	R: ATGCCACAGGATTCCATACC
Oxt	F: TTGACCAGATGAACGGAGTG
Oxt	R: AGCTACTCGGATACGGGAGA
Trpv1	F: GGGCGAGACTGTCAACAAGA
Trpv1	R: CGGCTCTATTGCTCCCTGAG
Adipoq	F: TGCCCTGTAACTTCTACCCCA
Adipoq	R: GGCAAGTGTCCTCAACTGTGTC
Phox2b	F: TACGCCGCAGTTCCATACAAACTC
Phox2b	R: TCTTTGAGCTGCGCGCTTGTGAAG
Abcg5	F: GTTCCAAGACTGCTTCTC
Abcg5	R: ATGACTGCCTCTACCTTC
Mogat2	F: CCAGGTTGAGAACACCCCTG
Mogat2	R: CTCGATGGGCTTCCCCACTAT
Dbh	F: AATCTGCAGCCTTTGCCTAA
Dbh	R: TTCAGCATCTGCCTCTGTTG
Plac8	F: TGGACTACAAAGACGATGACGA
Plac8	R: ACAAAAGGTCCCACAGAGGC
Neurog1	F: CGATCCCCTTTTCTCCTTTC
Neurog1	R: TGCAGCAACCTAACAAGTGG
Fos	F: TTTCAACGCGGACTACGAGG
Fos	R: GCGCAAAAGTCCTGTGTGTT
Dusp1	F: TGTGAAGCAGAGGAGGAGC
Dusp1	R: ACGCACGGCATGTTGGTC
Egr2	F: TTGACCAGATGAACGGAGTG
Egr2	R: AGCTACTCGGATACGGGAGA

图1 奥氮平给药引起的体重和摄食量变化趋势注：A，奥氮平给药引起的小鼠体重变化；B，奥氮平给药引起的小鼠摄食量变化。每组n=10。Ctrl，对照组（生理盐水灌胃）；Olz，奥氮平组（奥氮平灌胃）。与对照组相比，*P<0.05，**P<0.01。

Figure 1 Changes of body weight and food intake induced by olanzapine administrationNote：A, Changes in body weight induced by olanzapine administration; B, Changes in food intake induced by olanzapine administration. n=10 in each group. Ctrl, control group (intragastric injection with saline); Olz, olanzapine group (intragastric injection with olanzapine). Compared with control group, *P<0.05，**P<0.01.

图2 奥氮平给药后差异表达基因的转录组学分析（火山图和GO分析）注：A，差异表达基因分布的火山图（X、Y坐标轴均取筛选条件值的对数形式。图中每个点代表1个差异表达基因，橙色为上调基因，蓝色为下调基因，灰色为无明显变化的基因）。B，差异表达基因的GO富集分析结果（差异表达基因产物可能行使的分子功能、所处的细胞环境以及参与的生物学过程）。C，差异表达基因GO网络分析。

Figure 2 Transcriptomic analysis of differentially expressed genes after olanzapine administration （Volcano map and GO analysis）Note：A， Volcano map of differentially-expressed genes （logarithmic form of screening condition values were taken for the X and Y axes. Each point in the figure represents a differentially expressed gene. Orange， blue and gray indicate the up-regulated， down-regulated and unchanged genes， respectively）. B， GO analysis of differentially expressed genes （differentially-expressed genes-involved molecular functions， cellular environments and biological processes）. C， GO-net analysis of differentially expressed genes.

图3 奥氮平给药后差异表达基因的转录组学分析（KEGG分析和PPI网络分析）注：A，差异表达基因KEGG富集分析（不同的颜色代表了不同的通路群组或网络聚类）。B，蛋白互作（PPI）网络分析（各连接点标注为蛋白名称，蓝色为下调基因，红色为上调基因。圆点越大表示连接数越多，黑线代表两者间存在调节作用）。

Figure 3 Transcriptomic analysis of differentially expressed genes after olanzapine administration （KEEG analysis and PPI network analysis）Note：A， KEEG analysis of differentially expressed genes (different colors represent different path groups or network clusters). B, protein-protein interaction (PPI) network analysis (each connection point is labeled with the name of the proteins. Blue indicates the down-regulated genes, and red shows the up-regulated gene. The larger the dot, the more connections there are. The black line represents the regulatory effect between the two genes).

图4 奥氮平灌胃组与对照组小鼠全脑组织中差异基因的实时荧光定量PCR验证结果注：Ctrl，对照组（生理盐水灌胃）；Olz，奥氮平组（奥氮平灌胃）。每组n=10，与对照组相比，*P<0.05，**P<0.01。

Figure 4 Real-time quantitative fluorescence RCR verification results of differentially expressed genes in the whole brain tissues of mice in the olanzapine infusion group and control groupNote：Ctrl， control group （intragastric injection with saline）； Olz， olanzapine group （intragastric injection with olanzapine）. n= 10 in each group. Compared with the control group， *P<0.05， **P<0.01.

表2 奥氮平引起差异基因改变及可能产生的代谢效应

Table 2 Differential genes changes inced by olanzapine and possible metabolic effects

基因	奥氮平给药的影响	可能的效应
Oxt	↓	胃肠道厌食信号减少，享乐性进食增加，产热减少
Trpv1	↓	胃肠道厌食信号减少，享乐性进食增加，产热减少
Adipoq	↓	脂类合成增加、降解减少
Phox2b	↓	产热减少
Abcg5	↓	脂类吸收增加
Mogat2	↓	脂肪吸收的延迟
Dbh	↓	产热减少
Plac8	↑	产热增加
Neurog1	↑	未知

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奥氮平诱导体重增加小鼠的全脑转录组学分析

Whole-brain Transcriptomic Analysis of Weight Gain Mice induced by Olanzapine

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