Phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcoholism

Abstract

The invention discloses a phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcoholism, relates to the technical field of medicines, and has the technical key point that the inhibitor is BI-409306. The BI-409306 can influence the drinking behavior of a mouse with alcohol addiction by adjusting synaptic plasticity and intervene the formation of addiction. In addition, the BI can also reduce acute alcohol toxicity injury by recovering the GPX4 level and reducing inflammatory factors. The invention not only lays a research foundation for exploring the effect and mechanism of BI in alcohol addiction in the future, but also provides a new target and thought for clinically treating alcohol addiction.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcohol poisoning. Background Art

[0002] Some people who are addicted to alcohol may consume large doses of alcohol in a short period of time, which may induce acute poisoning symptoms such as severe physiological discomfort (such as respiratory imbalance, coma, cognitive confusion, vomiting, suffocation, hypothermia, etc.) and motor dysfunction, making acute alcohol poisoning another serious health problem of alcohol addiction, which is also an important cause of disability and death caused by alcoholism in drinkers. In addition, acute alcohol poisoning may serve as a trigger factor to promote or aggravate the occurrence and development of addictive behavior.

[0003] As a highly prevalent but severely undertreated disease, there are currently only three FDA-approved drugs for the treatment of alcohol addiction: disulfiram (alcohol sensitization drug), acamprosate (nitrogen-acetylhomotaurine, GABA receptor agonist) and naltrexone (opioid receptor antagonist Naltrexone, NTX). However, frequent side effects seriously reduce patient compliance and lead to treatment interruption; in addition, the efficacy of these types of drugs is still under debate, and some studies have even confirmed that there is no evidence that naltrexone and acetylhomotaurine have any effect on alcohol addiction. Therefore, it is urgent to develop new, more effective and diversified drugs for alcohol addiction. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcohol poisoning.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention provides a phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcohol poisoning. The inhibitor is BI-409306, and the structure is as follows:

[0007]

[0008] The present invention also provides an application of a phosphodiesterase 9A inhibitor

[0009] In the above technical scheme, BI-409306 inhibits PDE9A to increase cGMP levels, activates its downstream pathways to participate in the regulation of alcohol behavior, reduces the drinking behavior of mice, and intervenes in the formation of addiction. BI-409306 significantly restored the expression of PSD-95, GAP43 and Synapsin-1 proteins, and increased the density of dendritic spines, indicating that BI-409306 reduced the drinking behavior of mice by restoring synaptic plasticity. Pretreatment with BI-409306 can effectively restore the expression of GPX4, indicating that it alleviates the effects of oxidative stress by inhibiting PDE9A. BI-409306 treatment significantly reduced the level of the inflammatory factor IL-1β, but had no effect on the expression of iNOS.

[0010] Compared with the existing technology, the beneficial effects of this scheme are: BI-409306 can affect the drinking behavior of alcohol-addicted mice by regulating synaptic plasticity and intervene in the formation of addiction. In addition, BI can also reduce acute alcohol toxicity damage by restoring GPX4 levels and reducing inflammatory factors. This not only lays a research foundation for future exploration of the role and mechanism of BI in alcohol addiction, but also provides new targets and ideas for the clinical treatment of alcohol addiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 After treating SH-SY5Y cells with different concentrations of ethanol (62.5-1000 mM) for 24 h, CCK8 was used to detect cell viability;

[0012] Figure 2 SH-SY5Y cells were treated with different concentrations of BI in the present invention for 24 h, and the cell viability was detected;

[0013] Figure 3 The BI in the embodiment of the present invention alleviates the ethanol-induced SH-SY5Y cell damage, wherein A is SH-SY5Y cells pretreated with different concentrations of BI (0.125-2 μM) for 1 hour and then incubated with 500 mM ethanol for 24 hours, and the cell survival rate is determined by CCK8, and B is SH-SY5Y cells pretreated with different concentrations of BI (0.125-2 μM) for 1 hour and then incubated with 500 mM ethanol for 24 hours, and the cytotoxicity level is detected by LDH kit;

[0014] Figure 4 BI in the present invention reduces the intracellular ROS level of SH-SY5Y cells stimulated by ethanol, wherein A is SH-SY5Y cells pretreated with BI (0.5 μM) for 1 hour and then incubated with 500 mM ethanol for 24 hours, and the DCFH-DA staining method is used to detect the intracellular ROS level, and B is the fluorescence quantitative statistical result of the intracellular ROS level;

[0015] Figure 5 The BI in the present invention reduces the MDA level in SH-SY5Y cells elevated by ethanol stimulation;

[0016] Figure 6 BI in the present invention restores the GPX4 level in SH-SY5Y cells that is reduced by ethanol stimulation, wherein A is SH-SY5Y cells treated with 500 mM ethanol for different time periods, and the GPX4 protein level is detected by WB, B is the semi-quantitative result of GPX4 protein, C is BI (0.5 μM) pretreated for 1 h, and then incubated with 500 mM ethanol for 24 h, and the GPX4 protein level is detected, and D is the semi-quantitative result of GPX4 protein;

[0017] Figure 7 The mitochondrial membrane potential of SH-SY5Y cells reduced by BI ethanol stimulation in the embodiment of the present invention was alleviated by BI pretreatment, wherein A is the change of mitochondrial membrane potential detected by TRME staining after 1 hour of BI (0.5 μM) pretreatment of SH-SY5Y cells and co-incubation with 500 mM ethanol for 24 hours, and B is the statistical quantitative result of TMRE fluorescence intensity;

[0018] Figure 8 is siPDE in the embodiment of the present invention 9A Reducing SH-SY5Y cell death induced by ethanol stimulation, where A is SH-SY5Y cells treated with siPDE 9A PDE was detected by WB after 48h treatment 9A Expression level, B is PDE 9A Semi-quantitative statistical results, C is the level of live and dead cells detected by Calcein / PI staining, and D is the quantitative result of Calcein / PI staining;

[0019] Fig. 9 is siPDE in the embodiment of the present invention 9A Restoration of the increase in ROS and loss of mitochondrial membrane potential caused by ethanol stimulation, in the presence of siPDE 9A After 24 hours of transfection, 500 mM ethanol was used for stimulation for 24 hours, where A is the detection of ROS level by DCFH-DA, B is the detection of mitochondrial membrane potential level by TMRE staining, C is the quantitative statistical result of DCFH-DA staining, and D is the quantitative result of Calcein / PI staining;

[0020] Fig.10 This is the Two Bottle Choice alcohol addiction model in the embodiment of the present invention. AB is a schematic diagram of the change of alcohol preference and alcohol intake over time in female mice, CD is a schematic diagram of the change of alcohol preference and alcohol intake over time in male mice, E is a heat map of alcohol preference of male and female mice, and F is the alcohol consumption of male and female mice in the third week;

[0021] Fig.11 is the therapeutic effect of BI on female alcohol-addicted mice in the embodiment of the present invention, wherein A is the experimental design flow chart, BC is the alcohol intake and alcohol preference of each group of mice in the two-bottle selection experiment, D is the alcohol intake of mice in the dark drinking experiment, and E is the alcohol intake of mice that drink again after abstinence;

[0022] Fig.12 is the formation of BI intervention in alcohol addiction in mice in the embodiment of the present invention, wherein A is the experimental design flow chart, BC is the alcohol intake of each group of mice in the bottle selection experiment, DE is the alcohol preference of each group of mice in the two-bottle selection experiment, F is the total liquid intake of each group of mice, and G is the body weight of each group of mice;

[0023] Fig.13 It is that BI treatment in the embodiment of the present invention does not affect the taste preference of mice, wherein A and E are sugar water and quinine and preference, B and F are the intake of sugar water and quinine, C and G are the total fluid intake during the sugar water and quinine drinking period, and D and H are the weight of mice during the sugar water and quinine drinking period;

[0024] Fig.14 Chronic drinking significantly upregulates PDE in the prefrontal cortex and hippocampus of mice in the embodiment of the present invention 9A The expression of PDE was detected by Western blot in the prefrontal cortex of experimental mice. 9A Protein level, B is PDE in prefrontal cortex 9A Semi-quantitative results of the level. C is the hippocampus of experimental mice, and Western blot was used to detect PDE 9A Protein level, D is hippocampal PDE 9A Horizontal semiquantitative results;

[0025] Fig.15 Chronic drinking significantly upregulates PDE in the liver of mice 9A The expression of PDE was detected by Western blot. 9A Protein level, B is liver PDE 9A Horizontal semiquantitative results;

[0026] Fig.16 BI in the present invention restores the expression of synaptic-related proteins in the hippocampus of drinking mice, wherein A is the expression of postsynaptic density 95 (PSD95), B is the expression of synapsin-1, and C is the expression of growth-associated protein 43 (GAP-43);

[0027] Fig.17BI restores dendritic spine density in the hippocampus of drinking mice in the embodiment of the present invention, wherein A is the dendritic spine density detected by Golgi staining, and B is the statistical graph of dendritic spine density;

[0028] Fig.18 BI in the present invention restores the expression of GPX4 in the hippocampus of drinking mice, wherein A is the expression of GPX4 detected by WB, and B is the semi-quantitative result of GPX4;

[0029] Fig.19 BI restores the expression of GPX4 in the hippocampus of mice with acute alcohol intoxication model in the embodiment of the present invention, wherein A is the expression of GPX4 in the hippocampus detected by WB, and B is the semi-quantitative statistics of GPX4;

[0030] Fig. 20 It is the expression of IL-1β in the hippocampus of mice with acute alcohol poisoning restored by BI in the embodiment of the present invention, wherein A is the expression of iNOS in the hippocampus detected by WB, B is the semi-quantitative statistics of iNOS, and C is the expression of IL-1β in the hippocampus detected by WB. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. 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 should fall within the scope of protection of the present invention.

[0032] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0033] Example 1 Protective effect of BI-409306 on alcohol-induced SH-SY5Y cell damage:

[0034] 1. Experimental Materials

[0035] 1.1 Experimental cells

[0036] SH-SY5Y cells: human neuroblastoma cells, obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0037] 1.2 Experimental Reagents

[0038]

[0039]

[0040] 1.3 Preparation of experimental reagents

[0041] Preparation of BI-409306: Add 1 mg of BI-409306 to 32.1 μL of sterile DMSO to obtain a 100 mM stock solution, which is then aliquoted and stored. Dilute to the target concentration and filter with a microporous membrane before use. Preparation of Western Blot reagents:

[0042] 1) Preparation of electrophoresis buffer: 1 L of electrophoresis buffer is required for every four SDS-PAGE gels. The formula is 3.03 g Tris, 18.8 g glycine and 1 g SDS. Make up to 1 L with ultrapure water, stir thoroughly to dissolve, and prepare for use immediately.

[0043] 2) Preparation of transfer buffer: 1L of transfer buffer is required for every two SDS-PAGE gels. Add 3.03g of Tris to 14.4g of glycine and then add 800ml of ultrapure water. Stir to dissolve and then dilute to 1000mL with methanol.

[0044] 3) Preparation of TBST buffer: Add 3.03 g of Tris, 8.2 g of NaCl and 0.224 g of KCl to 1 L of ultrapure water, and finally add 1 mL of Tween 20 to dissolve all of them into a clear liquid.

[0045] 4) Preparation of blocking solution: Weigh 10 g skim milk powder, add 200 ml TBST, and place on a magnetic stirrer in advance to mix thoroughly.

[0046] 2. Experimental Methods

[0047] 2.1 Cultivation of human neuroblastoma cell line (SH-SY5Y)

[0048] 2.1.1 Cell recovery

[0049] Before the experiment begins, you need to take out the DMEM / F12 culture medium and fetal bovine serum from the refrigerator in advance, spray them with alcohol and place them on a sterile operating table, and turn on the ultraviolet light for 30 minutes of disinfection and preheating. In addition, preheat the water bath to 37°C in advance. After the temperature stabilizes, take out the frozen cells that need to be revived from the liquid nitrogen container and immediately place them in a 37°C water bath to accelerate the thawing process. When the cells are completely thawed, transfer them to a centrifuge, set the speed to 800rpm, and centrifuge for 3 minutes.

[0050] After centrifugation, remove the cryovial and spray 75% alcohol on its surface for disinfection, then move it to a sterile operating table. Carefully aspirate and discard the upper layer of liquid in the tube, then add 1 mL of complete culture medium to resuspend the cell pellet. Transfer the resuspended cells to a cell culture flask, add complete culture medium, and shake well to ensure that the cells are evenly distributed. Next, place the cells back in a 37°C, 5% CO 2 The cells were cultured in an incubator with a concentration of 1:1. The medium was changed after 4 hours to ensure that the residual DMSO was removed. After 24 hours, the cell growth was observed under a microscope and the cells were treated accordingly according to their density.

[0051] 2.1.2 Cell passaging

[0052] Observe the morphology and density of the cells under the microscope. When the cells are healthy and the density is appropriate, start the cell subculture. First, take out the required complete culture medium, trypsin solution, PBS and other reagents from the refrigerator, place them in the sterile operation area of ​​​​the cell culture, and turn on the ultraviolet lamp for 30 minutes of disinfection and preheating. Then, take out the culture bottle from the constant temperature incubator, discard the original culture solution, and inject 2mL PBS into the bottle, shake gently to wash the cells, and then discard PBS. Repeat this process twice. Next, add 1ml of trypsin digestion solution and put the culture bottle back into the incubator to digest the cells at 37°C for 1min. After the time is up, take out the culture bottle from the incubator and examine the cells under a microscope. When the cells are round and the transparency increases, move them to the sterile operation area and add 1mL of complete culture medium to stop digestion. After gently tapping the bottle wall to make the cells fall off, transfer the digested cell suspension to a centrifuge tube and centrifuge at 800rpm for 3min. After centrifugation, pour off the supernatant, resuspend the cells with new complete culture medium, dilute them according to a certain ratio and put them back into a new culture bottle. After gently shaking to evenly distribute the cells, put them back into the incubator for culture.

[0053] 2.1.3 Cell cryopreservation

[0054] First, observe the density and health of the cells under a microscope. When the cell morphology is normal and the cell density is appropriate, cryopreserve the cells. Take out the required culture medium, serum, PBS, trypsin and other reagents from the refrigerator in advance, place them in the sterile operating table, turn on the ultraviolet light for 30 minutes to sterilize and warm the reagents. At the same time, take out the cell freezing box and let it warm up at room temperature. Take out the cell culture bottle from the incubator, remove the old culture medium and wash the cells twice with PBS. Then add 1mL of trypsin digestion solution, put it back in the incubator, and let the cells digest for 1min. When the cells are observed to be round and translucent under the microscope, move them to the sterile operating table and add 1mL of complete culture medium to terminate the digestion process. Gently blow the culture bottle to remove the cells, transfer them to a 4mL centrifuge tube, and centrifuge them at 800rpm for 3min in a centrifuge. After centrifugation, discard the supernatant, add 1min of cell freezing solution (the ratio of fetal bovine serum to DMSO is 9:1) to the cell pellet to resuspend it, and then transfer the cell suspension to the cryopreservation tube. Label the cryovials with the cell type and date of freezing, then place them in a cell freezing box and store them in a -80°C freezer. The next day, transfer the cells to a liquid nitrogen tank for long-term storage.

[0055] 2.2 Detection of cell viability by CCK-8 method

[0056] CCK-8 reagent, full name Cell Counting Kit-8, is a simple and accurate tool for evaluating cell proliferation and toxicity. The water-soluble tetrazolium salt WST-8 contained in the reagent can be reduced by dehydrogenases in living cells with the help of the electron mediator 1-Methoxy PMS to generate a highly water-soluble yellow formazan product. The amount of formazan generated is positively correlated with the number of viable cells, which makes it an ideal choice for directly evaluating cell proliferation and toxicity. The number of viable cells can be indirectly determined by measuring the absorbance using a microplate reader at a wavelength of 450nm.

[0057] Observe the cell morphology and growth density, digest and centrifuge the cells in the logarithmic growth phase in good condition, and resuspend the cell pellet in DMEM / F12 containing 1% FBS. Count the cells before plating, dilute to the target concentration, and blow the cells several times to ensure that they are evenly mixed. Set up blank control wells, normal control wells, and wells treated with drugs at different concentrations. After adding 100 μL of pre-diluted cell suspension to each well, put it back into the incubator to adhere to the wall for 12 hours, and then perform the corresponding treatment after 12 hours. After the treatment time is over, add 10 μL of CCK8 detection solution to each empty space evenly, shake the well plate gently after adding to mix it, and incubate it in the dark for 30 minutes. After 30 minutes, use an enzyme reader to measure the absorbance of each well at a wavelength of 450nm. The cell survival rate can be obtained by calculating (absorbance of drug treatment group-absorbance of blank control group) / (absorbance of normal control group-absorbance of blank control group).

[0058] 2.3 Lactate dehydrogenase (LDH) detection of cytotoxicity

[0059] When cells are damaged and undergo apoptosis or necrosis, the cell membrane will be damaged, which will cause the lactate dehydrogenase (LDH) in the cytoplasm to leak into the culture medium. Therefore, the degree of LDH leakage is usually used as an indicator to evaluate cytotoxicity. The plating process is as described above. After the plate is laid, the well plate is placed in a cell culture incubator for continued culture. Similarly, after the cells adhere to the wall the next day, the corresponding drug treatment is carried out. After the treatment, the 96-well plate is taken out and centrifuged at 400rpm for 5min. After centrifugation, 120μL of supernatant is taken out from each treatment well and transferred to a new 96-well plate, and 60μL of the pre-prepared LDH working solution is added. After mixing evenly, react at room temperature in the dark for 30min. The absorbance of each well is measured at a wavelength of 490nm using a multifunctional microplate reader. The percentage of cytotoxicity can be calculated by the following formula: (absorbance of drug treatment group-absorbance of blank control group) / (absorbance of normal control group-absorbance of blank control group)×100%.

[0060] 2.4 Western blot detection of relative protein expression levels

[0061] 2.4.1 Extraction of total cell protein

[0062] After completing the cell intervention, remove the culture medium from the culture plate and rinse with PBS to remove any residual liquid. Make sure the entire operation is performed on ice, add 100 μL of the pre-prepared cell lysis buffer to each well, gently blow it several times, cover it with the lid, and fix the culture plate on the shaker and place it in a 4°C environment for 10 minutes. When the cells are completely lysed, the lysate containing the cell lysate is transferred to a pre-labeled centrifuge tube, placed in a pre-cooled ultra-high-speed centrifuge, and centrifuged at 12000 rpm and 4°C for 15 minutes. After centrifugation, transfer the supernatant to a new ep tube to obtain total cell protein.

[0063] 2.4.2 Protein quantification

[0064] Divalent copper ions can be reduced to monovalent copper ions by proteins under alkaline conditions, and monovalent copper ions interact with BCA to produce a sensitive color reaction, forming a purple reaction complex, which has strong absorbance at 562nm. The absorbance and protein concentration have a good linear relationship in a wide range, so the protein concentration can be calculated based on the absorbance. First, prepare a standard curve, take out the BCA standard with a concentration of 5mg / ml from the refrigerator, dilute 20μl of the standard with 180ul PBS to 0.5mg / ml, add 0, 1, 2, 4, 8, 12, 16, and 20μl of the standard dilution to the 96-well plate, and make up to 20μl with PBS. Each well represents the protein standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5mg / ml. Add a certain volume of sample to the remaining wells, and also make up to 20μl with PBS, mark the dilution multiple of the sample, and prepare 3 duplicate wells for each sample. After all samples are added, add 200μl of freshly prepared BCA working solution (A solution: B solution = 50:1) to each well, wrap with tin foil, and incubate at 37℃ for 30min. After incubation, use a microplate reader to measure the absorbance OD value at a wavelength of 562nm. Export the data, calculate the concentration of each sample based on the standard curve and the sample OD value, and dilute it to the target concentration with RIPA and loading buffer to make the concentration of each sample consistent. Vortex mix and centrifuge instantly so that the samples are at the bottom of the centrifuge tube. Use a 100℃ metal bath to heat for 10min to completely denature it, and then store it at -20℃ for later use.

[0065] 2.4.3 Western Blot

[0066] 2.4.3.1 Gel preparation

[0067] Take out the glass plate in advance, and add distilled water to the gap between the glass plates after installation to check for leaks. If the page inside the glass plate does not drop within 15 minutes, you can start preparing the gel. Usually, a gel with a thickness of 1.5 mm is selected, and the concentration of the separation gel is selected according to the molecular weight of the target protein. The specific formula is shown in Table 1-1 below. Vortex the prepared separation gel to mix evenly, and then let it stand for 1 minute to eliminate the bubbles during vortexing. Slowly add it to the gap between the long and short plates of the glass plate, and then fill it up with pure water. Shake it slightly from side to side a few times to make the interface between the separation gel and the pure water smoother. After about 30 minutes, a clear straight dividing line can be seen between the separation gel and the pure water, indicating that the lower gel has been completely solidified. At this time, prepare the concentrated gel. The formula of the concentrated gel is shown in Table 1-2. After preparing the concentrated gel, pour out the pure water, use a paper towel to absorb the moisture of the glass plate, carefully fill it with separation gel, add the comb vertically, and wait for it to solidify.

[0068] Table 1-1 Ratios of commonly used separation gels

[0069]

[0070] Table 1-2 Concentrated Glue Ratio Table

[0071]

[0072]

[0073] 2.4.3.2 Electrophoresis process

[0074] Prepare the corresponding volume of electrophoresis solution in advance according to the number of gel blocks to be electrophoresed. Generally, 4 gel blocks require 1L of electrophoresis solution. Separate the glass plate from the gel-making device and install it in the electrophoresis equipment. After adding the electrophoresis solution, carefully pull out the comb vertically to avoid skewness in the sample wells. Thaw the samples prepared in advance, vortex evenly and add the samples to the sample wells in sequence. Leave a hole on each side of the sample for adding pre-stained protein markers, and fill the volume with 1 times loading buffer. After all loading is completed, cover the motor, pay attention to black against black and red against red, adjust the electrophoresis time and voltage, and start electrophoresis. The general electrophoresis program is 80V, 30min, and then switch to 120V until electrophoresis reaches the bottom of the glass plate.

[0075] 2.4.3.3 Transfer steps

[0076] Prepare the transfer solution in advance. Generally, 1L of transfer solution is required for every two gels. Pre-cool the prepared transfer solution in a 4°C refrigerator in advance. Cut the corresponding volume of PDVF membrane according to the number of samples and gels, and soak it in formaldehyde for activation. After the electrophoresis is completed, take out the glass plate and open it, carefully take out the gel, gently rinse it twice in the transfer solution, and assemble the transfer clamp in the order of sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad. Use a roller to drive out bubbles. After ensuring that there are no bubbles between the gel and the filter paper, put it into the transfer device and pour in the transfer solution. After correctly connecting the electrodes, transfer the membrane to an ice bath, adjust the transfer time to a constant voltage of 90V for transfer, and adjust the time according to the molecular weight of the target protein.

[0077] 2.4.3.4 Closure Processing

[0078] Prepare the blocking solution in advance according to the number of membranes to be blocked. Generally, 25 ml of blocking solution is required for each membrane. After the transfer is completed, disconnect the power supply, take out the PDVF membrane, and place it in a blocking box with skim milk added, making sure the protein side is facing up. Incubate it on a shaker at room temperature and low speed for 1 hour. After the blocking is completed, discard the blocking solution, add TBST and continue to shake rapidly on the shaker for 4 times, 5 minutes each time. After the washing is completed, cut the band containing the target protein according to the molecular weight shown by the marker and proceed to the next incubation step.

[0079] 2.4.3.5 Antibody incubation

[0080] Refer to the antibody manual to dilute the antibody with antibody diluent, place the strip in the antibody diluent, and place in a 4°C refrigerator for rotation and incubation overnight, i.e. 16 hours. After the incubation, take out the strip and wash it 4 times with TBST on a shaker at room temperature, 5 minutes each time. After washing, use skim milk powder to prepare the corresponding secondary antibody diluent according to the species of the primary antibody, and the dilution multiple is generally 1:5000. Place the washed strip in the secondary antibody diluent and incubate at room temperature for 1 hour. After the incubation, wash it 4 times with TBST, 5 minutes each time.

[0081] 2.4.3.6 ECL detection

[0082] Turn on the multifunctional imager in advance to precool it to -25°C, and turn on the imaging software. Prepare the luminescent working solution according to the instructions of the ECL kit, use it immediately after preparation and avoid light. Take out the strips, remove the liquid on the strips as much as possible, spread the strips flat on the imaging system, evenly add the luminescent liquid, set the parameters, start exposure and capture images. Save the exposed protein band map and bright field image, export the data, and use imageJ software to analyze and count the obtained protein band map.

[0083] 2.5 Assessment of mitochondrial membrane potential by TMRE staining

[0084] When generating energy, mitochondria store electrochemical potential energy in the inner membrane of mitochondria. On both sides of the inner membrane, if the concentration of protons and other ions is asymmetrically distributed, mitochondrial membrane potential, or MMP, will be formed. MMP is one of the important parameters reflecting the functional state of mitochondria. When mitochondria are damaged, the membrane potential will decrease. The cationic dye tetramethylrhodamine ethyl ester (TMRE) can accumulate in the mitochondrial matrix, and its accumulation is positively correlated with MMP. Therefore, TMRE staining can be used to detect its fluorescence intensity to observe changes in the mitochondrial membrane potential of cells.

[0085] Prepare the confocal dish, digest and centrifuge the cells in the logarithmic growth phase, resuspend and count them in a medium containing 1% fetal bovine serum, then add 200μl of cell suspension to the center of the confocal dish, so that each confocal dish contains 3×10^4 cells. Return it to the incubator to let it stand and adhere to the wall. After 0.5h, add the culture medium to 2ml and return it to the incubator for further culture. The next day, after the cells are completely attached to the wall, perform the corresponding treatment. After the treatment, gently aspirate the culture medium, slowly add PBS to wash the cells, add the pre-prepared TMRE dye, and return it to the incubator for incubation for 30min. After the time is up, remove the dye, wash three times with PBS, and keep it away from light throughout the operation. After washing, you can take pictures and observe.

[0086] 2.6 DCFH-DA staining to detect intracellular reactive oxygen species (ROS) levels

[0087] DCFH-DA is a transmembrane dye that is not fluorescent. It crosses the cell membrane and is acted on by esterases inside the cell to generate DCFH, which accumulates in the cell. Reactive oxygen species inside the cell oxidize the non-fluorescent DCFH into fluorescent DCF. By measuring the fluorescence intensity of DCF, the level of intracellular reactive oxygen species can be quantitatively analyzed.

[0088] After digesting and centrifuging the cells, inoculate the cells in the confocal culture dish. The specific process is as described above. The next day, after the cells adhere to the wall, different drugs can be added for treatment. After the treatment, remove the old culture medium, add the diluted DCFH-DA dye and return to the cell culture incubator for 30 minutes. After 30 minutes, take out the confocal dish, remove the culture medium with the dye, and wash it three times with PBS. Protect from light throughout the operation. After washing, you can observe and take pictures.

[0089] 2.7 Calcein AM / PI staining to detect cell death

[0090] Calcein AM is a staining reagent that fluorescently labels living cells based on lipase hydrolysis. Calcein AM itself is non-fluorescent, but after being hydrolyzed by endogenous lipase to calcein, it exhibits green fluorescence in the cell. PI propidium iodide can emit red fluorescence by binding to the DNA of dead cells. The ratio of live cells to dead cells can be detected by analyzing the fluorescence level.

[0091] After digestion and centrifugation of the cells, inoculate the cells in the confocal culture dish, the specific process is as described above. The next day, after the cells adhere to the wall, different drugs can be added for treatment. After the treatment, remove the old culture medium, add calceinAM and PI dye diluted with staining buffer, incubate in the cell culture incubator for 20 minutes in the dark, and then observe and take pictures.

[0092] 2.7 Statistical analysis

[0093] Data analysis and graphing were performed using GraphPad Prism 8.0 software. Data were presented as mean ± standard error if they met the conditions of normal distribution and variance consistency. One-way analysis of variance (ANOVA) was used to determine whether there were significant differences between the groups. If statistically significant differences were found, we further used Tukey's post hoc test for comparative analysis among multiple groups. The statistical significance level was set at P < 0.05.

[0094] 3. Experimental results

[0095] 3.1 Ethanol treatment inhibits SH-SY5Y cell viability

[0096] Ethanol is the main component of alcohol and can directly damage nerve cells. In this study, we selected SH-SY5Y cells with dopaminergic neuron characteristics to explore the effect of ethanol on cell viability. We treated the cells with different concentrations of ethanol (0-1000mM) for 24 hours to construct a cell injury model. After the treatment, the CCK8 method was used to evaluate cell viability. Figure 1 As shown in the results, ethanol inhibited the viability of SH-SY5Y cells in a dose-dependent manner within the concentration range of 62.5-1000mM. At an ethanol concentration of 500mM, cell viability was steadily reduced to about 60% (65.75% ± 5.45%, p < 0.01). Therefore, in subsequent experiments, we chose an ethanol concentration of 500mM to establish a SH-SY5Y cell injury model.

[0097] 3.2BI-409306 (BI) has a protective effect on ethanol-induced SH-SY5Y cell damage

[0098] In order to evaluate whether BI has a protective effect on ethanol-induced cytotoxicity, we first evaluated whether BI itself is toxic to SH-SY5Y cells. To this end, we set up a blank control group and a BI concentration gradient treatment group (0.1, 0.5, 1, 5, 10 μM) to treat SH-SY5Y cells for 24 hours, and then used the CCK8 method to determine the viability of each group of cells. Figure 2 As shown in the results, BI did not significantly affect the viability of SH-SY5Y cells within the concentration range of 0.1 to 10 μM. Therefore, subsequent experiments will use BI in this concentration range to explore its potential protective effect on ethanol-induced SH-SY5Y cell damage.

[0099] After determining the safe range of BI, we stimulated SH-SY5Y cells with ethanol and pretreated them with different concentrations of BI to explore the effects of BI on cell viability and LDH release in ethanol-induced injury. Figure 3 As shown in A, we pretreated the cells with BI for 1 h (concentration range of 0.125-2 μM), then treated them with 500 mM ethanol for 24 h, and detected the effect of BI on SH-SY5Y cell viability by CCK8 method. The results showed that 500 mM ethanol treatment caused a significant decrease in cell viability to about 65.7% (p<0.0001). After BI treatment, cell viability increased significantly in a dose-dependent manner, and statistical differences appeared at a dose of 0.5 μM (P<0.01).

[0100] At the same time, SH-SY5Y cells were pretreated with different concentrations of BI (0.125-2 μM) for 1 h, and then treated with 500 mM ethanol for 24 h. After the treatment, the LDH release level in the culture medium was measured using an LDH detection kit to evaluate the cytotoxicity level. Figure 3 B, Compared with the blank control group, the intracellular toxicity level increased significantly after treatment with 500 mM ethanol (P < 0.0001), while the intracellular toxicity level decreased in a dose-dependent manner after BI treatment, indicating that BI pretreatment rescued the decreased cell viability caused by ethanol and reduced the cytotoxicity level.

[0101] 3.3BI-409306 reduces ethanol-induced oxidative stress in SH-SY5Y cells

[0102] Studies have reported that alcohol can directly upregulate the generation of intracellular ROS and promote oxidative stress. Continuous oxidative stress can lead to dendritic spine shedding and changes in synaptic plasticity. Excessive production of ROS is a key factor in causing oxidative stress. Therefore, in order to explore whether BI affects the level of intracellular ROS stimulated by ethanol, we applied the DCFH-DA staining method. The results are shown in Figure 2. Figure 3 As shown, compared with the control group, ethanol treatment significantly increased the green fluorescence intensity, indicating that the ROS level was significantly upregulated by ethanol, while the fluorescence intensity of the 0.5 μm BI pretreatment group was significantly reduced compared with the ethanol treatment group, suggesting that BI treatment inhibited ethanol-induced ROS generation.

[0103] When the production of ROS exceeds its clearance rate, lipid peroxidation will further occur to form lipid peroxidation products, such as malondialdehyde (MDA). Lipid peroxidation participates in the neuronal damage process through various mechanisms, including direct damage to cell membranes, proteins, and DNA, ultimately leading to functional and structural disorders of neurons. Therefore, one of the important detection indicators of oxidative stress is the change of intracellular malondialdehyde (MDA), which can reflect the level of oxidative damage to cells. Figure 5 As shown in the figure, compared with the blank control group, there was no significant change in the intracellular MDA level after 0.5 μM BI treatment. However, after 500 mM ethanol treatment, the MDA level increased significantly, indicating that the intracellular lipid peroxide level increased. However, after 1 hour of BI pretreatment, the MDA level was significantly reduced compared with the ethanol treatment group, suggesting that BI can effectively alleviate the cell lipid damage caused by ethanol stimulation.

[0104] In addition to the accumulation of lipid peroxides, oxidative stress is also related to the deficiency of the antioxidant system. Glutathione peroxidase 4 (GPX4) is a key regulatory enzyme of the cellular antioxidant system, which can inhibit intracellular lipid peroxidation to reduce oxidative stress. Our results show that ( Figure 6AB), the expression level of GPX4 protein in cells treated with 500mM EtOH showed a significant downward trend with the treatment time compared with the blank control group. The GPX4 level of cells treated with ethanol for 24h decreased by about 50% (P<0.01), suggesting that ethanol stimulation may increase the oxidative stress of SH-SY5Y cells by reducing GPX4 expression. However, when BI was pretreated for 1h, compared with the group treated with ethanol alone ( Figure 1-6 CD), the GPX4 level was significantly increased (p<0.05), suggesting that BI can counteract the oxidative stress induced by ethanol stimulation by restoring the GPX4 level.

[0105] 3.5BI-409306 restores ethanol-reduced mitochondrial membrane potential in SH-SY5Y cells

[0106] Alcohol can cause excessive oxidative stress, which involves the production of more reactive oxygen species (ROS). Mitochondria are the center of this process, so mitochondrial damage plays an important role in alcohol toxicity. Mitochondrial membrane potential (MMP) is a key factor in maintaining mitochondrial function, which reflects the potential difference between the inner and outer membranes of mitochondria. Under normal circumstances, MMP is maintained at a certain level, which contributes to the generation of ATP and the energy metabolism of cells. When the mitochondrial membrane potential changes, it usually means that mitochondrial function is affected, and the decrease in mitochondrial membrane potential is an important feature of mitochondrial damage. To explore the effect of ethanol on MMP, we used a TMRE kit for detection. The experimental results showed that the TMRE red fluorescence of SH-SY5Y cells was significantly weakened under ethanol stimulation, indicating that the mitochondrial membrane potential decreased. However, after pretreatment with 0.5 μM BI, the TMRE red fluorescence was significantly enhanced compared with the control group treated with ethanol alone, suggesting that BI can alleviate the decrease in mitochondrial membrane potential caused by ethanol to a certain extent.

[0107] 3.6siPDE 9A Reduces ethanol-induced SH-SY5Y cell death

[0108] Early findings: PDE 9A Inhibitor BI-409306 can significantly improve SH-SY5Y cell viability under ethanol stimulation-induced cell damage. 9A We knocked down PDE by siRNA to 9A First, the siPDE was transfected into 9A After 48 hours, we detected the knockdown efficiency by WB. Figure 8 SH-SY5Y cells were transfected with siPDE as shown in AB. 9A Compared with the NC group, its PDE 9AThe expression level was significantly reduced to about 50%, indicating good knockdown efficiency. We then investigated the 9A Whether it affects ethanol-induced cell death is shown by calceinAM / PI staining results ( Figure 8 CD), alcohol significantly increased the level of PI-positive cells, indicating that ethanol stimulation induced SH-SY5Y cell death, while siPDE 9A Compared with the model group, the proportion of PI-positive cells decreased significantly, indicating that inhibition of PDE 9A Can reduce ethanol-induced cell death.

[0109] 3.7siPDE 9A Restoration of ethanol-induced increase in ROS levels and loss of mitochondrial membrane potential

[0110] In determining siPDE 9A After having resistance to ethanol-induced cell death, we further examined its effects on alcohol-induced oxidative stress and mitochondrial damage. The study examined the effects of siPDE9A on the induction of ethanol-stimulated ROS levels and mitochondrial membrane potential loss by DCFH-DA and TMRE staining. Fig. 9 As shown in AD, siPDE 9A The increased ROS level under the model conditions was significantly reduced, and the mitochondrial membrane potential level that was reduced under the model conditions was restored. Therefore, consistent with the previous results of BI409306, the inhibition of PDE 9A Can reduce ethanol-induced cell death by reducing oxidative stress and protecting mitochondrial function

[0111] 4. Conclusion

[0112] 1) Ethanol dose-dependently reduced the viability of SH-SY5Y cells, and at 500 mM, the cell viability dropped to 65.7%. However, BI (0.1-10 μM) had no significant toxicity to cells, and after pretreatment, it could significantly increase cell viability and reduce LDH release.

[0113] 2) Ethanol treatment significantly increased intracellular ROS and MDA levels, and reduced mitochondrial membrane potential and GPX4 levels. BI effectively inhibited the increase of ROS, reduced MDA levels, and upregulated mitochondrial membrane potential and GPX4 expression, indicating that BI has the effect of alleviating oxidative damage and protecting mitochondrial function.

[0114] 3) siRNA knockdown of PDE 9A It significantly reduced ethanol-induced cell death and restored the increased ROS levels and loss of mitochondrial membrane potential caused by ethanol stimulation. 9A Important role in alcohol toxicity.

[0115] In summary, BI inhibits PDE 9A It effectively protected SH-SY5Y cells against ethanol-induced damage by inhibiting oxidative stress, improving antioxidant capacity and protecting mitochondrial function.

[0116] Example 2 Effects of BI-409306 on the treatment of alcohol addiction in chronic drinking mice and its intervention on the formation of addiction

[0117] 1. Experimental Materials

[0118] 1.1 Experimental animals

[0119] C57BL / 6 female and male mice, weighing 22-25g (8 weeks old), were obtained from Guangdong Zhiyuan Biopharmaceutical Technology Co., Ltd., and the experimental animal license number is SCXK (Guangdong) 2021-0057. They were kept in an environment with a temperature of 23-25°C and a humidity of 50-70%, and the indoor brightness was a 12 / 12h light-dark cycle, which was in line with the day and night law. All animal experiments and care procedures were carried out in accordance with the relevant guidelines for experimental animals of Southern Medical University.

[0120] 1.2 Experimental Reagents

[0121]

[0122] 1.3 Preparation of experimental reagents

[0123] Preparation of BI-409306 solution: Weigh 0.2 mg of BI powder and place it in a 4 ml centrifuge tube, add 100 ul DMSO to dissolve it, then add 800 ul PEG400, and add 0.9% NaCl solution to make up to 2 ml, thus obtaining a 0.1 mg / ml BI solution.

[0124] Preparation of NTX solution: Weigh 0.2 mg of NTX powder and place it in a 4 ml centrifuge tube, add 100 ul DMSO to dissolve it, then add 800 ul PEG400, and add 0.9% NaCl solution to make up to 2 ml, thus obtaining a 0.1 mg / ml BI solution.

[0125] Preparation of ethanol (v / v) solution: Measure the corresponding volume of anhydrous ethanol and dilute it to 2L with pure water to obtain an ethanol solution with the corresponding volume ratio.

[0126] Preparation of 2% sucrose solution: weigh 20 g of sucrose, add 1 L of pure water, and completely dissolve on a magnetic stirrer to obtain a 2% sucrose solution.

[0127] Preparation of 0.3 mM quinine solution: Weigh 195 mg of quinine, add 10 mL of DMSO to dissolve it, then add 30 mL of PEG-400 and 5 mL of Tween 20, dilute to 1 L with pure water, and stir on a magnetic stirrer to dissolve it.

[0128] 2. Experimental Methods

[0129] 2.1 Establishment of alcohol addiction model in female and male mice

[0130] Two-Bottle Choice (TBC): A continuous TBC experiment was used to construct a mouse alcohol addiction model. 8-10-week-old C57BL / 6J female and male mice (weight 19-21g) were housed in a single cage in a quiet environment, followed a 12-hour circadian rhythm, had free access to food, and were given two water bottles (50mL plastic bottles) to adapt to the environment for 7 days. Subsequently, the water in one of the bottles was replaced with a freshly prepared ethanol solution (10%, v / v) for 3 weeks (0-21 days). The bottles and mice were weighed every day, and the positions of the bottles were changed every day to avoid positional preference. Fresh water and alcohol were replaced every two days. The alcohol consumption (g / kg) was calculated as follows: (alcohol consumption × 10% alcohol mass fraction) / body weight, unit g / kg; the alcohol preference was calculated as follows: alcohol solution consumption / (alcohol solution consumption + water consumption) × 100%.

[0131] 2.2 Treatment process of mouse alcohol addiction model

[0132] Two-Bottle Choice (TBC): After the alcohol addiction model of female mice was established using the Two-Bottle Choice (TBC) paradigm for 21 days, they were randomly divided into 4 groups (n=8): blank group, model group, BI low-dose group and BI high-dose group (0.1 and 1 mg / kg, po). The drug-treated groups were orally administered once a day for 3 weeks (21-42 days). The consumption of ethanol and water was carefully measured every day, and the ethanol intake and preference of each mouse were calculated separately. The calculation formulas for drinking intake and alcohol preference were as described above.

[0133] Drink in dark (DID): Drinking in the dark (DID) is widely used to simulate the alcoholism stage of addiction. The drink in dark (DID) paradigm can be used to evaluate the alcoholism behavior of mice. The DID test started on the 43rd day after the end of the 42-day TBC test. The water bottle was removed 4 hours after the lights were turned off at night. Only a water bottle containing 20% ​​alcohol was provided, and drug or placebo treatment was given half an hour before the water bottle was removed. After that, the mice were allowed to drink freely for 4 hours. The changes in the weight of the bottle before and after drinking and the weight of the mice were weighed, and the process lasted for 3 days.

[0134] Drinking again after abstinence: After the DID experiment, the water bottle containing alcohol was removed, and each group of mice was allowed to abstain from alcohol for one week. After one week, drinking was resumed and the drug was administered for three days, and the amount of alcohol consumed was calculated.

[0135] 2.3 Interventional effect of BI on addiction formation in mice

[0136] After clarifying the therapeutic effect of BI on the drinking behavior of mice with established alcohol addiction, we further investigated the intervention effect of BI on the formation of addiction, that is, the effect of BI treatment on the addiction rate of mice. Here we introduced the positive control Naltrexone (NTX) to examine whether the two drugs affect the formation of addiction. We changed the dosing strategy and started our dosing treatment at the same time as the mice began to contact the alcohol solution. The specific process is as follows: 8-10 weeks old C57BL / 6J female mice (weighing 19-21g) are placed in a single cage in a quiet environment.

[0137] The mice were fed with a 12-hour circadian rhythm, free access to food, and given two water bottles (50 mL plastic bottles) to adapt to the environment for 7 days. After the adaptation, the mice were randomly divided into 5 groups (n = 9): blank group, model group, BI low-dose group and BI high-dose group (0.1 and 1 mg / kg, po), and positive control natriuretic acid NTX (1 mg / kg) group. At the same time, the water in one of the water bottles of the mice in the remaining groups except the blank group was replaced with freshly prepared ethanol solution. The concentration of the ethanol solution was in an increasing pattern of 3%, 6%, 12%, and 15%, and each concentration of ethanol solution was measured for 5 days. The bottles and mice were weighed every day, and the position of the bottles was changed every day to avoid position preference. Fresh water and alcohol were replaced every two days.

[0138] 2.4 Determination of preference for sugar water and quinine

[0139] Since alcohol is a substance with a special taste, and PDE 9A Regulated cGMP, as a second messenger, may be involved in the conduction of taste signals. Therefore, in order to explore whether BI affects alcohol intake by affecting the taste preference of mice, we measured the preference for quinine sugar water. We selected 2% sucrose solution and 0.3uM quinine solution and used the same two-bottle choice experiment for the measurement. Mice were housed in single cages, followed a 12-hour circadian rhythm, had free access to food, and were given two water bottles (50mL plastic bottles) filled with water to adapt to the environment for 7 days. Subsequently, the water in one of the bottles was replaced with a freshly prepared 2% sucrose solution, and the water bottles and mouse weights were weighed every day. To prevent position preference, the position of the water bottles was changed every day, and the measurement was continued for 5 consecutive days. After the measurement, the sucrose solution was replaced with a 0.3mM quinine solution and the measurement was continued. After 5 days of measurement, statistical analysis was performed.

[0140] 2.5 Statistical analysis

[0141] GraphPad prism was used for statistical analysis of the data. Data are presented as mean ± standard error (Mean ± SEM). Two-way repeated measures analysis of variance was used for the data of the two-bottle choice experiment with increasing alcohol concentrations; the drinking amount of male and female mice was compared using t-test; the remaining data were analyzed using one-way analysis of variance. Tukey's Post Hoc test was used to compare differences between groups. P < 0.05 was considered to be significantly different.

[0142] 3. Experimental results

[0143] 3.1 Establishment of the mouse alcohol addiction model

[0144] There are significant differences in the natural preference for alcohol between different mouse strains and mice of different sexes. Therefore, it is crucial to choose a strain and sex that is easy to establish alcohol addiction. Based on literature research, we chose the standard mouse voluntary drinking Two bottle choice (TBC) experiment to model C57 / BL6 mice, and included both female and male mice. First, we evaluated the drinking behavior of mice of both sexes separately. Fig.10 As shown in AB, the alcohol preference and alcohol intake of female mice gradually increased over time and stabilized at a high level after two weeks; Fig.10 CD, male mice alcohol preference and alcohol intake increased slightly in the first week, but showed irregular fluctuations in the long term, and their average alcohol intake after 6 weeks of modeling was still no different from the level at the beginning of modeling. Fig.10 EF shows that compared with male mice, the alcohol preference of female mice increased rapidly over time. In the third week, the alcohol preference of female mice was stable at above 0.6, and the alcohol intake was significantly higher than that of male mice (p<0.0001). Therefore, in order to ensure the implementation of the 3R principle, reduce the number of experimental animals, and reasonably reduce the duration of animal experiments, we selected female mice that are easy to model and have a relatively stable alcohol preference for subsequent experiments.

[0145] 3.2 BI reduces alcohol preference and alcohol intake in mice in the alcohol addiction model

[0146] Female mice were selected based on the comparison between males and females to construct an alcohol addiction model. After the alcohol addiction model was successfully established, the mice were randomly divided into groups and drug administration was started on the premise of ensuring the consistency of baseline alcohol preference and intake. The processing flow is as follows Fig.11 A. The data of each group in the last three days of treatment were statistically analyzed. Fig.11BC. After three weeks of treatment, compared with the model group, the average alcohol intake of the 0.1mg / kg BI group and the 1mg / kg BI group decreased by 4.14g / kg / 24h (p<0.05) and 5.43g / kg / 24h (p<0.01), respectively; 1mg / kg BI treatment significantly reduced the alcohol preference of alcohol-addicted mice (p<0.05); then we used the Drink in the dark (DID) test to evaluate the alcoholism behavior of each group of mice, and found that the 1mg / kg BI treatment group significantly reduced the amount of alcoholism (p<0.01); finally, we observed the relapse of drinking after abstinence in each group. After 7 days of abstinence, compared with the model group, BI treatment had a tendency to reduce the amount of relapse of mice, but there was no significant difference (p=0.08). The above experimental results show that BI can significantly improve the drinking behavior and reduce alcoholism of alcohol-addicted mice induced by chronic drinking.

[0147] 3.3BI intervention in the formation of alcohol addiction in mice

[0148] Alcohol addiction is a gradual process. Early addiction is mainly driven by rewards, while late addiction manifests as compulsive behavior. After we found that BI treatment can alleviate the compulsive drinking behavior of alcohol addiction model mice, we further explored the effect of BI treatment in the early stage of addiction on the formation of alcohol addiction in mice, and introduced positive control natriuretic acid to explore whether BI and natriuretic acid NTX affect the addiction process of non-addicted mice. The experimental process is designed as follows Fig.12 As shown in A. Fig.12 As shown in BC, with the continuous increase of alcohol concentration, the amount of alcohol consumed in each group also showed a trend of gradual increase (F(3,125)=92.79, p<0.0001). BI and naltrexone treatment significantly reduced the alcohol intake of mice, and there was no interaction (between groups F(3,125)=9.180, P<0.0001; interaction F(9,125)=1.017, P=0.4301). Compared with the model group, drug treatment significantly reduced the increase in alcohol preference of mice (between groups F(3,124)=6.054, p<0.0001, alcohol concentration (F(3,124)=4.576, p<0.01) interaction F(9,124)=1.062, P=0.3958), showing that the preference for alcohol in the BI and NTX treatment groups was significantly different from the gradually increasing trend in the model group. In addition, Fig.12 FG shows that there was no significant change in total fluid intake and body weight among the groups, and there was no statistical difference among the four groups. The above results show that both BI and NTX treatment can significantly reduce the increase in alcohol consumption and alcohol preference in mice during the modeling process, that is, they have an intervention effect on the formation of alcohol addiction in mice.

[0149] 3.4 BI treatment does not affect the taste preference of mice

[0150] Alcohol is a substance with a special bitter taste, and cyclic guanosine monophosphate (cGMP) as a second messenger may play an important role in the taste perception process, including the perception of bitterness. To explore whether BI directly affects the taste perception of mice and thus reduces their alcohol preference, we designed a sugar water preference experiment and a quinine preference experiment, using 2% sucrose solution and 0.3μM quinine solution to evaluate the changes in the taste preference of mice. Fig.13 As shown in A, in the sugar water preference experiment, natriuretic acid treatment significantly reduced the sugar water preference of mice (14.76% ± 4.031, p < 0.01), which is consistent with the findings reported in the literature that natriuretic acid reduces the euphoria induced by sugar water and reduces its intake. However, treatment with different concentrations of BI had no significant effect on sugar water preference and sugar water intake. Fig.13 As shown by EF, in the quinine preference test, neither naltrexone nor BI treatment affected the mice's quinine intake and preference. The total water intake and body weight of mice during the sugar water preference and quinine preference tests did not change significantly among the groups. The above results indicate that BI does not affect the mice's sugar water preference or bitterness sensitivity.

[0151] 4. Conclusion

[0152] 1) Female mice have significantly higher alcohol preference and alcohol intake than male mice, and can form a stable alcohol addiction model in a faster time. Based on the difference in drinking behavior, female mice were selected as the subjects of subsequent experiments.

[0153] 2) In the female alcohol addiction mouse model, compared with the solvent group, BI treatment can significantly reduce the alcohol intake and preference of alcohol-addicted mice, reduce their alcohol abuse, and has a tendency to reduce the amount of mice drinking again after withdrawal, indicating that BI has a therapeutic effect on the female mouse alcohol addiction model.

[0154] 3) BI also showed a positive effect in preventing the formation of alcohol addiction, significantly reducing the amount of alcohol consumed and the increase in preference in mice, which was similar to the effect of the positive control natriuretic peptide.

[0155] 4) BI did not affect the intake and preference of sucrose and quinine in female mice, suggesting that BI's regulation of drinking behavior in mice was not through changes in taste perception, which was different from naltrexone.

[0156] Example 3 Mechanism of BI-409306 in treating alcohol addiction and acute alcohol intoxication

[0157] 1. Experimental Materials

[0158] 1.1 Experimental animals

[0159] C57BL / 6 female mice, weighing 22-25 g (8 weeks old), were provided by Guangdong Zhiyuan Biopharmaceutical Technology Co., Ltd., with the experimental animal license number SCXK (Guangdong) 2021-0057. These mice were raised in an environment with a temperature of 23-25°C and a humidity of 50-70%, with a 12-h light-dark cycle, in line with the circadian rhythm. All animal experiments and care procedures followed the relevant guidelines for experimental animals of Southern Medical University.

[0160] 1.2 Experimental Reagents

[0161]

[0162] 1.3 Preparation of experimental reagents

[0163] Gelatin coating solution: Heat 250 mL of pure water to 60°C, add 2.5 g of gelatin, continue heating and stirring to dissolve, after all dissolved, add 0.25 g of potassium chromium sulfate, stir until clear, and then dilute to 500 mL. Filter and cool to room temperature.

[0164] 2. Experimental Methods

[0165] 2.1 Western blot detection of protein expression levels in animal tissues

[0166] 2.1.1 Collection of animal tissues

[0167] First, prepare the necessary experimental supplies, such as surgical instruments, insulation buckets, liquid nitrogen, labeled 1.5ml epoxies, ice cubes wrapped in tin foil, anesthetic drugs, etc. After the mice are over-anesthetized, they are perfused with PBS. After the blood is removed, the brain and liver tissues are quickly peeled off. The liver is cut into small pieces and quickly placed in liquid nitrogen. The brain tissue is placed on ice cubes wrapped in tin foil to cool it down and harden its texture. Then, the corresponding brain area is peeled off according to the experimental requirements. In this experiment, the hippocampus and prefrontal cortex are peeled off. The removed tissues are placed in the corresponding epoxies and quickly frozen in liquid nitrogen. After all the tissues are collected, all tissues are removed from the liquid nitrogen, marked with experimental information and date, and stored in a -80℃ refrigerator.

[0168] 2.3.2 Extraction of animal tissue protein

[0169] Prepare tissue lysis buffer in advance, i.e. RIPA: phosphatase inhibitor: protease inhibitor = 100:1:1. Take out the animal tissue from the refrigerator, add the corresponding volume of lysis buffer according to the size of the tissue block, and use an ultrasonic tissue disruptor in an ice bath to disrupt it until the ep tube is a transparent liquid and there is no visible floating material. After the ultrasound is finished, continue to incubate in the ice bath for 30 minutes to fully lyse it. Precool the high-speed centrifuge to 4°C in advance, put the ep tube after lysis into the precooled centrifuge, and centrifuge at 12000rpm for 10 minutes. After the centrifugation is completed, carefully aspirate the supernatant, transfer it to a new ep tube, mark it and put it in the refrigerator for use.

[0170] 2.3.3 Quantification of tissue proteins

[0171] Quantification was performed using the BCA quantification method as described previously.

[0172] 2.3.4 Protein immunoblotting and ECL development were performed as described above.

[0173] 2.2 Golgi staining

[0174] 2.2.1 Sample processing

[0175] After overdose anesthesia, remove the intact brain of the mouse as soon as possible. Do not perfuse. After washing the surface blood in PBS, put it into the A+B solution prepared one day in advance. After 24 hours at room temperature and away from light, replace it with fresh A+B solution, and then store it at room temperature and away from light for 14 days. Gently shake the sample 2-3 times a week. After the time is up, replace the liquid with C solution and continue to soak in the dark for 24 hours. After 24 hours, replace it with fresh C solution and store it at room temperature and away from light for 7 days. Prepare gelatin-coated slides in advance during the brain sample processing process. Place the dry adhesive slides in the prepared gelatin coating solution to avoid bubbles and incubate at 37 degrees for 2 hours. After the incubation time is over, dry it completely at room temperature and store it at 4°C.

[0176] 2.2.2 Cryosectioning

[0177] Adjust the slicer to -22℃ 4h in advance and prepare dry ice. After the sample is soaked, remove the brain tissue and gently dry it. Fix the tissue on the sample holder with distilled water, and then place the sample holder in dry ice for precooling. It takes about 1min. Quickly use a brush to embed a layer of distilled water on the surface of the sample, and then install the sample holder on the freezing slicer. After balancing the temperature, slice the slices with a thickness of 100μm. No anti-roll plate is required. Use a glass rod to transfer the slices to a gelatin-coated slide evenly coated with C liquid, then add a drop of C liquid on top of the sample, apply it evenly with a brush and remove bubbles, tilt the slide and absorb excess liquid. Dry the slices at room temperature and avoid light for 24h.

[0178] 2.2.3 Dyeing

[0179] First, wash the sample in distilled water twice, 4 minutes each time. Then place the slices in freshly prepared D+E solution for 10 minutes. Then wash with distilled water twice, 4 minutes each time, and perform gradient dehydration in 50%, 75%, and 95% ethanol solutions. After 4 minutes at each concentration, dehydrate in anhydrous ethanol 4 times, 4 minutes each time. After dehydration, make it transparent in xylene 3 times, 4 minutes each time. After completion, use neutral resin sealing agent to seal the slices. Note: During the staining process, ensure that the slices do not dry out and keep them away from light throughout the staining process.

[0180] 2.2.4 Sealing

[0181] Remove the slide from xylene, gently tilt it on absorbent paper and wipe off the liquid around it. Drop a drop of neutral resin on the cover slip and gently cover the sample, trying not to have bubbles. After covering it and wiping off the excess liquid around it, apply clear nail polish around the cover slip and wait for it to dry.

[0182] 3. Experimental results

[0183] 3.1 Chronic drinking alcohol addiction model

[0184] 3.1.1 Chronic alcohol consumption leads to PDE in female mice 9A High expression

[0185] Chronic drinking affects the function of the ventral tegmental area (VTA) of the midbrain mainly by affecting the activity of glutamate transmission and dopamine systems. The prefrontal cortex (mPFC) and hippocampus (HP), as its important downstream projection areas, play an important role in the reward pathway. Literature reports that cGMP levels in the brain decrease significantly after drinking, and the key regulatory enzyme PDE for cGMP levels 9A Therefore, we first investigated the PDE levels in the mPFC and HP brain regions of chronic alcohol addiction mice. 9A The protein levels were tested. Fig.14 As shown, compared with the blank group, PDE was found in the mPFC (p<0.05) and HP (p<0.01) of the model mice. 9A The level of PDE increased significantly. 9A The expression of PDE affects the metabolism of cGMP, thereby affecting the function of the downstream projection area of ​​the reward pathway, which may further affect the reward mechanism and addictive behavior of alcohol, suggesting that PDE 9A It may play an important role in the pathological mechanism of alcohol addiction.

[0186] In addition, since alcohol does not act on specific cells or receptors like traditional addictive substances such as opioids or nicotine, it can widely affect almost all organs in the body. Therefore, we further examined PDE in the liver, which is an important site of alcohol metabolism. 9A The changes in expression levels are as follows Fig.15 As shown, compared with the blank group, the PDE 9A This suggests that chronic drinking may not only affect cGMP metabolism in the central nervous system, but also in the periphery (such as the liver) by affecting PDE 9A Levels regulate cGMP-related physiological processes.

[0187] 3.1.2 BI restores the expression of synaptic-related proteins in the hippocampus of alcohol-consuming mice

[0188] Alcohol addiction is closely related to changes in synaptic plasticity in specific brain regions. Long-term alcohol exposure can lead to synapse-related abnormalities, which may occur without morphological damage, leading to cognitive and behavioral disorders. Therefore, we first detected the levels of synapse-related proteins in each group of mice. PSD95 (postsynaptic density protein-95), as a structural protein located at excitatory glutamate receptor synapses, plays a key role in synaptic signaling and behavioral plasticity. The results are as follows Fig.16 As shown in A, the PSD95 protein level in the hippocampus of mice drinking alcohol for a long time showed a downward trend, although it did not reach a significant difference (p=0.0638). In contrast, the PSD95 level in the hippocampus of model mice treated with 1 mg / kg BI was significantly increased.

[0189] We also evaluated the expression of Synapsin-1 and GAP-43. Synapsin-1 plays a key role in synaptic plasticity and is involved in neuronal axonogenesis and synaptic transmission. Fig.16 As shown in B, long-term chronic drinking significantly downregulated the level of Synapsin-1 in the hippocampus of mice, and BI restored its expression in a dose-dependent manner; GAP-43 (growth-associated protein-43) is mainly induced during the axon growth and synaptic network formation of neurons, and is closely related to synaptic plasticity phenomena such as long-term potentiation (LTP) and long-term depression (LTD). Fig.16 As shown in C, GAP43 levels were significantly downregulated in the chronic drinking group, while 1 mg / kg BI significantly rescued its expression level. These results suggest that long-term alcohol exposure can lead to abnormalities in synaptic-related proteins in the hippocampus, thereby affecting synaptic plasticity, leading to cognitive and behavioral disorders. BI intervention may improve alcohol-induced neurological dysfunction by restoring the expression of these synaptic-related proteins.

[0190] 3.1.3BI restores dendritic spine density in the hippocampus of alcohol-drinking mice

[0191] The dynamic changes in the morphology, size, and number of dendritic spines are closely related to the efficacy of synapses and are the main form of synaptic structural plasticity. During the addiction process, morphological changes such as the formation, shedding, expansion, and atrophy of dendritic spines are often accompanied by abnormal synaptic plasticity. Therefore, we further detected the changes in dendritic spine density in each group. Fig.17 As shown in the figure, compared with the normal group, the density of dendritic spines in the hippocampus of the drinking group was significantly decreased (p<0.001), and this change was significantly restored by 1 mg / kg BI treatment. This shows that BI may improve drinking behavior by protecting synaptic morphology and restoring synaptic plasticity.

[0192] 3.1.4BI restores GPX4 expression in the hippocampus of alcohol-consuming mice

[0193] As an important antioxidant enzyme, GPX4's main function is to protect cells from damage caused by lipid peroxidation. In alcohol-induced oxidative stress, the role of GPX4 is particularly important. Studies have shown that alcohol exposure can lead to changes in GPX4 activity, thereby affecting the cell's ability to respond to oxidative stress. This suggests that GPX4 may play a protective role in alcohol-induced oxidative stress, and its reduced level may be an important factor in alcohol-induced oxidative damage that ultimately leads to abnormal synaptic plasticity. We found that compared with the blank group, the level of GPX4 in the hippocampus of the chronic drinking group was significantly reduced, while BI treatment restored its expression in a dose-dependent manner. This shows that BI treatment can play a neuroprotective role by restoring GPX4 expression, improving the antioxidant response of cells, and alleviating alcohol-induced oxidative damage.

[0194] 3.2 Mouse model of acute alcohol intoxication

[0195] 3.2.1 BI restores GPX4 levels in the hippocampus of mice with acute alcohol intoxication

[0196] Previous results have shown that GPX4 was downregulated in the mouse hippocampus in the chronic drinking model. Alcohol is a potent oxidant, and its single-dose acute intake may also directly affect the activity and stability of GPX4. Therefore, we detected the expression of GPX4 in the acute toxicity model. Our study found that GPX4 was also significantly downregulated in the hippocampus of mice in the acute alcohol intoxication model (p<0.05), suggesting that acute alcohol may cause oxidative stress damage by affecting GPX4 levels; and 3mg / kg BI significantly upregulated the expression of GPX4 that was reduced by alcohol (p<0.01), indicating that BI may counteract acute alcohol toxicity by upregulating GPX4 levels.

[0197] 3.2.2 BI reduces the level of inflammatory factor IL-1β in the hippocampus of mice with acute alcohol intoxication model

[0198] Oxidative stress induced by alcohol toxicity can enhance inflammatory responses, while inflammatory responses can exacerbate oxidative stress by producing more ROS. This interaction may play a key role in the various tissue injuries caused by alcohol. Therefore, we further detected the expression levels of inflammatory factors in each group. The results showed that acute alcohol intoxication significantly upregulated the expression level of iNOS in the mouse hippocampus (p<0.001), indicating that alcohol induced the occurrence of neuroinflammation, and this change was not affected by BI treatment. However, we found that Fig. 20 As shown in C, alcohol intoxication induced significant expression of IL-1β in the mouse hippocampus, and 3 mg / kg BI treatment significantly reduced its level. The above results indicate that acute alcohol intoxication upregulated iNOS levels and the expression of inflammatory factor IL-1b, induced neuroinflammation, and BI treatment significantly reduced the upregulation of inflammatory factors, but its anti-inflammatory effect may be unrelated to the expression of iNOS.

[0199] 4. Conclusion

[0200] 1) In the prefrontal cortex, hippocampus, and liver of chronic alcohol-drinking mice, PDE 9A The protein level was significantly upregulated, suggesting that alcohol may synergistically increase PDE in the central and peripheral 9A Expression affects cGMP metabolism, which in turn affects addictive behavior and alcohol toxicity, suggesting that PDE 9A It may play an important role in both the central and peripheral toxicity of alcohol.

[0201] 2) Long-term drinking led to a significant downregulation of synaptic-related proteins such as PSD95, Synapsin-1 and GAP-43 in the hippocampus, and reduced the density of dendritic spines in the hippocampus, indicating that alcohol exposure may cause synaptic plasticity disorders, while BI treatment can upregulate synaptic-related proteins, restore dendritic spine density, and protect synaptic plasticity.

[0202] 3) Both chronic drinking and acute alcohol intoxication significantly reduced the level of GPX4 in the hippocampus, and BI treatment could restore its expression in a dose-dependent manner, suggesting that both acute and chronic drinking lead to abnormalities in the antioxidant system, and BI may protect neural tissue by improving antioxidant responses.

[0203] 4) Acute alcohol intoxication significantly upregulated the expression of iNOS and IL-1β, inducing neuroinflammation. BI treatment reduced IL-1β levels but had no effect on iNOS expression. This indicates that alcohol can induce neuroinflammation, and BI can reduce the expression of inflammatory factors, but its anti-neuroinflammatory effect may be unrelated to iNOS.

[0204] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A phosphodiesterase 9A inhibitor for treating alcohol addiction and acute alcohol poisoning, characterized in that: The inhibitor is BI-409306, and its structure is as follows:

2. Use of a phosphodiesterase 9A inhibitor in inhibiting ROS elevation and reducing MDA, characterized in that: The inhibitor is BI-409306.

3. A phosphodiesterase 9A inhibitor inhibits PDE 9A Improve the application of cGMP, its characteristics are: The inhibitor is BI-409306.

4. Use of a phosphodiesterase 9A inhibitor in restoring GPX4 expression, characterized in that: The inhibitor is BI-409306.

5. Use of a phosphodiesterase 9A inhibitor in reducing the inflammatory factor IL-1β, characterized in that: The inhibitor is BI-409306.

6. The use according to any one of claims 2 to 5, characterized in that: BI-409306 affects the drinking behavior of alcohol addicts by regulating synaptic plasticity.

7. The use according to any one of claims 2 to 5, characterized in that: BI-409306 reduces acute alcohol toxicity damage by restoring GPX4 levels and reducing inflammatory factors.

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