Method for screening drug targets based on LCN2 molecular mechanism and application

Abstract

The invention relates to the field of neurobiology and pharmacology, and provides a method for screening drug targets based on an LCN2 molecular mechanism and application. A mouse model of a mental stress-induced attack behavior is established through chronic unpredictable stress, and a mouse of which the attack behavior is remarkably enhanced is screened out. Differential expression genes are identified in combination with a high-throughput sequencing technology, and the key effect of the LCN2 in aggressive behaviors is found. The expression of LCN2 is regulated by using adeno-associated virus, and a neural circuit and a brain region activation mode regulated by the LCN2 are clarified in combination with behavioral testing, cFOS staining and molecular verification. A downstream signal channel of the LCN2 is explored through co-immunoprecipitation and protein spectrum technologies, related inflammatory factors and neurotransmitter regulation molecules are screened, and functions of the LCN2 are verified through in-vivo and in-vitro experiments. And screening potential drug targets and developing targeted drugs based on an LCN2 mechanism. The method provides a new target for treatment of mania, bidirectional affective disorder and other mental diseases, and has important scientific value and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of neurobiology and pharmacology, and specifically to a method and application for screening drug targets based on the LCN2 molecular mechanism. Background Art

[0002] Aggression is an important form of behavior in animal survival and competition, and is widely present in many species, including humans. In human society, abnormal aggressive behavior not only affects social order, but is also closely related to a variety of mental illnesses, such as mania, bipolar disorder, and post-traumatic stress disorder (PTSD). In particular, aggressive behavior caused by mental stress is often sudden, intense, and uncontrollable, which has a serious impact on the patient and society.

[0003] Studies have shown that the occurrence of aggressive behavior is closely related to specific neural circuits in the brain, mainly including the amygdala (CeA), prefrontal cortex (PFC), periaqueductal gray (PAG) and other areas. These brain regions participate in the initiation and regulation of aggressive behavior by regulating neurotransmitters (such as GABA, glutamate) and related signaling pathways. However, the molecular mechanism between aggressive behavior and mental stress is still not fully understood, especially at the molecular level, there is a lack of systematic research on how mental stress regulates aggressive behavior through neural circuits.

[0004] In recent years, studies have found that mental stress can induce abnormal expression of inflammatory factors and metabolic molecules, thereby regulating neural circuits. Lipocalin-2 (LCN2) is a molecule secreted by cells that is mainly involved in inflammatory responses, iron metabolism regulation, and intercellular signaling. In neurobiology, LCN2 has been found to have an important effect on synaptic activity and neurotransmitter release. However, no studies have yet clearly revealed the specific role of LCN2 in mental stress-induced aggressive behavior.

[0005] In addition, existing drug treatments for aggressive behavior have significant deficiencies. Current drugs mainly work by regulating overall neurotransmitter levels (such as dopamine and 5-HT), which often have limited effects and large side effects. For example, antidepressants and antipsychotics may be effective for aggressive behavior in some cases, but their non-specific mechanisms cause the efficacy to vary from person to person, and are accompanied by side effects such as emotional retardation and metabolic disorders. Therefore, new targets and specific drugs for the molecular mechanisms of aggressive behavior are urgently needed.

[0006] Therefore, we urgently need to design a method for screening drug targets based on the molecular mechanism of LCN2 and its application to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to provide a new drug development with LCN2 as a target for aggressive behaviors in existing mental illnesses, in order to reduce mental stress and aggressive behaviors associated with various mental disorders.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for screening drug targets based on the LCN2 molecular mechanism, comprising the following steps: Establish a mouse model of mental stress-induced aggressive behavior; Identify the role of LCN2 in mental stress-induced aggressive behavior; Explore the downstream signaling pathways of LCN2; Screening of potential drug targets based on LCN2-related mechanisms; The effectiveness of potential drug targets was verified through in vitro and in vivo experiments.

[0009] As a preferred technical solution of the present invention, the method for establishing a mouse model of mental stress-induced aggressive behavior comprises: Using chronic unpredictable stress to impose mental stress on experimental mice; Aggression in mice was recorded by standardized behavioral tests, including social interaction test and aggression potential test; Mice showing significantly enhanced aggressive behavior were screened to establish a stable aggressive behavior model.

[0010] As a preferred technical solution of the present invention, the method for identifying the role of LCN2 in mental stress-induced aggressive behavior comprises: High-throughput sequencing technology was used to screen genes differentially expressed in the brains of mice with significantly enhanced aggressive behavior after mental stress, with a focus on LCN2. Adeno-associated virus was used to upregulate or downregulate the expression level of LCN2 in the mouse brain and the effect on aggressive behavior was evaluated; Behavioral tests were used to verify the correlation between changes in LCN2 expression and the intensity of aggressive behavior.

[0011] As a preferred technical solution of the present invention, the method for verifying the effect of LCN2 in mental stress-induced aggressive behavior includes: qPCR and Western blot techniques were used to detect the mRNA and protein expression levels of LCN2 in different brain regions after mental stress; cFOS staining was used to analyze the activation of brain regions related to aggressive behavior induced by mental stress, including the central nucleus of the amygdala (CeA) and the periaqueductal gray (PAG); Zonal analysis was performed in different subregions of the PAG to clarify the spatial relationship between LCN2 expression and brain region activation.

[0012] As a preferred technical solution of the present invention, the method for exploring the downstream signaling pathway of LCN2 includes: The downstream molecules that may be regulated by LCN2 were screened by co-immunoprecipitation combined with protein profiling; Predict the interaction relationship between LCN2 and the screened signaling molecules; Verify the direct interaction between LCN2 and downstream signaling molecules through in vitro molecular interaction experiments (such as yeast two-hybrid or FRET experiments); Adeno-associated virus or CRISPR technology is used to target and regulate the expression levels of downstream molecules to evaluate their effects on aggressive behavior.

[0013] As a preferred technical solution of the present invention, the downstream signal molecules include but are not limited to inflammatory factors (such as IL-6, TNF-α), neurotransmitter regulatory molecules (such as GABA or glutamate receptor subtypes) or metabolism-related signal molecules.

[0014] As a preferred technical solution of the present invention, the method for screening potential drug targets based on LCN2-related molecular mechanisms includes: Screen candidate compounds that interfere with LCN2 expression and downstream signaling molecule activity through a drug screening platform; Test the intervention effects of candidate compounds on downstream key molecular functions and mental stress-induced aggressive behavior models; Comprehensively evaluate the selectivity, efficacy and improvement effect on aggressive behavior of candidate compounds.

[0015] As a preferred technical solution of the present invention, the in vitro verification method of the drug target includes: Testing candidate compounds for their regulatory effects on LCN2 expression and activity in in vitro cell models; Detect the regulatory effect of compounds on downstream signaling pathway-related molecules (such as inflammatory factors and neurotransmitter receptors); Neuronal co-culture models were used to investigate the effects of compounds on synaptic activity and neuronal excitability.

[0016] As a preferred technical solution of the present invention, the in vivo verification method of the drug target includes: Testing the therapeutic efficacy of candidate compounds in a mouse model of mental stress-induced aggression; Use brain region-specific gene regulation technology (such as PAG region viral expression system) to evaluate the brain region-specific effects of compounds; Detect the effects of compounds on behavioral indicators (such as aggressive behavior intensity, social interaction ability) and neural circuit activation levels.

[0017] As a preferred technical solution of the present invention, the application of the method for screening drug targets based on the molecular mechanism of LCN2 includes but is not limited to: Research and development of drugs related to mental illnesses (such as mania and bipolar disorder); Develop targeted therapeutic drugs for aggressive behaviors induced by mental stress; Study the potential role of LCN2-related neural regulatory mechanisms in other psychiatric disorders.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This study revealed for the first time the key role of lipocalin 2 (LCN2) in mental stress-induced aggressive behavior. By constructing a mouse model and using molecular biological methods, the molecular mechanism of LCN2 regulating aggressive behavior and its related neural circuits were clarified. This provides a new perspective for the study of the neural basis of aggressive behavior and fills the research gap in the field on the mechanism of action of LCN2.

[0019] By systematically studying LCN2 and its downstream signaling pathways, the present invention identified a variety of molecular targets related to the regulation of inflammatory factors and neurotransmitters, proving its key regulatory role in mental stress-induced aggressive behavior. These findings not only deepen the understanding of the mechanism of mental stress-induced neural circuit disorders, but also provide new molecular targets for the treatment of mental illnesses such as mania and bipolar disorder.

[0020] Screening potential drug targets based on the LCN2 mechanism and developing targeted therapeutic drugs provide new treatment options for patients with mental illnesses. The new drugs are highly targeted, highly effective, and have few side effects. They can be widely used in the precision treatment of mental illnesses and have significant clinical transformation value and market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flowchart of the method for screening drug targets based on the LCN2 molecular mechanism proposed in the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The following is combined with Figure 1 With reference to the accompanying drawings and a number of embodiments, specific embodiments of the present invention are described in detail.

[0024] Example 1: Establishment of a mouse model of mental stress-induced aggressive behavior; (1) Experimental animal preparation: 8-week-old healthy male C57BL / 6 mice weighing between 20-25g were selected. All mice were raised in a constant temperature (22±2°C) and constant humidity (50±10%) environment with a 12-hour day and night light cycle, 2-3 mice per cage, and free access to food and water. They were randomly divided into an experimental group and a control group, with 30 mice in each group.

[0025] (2) Chronic unpredictable stress management: The experimental group of mice was subjected to chronic unpredictable stress for 21 days, with one of the following stimuli randomly applied every day: Tilt the cage: Tilt the mouse cage at 45 degrees for 4 hours.

[0026] Wet bedding environment: Spray the mouse bedding wet and maintain a humid environment for 12 hours.

[0027] Electric shock stress: 0.3 mA electric shock was applied through the cage bottom grid at intervals of 5 seconds for 1 minute.

[0028] Fasting and water deprivation: deprivation of food and water for 12 hours.

[0029] Social isolation: Mice in the experimental group were housed individually for 24 hours.

[0030] The stress mode was changed randomly every day, and mice in the control group did not receive any stress treatment.

[0031] (3) Behavioral test: After the stress treatment, the aggressive behavior of mice was assessed by the following behavioral tests: Social interaction test: The experimental mice were placed in a cage containing a strange mouse, and the number and duration of aggressive behaviors (such as chasing, biting, and pressing) were observed within 5 minutes.

[0032] Aggression potential test: The mice were placed in the same cage with a non-resistance virtual opponent (mechanical model mouse), and the frequency of attack actions (such as biting and scratching) was recorded.

[0033] Open field test: Assess the activity and exploratory behavior of mice to exclude nonspecific motor influences of aggressive behavior.

[0034] Through behavioral tests, mice with significantly enhanced aggressive behavior were screened out and used as models for subsequent experiments.

[0035] Example 2: Identification of the role of LCN2 in mental stress-induced aggressive behavior; (1) Differential gene expression analysis: The brain tissues (including amygdala CeA and periaqueductal gray PAG) of the mice in the experimental group and the control group in Example 1 were collected, total RNA was extracted, and differentially expressed genes were screened using high-throughput sequencing technology. The results showed that the LCN2 gene in the brain of mice with aggressive behavior induced by mental stress was significantly upregulated (P<0.01).

[0036] (2) Gene regulation experiments: Adeno-associated virus expression system: AAV-LCN2 overexpression and interference virus vectors were designed and injected into the CeA and PAG brain regions of experimental mice using brain stereotaxic technology.

[0037] Experimental treatment: After virus injection, wait 3 weeks to ensure stable gene expression. Behavioral tests showed that LCN2 overexpression significantly enhanced the aggressive behavior of mice, while the aggressive behavior of mice with LCN2 expression interference was significantly reduced.

[0038] (3) Expression verification: Western blot and qPCR: The protein and mRNA levels of LCN2 in brain tissue were detected. The results showed that mouse models with high and low expression of LCN2 were successfully constructed.

[0039] cFOS activity analysis: The cFOS staining technique was used to detect neuronal activation in the brain after behavioral experiments, and it was found that the activation in the CeA and PAG regions of the LCN2 overexpression group was significantly enhanced.

[0040] Example 3: Exploring the downstream signaling pathway of LCN2; (1) Immunoprecipitation to screen binding proteins: The brain tissue of the LCN2 high-expressing mice in Example 2 was used to screen the possible binding proteins of LCN2 by immunoprecipitation technology combined with protein spectrum. The analysis results showed that LCN2 may bind to the inflammatory factor IL-6 receptor (IL-6R) and a specific subtype of the neurotransmitter GABA receptor.

[0041] (2) In vitro functional verification: Cell line validation: Co-express LCN2 and candidate downstream molecules (such as IL-6R or GABA receptor) in neuronal cell lines and validate direct interactions by FRET.

[0042] Signaling pathway analysis: Western blot was used to detect the activation levels of downstream molecules (such as STAT3 and NF-κB). The results showed that LCN2 promoted the phosphorylation of STAT3 through IL-6R, thereby activating the inflammatory pathway.

[0043] (3) In vivo functional verification: CRISPR / Cas9 was used to knock out the IL-6R gene in mice and detect changes in aggressive behavior. The results showed that IL-6R knockout significantly inhibited aggressive behavior and reduced neuronal activation in the PAG region.

[0044] Example 4: Screening of drug targets based on the LCN2 mechanism; (1) Screening of candidate compounds: Molecular docking technology was used to screen candidate compounds that could inhibit LCN2 or block the LCN2-IL-6R interaction. A total of 120 compounds were initially screened, and compound A, compound B, and compound C were selected through the LCN2 binding activity test.

[0045] (2) In vitro functional verification: LCN2 expression detection: Compounds A, B, and C were added to detect the expression level of LCN2 and its binding ability with downstream signaling molecules. Compound A can significantly inhibit LCN2 expression at a concentration of 10µM.

[0046] Synaptic function test: In the co-culture neuron model, the effects of the compounds on neuronal synaptic activity were tested, and the results showed that compound A reduced the hyperexcitability of neurons.

[0047] (3) In vivo validation Behavioral test: Compound A was injected into mice with aggressive behavior induced by mental stress, and it was found that the aggressive behavior was significantly attenuated. At the same time, cFOS staining showed that the activation of CeA and PAG was reduced.

[0048] Drug metabolism analysis: The half-life and brain distribution of compound A were determined, and the results showed that it could quickly cross the blood-brain barrier and maintain a stable concentration for a long time.

[0049] Example 5: Drug development for mental illness; Compound A was used to develop a new anti-aggression drug for the treatment of mania and bipolar disorder. An oral sustained-release formulation was designed and validated in a mouse model of psychiatric disease.

[0050] (1) Verification of treatment effect The drug significantly improved the aggressive behavior of mice and had a regulatory effect on abnormal mood swings in the mania model.

[0051] (2) Long-term toxicity test After 90 consecutive days of medication, no significant damage was found to the main organ functions (liver, kidney, and heart) of mice.

[0052] Example 6: Application of LCN2 mechanism in other psychiatric diseases; Compound A was applied to post-traumatic stress disorder (PTSD) and depression mouse models. The results showed: In the PTSD model, compound A significantly reduced excessive defensive behavior and stress-activated inflammatory levels.

[0053] In a depression model, compound A reduced despair behavior in mice (reduced immobility time in the forced swim test).

[0054] Summary: The above examples describe in detail the entire process from model establishment, LCN2 function research, downstream pathway exploration, drug screening to application, further expanding the potential application of LCN2 in various psychiatric diseases and providing a scientific basis for related drug research and development.

[0055] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in the field. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for screening drug targets based on the molecular mechanism of LCN2, characterized in that: The following steps are involved: Establish a mouse model of mental stress-induced aggressive behavior; Identify the role of LCN2 in mental stress-induced aggressive behavior; Explore the downstream signaling pathways of LCN2; Screening of potential drug targets based on LCN2-related mechanisms; The effectiveness of potential drug targets was verified through in vitro and in vivo experiments.

2. A method for screening drug targets based on the molecular mechanism of LCN2 according to claim 1, characterized in that: The method for establishing a mouse model of mental stress-induced aggressive behavior comprises: Using chronic unpredictable stress to impose mental stress on experimental mice; Aggression in mice was recorded by standardized behavioral tests, including social interaction test and aggression potential test; Mice showing significantly enhanced aggressive behavior were screened to establish a stable aggressive behavior model.

3. A method for screening drug targets based on the LCN2 molecular mechanism according to claim 1, characterized in that: The method for identifying the role of LCN2 in mental stress-induced aggressive behavior comprises: High-throughput sequencing technology was used to screen genes differentially expressed in the brains of mice with significantly enhanced aggressive behavior after mental stress, with a focus on LCN2. Adeno-associated virus was used to upregulate or downregulate the expression level of LCN2 in the mouse brain and the effect on aggressive behavior was evaluated; Behavioral tests were used to verify the correlation between changes in LCN2 expression and the intensity of aggressive behavior.

4. A method for screening drug targets based on the LCN2 molecular mechanism according to claim 3, characterized in that: The method for verifying the effect of LCN2 in mental stress-induced aggressive behavior includes: qPCR and Western blot techniques were used to detect the mRNA and protein expression levels of LCN2 in different brain regions after mental stress; cFOS staining was used to analyze the activation of brain regions related to aggressive behavior induced by mental stress, including the central nucleus of the amygdala CeA and the periaqueductal gray matter PAG; Zonal analysis was performed in different subregions of the PAG to clarify the spatial relationship between LCN2 expression and brain region activation.

5. A method for screening drug targets based on the LCN2 molecular mechanism according to claim 1, characterized in that: The method for exploring the LCN2 downstream signaling pathway comprises: The downstream molecules that may be regulated by LCN2 were screened by co-immunoprecipitation combined with protein profiling; Predict the interaction relationship between LCN2 and the screened signaling molecules; The direct interaction between LCN2 and downstream signaling molecules was verified by in vitro molecular interaction experiments; Adeno-associated virus or CRISPR technology is used to target and regulate the expression levels of downstream molecules to evaluate their effects on aggressive behavior.

6. A method for screening drug targets based on the LCN2 molecular mechanism according to claim 5, characterized in that: The downstream signaling molecules include, but are not limited to, inflammatory factors, neurotransmitter regulatory molecules, or metabolism-related signaling molecules.

7. A method for screening drug targets based on the molecular mechanism of LCN2 according to claim 1, characterized in that: Methods for screening potential drug targets based on LCN2-related molecular mechanisms include: Screen candidate compounds that interfere with LCN2 expression and downstream signaling molecule activity through a drug screening platform; Test the intervention effects of candidate compounds on downstream key molecular functions and mental stress-induced aggressive behavior models; Comprehensively evaluate the selectivity, efficacy and improvement effect on aggressive behavior of candidate compounds.

8. A method for screening drug targets based on the molecular mechanism of LCN2 according to claim 7, characterized in that: The in vitro validation method of the drug target comprises: Testing candidate compounds for their regulatory effects on LCN2 expression and activity in in vitro cell models; Detect the regulatory effect of compounds on molecules related to downstream signaling pathways; Neuronal co-culture models were used to investigate the effects of compounds on synaptic activity and neuronal excitability.

9. A method for screening drug targets based on the LCN2 molecular mechanism according to claim 7, characterized in that: The in vivo verification method of the drug target comprises: Testing the therapeutic efficacy of candidate compounds in a mouse model of mental stress-induced aggression; Using brain region-specific gene regulation technology to evaluate the brain region-specific effects of compounds; Detect the effects of compounds on behavioral indicators and neural circuit activation levels.

10. The method for screening drug targets based on the LCN2 molecular mechanism according to claim 1, characterized in that: The application of the method for screening drug targets based on the molecular mechanism of LCN2 includes but is not limited to: Research and development of drugs related to mental illness; Develop targeted therapeutic drugs for aggressive behaviors induced by mental stress; Study the potential role of LCN2-related neural regulatory mechanisms in other psychiatric disorders.

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