Application of NR4A1 gene expression inhibitor in preparation of medicine for treating wine-dependent withdrawal

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an expression inhibitor of an NR4A1 gene in preparation of a medicine for treating wine-dependent withdrawal. It is found that after wine-dependent withdrawal, mice have anxiety behaviors, the NR4A1 level of the prefrontal lobe is increased, and apoptosis-related protein in the prefrontal lobe is increased. Knockout of the forehead NR4A1 gene intervenes the expression level of the NR4A1 gene in the forehead lobe of the mouse, it can be proved that down-regulation of the expression of the forehead lobe NR4A1 gene can rescue apoptosis and damage, caused by alcohol, of cortical neurons of the forehead lobe, relieve anxiety behaviors caused by alcohol withdrawal of the mouse, and have the effect of protecting the brain of the mouse; the inhibitor DIM-C-pPhOH of the NR4A1 protein can reduce the expression of the NR4A1 gene of the prefrontal lobe of an alcohol withdrawal mouse and anxiety-like behaviors caused by alcohol withdrawal, and provides related therapeutic targets and new therapeutic drugs for anxiety dysfunction caused by clinical alcohol dependence and alcohol dependence withdrawal.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of an NR4A1 gene expression inhibitor in the preparation of a drug for treating alcohol dependence withdrawal. Background Art

[0002] Alcohol dependence (AD) is a chronic recurrent mental illness caused by long-term drinking. It is also called alcohol addiction. It is characterized by compulsive alcohol seeking, continuous or regular drinking. Its core symptoms include physical dependence with increased alcohol tolerance and withdrawal reactions, and psychological dependence with craving for alcohol. The number of people suffering from alcohol dependence has reached 140 million worldwide. Alcohol dependence seriously threatens individual health, family happiness and social stability. Alcohol dependence is characterized by chronicity and recurrence. Withdrawal symptoms are the core symptoms of alcohol dependence and the main reason for failure to quit drinking. Alcohol-dependent patients will have reduced tolerance for drinking, leading to physiological dependence, and thus obvious irritability, restlessness, hand tremors, reduced sleep, depression, anxiety, etc. Alcohol dependence withdrawal can lead to negative emotional states and abnormal behaviors, including anxiety and depression. These mental illnesses, as negative reinforcers, will promote relapse of drinking. Anxiety caused by alcohol dependence withdrawal is a key issue in the treatment of alcohol dependence.

[0003] The prefrontal cortex (PFC) is one of the important links in the emotional central pathway. Anxiety can cause structural degeneration and functional damage to the prefrontal cortex. Studies on humans and animal models have shown that difficulties in drinking and emotional interruption during alcohol withdrawal are related to the prefrontal cortex. It has been found that the prefrontal cortex can regulate functions related to alcohol dependence and anxiety, and it is a brain area that is very susceptible to the effects of chronic alcohol. The effect of alcohol-induced anxiety is more obvious in adolescents. Studies have found that intermittent drinking during adolescence can cause changes in the excitability of the prefrontal cortex, and changes in histone methylation, which are likely to cause some persistent mental illnesses and behavioral abnormalities in adulthood. These studies all suggest that anxiety dysfunction caused by alcohol withdrawal is closely related to the prefrontal cortex.

[0004] Nuclear receptor NR4A1 (Nuclear receptor subfamily 4, group A-1, NR4A1) is an important member of the NR4A family, and it is involved in the regulation of cell proliferation and apoptosis. B-cell lymphoma / leukemia-2 (BCL-2) gene was first discovered as an oncogene in the study of B-cell lymphoma, which can inhibit cell apoptosis. BCL-2 family members play a very critical role in the regulation of cell apoptosis. In recent years, the study of non-genomic activity of NR4A1 has received increasing attention. It is mainly manifested in that NR4A1 can directly bind to anti-apoptotic BCL-2, thereby converting it into a pro-apoptotic protein. In response to certain apoptotic stimuli, NR4A1 can translocate from the cell nucleus to the outer membrane of the mitochondria and bind to BCL-2, thereby causing cell apoptosis. Therefore, NR4A1 and BCL-2 play a very important role in regulating cell apoptosis and proliferation. However, the prior art does not disclose the role of NR4A1 gene in the treatment of alcohol dependence. Summary of the invention

[0005] In order to solve the defect that the role of NR4A1 gene in treating alcohol dependence is not disclosed in the prior art, the present invention provides a new use of NR4A1 gene, specifically relates to the use of an expression inhibitor of NR4A1 gene in the preparation of a drug for treating alcohol dependence withdrawal, and provides a new therapeutic drug for the clinical treatment of alcohol dependence and anxiety dysfunction caused by alcohol dependence withdrawal.

[0006] In some embodiments of the present invention, the NR4A1 gene expression inhibitor includes: interfering RNA targeting NR4A1 gene expression or an NR4A1 protein expression inhibitor.

[0007] In the present invention, by knocking out the forebrain NR4A1 gene or intervening the expression level of the NR4A1 gene and NR4A1 protein in the prefrontal lobe of mice with an NR4A1 protein expression inhibitor, it can be proved that down-regulating the expression of the prefrontal NR4A1 gene and NR4A1 protein can rescue the apoptosis and damage of prefrontal cortical neurons caused by alcohol, alleviate the anxiety-like behavior caused by alcohol withdrawal in mice, and has the effect of protecting the mouse brain.

[0008] In some embodiments of the present invention, the DNA sequence of the interfering RNA is shown as SEQ ID NO:1.

[0009] SEQ ID NO: 1: 5'-TCGAGGACTTCCAGGTGTA-3'.

[0010] In some embodiments of the present invention, the NR4A1 protein expression inhibitor comprises DIM-C-pPhOH.

[0011] The present invention also provides a medicine for treating alcohol dependence withdrawal, wherein the medicine comprises the expression inhibitor of the NR4A1 gene.

[0012] In some embodiments of the present invention, the medicament is used to relieve anxious behavior in a patient.

[0013] In some embodiments of the present invention, the content of the NR4A1 gene expression inhibitor in the drug is 1w% to 99w%.

[0014] In some embodiments of the present invention, a pharmaceutically acceptable excipient is also included.

[0015] In some embodiments of the present invention, the excipients are selected from one or more of propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0016] In some embodiments of the present invention, the drug is an oral preparation or an injectable preparation.

[0017] In some embodiments of the present invention, the drug is used to reduce the expression of apoptosis protein in the patient's prefrontal cortex.

[0018] In some embodiments of the present invention, the medicament is used to reduce apoptosis of prefrontal neurons in a patient.

[0019] In some embodiments of the present invention, the medicament is used to reduce damage to prefrontal neurons in a patient.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention found that after alcohol dependence withdrawal, mice showed anxiety-like behavior, and the expression level of NR4A1 gene increased, which could reverse the anti-apoptotic function of BCL-2, leading to an increase in BCL-2 and apoptosis-related proteins in the prefrontal lobe. Knocking out forebrain NR4A1 or intervening with the NR4A1 protein expression inhibitor DIM-C-pPhOH (CDIM8) in the expression level of NR4A1 gene and NR4A1 protein in the prefrontal lobe of mice can prove that down-regulating the expression of NR4A1 in the prefrontal lobe can rescue the apoptosis and damage of prefrontal cortical neurons caused by alcohol, alleviate the anxiety-like behavior caused by alcohol withdrawal in mice, and protect the mouse brain, providing relevant therapeutic targets and new therapeutic drugs for clinical alcohol dependence and anxiety dysfunction caused by alcohol dependence withdrawal. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram of the gene identification results of knockout mice; Figure 1 A in the figure indicates the identification result of mouse NR4A1 gene with or without Loxp site; Figure 1 B in the figure indicates the identification result of whether the mice have the Camk2a-Cre gene or not.

[0022] Figure 2 The results of knocking out mouse protein and three groups of CPP modeling; among them, Figure 2 A in the figure is the result of protein detection in specific knockout mice, n=3; Figure 2 B in the figure is the CPP test results of mice in the control group, alcohol treatment group and knockout group; Con represents saline control group + 16h withdrawal, n = 10; EtOH represents 2g / kg / day alcohol injection group + 16h alcohol withdrawal, n = 10; KO-EtOH represents NR4A1 forebrain-specific knockout + 2g / kg / day alcohol injection group + 16h alcohol withdrawal, n = 8; values ​​are expressed as mean ± standard error Indicates, * indicates P<0.05, ns indicates P>0.05.

[0023] Figure 3 This is a graph showing the results of the mouse behavioral experiment analysis; Figure 3 A in the figure represents the discrimination index result graph of the novel object recognition experiment; Figure 3 B in the figure represents the number of times the new arm is entered in the Y-maze experiment; Figure 3 CE in the figure represent the time of entering the central area, the number of times entering the central area and the total movement distance in the open field experiment; Figure 3 The F in the figure represents the result of shuttling times in the black-white box experiment; Figure 3 GH in the figure represent the number of entries into the open arm and the time spent in the open arm of the elevated plus maze.

[0024] Figure 4 This is the result of some behavioral trajectory of mice; among them, Figure 4 A in the figure represents the movement trajectory of mice in the open field experiment; Figure 4 B in the figure shows the movement trajectory of mice in the elevated plus maze experiment.

[0025] Figure 5 is the result diagram of real-time fluorescence quantitative analysis; Figure 5 A in the figure represents the results of real-time fluorescence quantitative analysis of NR4A1 in three groups of mice; Figure 5 B in the figure represents the results of real-time fluorescence quantitative analysis of BCL-2 (B lymphocytoma / leukemia-2) in three groups of mice; Figure 5 C in the figure shows the results of real-time fluorescence quantitative analysis of BAX in three groups of mice.

[0026] Figure 6 The expression results of NR4A1 and apoptosis-related proteins in the prefrontal cortex of alcohol-dependent withdrawal mice are shown in Figure 2. Figure 6 A in the figure represents the WB results of the prefrontal lobes of the three groups of mice; Figure 6 B in the figure shows the results of semi-quantitative WB analysis of 3 groups of mice; Figure 6 C in the figure represents the BCL-2 result graph; Figure 6 D in the figure indicates the BAX result diagram; Figure 6 E in the figure represents the result of Cleaved-Caspase-3; n = 3, values ​​are expressed as mean ± standard error Indicates, * indicates P<0.05, ** indicates P<0.01.

[0027] Figure 7 The results of TUNEL staining and Nissl staining are shown in Figure 2. Figure 7 A in the figure shows the results of TUNEL staining, the magnification is 200X, and the scale bar is 100 μm; Figure 7 B in the figure shows the statistical results of Nissl staining and the number of neurons, with a magnification of 400X and a scale of 50 μm; the values ​​are expressed as mean ± standard error indicates, n=3, * indicates P<0.05.

[0028] Figure 8 Figure 2 is the result of immunofluorescence; Figure 8 A in the figure represents the 200X prefrontal immunofluorescence result; Figure 8 B in the figure shows the semi-quantitative analysis result of the number of co-stained positive cells, with a magnification of 200X.

[0029] The scale bar is 50 μm, and the values ​​are expressed as mean ± standard error. indicates, n=3, * indicates P<0.05, ** indicates P<0.01.

[0030] Fig. 9 Figure 1 is the result of immunoprecipitation; IgG is the result of negative control group, Input is the result of positive control group, and IB is the result of protein blotting.

[0031] Fig.10 This is the WB result of the prefrontal cortex of alcohol withdrawal mice treated with inhibitors and the behavioral statistical results of mice; Fig.10 A in the figure represents the WB results of the mouse prefrontal cortex; Fig.10 B in the figure represents the total distance traveled by the two groups in the open field of mice; Fig.10 C in the figure represents the distance that the two groups moved in the center area of ​​the mouse open field; Fig.10 D represents the number of times the two groups of mice shuttled in the central area; Fig.10 E in the figure indicates the number of shuttling between the black and white boxes by the two groups of mice; Fig.10F in the figure represents the number of entries of the two groups of mice into the open arms of the elevated plus maze; Fig.10 G in the figure represents the time that the two groups of mice stayed in the open arm of the elevated plus maze. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] Alcohol dependence (AD) is a chronic relapsing mental illness caused by long-term drinking, also known as alcohol addiction, which is manifested by compulsive alcohol seeking, continuous or regular drinking. Its core symptoms include physical dependence with increased alcohol tolerance and withdrawal reactions, and psychological dependence with craving for alcohol. Withdrawal from alcohol dependence can lead to negative emotional states and abnormal behaviors, including anxiety and depression. These mental illnesses, as negative reinforcers, can promote relapse of drinking. Anxiety caused by withdrawal from alcohol dependence is a key issue in the treatment of alcohol dependence.

[0034] Nuclear receptor NR4A1 (Nuclear receptor subfamily 4, group A-1, NR4A1) is an important member of the NR4A family, and it is involved in the regulation of cell proliferation and apoptosis. B-cell lymphoma / leukemia-2 (B-cell lymphoma / leukemia-2, BCL-2) gene was first discovered as an oncogene in the study of B-cell lymphoma, which can inhibit cell apoptosis. NR4A1 can directly bind to anti-apoptotic BCL-2, thereby converting it into a pro-apoptotic protein. In response to certain apoptotic stimuli, NR4A1 can translocate from the nucleus to the outer membrane of mitochondria and bind to BCL-2, thereby causing cell apoptosis. Therefore, NR4A1 and BCL-2 play a very important role in regulating cell apoptosis and proliferation. However, the role of NR4A1 gene in the treatment of alcohol dependence is not disclosed in the prior art.

[0035] The present invention provides an application of an NR4A1 gene expression inhibitor in the preparation of a drug for treating alcohol dependence and withdrawal. Knocking out forebrain NR4A1 or intervening with the NR4A1 protein expression inhibitor DIM-C-pPhOH (CDIM8) on the expression levels of the NR4A1 gene and NR4A1 protein in the prefrontal lobe of mice can prove that down-regulating the expression of NR4A1 in the prefrontal lobe can rescue the apoptosis and damage of prefrontal cortical neurons caused by alcohol, relieve the anxiety-like behavior caused by alcohol withdrawal in mice, and protect the mouse brain, providing relevant therapeutic targets for clinical alcohol dependence and anxiety dysfunction caused by alcohol dependence and withdrawal.

[0036] 1. Experimental Materials

[0037] 1. Experimental Animals

[0038] (1) The present invention uses the Loxp-Cre system to create forebrain-specific knockout mice of the NR4A1 gene (Gene ID: 15370) (knockout of the gene in brain regions such as the cortex and hippocampus), and purchases NR4A1f from Saiye Biotechnology. lox / flox The mice and CamK2a-cre mice were crossed to obtain the first generation of offspring, and the genotype of NR4A1 was screened from the first generation of offspring. flox / - -CamK2a-cre animals, the genotype animals were then mated and bred, and finally the genotype of NR4A1 was screened out flox / flox -CamK2a-cre animals, namely NR4A1 forebrain-specific knockout mice. A sufficient number of knockout mice have been obtained, and a conditioned place preference alcohol dependence mouse model has been established to prepare for behavioral and molecular expression testing after withdrawal.

[0039] (2) The present invention uses 20 SPF-grade male C57BL / 6 mice, aged 7-8 weeks, purchased from Spaif Biotechnology Co., Ltd. The entire experiment was completed in the Henan Provincial Key Laboratory of Biological Psychiatry. The experimental mice were raised individually in an animal room with a ventilation system, so that the mice were in an alternating light and dark environment for 12 hours each day, the temperature was set at 22°C ± 2°C, and the relative humidity was set at 57% ± 10%. The mice can move freely in the cage and can freely eat feed and water. This experiment was approved by the Animal Ethics Committee of the Henan Provincial Key Laboratory of Biological Psychiatry (Ethics Number: HMH.No20220727AEC001).

[0040] (3) Twenty SPF male C57BL / 6 mice were equally divided into a control group (Con, n = 10) and an alcohol dependence group (EtOH, n = 10). Eight male mice with forebrain (mainly including the prefrontal lobe and hippocampus) specific knockout of the NR4A1 gene (KO-EtOH) were selected. The mice in the EtOH and KO-EtOH groups were intraperitoneally injected with 20% alcohol solution (v / v) at a dose of 2 g / kg / d; the Con group was intraperitoneally injected with an equal volume of normal saline. The three groups of mice were all used to establish the alcohol dependence model using conditioned place preference.

[0041] 2. Reagents

[0042] Table 1 Reagents

[0043]

[0044]

[0045] 3. Experimental instruments

[0046] Table 2 Instruments

[0047] instrument factory Cryostat Leica Corporation ELISA reader Thermo Fisher Scientific (China) Co., Ltd. Upright microscope Nikon Corporation Smart3.0 Image Acquisition and Analysis System Spanish Panlab SIGMA SDS-PAGE gel electrophoresis apparatus Thermo Fisher Scientific (China) Co., Ltd. Conditioned Place Preference Box Shanghai Xinruan Technology Co., Ltd. Open Field Test Chamber Shanghai Xinruan Technology Co., Ltd. Fluorescence quantitative PCR instrument Thermo Fisher Scientific (China) Co., Ltd. Amersham Imager 600 (AI600) GE

[0048] 4. Main solution

[0049] (1) 1× PBS: Take 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4, and dissolve them in 800 mL of ultrapure water. Then make up to 1 L with ultrapure water, adjust the pH to 7.2, and store in a sealed container at room temperature. Prepare it before use.

[0050] (2) 4% paraformaldehyde: Take 40 g of paraformaldehyde, add it to 500 mL of 1×PBS solution and heat it to about 60°C. Stir it with a magnetic stirrer and add a few drops of NaOH until the solution becomes clear. After the paraformaldehyde is dissolved, the solution should be cooled and filtered. Use 1×PBS to make the solution 1 L. Measure the pH of the solution with a pH meter, add concentrated hydrochloric acid to adjust the pH of the solution to 7.2, and store it at room temperature.

[0051] (3) 5% skim milk powder: Take 2.5 g of skim milk powder and add 1× TBST solution to make up to 50 mL. Shake well and store in a sealed container at 4°C. Prepare before use.

[0052] (4) 10× electrophoresis buffer: Weigh 10 g of SDS, 30.3 g of Tris base, and 144.4 g of glycine, dissolve them in 800 mL of ultrapure water, mix well, and make up to 1 L with ultrapure water. Store in a sealed container at room temperature.

[0053] (5) 1× electrophoresis working solution: Take 100 mL of 10X electrophoresis solution, add 900 mL of ultrapure water, shake well, seal and store at room temperature, and prepare before use.

[0054] (6) 10× electroporation working solution: Weigh 30.3 g of Tris base and 144.4 g of glycine, dissolve them in 800 mL of ultrapure water, mix well, and make up to 1 L with ultrapure water. Store in a sealed container at room temperature.

[0055] (7) 1× electrotransfer working solution: Take 100 mL of 10× electrotransfer working solution, add 700 mL of ultrapure water and 200 mL of methanol, shake well, and store in a sealed container at 4°C. Prepare before use.

[0056] (8) 10× TBST buffer: Take 80 g of NaCl, 2 g of KCl, and 30 g of Tris base, add them to ultrapure water, and after they are completely dissolved, make up to 1,000 mL with ultrapure water. Adjust the pH to 7.4 with concentrated hydrochloric acid and store in a sealed container at room temperature.

[0057] (9) 1× TBST buffer: Take 100 mL of 10× TBS buffer, add 900 mL of ultrapure water, and then add 1 mL of Tween and mix thoroughly. Store in a sealed container at room temperature and prepare before use.

[0058] (10) 20% (v / v) alcohol solution: Place 2.1 mL of 95% pharmaceutical grade alcohol and 7.9 mL of 0.9% saline in a centrifuge tube. Mix well and store at room temperature. Prepare before use.

[0059] Embodiment 1:

[0060] 1. Preparation of CPP model:

[0061] The conditioned place preference box is a common experimental instrument used to evaluate alcohol dependence and drug abuse. It establishes a connection between drugs and the environment to determine whether the animal has formed a preference for the environment. The first is the pre-adaptation period (D1-D3): From the 1st to the 3rd day, the mice were first placed in the black and white box, and then the partition between the two chambers was removed, and the 3 groups of mice were allowed to shuttle between the black and white boxes for a total of 3 days, 15 minutes a day. Screening period (D4): After the pre-adaptation period, 28 C57BL / 6 mice that naturally preferred the black box were screened and divided into 3 groups, namely the control group (Con, n=10), the alcohol dependence group (EtOH, n=10) and the knockout group (KO-EtOH, n=10). Adaptation period (D5-D19): From the 5th to the 19th day, the mice in the EtOH group and the KO-EtOH group were intraperitoneally injected with 20% alcohol solution every day, with an injection dose of 2g / kg / d, and the mice in the Con group were intraperitoneally injected with an equal volume of 0.9% saline every day. Adaptation period (D20): On the 20th day, the partition was opened to allow the three groups of mice to shuttle between the black and white boxes for 15 minutes. Baseline value determination (D21): After the adaptation, the Smart Version3.0 trajectory image analysis system was used to track the activities of the experimental mice in the two boxes, and the recording time was 15 minutes. Training period (D22-D29): On the 22nd day, the mice in the EtOH group and the KO-EtOH group were injected with alcohol on the white box side in the morning and with saline on the black box side in the afternoon. On the 23rd day, the mice in the EtOH group and the KO-EtOH group were injected with saline on the black box side in the morning and with alcohol on the white box side in the afternoon, and so on. The treatment time and method of the Con group mice were consistent with the other two groups, and it was only necessary to ensure that the Con group mice were given an equal volume of saline each time. Test period (D30): On the 30th day, at the beginning of the test, all three groups of mice were first placed in the white box, facing the same direction, and recorded for 15 minutes using the Smart Version3.0 trajectory image analysis system.

[0062] 2. Animals treated with NR4A1 inhibitor CDIM8

[0063] C57BL / 6J male mice aged 7-8 weeks were divided into two groups: a control group and an inhibitor group, with 10 mice in each group. According to the experimental protocol, an alcohol-dependent mouse model was established, and both groups of mice were intraperitoneally injected with 20% alcohol (v / v) at a dose of 2g / kg / day. After successful modeling, the inhibitor group was gavaged with 50mg / kg of NR4A1 protein expression inhibitor DIM-C-pPhOH (CDIM8) suspension, and the control group was gavaged with normal saline, and the gavage time was 1 week (during the treatment, intraperitoneal injection of alcohol was continued). The inhibitor intervened for 1 week, and after 16h of alcohol withdrawal, the anxiety-like behavior of the mice was tested by open field, cross elevated maze and black and white box.

[0064] 3. Mouse genotype identification

[0065] At 20 days old, the mouse tails were cut with scissors to 3-5 mm in length, numbered and marked, and then separated into cages after the gene identification was completed. The specific methods for cleaving the mouse tails and extracting DNA are as follows:

[0066] (1) Lyse the rat tail as follows:

[0067] First, add 50 μL Tris-HCl, 5 μL 0.5 M EDTA (pH 8.0), 10 μL SDS (10%), 20 μL 5 M NaCl, 5 μL proteinase K (10 mg / mL) and 410 μL ultrapure water to a 1.5 mL EP tube, mix them in proportion, and place them in a constant temperature drying oven at 55°C for overnight digestion until the rat tail in the EP tube is observed to be completely dissolved.

[0068] (2) Purify mouse tail DNA as follows:

[0069] 1) Centrifugation: Turn on the centrifuge, set to 4°C, speed to 13,000 rpm, time for 10 minutes, then take the supernatant and place it in a new 1.5 mL EP tube;

[0070] 2) Add an equal volume of isopropanol to each EP tube (500 μL per tube) and shake well at room temperature;

[0071] 3) Centrifugation: set to 4°C, 13,000 rpm, 10 minutes, then discard the supernatant;

[0072] 4) Add 75% alcohol and wash twice;

[0073] 5) Centrifugation: 4°C, 13,000 rpm, 2 min, then discard the supernatant;

[0074] 6) Place in a fume hood and let dry for 30 minutes;

[0075] 7) Add 120 μL ultrapure water to dissolve, and mix well by pipetting with a 200 μL pipette.

[0076] (3) Measure DNA concentration

[0077] 1) Turn on the UV spectrophotometer;

[0078] 2) Firstly, use ultrapure water to adjust the zero value;

[0079] 3) Then add 1 μL of DNA and use a UV spectrophotometer to detect the DNA mouse concentration.

[0080] 3. PCR reaction

[0081] PCR is a molecular technique used to amplify DNA fragments.

[0082] (1) The DNA template used in this experiment is mouse tail DNA extracted by the above method.

[0083] (2) The PCR system used in this experimental method is 10 μL, and the required gene identification primers are shown in Table 3 as SEQ ID NO: 2 to SEQ ID NO: 5.

[0084] Table 3 Gene identification primers

[0085] name sequence serial number Camk2a-F 5'-GTTCTCCGTTTGCACTCAGG-3' SEQ ID NO:2 Camk2a-R 5'-CAGGTTCTTGCGAACCTCAT-3' SEQ ID NO:3 NR4A1-F 5'-CCTGTCATGGATTTTACTGTGCG-3' SEQ ID NO:4 NR4A1-R 5'-CAGCTCCTGGTCTTGATCTACTCA-3' SEQ ID NO:5

[0086] (3) The experimental reaction system is configured as follows:

[0087] PCR reaction system (10 μL system): 2X Bio Gold TaqPlus PCR Mix, 5.0 μL; 10 μM F1, 0.5 μL; 10 μM R1, 0.5 μL; sterile enzyme-free water, 3.0 μL; mouse tail DNA, 1.0 μL.

[0088] (4) The reaction procedure is as follows:

[0089] 95°C, 5 min, 1.0 cycle; 95°C, 30 s, 1.0 cycle; 60°C, 30 s, 30 cycles; 72°C, 40 s, 30 cycles; 72°C, 20 min, 1.0 cycle, 4.0°C, store.

[0090] 4. Agarose gel electrophoresis

[0091] (1) Preparation: Prepare appropriate electrophoresis gel molds and gel combs and install them.

[0092] (2) Gel preparation: Take 1.5 g of agarose and place it in a 75 mL Erlenmeyer flask. Add 1X TAE buffer and heat the prepared gel in a microwave oven until large bubbles are observed.

[0093] (3) Take the gel out of the microwave, add an appropriate amount of nucleic acid dye, shake well, and pour it into the gel mold. Wait for the gel to solidify for about 30 minutes.

[0094] (4) Sample loading: Place the prepared gel in the electrophoresis tank, first load 5 μL of DNAMaker, and then add 10 μL of the above PCR products into the gel wells one by one.

[0095] (5) Start running the gel: First, connect the gel tank to the electrophoresis instrument, then turn on the electrophoresis instrument and set the voltage to 150 V. Electrophoresis begins when bubbles are seen moving.

[0096] (6) Stop running the gel: When the band is observed to run 2 / 3 of the way through the gel, stop running the gel and observe the results.

[0097] 5. Behavioral assessment methods

[0098] The three groups of mice were subjected to alcohol withdrawal for 16 hours after the CPP model test. The cognitive impairment of the mice was first detected using novel object recognition and Y-maze, and then the anxiety-like behavior of the mice was detected using black-and-white box, open field and elevated plus maze behavioral tests.

[0099] (1) New Object Recognition (NOR)

[0100] The novel object recognition experiment is mainly used to test the learning and memory ability of mice. This experiment is mainly divided into two periods. During the familiarization period, two identical objects (A and B) are placed in the device, and the mice are gently placed in the device for 5 minutes to get familiar with it. Each time, the mice are placed facing the same direction. The test period usually starts 2 hours after the end of the familiarization period. One of the two identical objects (A or B) is replaced with a new object (C), and the mice are placed facing the same direction. The mice are allowed to adapt for 30 seconds, and the experiment is recorded for 5 minutes.

[0101] (2) Y-maze Test

[0102] The Y-maze experiment is used to test the learning and spatial memory abilities of mice. The experiment is mainly divided into two periods. During the adaptation period, the novel arm is first closed, and the mice are placed in the starting arm facing the same direction, and the mice are allowed to move around in it for 10 minutes. During the test period, 2 hours after the adaptation period, the novel arm of the Y-maze is opened, and the mice are placed in the starting arm facing the same direction. The test is recorded for a total of 5 minutes and 30 seconds, with the first 30 seconds being the adaptation time for the mice and the last 5 minutes being the test time.

[0103] (3) Light / Dark Box Test (LDB)

[0104] In the black and white box, mice instinctively like to move in the black box, but their natural exploratory habits will prompt them to explore the white box. At the beginning of the experiment, the mice were gently placed in the black and white box, and each time the mice were placed in the white box, they faced the same direction. Let the mice adapt for 30 seconds. After that, the number of times they entered the white box and the time they stayed in the white box were recorded, and the total observation and recording time was 15 minutes. After each mouse was tested, the black and white box was wiped. The relevant data of the mice in the black and white box was recorded and analyzed after the experiment. The more times the mice shuttled between the black and white boxes and the longer they stayed in the white box, the lower their anxiety level.

[0105] (4) Open Field Test (OFT)

[0106] OFT is a common method for evaluating the exploratory behavior, depression and anxiety of mice in a novel environment. The experimental device mainly consists of an open field test box (Xinruan, Shanghai) and a Smart 3.0 software image acquisition and analysis system. The mice were placed in the mouse open field test box, and each time they were placed in the central area of ​​the open field with their backs to the experimenter. After that, the experimenter quickly left and let the mice move freely in the test box. Then the video acquisition and data recording system of Smart 3.0 software was quickly turned on to record the activities of the mice in the open field for 5 minutes. After the end, the mice were taken out and put back in the original cage. The open field test box was wiped with alcohol after each test. The test was recorded for a total of 5 minutes and 30 seconds, the first 30 seconds was the adaptation time of the mice, and the last 5 minutes was the test time.

[0107] (5) Elevated Plus-Maze Test (EPMT)

[0108] EPMT is a common experimental device for evaluating anxiety-like behavior in mice. It mainly uses the fear of open arms and exploration of new environments of rodents to evaluate the anxiety of animals. At the beginning of the experiment, the mice were placed in the center of the plus maze each time, facing the direction of the open arms each time. Then, the Smart Version3.0 trajectory image analysis software was opened to record the movement trajectory of the mice. It mainly records the time the mice stayed in the open arms and the number of times they entered the open arms in the elevated plus maze. The test was recorded for a total of 5 minutes and 30 seconds, with the first 30 seconds being the adaptation time of the mice and the last 5 minutes being the test time.

[0109] 6. Collection of Mouse Brain Tissue Samples

[0110] The mice were anesthetized by intraperitoneal injection of isoflurane and then decapitated to remove the brain. A portion of the brain tissue was fully fixed in 4% paraformaldehyde for frozen sections, and the other portion was freshly sampled. The prefrontal cortex, hippocampus, striatum and other brain regions of the mice were respectively obtained and stored in a -80°C refrigerator for the determination of protein and RT-qPCR expression levels.

[0111] 7. Western Blotting

[0112] (1) Extraction of total protein from prefrontal cortex:

[0113] 1) Prepare RIPA lysis buffer: add 20 μL of Na3VO4, 100 μL of NaF and 100 μL of phenylmethylsulfonyl fluoride (PMSF) to 10 mL of RIPA solution to prevent protein degradation during extraction.

[0114] 2) Weigh an appropriate amount of prefrontal lobe tissue and add it to a sterile grinding tube containing magnetic beads, and add RIPA lysis buffer to the grinding tube at a ratio of 1:10.

[0115] 3) Place the grinding tubes symmetrically in the grinder and set the parameters as follows: 4°C, rotation speed 3,500 rpm, running time 10 seconds, interval 10 seconds, and repeat grinding 3 times.

[0116] 4) Place the cells on ice for 30 minutes and transfer the solution into an EP centrifuge tube.

[0117] 5) Centrifugation: Place the fully lysed tissue in a low-temperature centrifuge for centrifugation at 4°C, 12,000 rpm, and 15 minutes. The protein in the supernatant is the total protein extracted.

[0118] (2) Determination of supernatant protein concentration by BCA method

[0119] 1) Prepare a protein standard solution with gradient concentrations: add 1.2 mL of protein standard solution to 30 mg of BSA and fully dissolve it to make a protein standard solution of 25 mg / mL. Then dilute it with 1X PBS to make 8 concentration gradients of protein standards for use.

[0120] 2) Dilute the supernatant of the protein to be tested: Take 3 μL of the protein solution to be tested and add it to 57 μL of PBS diluent to dilute its concentration 20 times.

[0121] 3) Prepare BCA working solution: Take appropriate amount of reagent A and reagent B in the BCA kit according to the volume ratio of reagent A to reagent B of 50:1, then mix thoroughly and set aside.

[0122] 4) Determination of protein concentration: Add 180 μL of the prepared BCA working solution to a 96-well plate, then add 20 μL of the diluted sample to be tested and 20 μL of the prepared protein standard. After adding, place it in a constant temperature box at 37°C for 30 minutes, and then detect it with an ELISA instrument to calculate the protein concentration of the supernatant to be tested.

[0123] 5) Set the target concentration, and then prepare samples of equal concentration and volume, add protein loading buffer, and then divide them into packages. Temporary storage can be placed in a -20℃ refrigerator, and long-term storage can be placed in a -80℃ refrigerator.

[0124] (3) Western blotting

[0125] 1) Prepare 12.5% ​​SDS-PAGE gel according to the instructions of 12.5% ​​SDS-PAGE fast gel preparation kit.

[0126] 2) Sample loading and electrophoresis: The loading amount of total protein for each sample is 20 μg, and the protein molecular weight standard is from Thermo Fisher Scientific (Cat. No. 26616). 80V, 30 minutes; 120V, 60 minutes.

[0127] 3) Transfer: Take a 0.2μm PVDF membrane, cut it into appropriate sizes and activate it with methanol. Take out the gel after electrophoresis and install the transfer clip from negative electrode to positive electrode in the following order: sponge-filter paper-gel-membrane-filter paper-sponge. Add the pre-cooled 1× transfer solution into the electrophoresis tank and place it in the electrophoresis tank. 280mA, 60min. During the transfer process, place the electrophoresis tank in an ice-water mixture to cool it down.

[0128] 4) Blocking: After electroporation, wash the membrane with TBST for 5 minutes and then block with 5% skim milk powder at room temperature for 1 hour.

[0129] 5) Incubate with primary antibody: After blocking, wash the membrane with TBST three times, 10 minutes each time. Then dilute the antibody with primary antibody diluent at an appropriate ratio (the dilution ratio of rabbit anti-NR4A1 antibody (Cat. No. 12235-1-AP), mouse anti-BCl-2 antibody (Cat. No. 66799-1-Ig), mouse anti-BAX antibody (Cat. No. 60267-1-Ig) and rabbit anti-cysteine ​​protease-3 is 1:2000; the dilution ratio of mouse anti-β-actin (Cat. No. 12235-1-AP) is 1:10000), and then cut the PVDF membrane into corresponding strips according to the size of the target protein to be detected. Place the strips in the corresponding primary antibody and incubate overnight at 4°C.

[0130] 6) Incubate with secondary antibody: After the primary antibody incubation is completed, wash 3 times with 1×TBST solution, 10 minutes each time. Then select appropriate rabbit secondary antibody or mouse secondary antibody for incubation according to the source of the primary antibody, place it on a shaker, and incubate at room temperature for 1 hour.

[0131] 7) ECL color development and analysis: After the secondary antibody incubation, wash 3 times with 1×TBST, 10 min each time. Then prepare the luminescent solution according to the ratio of solution A to solution B in a ratio of 1:1. Add about 200 μL of luminescent solution to each membrane to ensure that the strip is completely covered, expose with the AI600 equipment of GE in the United States, and perform corresponding analysis with Image J.

[0132] 9. Co-immunoprecipitation

[0133] (1) Extraction of tissue proteins

[0134] 1) Place the prefrontal lobe tissue in the homogenization tube and add the corresponding amount of IP lysis buffer according to 20 μL IP lysis buffer per mg of prefrontal lobe tissue.

[0135] 2) Place the tissue fragments in a homogenization tube and place in a pre-cooled homogenizer, then homogenize and lyse on ice for 30 minutes.

[0136] 3) Centrifuge at 4°C, 10,000 g for 15 minutes, then pour the supernatant into a new 1.5 mL EP tube for later use and discard the precipitate.

[0137] 4) The total protein concentration of the lysed tissue was determined by the BCA method.

[0138] 5) Determine the concentration of the lysed tissue for subsequent co-immunoprecipitation experiments.

[0139] (3) Immunoprecipitation

[0140] The immunoprecipitation kit (Cat. No. P2197M) of Biotech was used for the analysis. The specific method was described in the instruction manual of the immunoprecipitation kit.

[0141] (4) Elution

[0142] 1) Collect the above product with 1.5 mL EP, and then elute the product again with 40 μL Elution buffer. After that, place it at room temperature for 10 minutes, put it in a centrifuge, set the centrifugation parameters to 4°C, 10,000 rpm, centrifuge for 1 minute, and then repeat the elution once with a new 40 μL Elution buffer. Elute twice in total and collect the eluted products twice.

[0143] 2) Take an appropriate amount of Alkali neutralization buffer and an appropriate amount of 5X Sample buffer and add them to the eluted product. Mix well and heat in a water bath with boiling water for 10 minutes.

[0144] (5) Western Blot analysis

[0145] 1) Add 20 μL of the immunoprecipitation sample to the corresponding wells of the 10% SDS-PAGE gel. The remaining immunoprecipitation sample can be stored in a -80°C refrigerator for later use.

[0146] 2) Separate the immunoprecipitated samples by SDS-PAGE using the same method as western blot.

[0147] 10. RT-qPCR

[0148] (1) Total RNA extraction:

[0149] Preparation: First, turn on the ultra-clean bench UV lamp for 30 minutes in advance, then turn on the low-temperature ultracentrifuge to precool it to 4°C, and take an appropriate amount of ice.

[0150] 1) After the preparation is completed, the prefrontal lobe tissue can be taken out of the -80℃ freezer and placed on ice prepared in advance, and the same amount of tissue can be placed in each grinding tube.

[0151] 2) Working in a fume hood, place 1 mL of Trizol into each grinding tube, then cover and label.

[0152] 3) Turn on the homogenizer and homogenize the prefrontal lobe tissue three times.

[0153] 4) The ground prefrontal lobe tissue was placed on ice for 10 minutes and then transferred to a 1.5 mL EP tube.

[0154] 5) Centrifugation: Set the centrifugation parameters to 4°C, 12,000 rpm, and time to 15 minutes.

[0155] 6) Place the supernatant after centrifugation in new 1.5 mL EP tubes, about 800 μL per tube, then add 160 μL of chloroform to each tube, about 20% of the supernatant volume, manually shake and mix for 15 seconds, let stand at room temperature for 5 minutes, then place in a low-temperature high-speed centrifuge, set the centrifugation parameters to 4°C, 12,000 rpm, and time for 10 minutes (be careful to handle gently).

[0156] (7) After centrifugation, the above liquid is separated into three layers. Use a new 1.5 mL EP tube to collect the clear RNA supernatant, about 280 mL. At the same time, add isopropanol to each tube to a volume of 280 mL, then use a shaker to mix, and leave at room temperature for 10 minutes after mixing.

[0157] 8) Place the mixed EP tube into a centrifuge and set the centrifugation parameters to 4°C, 12,000 rpm, and 5 minutes. A white precipitate can be observed at the bottom of the tube. Discard the supernatant and keep the precipitate for later use.

[0158] 9) Add 1 mL of 75% ethanol to the precipitate to wash the precipitate, gently flick the precipitate, and manually invert the liquid 5 times. Then centrifuge at 4°C, 12,000 rpm for 5 minutes.

[0159] 10) After centrifugation, carefully pour out the ethanol, discard the supernatant, keep the precipitate, and then blow dry the precipitate in a fume hood. Then add 50 μL of sterile enzyme-free water to each tube, and use a 200 μL pipette to mix each tube to fully dissolve the RNA.

[0160] 11) Measure and record the RNA concentration using a UV spectrophotometer.

[0161] (2) Reverse transcription was performed using a kit from Nearshore Products (Cat. No. E047).

[0162] (3) RT-qPCR

[0163] After reverse transcription, the product was diluted 10 times and used for RT-qPCR.

[0164] 1) Prepare the reaction system, see Table 4 for details, and see Table 5 for specific primers.

[0165] Table 4 Real-time fluorescence quantitative PCR reaction system

[0166] Reagent name Dosage (10μL system) PowerUp™ SYBR™ Green Master Mix 5.0μL Primer F1 0.5μL Primer R1 0.5μL cDNA 1.0μL Sterile enzyme-free water 3.0μL

[0167] Table 5 Real-time fluorescence quantitative PCR primers

[0168] Primer name sequence serial number NR4A1-F 5'-CTTCGGCGTCCTTCAAGTTTG-3' SEQ ID NO:6 NR4A1-R 5'-GGCTGGAAGTTGGGTGTAGA-3' SEQ ID NO:7 BCL-2-F 5'-GCTACCGTCGTGACTTCGC-3' SEQ ID NO:8 BCL-2-R 5'-CCCCACCGAACTCAAAGAAGG-3' SEQ ID NO:9 BAX-F 5'-CCGGCGAATTGGAGATGAACT-3' SEQ ID NO:10 BAX-R 5'-CCAGCCCATGATGGTTCTGAT-3' SEQ ID NO:11 GAPDH-F 5'-AATGGATTTGGACGCATTGGT-3' SEQ ID NO:12 GAPDH-R 5'-TTTGCACTGGTACGTGTTGAT-3' SEQ ID NO:13

[0169] 2) Turn on the fluorescence quantitative PCR instrument and run the reaction program, which includes pre-denaturation at 95°C for 5 minutes, one cycle, denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 20 seconds, wherein the denaturation, annealing and extension are repeated for 40 cycles.

[0170] 11. Nissl staining

[0171] (1) Preparation of frozen sections

[0172] First, the brain tissue was fully fixed with 4% paraformaldehyde, and then immersed in 15% sucrose solution, 20% sucrose solution and 30% sucrose solution for gradient dehydration. After dehydration, the residual water on the surface of the brain tissue was gently and fully absorbed with absorbent paper, and the brain tissue was embedded with OCT embedding agent. The generation of bubbles should be reduced during the embedding process. After embedding, it was quickly placed on an iron block surrounded by liquid nitrogen until the OCT embedding agent changed from transparent to white, then taken out and stored in a -80℃ freezer. When frozen sections were made, the tissue was taken out of the -80℃ freezer, and when it was balanced to about -20℃, it could be sliced ​​on the coronal plane on the frozen slicer, and the slice thickness was set to 10μm. Finally, the prepared frozen sections were placed on a 55℃ baking machine, and after 30 minutes, they were removed and placed at 4℃ for storage.

[0173] (2) Nissl staining

[0174] 1) Sample processing: After the frozen sections were restored to room temperature, they were fixed in 4% paraformaldehyde for 10 minutes, washed with distilled water for 2 minutes, and then washed with new distilled water for another 2 minutes.

[0175] 2) Staining: The brain tissue on the slice was circled with an immunohistochemical pen in advance to prevent the staining solution from flowing out. After adding an appropriate amount of Nissl staining solution to the brain tissue, the slice was placed in a wet box, and then the wet box was placed in a 37°C incubator for 50 minutes. After that, the slice was taken out and washed twice with distilled water, each time for 5 seconds, and then immersed in 95% ethanol for 5 seconds.

[0176] 3) Dehydration, transparency and sealing: First, dehydrate the slices with 95% anhydrous ethanol for two times, two minutes each time, then transparentize them in xylene for two times, five minutes each time. Finally, seal the slices with neutral gum. All operations need to be performed in a fume hood. After sealing, the slices can be observed and photographed under an upright microscope.

[0177] 12. Immunofluorescence staining

[0178] (1) Sample processing: After taking out the frozen sections from 4°C, equilibrate them to room temperature and then bake them at 55°C on a slide baking machine for 30 minutes.

[0179] (2) Antigen repair: Soak the slices in sodium citrate antigen repair solution, then heat them in a microwave oven. Stop heating immediately when bubbles appear. Take them out and leave them at room temperature for 5 minutes before putting them in a microwave oven and heating them until boiling. Boil them 3 times in total. Take them out and cool them to room temperature, then wash them 3 times with PBS, 5 minutes each time.

[0180] (3) Membrane permeabilization: Place the slices in 1% Triton X-100 solution for 15 minutes. After soaking, wash them with PBS three times, each time for 5 minutes.

[0181] (4) Blocking: Shake the slices dry, wipe them dry with absorbent paper, and then draw circles with an immunohistochemistry pen to prevent liquid from flowing out and causing insufficient incubation. Then add goat serum to each brain tissue and block it at room temperature for 30 minutes.

[0182] (5) Incubation with primary antibody: First, recover the blocking solution on each slice, and then use absorbent paper to absorb the blocking solution on the slice. Take an appropriate amount of rabbit anti-NR4A1 antibody solution (dilution ratio of 1:100) and mouse anti-BCl-2 antibody solution (dilution ratio of 1:800) and drip it on each brain tissue, then put the slice into a moisturizing box and put the moisturizing box in a refrigerator at 4°C overnight.

[0183] (6) Incubation with secondary antibody: After the primary antibody incubation is completed, the primary antibody is recovered, and then the slices are washed 3 times with PBS solution, each time for 5 minutes. Then, appropriate amounts of corresponding secondary antibodies anti-rabbit Cy3 and anti-mouse FITC (dilution ratio is 1:500) are added dropwise to the brain tissue and incubated in a constant temperature oven at 37°C in the dark for 1 hour. All subsequent operations must be performed in the dark.

[0184] (7) Nuclear staining: After the incubation with the secondary antibody is completed, the secondary antibody is discarded, and the sections are washed three times with PBS solution, each washing time is 5 minutes. After that, the sections are dried, and an appropriate amount of DAPI staining solution is added to each brain tissue to stain the cell nucleus. The sections are placed in a dark place at room temperature for 5 minutes.

[0185] (8) Sealing and observation: After DAPI nuclear staining, the slices were dried and then washed with PBS three times, 5 minutes each time. After that, the slices were dried, anti-fluorescence quenching solution was added to the slices, and coverslips were placed on the slices for sealing. Then, the slices were observed under a fluorescence microscope in a dark environment.

[0186] 13. TUNEL staining

[0187] The TUNEL staining kit (C1086) was used for observation and photography under a fluorescence microscope. For detailed operation, please refer to the instruction manual.

[0188] 14. Statistical analysis

[0189] The experimental data of this study were analyzed using GraphPad Prism 8 statistical software and the graphs were prepared using the mean ± standard error. The independent sample t test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. The difference was considered statistically significant if P < 0.05.

[0190] 15. Results

[0191] After alcohol withdrawal, the levels of NR4A1 and BCL-2 in the prefrontal cortex increase and promote the combination of the two, thereby reversing the anti-apoptotic function of BCL-2, leading to apoptotic damage to neurons, and then causing anxiety-like behavior in mice. Specific knockout of the NR4A1 gene in the forebrain (including the prefrontal lobe) can rescue the apoptosis and damage of prefrontal cortical neurons caused by alcohol, alleviate the anxiety-like behavior caused by alcohol withdrawal in mice, and have a neuroprotective effect.

[0192] (1) Construction and identification of NR4A1 forebrain-specific knockout mice

[0193] In the preliminary experimental results, it was found that the NR4A1 level in the prefrontal cortex of alcohol-dependent mice was increased, and alcohol-dependent withdrawal would lead to the appearance of anxiety-like behaviors in mice. It was speculated that NR4A1 was involved in the generation of anxiety-like behaviors caused by alcohol-dependent withdrawal. The Loxp-Cre system was used to construct forebrain-specific knockout mice, and conditional place preference was used to create an alcohol-dependent mouse model in preparation for subsequent behavioral and molecular expression testing after withdrawal. The Loxp-Cre system was used to create NR4A1 forebrain-specific knockout mice (knockout of the gene in brain areas such as the prefrontal cortex and hippocampus), and a sufficient number of knockout mice have been obtained. The identification results of the knockout mice can be found in Figure 1 A and Figure 1 B. As can be seen from the figure, all NR4A1 genes carry homozygous Loxp sites and Camk2a-Cre genes.

[0194] (2) Knockout of the NR4A1 gene in the forebrain of alcohol-dependent mice did not alter alcohol-induced CPP

[0195] The protein detection results also showed that the NR4A1 protein level in the prefrontal cortex of the knockout mice was decreased (see Figure 2 A in the figure), which indicates that the NR4A1 gene forebrain knockout mouse was successfully constructed. Male mice aged 7-8 weeks were divided into 3 groups: control group (Con group, 10 C57BL / 6J mice), alcohol treatment group (EtOH group, 10 C57BL / 6J mice) and knockout group (KO-EtOH group, 8 NR4A1 gene forebrain-specific knockout mice). According to the experimental plan, the alcohol-dependent mouse model was established. The mice in the alcohol-treated group and forebrain-specific knockout group were intraperitoneally injected with 20% alcohol (v / v) at a dose of 2g / kg / day, and the mice in the control group were intraperitoneally injected with the same dose of normal saline. After waiting for the model to be successfully established, the behavior was measured and the samples were collected after 16 hours of alcohol withdrawal. The results showed that alcohol treatment caused CPP in mice, while knocking out the NR4A1 gene in the forebrain of mice did not change the alcohol-induced CPP (see Figure 2 B).

[0196] (3) Alcohol-dependent mice showed anxiety-like behaviors after withdrawal, but knocking out the NR4A1 gene in the forebrain alleviated the anxiety-like behaviors

[0197] The alcohol-dependent mice successfully modeled above were subjected to behavioral tests such as open field and elevated frame tests 16 hours after withdrawal (because the CPP training box used a black-and-white box, in order to avoid the influence of training on the black-and-white box results, the black-and-white box test was performed before CPP training, that is, the black-and-white box test was performed 16 days after alcohol injection and 16 hours after withdrawal) to reflect the anxiety of the mice.

[0198] The behavioral results are as follows: The novel object recognition experiment showed that there was no significant statistical difference in the discrimination index between the EtOH group and the Con group (P>0.05), and there was no statistical difference in the discrimination index between the KO-EtOH group and the EtOH group (P>0.05), indicating that the mice did not have obvious learning and memory impairment (see Figure 3 The Y-maze test showed that there was no statistical difference in the number of times the mice in the EtOH group entered the novel arm compared with the Con group (P>0.05), and there was no statistical difference in the number of times the mice in the KO-EtOH group entered the novel arm compared with the EtOH group (P>0.05), indicating that there was no significant change in the cognitive ability of the mice (see Figure 3 B).

[0199] The open field test showed that the time that mice in the EtOH group spent in the central area was significantly shorter than that in the Con group (P<0.01), and the time that mice in the KO-EtOH group spent in the central area was longer than that in the EtOH group (P<0.05). Figure 3 C and Figure 4 A in the figure); compared with the Con group, the number of times the mice in the EtOH group entered the central area of ​​the open field decreased, and the difference was statistically significant (P<0.05). Compared with the EtOH group, the number of times the mice in the KO-EtOH group entered the central area increased significantly, and the difference was statistically significant (P<0.05), indicating that the mice in the EtOH group had obvious anxiety-like behaviors, and the anxiety-like behaviors of the mice in the KO-EtOH group were significantly alleviated (see Figure 3 D and Figure 4 A in the figure); the total distance of open field movement of the three groups of mice did not change significantly, and the difference was not statistically significant (P>0.05), indicating that alcohol had no significant effect on the free movement of mice (see Figure 3 E and Figure 4 A).

[0200] The black-and-white box experiment showed that compared with the Con group, the number of shuttles in the EtOH group was significantly reduced, and the difference was statistically significant (P<0.05). Compared with the EtOH group, the number of shuttles in the KO-EtOH group was significantly increased, and the difference was statistically significant (P<0.01). This suggests that the EtOH group mice had obvious anxiety-like behaviors, and the anxiety-like behaviors of the KO-EtOH group mice were significantly alleviated (see Figure 3 F).

[0201] The elevated plus maze test showed that the number of times the mice in the EtOH group entered the open arm was significantly reduced compared with the Con group, and the difference was statistically significant (P<0.01). Compared with the EtOH group, the number of times the mice in the KO-EtOH group entered the open arm increased, but there was no statistical difference (P=0.57) (see Figure 3 G and Figure 4 B); Compared with the Con group, the time that mice in the EtOH group stayed in the open arms was reduced, which was statistically significant (P<0.05). Compared with the EtOH group, the time that mice in the KO-EtOH group stayed in the open arms was increased, but it was not statistically significant (P=0.08), indicating that mice in the EtOH group had obvious anxiety-like behaviors, and the anxiety-like behaviors of mice in the KO-EtOH group were alleviated compared with the previous group (see Figure 3 H and Figure 4 B).

[0202] The above behavioral test results show that alcohol-dependent mice have no obvious cognitive impairment after withdrawal, and mainly manifest as anxiety-like behavior. After knocking out NR4A1, the anxiety-like behavior of mice is alleviated.

[0203] (4) NR4A1 protein and apoptosis-related proteins increased in the prefrontal cortex of mice with alcohol withdrawal; knocking out the NR4A1 gene in the prefrontal cortex rescued the increase in apoptosis-related proteins caused by alcohol

[0204] After the behavioral test, the mice were anesthetized and the prefrontal lobe was removed. The expression of apoptosis-related proteins BCL-2, BAX and Cleaved-caspase-3 was detected by RT-qPCR and WB. The experimental results showed (see Figure 5 AC in Figure 6 AD in the study): The mRNA and protein levels of BAX, which is related to apoptosis, increased in mice treated with alcohol, and the protein level of Cleaved-Caspase-3 (which reflects cell apoptosis) also increased; after knocking out NR4A1, the mRNA and protein of apoptosis-related genes decreased. The above results indicate that alcohol withdrawal increases apoptosis-related proteins in prefrontal neurons, and knocking out the NR4A1 gene can rescue alcohol-induced apoptosis-related proteins.

[0205] The anti-apoptotic protein BCL2 is also upregulated during alcohol withdrawal, but is reduced when the NR4A1 protein is specifically knocked out in the prefrontal cortex; according to literature reports, after the NR4A1 protein binds to the BCL2 protein, BCl2 changes from anti-apoptotic to pro-apoptotic. Therefore, these results show that alcohol promotes the expression of apoptosis-related proteins by increasing the expression of NR4A1; and promotes the binding of NR4A1 protein to BCL2 protein, turning it from anti-apoptotic to pro-apoptotic, thereby increasing cell apoptosis.

[0206] The above results show that alcohol treatment increases the expression of NR4A1 gene, promotes apoptosis of prefrontal neurons, and reduces the number of neurons; while knocking out the NR4A1 gene can restore the apoptosis of prefrontal neurons to a certain extent, and the reduction in the number of neurons caused by alcohol can be rescued to a certain extent. Although knocking out the NR4A1 gene has no effect on alcohol-induced CPP, it can reduce apoptosis of prefrontal neurons and has a protective effect on the brain.

[0207] (5) Alcohol treatment increased apoptosis and reduced the number of prefrontal neurons in mice

[0208] Neuronal apoptosis and neuronal cell number were detected by TUNEL staining and Nissl staining. The results showed that in the alcohol-treated mice, prefrontal neuronal apoptosis increased, while when the NR4A1 gene was knocked out, neuronal apoptosis was rescued to a certain extent (see Figure 7 A in the figure). The number of neurons in the prefrontal cortex of the alcohol-treated group was significantly reduced, but after knocking out the NR4A1 gene, the number of neurons was restored to a certain extent (see Figure 7 B).

[0209] (6) Alcohol treatment caused colocalization of NR4A1 and BCL-2 proteins in prefrontal neurons, while NR4A1 gene knockout reduced colocalization with BCL-2

[0210] Immunofluorescence detection results of frozen sections of mouse prefrontal cortex: the number of cells with co-localization of NR4A1 protein and BCL-2 protein increased, while when the NR4A1 gene was specifically knocked out in the prefrontal cortex, its co-localization with BCL-2 decreased (see Figure 8 A and Figure 8 B).

[0211] (7) Immunoprecipitation method verifies the interaction between NR4A1 protein and BCL-2 protein

[0212] The immunoprecipitation kit (Cat. No.: P2197M) of Biotech was used. The specific method was carried out according to the instruction manual. The immunoprecipitation results further proved that NR4A1 protein interacted with BCL-2 and NR4A1 protein could precipitate it (see Fig. 9 ).

[0213] (8) Oral administration of the NR4A1 protein inhibitor CDIM8 can alleviate the anxiety-like behavior caused by withdrawal in mice

[0214] C57BL / 6J male mice aged 7-8 weeks were divided into two groups: a control group and an inhibitor group, with 10 mice in each group. According to the experimental plan, an alcohol-dependent mouse model was established, and both groups of mice were given an intraperitoneal injection of 20% alcohol (v / v) at a dose of 2g / kg / day. After the model was successfully established, the inhibitor group was gavaged with 50mg / kg of CDIM8, and the control group was gavaged with normal saline, and the gavage time was 1 week (during the treatment, intraperitoneal injection of alcohol was continued). The inhibitor intervened for 1 week, and after 16 hours of alcohol withdrawal, the anxiety-like behavior of the mice was tested by open field, cross elevated maze and black and white box. Western blot results showed that the protein expression of NR4A1 in the prefrontal cortex of mice in the inhibitor group was reduced, and the inhibitor had an inhibitory effect on the NR4A1 protein in the prefrontal cortex (see Fig.10 The behavioral results showed that the total distance traveled by the two groups of mice in the open field did not change significantly (see Fig.10 A), the inhibitor group mice stayed in the central area for a longer time and entered the central area more frequently, with a statistically significant difference (P<0.05) (see Fig.10 C and Fig.10 D in the figure). The black-and-white box experiment showed that the inhibitor group mice stayed in the white box for a longer time and shuttled more frequently (see Fig.10 E), the difference was statistically significant (P<0.05); the elevated plus maze test showed that the inhibitor group mice entered the open arm more times and stayed longer (P<0.05) (see Fig.10 F and Fig.10 G in the figure); the open arm residence time of mice in the inhibitor group was significantly increased (P<0.05), indicating that the anxiety-like behavior caused by alcohol withdrawal in mice in the inhibitor group was alleviated. The inhibitor has a therapeutic effect on the anxiety-like behavior caused by alcohol withdrawal in mice.

[0215] In the present invention, it is found that mice with alcohol dependence develop anxiety-like behaviors after withdrawal, and the expression level of NR4A1 gene is increased, which can reverse the anti-apoptotic function of BCL-2 protein, leading to an increase in BCL-2 protein and apoptosis-related proteins in the prefrontal lobe. Knocking out the NR4A1 gene in the forebrain and intervening in the expression level of the NR4A1 gene in the prefrontal lobe of mice can prove that down-regulating the expression of the NR4A1 gene in the prefrontal lobe can rescue the apoptosis and damage of prefrontal cortical neurons caused by alcohol, relieve the anxiety-like behaviors caused by alcohol withdrawal in mice, and have the effect of protecting the mouse brain; intragastric administration with CDIM8, an inhibitor of NR4A1 protein, can reduce the anxiety-like behaviors of mice with alcohol withdrawal.

[0216] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0217] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of NR4A1 gene expression inhibitor in the preparation of drugs for treating alcohol dependence withdrawal.

2. The use according to claim 1, characterized in that The NR4A1 gene expression inhibitor includes: interfering RNA targeting NR4A1 gene expression or an NR4A1 protein expression inhibitor.

3. The use according to claim 2, characterized in that The DNA sequence of the interfering RNA is shown in SEQ ID NO: 1; The NR4A1 protein expression inhibitor includes DIM-C-pPhOH.

4. A drug for treating alcohol dependence withdrawal, characterized in that: The drug contains the NR4A1 gene expression inhibitor described in claim 1 as the only active ingredient.

5. The drug according to claim 4, characterized in that The drug is used to alleviate the anxiety behavior caused by withdrawal in alcohol-dependent patients.

6. The drug according to claim 4, characterized in that In the medicine, the content of the NR4A1 gene expression inhibitor is 1w% to 99w%.

7. The drug according to claim 4, characterized in that Pharmaceutically acceptable excipients are also included.

8. The drug according to claim 7, characterized in that The auxiliary materials are selected from one or more of propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

9. The drug according to claim 8, characterized in that The medicine is an oral preparation or an injection preparation.

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