Living screening method of high-throughput addiction inhibitor

Abstract

The invention discloses a living body screening method of a high-throughput addiction inhibitor, which comprises the following steps: acquiring addictive nematodes with a plurality of addiction behavioral phenotypes; applying different addiction inhibitors to the addictive nematodes; the screening of the addiction inhibitors is realized according to the inhibition efficacy of the addiction inhibitors on the addiction behavioral phenotypes of the addiction nematodes, and based on the steps, the drug screening of the addiction inhibitors can be realized through the inhibition efficacy of the addiction inhibitors on the addiction behavioral phenotypes of the addiction nematodes in a high-throughput manner. Compared with the prior art, the medicine screening method of a traditional cell function system is abandoned, and medicine screening is carried out on the living animal behavioristics level in a brand new mode. Meanwhile, the advantages of easiness in large-scale culture, abundant behavioral modes and the like of the nematodes are utilized, and compared with expensive mammal feeding and experimental operation for drug screening, the method has higher cost performance, practical operability and high throughput.

Description

Technical Field

[0001] This invention relates to the field of addiction inhibitor screening technology, and more particularly to a high-throughput in vivo screening method for addiction inhibitors. Background Technology

[0002] Smoking poses a serious and comprehensive threat to public health. It is a major contributing factor to many types of cancer, including lung, oral, laryngeal, esophageal, stomach, and bladder cancer. Smoking significantly increases the risk of cardiovascular diseases, including coronary heart disease, hypertension, and stroke. It accelerates the process of arteriosclerosis, increases the probability of thrombosis, and causes fatal damage to the heart and brain. Data shows that smoking at least doubles the risk of coronary heart disease and stroke in adults. Smoking addiction leads to chronic obstructive pulmonary disease (COPD), exacerbates asthma, and is also a high-risk factor for pneumonia and other respiratory infections. Nicotine is the primary culprit in smoking addiction. Currently, prescription drugs for treating nicotine addiction are very scarce, and all have varying degrees of side effects. Therefore, screening and developing more nicotine addiction inhibitors is urgently needed. While traditional cell line screening methods can achieve a certain high throughput, their effectiveness differs significantly from that of in vivo physiological efficacy. On the other hand, it is difficult to achieve high throughput when using live mammals for drug screening, and the cost of raising them is expensive. Screening tens of thousands of small molecule drug precursors would be time-consuming and costly.

[0003] Based on the above analysis, existing technologies have the following technical problems: 1. There are very few prescription nicotine addiction inhibitors on the market, and many have side effects. Screening and developing new inhibitors is beneficial to reducing the smoking population. 2. Nicotine addiction can form typical behavioral phenotypes. Previous drug screening methods relied on cellular physiological functions, resulting in low screening efficiency and high time and labor costs. 3. Large-scale screening of drug precursor molecules using mammals is very costly. Although nicotine can induce strong addictive behavior in mice, the experimental procedures such as mouse rearing, drug injection, and behavioral monitoring require significant manpower and time, and high-throughput screening is not possible. Therefore, there are no successful cases of using mammalian behavioral models for addiction drug screening. 4. Currently, there is no large-scale modular monitoring, statistical analysis, and artificial intelligence analysis of relevant behavioral patterns (e.g., addictive behavior) in model animals. This is because mammalian behavior is complex, making it difficult to form a single statistical factor, and also difficult to achieve large-scale, uniform condition and environmental monitoring. Understandably, there is currently no feasible and efficient live animal screening system for small-molecule nicotine addiction inhibitors, which is why the discovery of addiction inhibitors has been very limited over the decades. Summary of the Invention

[0004] The purpose of this invention is to address the technical problems existing in the prior art by proposing a live screening method for high-throughput addiction inhibitors, comprising the following steps: Obtain addictive nematodes, which possess a variety of addictive behavioral phenotypes; Different addiction inhibitors were administered to addictive nematodes; Based on the inhibitory efficacy of different addiction inhibitors on the addictive behavioral phenotypes of addictive nematodes, the screening of different addiction inhibitors can be achieved.

[0005] Furthermore, the step of "obtaining addictive nematodes" includes the following steps: Add culture medium and larval nematodes to the culture dish; Numerous nematodes are cultured into adult nematodes; Addiction was induced in adult nematodes by administering addictive agents.

[0006] Furthermore, the culture vessel includes several culture wells, and the step of "adding culture medium and larval nematodes to the culture vessel" includes the following steps: Add culture medium and nematode food to each culture well; Add several juvenile nematodes to each culture well.

[0007] Furthermore, the following steps are included after the step of "inducing addiction by administering an addictive agent to adult nematodes": The adult nematodes in each culture well were monitored in real time, and their behavioral characteristics were recorded after they were given an addictive agent. Different behavioral characteristics were summarized and statistically analyzed to construct a cluster of phenotypes of addictive nematodes.

[0008] Furthermore, behavioral characteristics include head-shaking characteristics, twisting characteristics, and swimming motion characteristics.

[0009] Furthermore, after obtaining the addictive nematodes, they are kept in an environment with the corresponding addictive reagents.

[0010] Furthermore, the step of "administering addiction inhibitors to addictive nematodes" includes the following steps: The addictive nematodes were divided into an experimental group and a control group; Addictive agents were removed from the living environment of the experimental and control groups to induce addictive behavioral phenotypes in addictive nematodes. Addiction inhibitors were administered to the experimental group's living environment.

[0011] Furthermore, the step of "screening different addiction inhibitors based on their inhibitory efficacy against addictive behavioral phenotypes in addictive nematodes" includes the following steps: Record the inhibitory intensity of different addiction inhibitors on the addictive behavioral phenotype of addictive nematodes at a certain time point; Record the duration of inhibition required for different addiction inhibitors to induce addictive behavioral phenotypes in addictive nematodes at a given level of inhibition; The goal is to screen for the addiction inhibitor with the strongest inhibitory effect at a given time point and / or the addiction inhibitor with the shortest required duration of inhibition at a given inhibitory effect.

[0012] Furthermore, the inhibition strength is based on the characteristic response inhibition strength of the addictive nematode addiction behavioral phenotype, which includes the number and / or frequency of head shaking, the angle and / or frequency of twisting, and the swimming speed.

[0013] Furthermore, addiction inhibitors include nicotine, and nematodes include Caenorhabditis elegans.

[0014] Compared with existing technologies, this invention offers the following advantages: It is the first drug screening technology based on high-throughput in vivo animal behavioral phenotypes. This method, based on establishing a nematode nicotine addiction behavioral paradigm model, achieves rapid and accurate behavioral judgment through the collection of key behavioral parameter data and artificial intelligence classification analysis. Based on the inhibitory efficacy of drug molecules on nematode nicotine addiction behavior, high-throughput drug screening of inhibitors is performed. This technology abandons traditional cell-based drug screening methods, novelly establishing drug screening at the in vivo animal behavioral level. Furthermore, leveraging the ease of large-scale cultivation and rich behavioral patterns of nematodes, it offers higher cost-effectiveness, practicality, and high throughput compared to expensive mammalian breeding and experimental procedures for drug screening. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the 96-well plate high-throughput behavioral screening system in a specific implementation method; Figure 2 This is a schematic diagram illustrating the behavioral monitoring of nematodes during their first exposure to nicotine and after addicted nematodes leave nicotine, as described in a specific implementation method. Figure 3 This is a schematic diagram illustrating the behavioral monitoring of nematodes during the screening of addiction inhibitors in a specific implementation method. Detailed Implementation

[0016] The present invention will be further illustrated by the following embodiments. These embodiments are purely illustrative and are used only to specifically describe the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product instructions. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased through legitimate channels. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0017] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0018] This invention proposes a method for in vivo screening of high-throughput addiction inhibitors, comprising the following steps: S1: Obtain addictive nematodes, which possess a variety of addictive behavioral phenotypes; S2: Administer different addiction inhibitors to addictive nematodes; S3: Based on the inhibitory efficacy of different addiction inhibitors on the addictive behavioral phenotypes of addictive nematodes, different addiction inhibitors can be screened.

[0019] Optionally, the nematode in this embodiment is *C. elegans*, which is based on nicotine-induced addiction. Under nicotine stimulation, the nematode produces a series of addictive behavioral phenotypes. These addictive behavioral phenotypes can be obtained through monitoring devices and data analysis, digitizing the behavioral characteristics of the nematode under nicotine stimulation. Furthermore, when an addictive agent is applied, the nematode produces a series of addictive behavioral phenotypes. As time progresses and the nematode gradually adapts to the addiction, its addictive behavioral phenotypes will gradually fade. When the addictive agent is removed, due to withdrawal symptoms, the addicted nematode will also exhibit a series of addictive behavioral phenotypes. It is understood that this invention can add different addiction inhibitors when the nematode produces a series of addictive behavioral phenotypes, and by observing the inhibitory efficacy of different addiction inhibitors on the addictive behavioral phenotypes of the nematode, the screening of different addiction inhibitors can be achieved. In some embodiments, the present invention may add a variety of existing addiction inhibitors to the liquid culture environment of addicted nematodes. Based on the effect of the addiction inhibitors on the addictive behavioral phenotype of addicted nematodes, specifically, the addiction inhibitors may be rapidly determined by real-time monitoring and screening of nematode behavioral characteristics based on artificial intelligence.

[0020] Furthermore, in some embodiments, step S1: "obtaining addictive nematodes" includes the following steps: S11: Add culture medium and larval nematodes to a culture dish; S12: Cultivate larval nematodes into adult nematodes; S13: Induce addiction in adult nematodes by administering an addictive agent.

[0021] Furthermore, in some embodiments, the culture vessel includes several culture wells, and step S11: "Adding culture medium and larval nematodes to the culture vessel" includes the following steps: S111: Add culture medium and nematode food to each culture well; S112: Add several juvenile nematodes to each culture well.

[0022] Optionally, the culture vessel is a 96-well plate. The nematodes are cultured using 96-well plate liquid culture technology, specifically by adding a certain amount of OP50 food to M13 culture medium, and culturing 3-5 individual nematodes synchronously in each well. On Day 1, after the nematodes have reached adulthood, a certain concentration of nicotine is added to the well plate to induce addiction for 24-48 hours. Based on the above steps, this invention can efficiently detect and screen the efficacy of tens of thousands of addiction inhibitors based on the inhibitory efficacy of addictive behavioral phenotypes and the high-throughput drug screening of 96-well plates, thereby obtaining effective addiction inhibitors for nicotine addiction.

[0023] Furthermore, in some embodiments, step S13, "inducing addiction by administering an addiction agent to adult nematodes," further includes the following steps: S131: Real-time monitoring of adult nematodes in each culture well and recording of the behavioral characteristics of adult nematodes after being given addictive agents; S132: Inductively summarize and statistically analyze different behavioral characteristics to construct a cluster of phenotypes of addictive nematodes.

[0024] Optionally, this invention utilizes a laboratory-developed nematode behavioral detection system to monitor nematodes in 96-well plates for 72-96 hours, statistically analyzing multiple sets of key behavioral characteristics (e.g., head shaking frequency, C-shaped bending, figure-eight bending, and no-swimming state). Through system analysis and artificial intelligence, the main characteristics of addictive behavior are summarized. A large amount of monitoring data is systematically analyzed to construct an addictive behavioral data cluster, and artificial intelligence algorithms are used to analyze and identify key addictive behavioral characteristics in nematodes. These characteristics enable rapid, efficient, and accurate determination of addiction, establishing a powerful data analysis capability for inhibitor screening. It should be noted that the nematode behavioral detection system and artificial intelligence algorithm in this embodiment can employ existing technologies, which will not be elaborated upon here.

[0025] Furthermore, in some embodiments, the addictive nematodes obtained by the present invention need to be kept in a living environment with the corresponding addictive agent before being treated with addiction inhibitors. Correspondingly, step S2: "treating the addictive nematodes with different addiction inhibitors" includes the following steps: S21: The addictive nematodes were divided into an experimental group and a control group; S22: Remove addictive agents from the living environment of the experimental and control groups to induce addictive behavioral phenotypes in addictive nematodes; S23: Apply addiction inhibitors to the living environment of the experimental group.

[0026] It is understandable that addictive nematodes will not exhibit addictive behavioral phenotypes in the environment of addictive reagents. Therefore, before applying addiction inhibitors, it is necessary to remove addictive reagents from the environment of addictive nematodes in order to induce addictive behavioral phenotypes in addictive nematodes. Specifically, the removal of addictive reagents can be achieved by replacing the culture medium with the same one that does not contain addictive reagents.

[0027] Furthermore, in some embodiments, step S3: "Screening different addiction inhibitors based on their inhibitory efficacy against addictive behavioral phenotypes in addictive nematodes" includes the following steps: S31: Record the inhibitory intensity of different addiction inhibitors on the addictive behavioral phenotype of addictive nematodes at a certain time point; S32: Record the duration of inhibition required for different addiction inhibitors to induce addictive behavioral phenotypes in addictive nematodes at a given level of inhibition; S33: Screen for the addiction inhibitor with the strongest inhibitory strength at a certain time point and / or the addiction inhibitor with the shortest required inhibition time at a certain inhibitory strength.

[0028] Optionally, the intensity of inhibition may be based on the intensity of inhibitory responses characteristic of the addictive nematode behavioral phenotype, including the number and / or frequency of head shaking, the angle and / or frequency of twisting, swimming speed, etc.

[0029] Furthermore, in some embodiments, such as Figures 1-3 As shown, a specific embodiment of a live screening method for high-throughput addiction inhibitors is provided: 1. Nematodes cultured on NGM plates were synchronized to the L4 larval stage, with an average of 3-5 nematode individuals per well for 96-well plate partitioning. The culture medium in each well was standard M13 solution, 500 μL in volume, with 10 μL of concentrated OP50 nematode food added.

[0030] 2. After 24 hours, most nematodes were in their adult DAY1 stage. An experimental concentration of 100 μmol of nicotine was added to a 96-well plate to induce excitement and addiction in the nematodes. Simultaneously, they were placed in a real-time nematode behavior monitoring system to record behavioral data such as swimming speed, head wagging frequency, twisting angle, and frequency. Recording time was 24-48 hours. Figure 1 As shown in the figure. The experimental data shows that nematodes are abnormally excited upon initial exposure to nicotine, but after a prolonged exposure (about 1 hour), they adapt to the presence of nicotine, and their behavior returns to normal. Figure 2 As shown.

[0031] 3. After the nematodes were exposed to nicotine, the nicotine-containing culture medium was removed and replaced with a nicotine-free culture medium under the same conditions. Behavioral parameters of the nematodes in the absence of nicotine were recorded. The experimental data showed that the nematodes exhibited abnormal hyperactivity even after being deprived of nicotine, which is the addiction model of nicotine in nematodes. Furthermore, their behavior was difficult to calm down for a considerable period (approximately 2 hours). Figure 2 As shown.

[0032] 4. Using the established 96-well plate high-throughput behavioral screening system for nematode nicotine addiction, the small molecule drugs to be screened were added to 96-well plates of nematodes that had become addicted and had been released from nicotine, according to different working concentration gradients of 1-100 μmol. Simultaneously, behavioral data of the nematodes were collected and analyzed to screen for candidate small molecule drugs with reduced intensity and duration of addictive behavior, such as... Figure 3 As shown.

[0033] This invention represents the first high-throughput drug screening technology based on behavioral phenotypic patterns in live animals. It is the first method to establish a behavioral paradigm model of nicotine addiction in nematodes, enabling rapid and accurate behavioral assessment through the collection of key behavioral parameters and artificial intelligence classification analysis. Based on the inhibitory efficacy of drug molecules on nicotine addiction behavior in nematodes, high-throughput drug screening of inhibitors is conducted. This technology abandons traditional cell-based drug screening methods, establishing a novel approach at the level of live animal behavior. Furthermore, leveraging the ease of large-scale cultivation and rich behavioral patterns of nematodes, it offers higher cost-effectiveness, practicality, and high throughput compared to expensive mammalian breeding and experimental procedures for drug screening.

[0034] The above descriptions provide one or more embodiments in conjunction with specific details, but do not imply that the specific implementation of the present invention is limited to these descriptions. Any methods or structures that are similar to or identical to those of the present invention, or any technical deductions or substitutions made based on the concept of the present invention, should be considered within the scope of protection of the present invention.

Claims

1. A method for in vivo screening of high-throughput addiction inhibitors, characterized in that, Includes the following steps: Obtain addictive nematodes, which possess addictive behavioral phenotypes; The addictive nematodes were treated with addiction inhibitors; The addiction inhibitors are screened based on their inhibitory efficacy against the addictive behavioral phenotypes of the addictive nematodes.

2. The in vivo screening method for a high-throughput addiction inhibitor according to claim 1, characterized in that, The step "obtaining addictive nematodes" includes the following steps: Add culture medium and larval nematodes to the culture dish; The larval nematodes were cultured into adult nematodes; The adult nematodes were induced to become addicted by being given an addictive agent.

3. The in vivo screening method for a high-throughput addiction inhibitor according to claim 2, characterized in that, The culture vessel includes several culture wells, and the step of "adding culture medium and larval nematodes to the culture vessel" includes the following steps: Add culture medium and nematode food to each of the culture wells; Several juvenile nematodes were added to each of the culture wells.

4. The in vivo screening method for a high-throughput addiction inhibitor according to claim 2, characterized in that, Following the step of "inducing addiction by administering an addiction agent to the adult nematodes", the following steps are also included: The adult nematodes in each culture well are monitored in real time, and their behavioral characteristics are recorded after being treated with the addictive agent. The different behavioral characteristics are summarized and statistically analyzed to construct a cluster of phenotypes of addictive nematodes.

5. The in vivo screening method for a high-throughput addiction inhibitor according to claim 4, characterized in that, Examples of behavioral characteristics include head swaying, twisting, and swimming motion.

6. The in vivo screening method for a high-throughput addiction inhibitor according to claim 1, characterized in that, After obtaining the addictive nematodes, they are preserved in an environment containing the corresponding addictive reagents.

7. The in vivo screening method for a high-throughput addiction inhibitor according to claim 6, characterized in that, The step of "administering an addiction inhibitor to the addictive nematode" includes the following steps: The addictive nematodes were divided into an experimental group and a control group; The addictive reagents were removed from the living environment of the experimental group and the control group to induce the addictive behavioral phenotype of the addictive nematodes; Addiction inhibitors were applied to the living environment of the experimental group.

8. The in vivo screening method for a high-throughput addiction inhibitor according to claim 1, characterized in that, The step of "screening the addiction inhibitor based on its inhibitory efficacy against the addictive behavioral phenotype of the addictive nematode" includes the following steps: Record the inhibitory intensity of different addiction inhibitors on the addictive behavioral phenotype of the nematode at a certain time point; Record the duration of inhibition required at a given intensity for different addiction inhibitors to induce the addictive behavioral phenotype of the nematode. The addiction inhibitor with the strongest inhibitory effect at a certain time point and / or the addiction inhibitor with the shortest required inhibition time at a certain inhibitory effect are selected.

9. The in vivo screening method for a high-throughput addiction inhibitor according to claim 8, characterized in that, The inhibition intensity is based on the characteristic response inhibition intensity of the addictive nematode addiction behavioral phenotype, the characteristic responses of which include the number and / or frequency of head shaking, the angle and / or frequency of twisting, and the swimming speed.

10. The in vivo screening method for a high-throughput addiction inhibitor according to claim 1, characterized in that, The addiction inhibitors include nicotine, and the nematodes include Caenorhabditis elegans.

Images