WO2024036787A1 - 一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用 - Google Patents

一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用 Download PDF

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WO2024036787A1
WO2024036787A1 PCT/CN2022/132209 CN2022132209W WO2024036787A1 WO 2024036787 A1 WO2024036787 A1 WO 2024036787A1 CN 2022132209 W CN2022132209 W CN 2022132209W WO 2024036787 A1 WO2024036787 A1 WO 2024036787A1
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refreshing
group
zebrafish
brain
screening
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French (fr)
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靳梦
刘可春
张轩铭
屠鹏飞
任擎宇
李宁
王利振
王荣春
张姗姗
孙晨
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山东省科学院生物研究所
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    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the present invention relates to the technical field of drug screening, specifically a method for screening drugs for refreshing and refreshing, and the application of trifoliate in refreshing and refreshing, wherein the screening is based on behavioral indicators and refreshing marker genes.
  • Current drug screening methods mainly include molecular-level screening, virtual screening, cell-level screening, and animal model drug screening.
  • drugs that are effective in molecular-level screening have strong binding to the target protein, but may not work in the cellular environment; virtual screening Screening refers to screening out potentially effective drugs through computer simulation of the interaction between known targets and drug structures. It is low-cost and fast, but the screened drugs require further biological experiments for verification; cell-level screening is It refers to using candidate drugs to treat target cells, and then testing the effect of the drug on the target cells through biochemical experiments.
  • the screened drugs can effectively act on the target cells, the experimental process is complex, the requirements for the experimental environment are high, and the drug's effect on the target cells cannot be evaluated.
  • animal model drug screening generally requires the establishment of primate models such as monkeys, pigs, and mouse models.
  • primate models such as monkeys, pigs, and mouse models.
  • construction process of animal disease models is a huge project and requires systematic theory. It requires complex technical means to achieve effective construction, and has strict requirements for the animal's growth environment.
  • zebrafish Due to its high genetic homology with humans, zebrafish can form various tissues and organs within 24 hours after fertilization, has a short development cycle, and is small in size, which makes it possible to use it as an animal model for screening active drugs. At present, there is no scientific, fast and effective screening method for refreshing drugs.
  • the purpose of the present invention is to provide a method for screening refreshing and refreshing drugs and the application of trifoliolin in refreshing and refreshing, wherein the screening is based on behavioral indicators and refreshing and refreshing marker genes.
  • the present invention also includes a method for screening refreshing drugs based on behavioral indicators and the refreshing marker gene described in claim 1, which includes the following steps:
  • zebrafish juveniles were transferred to a 48-well plate, with one fish in each well.
  • the above-mentioned zebrafish juveniles were divided into a control drug group, a blank group and several experimental groups.
  • the control drug group added the maximum safety concentration of caffeine, 1mL of fresh fish farming water was added to the blank group, and 1mL of the maximum safe concentration of a drug to be screened was added to each experimental group; three parallel experiments were set up for each group;
  • nr1d4a-RT-F AGATATCGCATCAGGGTTCC
  • Collect the zebrafish after behavioral recording in different treatment groups wash them two to three times with fish culture water, transfer them to a centrifuge tube, centrifuge at high speed and drain the upper liquid, leaving the bottom zebrafish tissue; use an RNA extraction kit to extract the zebrafish For the total RNA of different experimental groups, use an ultra-micro spectrophotometer to measure the RNA concentration, and use a reverse transcription kit to reverse the RNA into cDNA.
  • the total swimming distance of the brain-refreshing drugs became larger, the swimming speed increased, and the reaction ability was enhanced; the changes in the expression of the brain-refreshing marker genes of the brain-refreshing drugs and the control drug caffeine were the same , all are downward changes.
  • the specific steps to determine the maximum safe concentration of the drug to be screened and the control drug caffeine on zebrafish juveniles are:
  • the 3-day-old zebrafish juveniles were randomly divided into a blank group, a control drug group and several experimental groups. The number of fish in each group was the same.
  • the zebrafish juveniles were placed in a 24-well cell culture plate, with 8 to 12 fish in each well. , put different gradient concentrations of the drug to be screened into the 24-well cell culture plate and soak it for 3 days. Each concentration test was repeated three times. The mortality rate of the zebrafish was recorded.
  • the maximum value of the experimental group drug and the control group drug was calculated through SPSS software. safe concentration.
  • the drugs to be screened are Polygala extract, Ziziphus seed extract, Eucommia male flower extract, Calendula extract and trifoliate.
  • Trifolium a refreshing drug that refreshes the brain, was screened out using the above method.
  • the present invention has the following advantages:
  • the present invention's method for screening refreshing drugs based on behavioral indicators and refreshing marker genes uses transcriptomics methods for the first time to clarify that nr1d4a and nr1d4b are refreshing marker genes.
  • nr1d4a and nr1d4b are refreshing marker genes.
  • the method for screening refreshing drugs based on behavioral indicators and refreshing marker genes of the present invention has a short operation process, is easy to operate, and the screening results are accurate and efficient. It combines behavioral characterization and gene expression levels to provide future refreshing drugs. Drug screening provides new methods.
  • the screening method of the present invention clarifies the refreshing effect of the drug trifoliolin to be screened.
  • Figure 1 is a schematic diagram of the total distance swimming by zebrafish larvae in each group during the detection of light stimulation response; (*P ⁇ 0.05vs blank group, **P ⁇ 0.01vs blank group);
  • Figure 2 is a schematic diagram of the swimming speed of zebrafish larvae in the blank group during light stimulus response detection
  • Figure 3 is a schematic diagram of the swimming speed of zebrafish larvae in the control drug group (caffeine group) during light stimulation response detection;
  • Figure 4 is a schematic diagram of the swimming speed of zebrafish larvae in the trifoliolin group during light stimulation response detection
  • Figure 5 is a schematic diagram of the expression levels of the refreshing and refreshing iconic gene nr1d4a in zebrafish larvae in each group (***P ⁇ 0.001 vs blank group);
  • Figure 6 is a schematic diagram of the expression levels of the refreshing and refreshing iconic gene nr1d4b in zebrafish larvae of each group (**P ⁇ 0.01vs blank group, ***P ⁇ 0.001vs blank group);
  • the purpose of the present invention is to provide a method for screening refreshing and refreshing drugs and the application of trifoliate in refreshing and refreshing drugs, wherein the screening is based on behavioral indicators and refreshing and refreshing marker genes, and is achieved through the following technical solutions:
  • the present invention also includes a method for screening refreshing drugs based on behavioral indicators and the refreshing marker gene described in claim 1, which includes the following steps:
  • zebrafish juveniles were transferred to a 48-well plate, with one fish in each well.
  • the above-mentioned zebrafish juveniles were divided into a control drug group, a blank group and several experimental groups.
  • the control drug group added the maximum safety concentration of caffeine, 1mL of fresh fish farming water was added to the blank group, and 1mL of the maximum safe concentration of a drug to be screened was added to each experimental group; three parallel experiments were set up for each group;
  • the above steps use behavioral testing to measure the drug's ability to promote autonomous activities and stimulate the central nervous system, or detect the sleep duration of experimental animals to measure the drug's refreshing effect;
  • nr1d4a-RT-F AGATATCGCATCAGGGTTCC
  • Collect the zebrafish after behavioral recording in different treatment groups wash them two to three times with fish culture water, transfer them to a centrifuge tube, centrifuge at high speed and drain the upper liquid, leaving the bottom zebrafish tissue; use an RNA extraction kit to extract the zebrafish For the total RNA of different experimental groups, use an ultra-micro spectrophotometer to measure the RNA concentration, and use a reverse transcription kit to reverse the RNA into cDNA.
  • the total swimming distance of the brain-refreshing drugs became larger, the swimming speed increased, and the reaction ability was enhanced; the changes in the expression of the brain-refreshing marker genes of the brain-refreshing drugs and the control drug caffeine were the same , all are downward changes.
  • the specific steps to determine the maximum safe concentration of the drug to be screened and the control drug caffeine on zebrafish juveniles are:
  • the 3-day-old zebrafish juveniles were randomly divided into a blank group, a control drug group and several experimental groups. The number of fish in each group was the same.
  • the zebrafish juveniles were placed in a 24-well cell culture plate, with 8 to 12 fish in each well. , put different gradient concentrations of the drug to be screened into the 24-well cell culture plate and soak it for 3 days. Each concentration test was repeated three times. The mortality rate of the zebrafish was recorded.
  • the maximum value of the experimental group drug and the control group drug was calculated through SPSS software. safe concentration.
  • the method is based on behavioral indicators and the use of refreshing and refreshing marker genes to screen refreshing and refreshing drugs.
  • the drugs to be screened are Polygala extract, Ziziphus seed extract, Eucommia male flower extract, Calendula extract and Wooden leaf. glycosides.
  • Trifolium a refreshing drug that refreshes the brain, was screened out using the above method.
  • the qRT-PCR instrument in the present invention is a fluorescence real-time quantitative PCR instrument.
  • the manufacturer and model of the RNA extraction kit is Beijing Sydney Biotechnology Co., Ltd. RN2802; the manufacturer and model of the reverse transcription kit is Takara-RR036A; the manufacturer and model of the qRT-PCR instrument is Takara-RR091Q.
  • zebrafish samples and the drugs to be screened need to be prepared.
  • the traditional Chinese medicine raw materials to be screened in the present invention are: polygala root, jujube seed, eucommia male flower and marigold; at the same time, trifoliate is purchased from outside.
  • the effective substances after water extraction of Polygala root, Ziziphus kernel, Eucommia male flower and Calendula are Polygala root extract, Ziziphus kernel extract, Eucommia male flower extract and Calendula extract respectively.
  • the traditional Chinese medicine extracts used in the embodiments of the present invention are distilled and purified with water that is 5 times the weight of the traditional Chinese medicine raw material powder.
  • the method of screening refreshing drugs based on behavioral indicators and using refreshing marker genes includes the following steps:
  • Three-day-old zebrafish larvae were randomly divided into a blank group, a control drug group (caffeine) and several experimental groups (Polygala extract, Ziziphus seed extract, Eucommia male flower extract, Calendula extract, Trichosanthes leaf extract Grass glycosides), the number of fish in each group is the same. Place the zebrafish juveniles in a 24-well cell culture plate, with 8 to 12 fish in each well, preferably 10 fish. Put different gradient concentrations of zebrafish into the 24-well cell culture plate. The screening drugs were soaked for 3 days, and each concentration test was repeated three times. The mortality rate of zebrafish was recorded, and the maximum safe concentrations of the experimental group drugs and the control group drugs were calculated and determined through SPSS software.
  • Drug name Maximum safe concentration table Polygala extract 209 ⁇ g/mL Ziziphus seed extract 30 ⁇ g/mL Eucommia male flower extract 206 ⁇ g/mL Calendula Extract 26 ⁇ g/mL Trifoliate 10 ⁇ g/mL caffeine 200 ⁇ mol/L
  • nr1d4a and nr1d4b are marker genes for refreshing and refreshing, which clarified the characteristics of refreshing and refreshing at the genetic level;
  • zebrafish juveniles were transferred to a 48-well plate, with one fish in each well.
  • the above-mentioned zebrafish juveniles were divided into a control drug group (caffeine group), a blank group and several experimental groups (Polygala extract group, Ziziphus jujube kernel extract group, Eucommia male flower extract group, Calendula officinalis extract group, Trifoliate group), in which the maximum safe concentration of caffeine was added to the control drug group, and 1 mL of fresh fish farming water was added to the blank group.
  • Each experimental group was added with 1 mL of a drug to be screened at the maximum safe concentration; each group was set up with three parallel experiments;
  • the schematic diagram of the total swimming distance results of each group of zebrafish juveniles in the light stimulation response test compared with the blank group, under the sleep time, the Polygala group, Eucommia male group and Calendula group in the drug screening group group had a certain light stimulation response, but the difference from the blank group was not statistically significant.
  • the P value of the caffeine group was 0.002
  • the P value of the Ziziphus jujuba kernel extract group was 0.04
  • the P value of the trifoliate group was 0.008.
  • the P values of these three groups were all less than 0.05, which was statistically significant.
  • the schematic diagram of the swimming speed per minute of zebrafish juveniles in each group in the light stimulation response test shows that compared with the blank group, the swimming speed of zebrafish in the control group and trifoliolin group increased. , the reaction ability is enhanced, indicating that trifoliolin has a refreshing effect.
  • nr1d4a-RT-F AGATATCGCATCAGGGTTCC
  • zebrafish tissue use an RNA extraction kit to extract total RNA from different groups of zebrafish, use an ultra-micro spectrophotometer to measure the RNA concentration, use a reverse transcription kit to reverse the RNA into cDNA, refer to the real-time fluorescence quantitative PCR instructions, and use each
  • the cDNA after group inversion was used as a template, and the primers to be tested were added for amplification in a qRT-PCR instrument.
  • Three parallel experiments were set up for each group to detect the expression levels of the refreshing and refreshing marker genes nr1d4a and nr1d4b in each group;

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Abstract

本发明公开了提神醒脑标志基因nr1d4a和nr1d4b在筛选提神醒脑药物中的应用，涉及药物筛选技术领域，具体为基于行为学指标和上述提神醒脑标志基因进行提神醒脑药物筛选的方法，包括以下步骤：①确定待筛选药物和对照药咖啡因对5日龄斑马鱼幼鱼的最大安全浓度；②基于光照刺激反应下的斑马鱼行为学表现，筛选提神醒脑药物；③基于Real-time PCR技术，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平，筛选出提神醒脑药物；④提神醒脑药物的判断标准；本发明的筛选方法，操作流程短，易操作，筛选结果准确高效，将行为学表征和基因表达水平相结合，为以后提神醒脑药物的筛选提供了新的方法。

Description

一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用 技术领域
本发明涉及药物筛选技术领域，具体说是一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用，其中筛选是基于行为学指标和提神醒脑标志基因进行。
背景技术
目前药物筛选的方法主要包括分子水平筛选、虚拟筛选、细胞水平筛选和动物模型药物筛选等，其中分子水平筛选筛选有效的药物与目的蛋白结合强，但是在细胞环境中却可能无法发挥作用；虚拟筛选是指通过计算机模拟已知靶点和药物结构之间的相互作用，筛选出可能有效的药物，成本低、速度快，但是筛选出的药物还需要进一步的生物实验进行验证；细胞水平筛选是指用候选药物处理靶细胞，再通过生化实验检验药物对靶细胞的作用，筛选得到的药物虽然能够有效地作用于靶细胞，但是实验流程复杂，对实验环境的要求高，且无法评估药物对整体生物是否有毒副作用，因此也限制了其使用；动物模型药物筛选一般需建立猴等灵长类动物模型、猪和小鼠模型，但是动物疾病模型构建过程是一个巨大的工程，需要系统的理论和复杂的技术手段才能获得有效构建，对动物的生长环境有严格的要求。
斑马鱼由于和人类基因同源性高，各组织器官在受精后24小时即可形成，发育周期短，个体小等优势，为其作为筛选活性药物的动物模型提供了可能。目前对于提神醒脑药物还没有科学、快速有效的筛选方法。
发明内容
为解决上述问题，本发明的目的是提供一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用，其中筛选是基于行为学指标和提神醒脑标志基因进行。
本发明为实现上述目的，通过以下技术方案实现：
提神醒脑标志基因nr1d4a和nr1d4b在筛选提神醒脑药物中的应用。
本发明还包括基于行为学指标和权利要求1所述的提神醒脑标志基因进行提神醒脑药物筛选的方法，包括以下步骤：
①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度；
②基于光照刺激反应下的斑马鱼行为学表现，筛选提神醒脑药物：
实验当晚24时，将5dpf的斑马鱼幼鱼转移至48孔板中，每孔一条鱼，将上述斑马鱼幼鱼分为对照药物组、空白组和若干实验组，其中对照药物组加入最大安全浓度的咖啡因，空白组加入1mL新鲜养鱼水，每个实验组加入1mL最大安全浓度的一种待筛选药物；每组均设置三个平行试验；
将上述48孔板放入Zebrabox斑马鱼行为分析仪暗箱中，开始检测斑马鱼对光照刺激反应，检测时间为60分钟，每分钟记录一次数据，运用Zeblab软件进行数据处理，计算斑马鱼游动总距离和游动速度，测试不同组别斑马鱼在沉睡时间下的光照刺激反应；
③基于Real-time PCR技术，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平，筛选出提神醒脑药物：
对提神醒脑标志基因nr1d4a和nr1d4b设计引物序列：
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
收集不同处理组行为学记录后的斑马鱼，用养鱼水清洗两到三次，转移至离心管中，高速离心后吸干上层液体，留下底层斑马鱼组织；使用RNA提取试剂盒提取斑马鱼不同实验组的总RNA，利用超微量分光光度计测定RNA浓度，使用反转录试剂盒将RNA反转成cDNA，参照实时荧光定量PCR说明书，以各组反转完成后的cDNA为模板，加入待测引物在qRT-PCR仪器中扩增，每组设置三个平行试验，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平；
④提神醒脑药物的判断标准：
在沉睡时间下，与空白组相比，提神醒脑药物的游动总距离变大，游动速度增加，反应能力增强；提神醒脑药物和对照药咖啡因的提神醒脑标志基因表达变化相同，均为下调变化。
优选的，①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度的具体步骤为：
将3日龄斑马鱼幼鱼随机分为空白组、对照药物组和若干实验组，每组鱼的数量相同，将斑马鱼幼鱼置于24孔细胞培养板中，每孔8～12条鱼，对24孔细胞培养板投放不同梯度浓度的待筛选药物浸泡3天，每个浓度测试重复3次，对斑马鱼的死亡率进行记录，通过SPSS软件计算确定实验组药物和对照组药物的最大安全浓度。
优选的，待筛选药物为远志提取物、酸枣仁提取物、杜仲雄花提取物、金盏菊提取物和车叶草苷。
一种提神醒脑药物车叶草苷，采用上述方法筛选出来。
本发明相比现有技术具有以下优点：
本发明的基于行为学指标和提神醒脑标志基因进行提神醒脑药物筛选的方法，首次采用用转录组学方法明确了nr1d4a和nr1d4b为提神醒脑的标志基因，在筛选过程中，首先通过行为学对各组斑马鱼进行初筛，然后对提神醒脑标志基因nr1d4a和nr1d4b设计上下游引物，进行RT-qPCR验证，由于车叶草苷组和对照组的提神醒脑标志基因nr1d4a和nr1d4b的表达趋势一致，从而确定了待筛选药物车叶草苷具有提神醒脑作用。本发明的基于行为学指标和提神醒脑标志基因进行提神醒脑药物筛选的方法，操作流程短，易操作，筛选结果准确高效，将行为学表征和基因表达水平相结合，为以后提神醒脑药物的筛选提供了新的方法。
本发明的筛选方法，明确了待筛选药物车叶草苷的提神醒脑功效。
附图说明
图1为各组的斑马鱼幼鱼在光照刺激反应检测中游动的总距离示意图；(*P＜0.05vs空白组，**P＜0.01vs空白组)；
图2为空白组的斑马鱼幼鱼在光照刺激反应检测中游动速度示意图；
图3为对照药物组(咖啡因组)的斑马鱼幼鱼在光照刺激反应检测中游动速度示意图；
图4为车叶草苷组的斑马鱼幼鱼在光照刺激反应检测中游动速度示意图；
图5为各组斑马鱼幼鱼提神醒脑标志性基因nr1d4a表达水平示意图(***P＜0.001vs 空白组)；
图6为各组斑马鱼幼鱼提神醒脑标志性基因nr1d4b表达水平示意图(**P＜0.01vs空白组，***P＜0.001vs空白组)；
附图标记：
1空白组，2对照药物组(咖啡因组)，3远志提取物组，4酸枣仁提取物组，5杜仲雄花提取物组，6金盏菊提取物组，7车叶草苷组。
具体实施方式
本发明的目的是提供一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用，其中筛选是基于行为学指标和提神醒脑标志基因进行，通过以下技术方案实现：
提神醒脑标志基因nr1d4a和nr1d4b在筛选提神醒脑药物中的应用。
本发明还包括基于行为学指标和权利要求1所述的提神醒脑标志基因进行提神醒脑药物筛选的方法，包括以下步骤：
①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度；
②基于光照刺激反应下的斑马鱼行为学表现，筛选提神醒脑药物：
实验当晚24时，将5dpf的斑马鱼幼鱼转移至48孔板中，每孔一条鱼，将上述斑马鱼幼鱼分为对照药物组、空白组和若干实验组，其中对照药物组加入最大安全浓度的咖啡因，空白组加入1mL新鲜养鱼水，每个实验组加入1mL最大安全浓度的一种待筛选药物；每组均设置三个平行试验；
将上述48孔板放入Zebrabox斑马鱼行为分析仪暗箱中，开始检测斑马鱼对光照刺激反应，检测时间为60分钟，每分钟记录一次数据，运用Zeblab软件进行数据处理，计算斑马鱼游动总距离和游动速度，测试不同组别斑马鱼在沉睡时间下的光照刺激反应；
上述步骤通过行为学检测药物的促进自主活动、兴奋中枢神经系统能力，或检测实验动物的睡眠时长，用以衡量药物的提神醒脑功效；
③基于Real-time PCR技术，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达 水平，筛选出提神醒脑药物：
对提神醒脑标志基因nr1d4a和nr1d4b设计引物序列：
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
收集不同处理组行为学记录后的斑马鱼，用养鱼水清洗两到三次，转移至离心管中，高速离心后吸干上层液体，留下底层斑马鱼组织；使用RNA提取试剂盒提取斑马鱼不同实验组的总RNA，利用超微量分光光度计测定RNA浓度，使用反转录试剂盒将RNA反转成cDNA，参照实时荧光定量PCR说明书，以各组反转完成后的cDNA为模板，加入待测引物在qRT-PCR仪器中扩增，每组设置三个平行试验，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平；
④提神醒脑药物的判断标准：
在沉睡时间下，与空白组相比，提神醒脑药物的游动总距离变大，游动速度增加，反应能力增强；提神醒脑药物和对照药咖啡因的提神醒脑标志基因表达变化相同，均为下调变化。
优选的，①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度的具体步骤为：
将3日龄斑马鱼幼鱼随机分为空白组、对照药物组和若干实验组，每组鱼的数量相同，将斑马鱼幼鱼置于24孔细胞培养板中，每孔8～12条鱼，对24孔细胞培养板投放不同梯度浓度的待筛选药物浸泡3天，每个浓度测试重复3次，对斑马鱼的死亡率进行记录，通过SPSS软件计算确定实验组药物和对照组药物的最大安全浓度。
优选的，基于行为学指标和利用提神醒脑标志基因进行提神醒脑药物筛选的方法，待筛选药物为远志提取物、酸枣仁提取物、杜仲雄花提取物、金盏菊提取物和车叶草苷。
一种提神醒脑药物车叶草苷，采用上述方法筛选出来。
以下结合具体实施例来对本发明作进一步的描述。
本发明中的qRT-PCR仪器即为荧光实时定量PCR仪。
RNA提取试剂盒的生产厂家和型号为北京艾德莱生物科技有限公司RN2802；反转录试剂盒的厂家和型号为Takara-RR036A；qRT-PCR仪器的厂家和型号为Takara-RR091Q。
实施例1
在进行药物筛选前，需要准备斑马鱼样品和待筛选的药物。
(一)准备斑马鱼样品具体过程如下：
取健康性成熟斑马鱼雌雄1:1于当日16：00时左右放入产卵缸中，次日8：30时抽取隔板，2h后收取受精卵，剔除死胚，养殖水反复冲洗3次，移入含2mg/L亚甲基蓝的养殖水中，置于28℃培养箱中孵化培养。4小时后将胚胎置于显微镜下观察，选取发育正常的胚胎孵化培养至受精后5天(day post fertilization，dpf)，期间每天及时剔除死胚。
(二)准备待筛选的药物
本发明准备待筛选的中药原料为：远志、酸枣仁、杜仲雄花和金盏菊；同时外购车叶草苷。
以上中药原料均采用下述方法进行有效物质的提取：
将上述中药原料研磨成粉末，加入圆底烧瓶中，向其中加入中药原料粉末重量3～5倍的水，蒸馏提纯至少5小时，冷却后离心，收集上清液，对下部液体进行抽滤，得到滤液，将上清液和滤液旋蒸去除水分，得到中药提取物；
其中远志、酸枣仁、杜仲雄花和金盏菊水提后的有效物质分别为远志提取物、酸枣仁提取物、杜仲雄花提取物和金盏菊提取物。
本发明实施例采用的中药提取物在提取过程中均采用向中药原料粉末重量5倍的水进行蒸馏提纯。
基于行为学指标和利用提神醒脑标志基因进行提神醒脑药物筛选的方法，包括以下步骤：
①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度；
将3日龄斑马鱼幼鱼随机分为分为空白组、对照药物组(咖啡因)和若干实验组(远志提取物、酸枣仁提取物、杜仲雄花提取物、金盏菊提取物、车叶草苷)，每组鱼的数量相同，将斑马鱼幼鱼置于24孔细胞培养板中，每孔8～12条鱼，优选10条鱼，对24孔细胞培养板投放不同梯度浓度的待筛选药物浸泡3天，每个浓度测试重复3次，对斑马鱼的死亡率进行记录，通过SPSS软件计算确定实验组药物和对照组药物的最大安全浓度。
远志提取物、酸枣仁提取物、杜仲雄花提取物、金盏菊提取物、车叶草苷、咖啡因的最大安全浓度如表1所示。
表1 各物质的最大安全浓度表
药物名称 最大安全浓度表
远志提取物 209μg/mL
酸枣仁提取物 30μg/mL
杜仲雄花提取物 206μg/mL
金盏菊提取物 26μg/mL
车叶草苷 10μg/mL
咖啡因 200μmol/L
②转录组学明确提神醒脑的标志基因
实验当晚24时，将5dpf的斑马鱼幼鱼转移至48孔板中，每孔一条鱼，设置空白组和咖啡因组，空白组加入1mL新鲜养鱼水，阳性药咖啡因组加入最大安全浓度的咖啡因药液，静置60分钟，用养鱼水清洗斑马鱼2～3次，转移至离心管中，分别提取两组斑马鱼的总RNA；
对提取后的两组斑马鱼的总RNA进行转录组学研究，对两组斑马鱼的基因表达差异进行显著性分析，使用统计学方法，进行信息分析，比较两组斑马鱼的基因表达差异，找出与条件相关的特异性基因。经分析转录组测序结果，nr1d4a和nr1d4b为提神醒脑的标志基因，从基因层面明确了提神醒脑表征；
③基于光照刺激反应下的斑马鱼行为学表现，筛选提神醒脑药物：
实验当晚24时，将5dpf的斑马鱼幼鱼转移至48孔板中，每孔一条鱼，将上述斑马鱼幼鱼分为对照药物组(咖啡因组)、空白组和若干实验组(远志提取物组、酸枣仁提取物组、杜仲雄花提取物组、金盏菊提取物组、车叶草苷组)，其中对照药物组加入最大安全浓度的咖啡因，空白组加入1mL新鲜养鱼水，每个实验组加入1mL最大安全浓度的一种待筛选药物；每组均设置三个平行试验；
将上述48孔板放入Zebrabox斑马鱼行为分析仪暗箱中，开始检测斑马鱼对光照刺激反应，检测时间为60分钟，每分钟记录一次数据，运用Zeblab软件进行数据处理，计算斑马鱼游动总距离和游动速度，测试不同组别斑马鱼在沉睡时间下的光照刺激反应；
如图1各组斑马鱼幼鱼在光照刺激反应检测中游动总距离结果示意图所示，与空白组相比，在沉睡时间下，药物筛选组中的远志组、杜仲雄花组和金盏菊组的具有一定光照刺激反应，但与空白组的差异不具有统计学意义。咖啡因组的P值为0.002，酸枣仁提取物组的P值为0.04，车叶草苷组的P值为0.008，这三组P值均小于0.05，具有统计学意义，远志提取物组、杜仲雄花提取物组、金盏菊提取物组三组的P值均大于0.05，不具有统计学意义。因此成功筛选出酸枣仁提取物和车叶草苷能够提高斑马鱼幼鱼的游动总距离，因此具有提神醒脑效果，并且车叶草苷的效果最佳，以下只对车叶草苷的数据进行下一步处理。
如图2～图4各组斑马鱼幼鱼在光照刺激反应检测中每分钟游动速度结果示意图所示，相比于空白组，对照组和车叶草苷组斑马鱼游动速度有所增加，反应能力增强，说明车叶草苷具有提神醒脑作用。
④基于Real-time PCR技术，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平，筛选出提神醒脑药物：
对提神醒脑标志基因nr1d4a和nr1d4b设计引物序列：
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
收集上述空白组，对照组和车叶草苷组斑马鱼进行行为学记录后的斑马鱼，用养鱼水清洗两到三次，转移至离心管中，高速离心后吸干上层液体，留下底层斑马鱼组织；使用RNA提取试剂盒提取斑马鱼不同组的总RNA，利用超微量分光光度计测定RNA浓度，使用反转录试剂盒将RNA反转成cDNA，参照实时荧光定量PCR说明书，以各组反转完成后的cDNA为模板，加入待测引物在qRT-PCR仪器中扩增，每组设置三个平行试验，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平；
结果如图5和图6所示，与空白组相比，车叶草苷组的nr1d4a和nr1d4b的基因表达均下调，与对照组的表达方向一致，可以得出车叶草苷可以作为提神醒脑药物。

Claims (5)

  1. 提神醒脑标志基因在筛选提神醒脑药物中的应用，其特征在于：所述提神醒脑标志基因为nr1d4a和nr1d4b。
  2. 基于行为学指标和权利要求1所述的提神醒脑标志基因进行提神醒脑药物筛选的方法，其特征在于：包括以下步骤：
    ①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度；
    ②基于光照刺激反应下的斑马鱼行为学表现，筛选提神醒脑药物：
    实验当晚24时，将5dpf的斑马鱼幼鱼转移至48孔板中，每孔一条鱼，将上述斑马鱼幼鱼分为对照药物组、空白组和若干实验组，其中对照药物组加入最大安全浓度的咖啡因，空白组加入1mL新鲜养鱼水，每个实验组加入1mL最大安全浓度的一种待筛选药物；每组均设置三个平行试验；
    将上述48孔板放入Zebrabox斑马鱼行为分析仪暗箱中，开始检测斑马鱼对光照刺激反应，检测时间为60分钟，每分钟记录一次数据，运用Zeblab软件进行数据处理，计算斑马鱼游动总距离和游动速度，测试不同组别斑马鱼在沉睡时间下的光照刺激反应；
    ③基于Real-time PCR技术，检测各组提神醒脑标志基因nr1d4a和nr1d4b的表达水平，筛选出提神醒脑药物：
    对提神醒脑标志基因nr1d4a和nr1d4b设计引物序列：
    nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
    nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
    nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
    nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
    收集不同处理组行为学记录后的斑马鱼，用养鱼水清洗两到三次，转移至离心管中，高速离心后吸干上层液体，留下底层斑马鱼组织；使用RNA提取试剂盒提取斑马鱼不同实验组的总RNA，利用超微量分光光度计测定RNA浓度，使用反转录试剂盒将RNA反转成cDNA，参照实时荧光定量PCR说明书，以各组反转完成后的cDNA为模板，加入待测引物在qRT-PCR仪器中扩增，每组设置三个平行试验，检测各组提 神醒脑标志基因nr1d4a和nr1d4b的表达水平；
    ④提神醒脑药物的判断标准：
    在沉睡时间下，与空白组相比，提神醒脑药物的游动总距离变大，游动速度增加，反应能力增强；提神醒脑药物和对照药咖啡因的提神醒脑标志基因表达变化相同，均为下调变化。
  3. 根据权利要求2所述的基于行为学指标和利用提神醒脑标志基因进行提神醒脑药物筛选的方法，其特征在于：①确定待筛选药物和对照药咖啡因对斑马鱼幼鱼的最大安全浓度的具体步骤为：
    将3日龄斑马鱼幼鱼随机分为空白组、对照药物组和若干实验组，每组鱼的数量相同，将斑马鱼幼鱼置于24孔细胞培养板中，每孔8～12条鱼，对24孔细胞培养板投放不同梯度浓度的待筛选药物浸泡3天，每个浓度测试重复3次，对斑马鱼的死亡率进行记录，通过SPSS软件计算确定实验组药物和对照组药物的最大安全浓度。
  4. 根据权利要求2所述的基于行为学指标和利用提神醒脑标志基因进行提神醒脑药物筛选的方法，其特征在于：待筛选药物为远志提取物、酸枣仁提取物、杜仲雄花提取物、金盏菊提取物和车叶草苷。
  5. 一种提神醒脑药物，采用权利要求2的方法筛选出来，其特征在于：所述提神醒脑药物为车叶草苷。
PCT/CN2022/132209 2022-08-17 2022-11-16 一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用 WO2024036787A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005076004A1 (en) * 2004-02-10 2005-08-18 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human nuclear receptor nr1d1 (nr1d1)
JP2009209088A (ja) * 2008-03-04 2009-09-17 Kobayashi Pharmaceut Co Ltd アスペルロシドまたはその類縁体を含む筋肉増強剤
CN106902128A (zh) * 2017-01-19 2017-06-30 江苏大学 车叶草苷在制备防治神经退行性疾病药物中的应用
CN115247211A (zh) * 2022-08-17 2022-10-28 山东省科学院生物研究所 一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100425243C (zh) * 2005-08-19 2008-10-15 李斌 车叶草苷在制备治疗类风湿性关节炎药物中的应用
CN101606062B (zh) * 2006-08-15 2013-11-13 宾夕法尼亚大学理事会 用于治疗和诊断自身免疫性脑炎或癫痫的方法和组合物
CN102100913B (zh) * 2009-12-16 2013-06-05 中国科学院上海生命科学研究院 脑损伤治疗的靶点和药物
CN104450602B (zh) * 2013-09-17 2020-08-07 中国科学院遗传与发育生物学研究所 非人哺乳动物神经精神疾病动物模型及其制备方法和用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005076004A1 (en) * 2004-02-10 2005-08-18 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human nuclear receptor nr1d1 (nr1d1)
JP2009209088A (ja) * 2008-03-04 2009-09-17 Kobayashi Pharmaceut Co Ltd アスペルロシドまたはその類縁体を含む筋肉増強剤
CN106902128A (zh) * 2017-01-19 2017-06-30 江苏大学 车叶草苷在制备防治神经退行性疾病药物中的应用
CN115247211A (zh) * 2022-08-17 2022-10-28 山东省科学院生物研究所 一种提神醒脑药物筛选的方法和车叶草苷在提神醒脑方面中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LING YANG, YAN JUN-JUN; LONG YONG; CUI ZONG-BIN; BAO CHUAN-HE: "Expression of nr1d4a and nr1d4b Genes in Zebrafish and Their Responses to Different Environmental Stimuli", SHUISHENG-SHENGWU-XUEBAO = ACTA HYDROBIOLOGICA SINICA, KEXUE CHUBANSHE, CHINA, vol. 38, no. 1, 10 February 2014 (2014-02-10), CHINA , pages 100 - 107, XP093139650, ISSN: 1000-3207, DOI: 10.7541/2014.13 *

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