目的:基于网络药理学方法探究乳酸改善焦虑的主要靶点及信号通路,结合行为学初步研究其潜在作用机制方法:通过Swiss Target Prediction、Target Net、Sea数据库获得乳酸作用靶点;通过Gene Cards、OMIM、CTD数据库获得焦虑靶点;通过Gene Cards和OMIM建立乳酸和焦虑症的靶基因集用韦恩图显示;通过STRING数据库构建乳酸与焦虑蛋白互作PPI网络图,利用DAVID数据库进行GO功能富集与KEGG通路富集分析。基于网络药理学预测的通路结果,将雄性ICR小鼠随机成溶媒组、乳酸组,每组10只,进行旷场实验,在实验前1 h腹腔注射乳酸或溶媒,记录在旷场实验中进入中间区域的时间和活动度并计算辨别指数。结果:通过Swiss Target Prediction、Target Net、Sea数据库共获得乳酸作用靶点248个;通过Gene Cards、OMIM、CTD数据库共获得焦虑靶点717个;靶点经与Gene Cards和OMIM综合数据库比对,共获取28个乳酸与焦虑的交集基因。结论:乳酸主要通过调控MAO-A信号通路进一步调节5-HT代谢,以及调控苯丙氨酸和酪氨酸代谢与cAMP信号通路及相应的基因表达进一步调节儿茶酚胺类神经递质代谢发挥抗焦虑作用。 Objective: To explore the main targets and signal pathways of lactic acid for improving anxiety based on the method of network pharmacology, and to study its potential mechanism of action in combination with behavioral studies. Methods: Obtain lactic acid targets through Swiss Target Prediction, Target Net, and Sea databases; obtain anxiety targets through Gene Cards, OMIM, and CTD databases; establish lactic acid and anxiety target gene sets through Gene Cards and OMIM; display with Venn diagram; construct lactic acid through STRING database. It interacts with anxiety protein as a PPI network diagram, and uses DAVID database for GO function enrichment and KEGG pathway enrichment analysis. Based on the results of network pharmacology, Male ICR mice were randomly divided into a vehicle group and a lactic acid group, each with 10 mice, and an open field experiment was performed. Lactic acid or vehicle was injected into the abdominal cavity 1 hour before the experiment and the time and activity of entering the middle area in the open field experiment were recorded and the discrimination was calculated. Index. Results: A total of 248 lactic acid targets were obtained through Swiss Target Prediction, Target Net, and Sea databases; a total of 717 anxiety targets were obtained through Gene Cards, OMIM, and CTD databases; a total of 28 lactic acid targets were obtained by comparison with Gene Cards and OMIM comprehensive databases. The intersection gene with anxiety. Conclusion: Lactic acid mainly regulates 5-HT metabolism by regulating the MAO-A signaling pathway, as well as regulating the metabolism of phenylalanine and tyrosine, the cAMP signaling pathway and the corresponding gene expression to further regulate the metabolism of catecholamine neurotransmitters to exert anti-anxiety effects.
目的:基于网络药理学方法探究乳酸改善焦虑的主要靶点及信号通路,结合行为学初步研究其潜在作用机制方法:通过Swiss Target Prediction、Target Net、Sea数据库获得乳酸作用靶点;通过Gene Cards、OMIM、CTD数据库获得焦虑靶点;通过Gene Cards和OMIM建立乳酸和焦虑症的靶基因集用韦恩图显示;通过STRING数据库构建乳酸与焦虑蛋白互作PPI网络图,利用DAVID数据库进行GO功能富集与KEGG通路富集分析。基于网络药理学预测的通路结果,将雄性ICR小鼠随机成溶媒组、乳酸组,每组10只,进行旷场实验,在实验前1 h腹腔注射乳酸或溶媒,记录在旷场实验中进入中间区域的时间和活动度并计算辨别指数。结果:通过Swiss Target Prediction、Target Net、Sea数据库共获得乳酸作用靶点248个;通过Gene Cards、OMIM、CTD数据库共获得焦虑靶点717个;靶点经与Gene Cards和OMIM综合数据库比对,共获取28个乳酸与焦虑的交集基因。结论:乳酸主要通过调控MAO-A信号通路进一步调节5-HT代谢,以及调控苯丙氨酸和酪氨酸代谢与cAMP信号通路及相应的基因表达进一步调节儿茶酚胺类神经递质代谢发挥抗焦虑作用。
乳酸,焦虑,网络药理学,旷场实验
Jinlong Zhao*, Zixuan Deng, Huanyu Cai, Puyuan Chen, Zhiming He#
Basic Medical School Shaoyang University, Shaoyang Hunan
Received: Sep. 26th, 2021; accepted: Oct. 26th, 2021; published: Nov. 3rd, 2021
Objective: To explore the main targets and signal pathways of lactic acid for improving anxiety based on the method of network pharmacology, and to study its potential mechanism of action in combination with behavioral studies. Methods: Obtain lactic acid targets through Swiss Target Prediction, Target Net, and Sea databases; obtain anxiety targets through Gene Cards, OMIM, and CTD databases; establish lactic acid and anxiety target gene sets through Gene Cards and OMIM; display with Venn diagram; construct lactic acid through STRING database. It interacts with anxiety protein as a PPI network diagram, and uses DAVID database for GO function enrichment and KEGG pathway enrichment analysis. Based on the results of network pharmacology, Male ICR mice were randomly divided into a vehicle group and a lactic acid group, each with 10 mice, and an open field experiment was performed. Lactic acid or vehicle was injected into the abdominal cavity 1 hour before the experiment and the time and activity of entering the middle area in the open field experiment were recorded and the discrimination was calculated. Index. Results: A total of 248 lactic acid targets were obtained through Swiss Target Prediction, Target Net, and Sea databases; a total of 717 anxiety targets were obtained through Gene Cards, OMIM, and CTD databases; a total of 28 lactic acid targets were obtained by comparison with Gene Cards and OMIM comprehensive databases. The intersection gene with anxiety. Conclusion: Lactic acid mainly regulates 5-HT metabolism by regulating the MAO-A signaling pathway, as well as regulating the metabolism of phenylalanine and tyrosine, the cAMP signaling pathway and the corresponding gene expression to further regulate the metabolism of catecholamine neurotransmitters to exert anti-anxiety effects.
Keywords:Lactic Acid, Anxiety Disorder, Network Pharmacology, Open Field Experiments
Copyright © 2021 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
焦虑症(anxiety disorder)是一种精神性疾病,随着社会的发展,人类生活节奏加快,焦虑症的发病率逐年升高,焦虑症的临床表现主要为无明确客观事物的紧张、恐惧、不安等,伴有自主神经紊乱、肌肉紧张、运动不安等症状 [
乳酸(lactic acid)是哺乳动物体内最常见的代谢物之一 [
通过采集化合物作用靶点,探究化合物-靶点相互作用关系从而研究药物的潜在作用机制。首先登录Pub Chem Compound (网址:http://en.chembase.cn/company-17.html)数据库 [
Gene Cards (网址:https://www.genecards.org/)、OMIM (网址:https://www.omim.org/)等数据库 [
登录Venny 2.1.0网站,将下载好的药物靶点与疾病靶点基因导入得到相应的交集基因,将所得的Venny图与交集基因保存用于后续分析工作。
STRING (网址:https://string-db.org/)数据库 [
生物学信息注释数据库(David网址:https://david.ncifcrf.gov/) [
雄性ICR小鼠(7周龄)购于中国湖南长沙的湖南斯莱克景达实验动物有限公司。小鼠饲养在恒温23℃ ± 2℃的笼子中(每个笼子1只小鼠),并在12小时的明暗交替环境中自由进食和饮水。实验开始前一周,实验者每天对小鼠进行抚摸(5 min/只) [
旷场实验可以评估小鼠运动活动和焦虑样行为,旷场实验装置由带有暗光(25 lx)和通风扇(30 dB)的木质隔音箱(50 cm × 50 cm × 70 cm)和置于隔音箱中心上方的数码相机组成,隔音箱覆盖黑色薄膜,用白色标记笔画出中间区域(30 cm × 30 cm)。实验时将小鼠放置于箱子中自由探索10分钟然后放回鼠笼,每次实验后,喷洒75%的酒精用抹布擦拭装置去除遗留的气味,用ANYmaze统计进入中间区域的时间以及总活动度。
将乳酸钠(厂商)溶解在生理盐水中,该生理盐水也用作溶媒。本研究中使用的乳酸(1 g/kg)的剂量是根据以前的研究选择的。每只小鼠以10 ml/kg的体积腹腔给药。
统计分析均使用Sigma Stat 3.1完成,实验数据均采用单向方差分析(one-way ANOVA)和非配对的t检验,所有数据均表示为平均值 ± 标准误。
通过Swiss Target Prediction、Target Net数据库获得乳酸作用靶点共有248个,通过Gene Cards、OMIM数据库获得焦虑靶点共有717个,通过与Gene Cards和OMIM数据库中乳酸与焦虑症相关的基因进行比对,得到28个可能与乳酸改善焦虑相关的作用靶点。登录Venny 2.1.0网站,将采集的药物靶点与疾病靶点基因导入得到相应的交集基因,导出Venny图(图1)。
图1. 乳酸与焦虑交集基因的韦恩图
将乳酸与焦虑症交集靶点基因名导入STRING数据库,物种限定为人类,分析蛋白相互作用关系,得到乳酸与焦虑症交集基因靶点的蛋白互作网络图(图2(A)),该网络图含有28个节点和79条边线,不同节点分别代表不同的靶点蛋白,不同颜色的连线表示各节点之间的相互作用关系,将结果导入Cytoscape软件绘制乳酸与焦虑症交集基因核心靶点蛋白相互作用网络图(图2(B))以及核心靶点(表1,Degree > 7),节点大小,字号大小与Degree呈正相关。
图2. 乳酸与焦虑交集基因靶点蛋白相互作用关系图
Number | Degree | Name |
---|---|---|
1 | 12 | APP |
2 | 12 | MAOB |
3 | 11 | ACHE |
4 | 10 | MAOA |
5 | 10 | SLC6A3 |
6 | 9 | DRD1 |
7 | 8 | DRD5 |
8 | 8 | ESR1 |
9 | 7 | MAPT |
10 | 7 | SIRT1 |
表1. 乳酸与焦虑交集基因核心靶点
利用David数据库进行GO功能富集分析,对乳酸改善焦虑症涉及的28交集基因的功能作用进行了分析(图3)。主要涉及多巴胺能传递、神经系统过程、突触传递、代谢过程,信号调节等方面;分子功能结果主要包括配体激活的转录因子活性,核受体活性,类固醇羟化酶活性,氧化还原酶活性,单加氧酶活性,神经递质受体活性,血红素结合等,细胞组成主要结果有突触膜区、膜微区、膜筏,神经元棘突、树突棘等方面。
图3. 乳酸与焦虑交集基因GO富集
利用DAVID数据库对乳酸与焦虑症的交集基因进KEGG通路富集分析(图4),共获得条富集结果共20条,其中主要包括环磷腺苷信号通路,钙信号通路等信号传导相关通路,涉及成瘾,帕金森综合症,神经退行性疾病通路,内分泌抵抗等生物系统相关通路,以及药物代谢–细胞色素p450,色氨酸、精氨酸、脯氨酸、苯丙氨酸、组氨酸、亚油酸、酪氨酸代谢。此外还包括多巴胺能突触,血清素突触。
如图所示,各组之间的总活动度没有显著性(P > 0.05;图5(A))。尿刊酸组进入中间区域的总时间(P > 0.05;图5(B))有显著性差异,上述结果表明,乳酸改善了旷场实验中小鼠的焦虑症状。
通过网络药理学,本研究对乳酸改善焦虑的主要靶点及信号通路进行了预测,本研究结果显示,乳酸与焦虑症交集靶点基因共有28个,通过STRING数据绘制蛋白互作网络图,直观地展现了乳酸与焦虑的多重机制与相互关联。对酸与焦虑症交集靶点基因进行生物信息富集分析的结果显示,乳酸改善焦虑可能与巴胺能传递、神经系统过程、突触传递、代谢过程,信号调节,受体及酶活性等方面密切相关。
图4. 乳酸与焦虑交集基因KEGG富集
图5. 在旷场实验中测试前1 h注射乳酸可以改善焦虑。(A) 各组小鼠旷场测试中进入中间区域总时间。(B) 各组小鼠旷场测试中活动度。*P < 0.05与溶媒组相比
研究表明,焦虑症的发病与突触间隙神经递质浓度的改变密切相关,5-HT,多巴胺,去甲肾上腺素,γ-氨基丁酸,神经肽Y,谷氨酸等含量变化与焦虑症的发生发展密切相关当各种原因导致神经递质浓度异常、递质传递功能紊乱时,个体将会表现出焦虑样症状 [
研究表明,多巴胺激动杏仁核中的D1,D2受体,可以抑制腺苷酸环化酶(AC)活性,使环磷腺苷(cAMP)减少,进一步抑制蛋白激酶A (PKA)和环磷腺苷效应元件结合蛋白(CREB)活性,从而参与焦虑调节 [
本研究通过旷场测试来评价乳酸的抗焦虑作用,研究发现乳酸组与相比,乳酸在旷场实验中能明显改善小鼠的焦虑行为。结合网络药理学与行为学测试的结果,乳酸可能通过调节单胺类神经递质的代谢发挥抗焦虑作用,主要通过乳酸主要通过调控MAO-A信号通路进一步调节5-HT代谢,以及调控苯丙氨酸和酪氨酸代谢与cAMP信号通路及相应的基因表达进一步调节儿茶酚胺类神经递质代谢发挥抗焦虑作用。本研究通过网络药理学进行通路预测与行为学实验验证了乳酸改善焦虑的作用,为乳酸改善焦虑分子机制的进一步研究奠定了基础。
省级大学生创新创业训练计划项目(编号:S202010547016)。
赵金龙,邓子璇,蔡焕宇,陈莆苑,贺志明. 基于整合药理学的乳酸改善小鼠焦虑作用及机制研究Research on the Effect and Mechanism of Lactic Acid on Improving Anxiety in Mice Based on Integrated Pharmacology[J]. 国际神经精神科学杂志, 2021, 10(04): 99-107. https://doi.org/10.12677/IJPN.2021.104013