Biological agent for inhibiting alcohol drinking amount of alcohol-dependent person and application of biological agent

Abstract

The invention relates to the field of medical biology, in particular to a biological preparation for inhibiting the alcohol drinking amount of an alcohol-dependent person and application of the biological preparation. The invention discloses a biological agent for inhibiting the alcohol drinking amount of an alcohol-dependent person. The biological agent is lactobacillus plantarum extracellular vesicles. The lactobacillus plantarum extracellular vesicles play a role in regulating and controlling the drinking amount of alcohol-dependent rats by regulating the expression of the BNDF, the lactobacillus plantarum extracellular vesicles can increase the expression of the BDNF of PC12 cells in an in-vitro cell culture experiment, alcohol reduces the expression of the BDNF of the PC12 cells, and the lactobacillus plantarum extracellular vesicles achieve the inhibition effect by reversing the expression of the alcohol on the BDNF of the PC12 cells. According to the biological preparation for inhibiting the alcohol drinking amount of the alcohol-dependent person and the application of the biological preparation, SD male rats serve as research objects, the inhibiting effect of extracellular vesicles derived from intestinal lactobacillus plantarum on the alcohol drinking amount of the alcohol-dependent rats and related mechanisms are researched, and a new thought is provided for clinical prevention and treatment of alcohol dependence.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biology, specifically to a biological agent that inhibits alcohol consumption in alcohol-dependent individuals and its application. Background Technology

[0002] Alcohol dependence is a complex chronic disease. From first-time drinking to continued drinking and finally developing into alcohol dependence, this process is accompanied by neuroadaptive changes in the function of neurotransmitters, ion channels, and receptors, as well as the signaling pathways they mediate. Furthermore, alcohol dependence is a chronic relapsing encephalopathy that can cause a wide range of adverse health consequences. The high relapse rate and increased alcohol consumption after withdrawal treatment are currently major challenges in clinical practice. No reports have been found regarding drugs that inhibit alcohol consumption in patients with alcohol dependence or their mechanisms of action. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide a biological agent and its application to inhibit alcohol consumption in alcohol-dependent individuals.

[0004] To achieve this objective, the present invention employs the following technical solution: a biological agent for inhibiting alcohol consumption in individuals with alcohol dependence, wherein the biological agent is Lactobacillus plantarum derived extracellular vesicles (L-EVs).

[0005] On the other hand, the present invention provides the application of a biological agent in the preparation of a drug to inhibit alcohol consumption in alcohol-dependent individuals, wherein the biological agent is the *Lactobacillus plantarum* extracellular vesicle described in the present invention.

[0006] In some embodiments of the present invention, the Lactobacillus plantarum extracellular vesicles can enhance the expression of BDNF in PC12 cells.

[0007] In some embodiments of the present invention, K252a has an antagonistic effect on the regulation of alcohol consumption by extracellular vesicles of Lactobacillus plantarum, wherein K252a is a receptor-specific antagonist of brain-derived neurotrophic factor (BDNF).

[0008] In some embodiments of the present invention, the extracellular vesicles of *Lactobacillus plantarum* can enhance the expression of BDNF in the ventral tegmental area of ​​the midbrain.

[0009] In some embodiments of the present invention, the concentration of the Lactobacillus plantarum extracellular vesicles is 15 μg / 0.5 mL or 30 μg / 0.5 mL.

[0010] In some embodiments of the present invention, the Lactobacillus plantarum extracellular vesicles can enhance the expression of BDNF in PC12 cells.

[0011] In some embodiments of the present invention, the concentration of the extracellular vesicles of Lactobacillus plantarum is 0 μg / ml, 20 μg / ml or 30 μg / ml.

[0012] In some embodiments of the present invention, the extracellular vesicles of *Lactobacillus plantarum* regulate the amount of alcohol consumed by alcohol-dependent individuals by modulating BNDF expression.

[0013] In another aspect, the present invention provides a pharmaceutical composition for inhibiting alcohol consumption in alcohol-dependent individuals, the composition comprising the *Lactobacillus plantarum* extracellular vesicles described in the present invention, and further comprising glycine reuptake inhibitor N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catechyl]methylglycine.

[0014] In another aspect, the present invention provides an inhibitor to suppress alcohol consumption in alcohol-dependent individuals, the inhibitor comprising the Lactobacillus plantarum extracellular vesicles as described in claim 1.

[0015] The beneficial effects of this invention are as follows:

[0016] (1) This invention provides a plant-derived extracellular vesicle of Lactobacillus plantarum that inhibits alcohol consumption in alcohol-dependent individuals. It plays a regulatory role in alcohol consumption in alcohol-dependent rats by regulating BNDF expression. In in vitro cell culture experiments, plant-derived extracellular vesicles of Lactobacillus plantarum can increase BDNF expression in PC12 cells, while alcohol reduces BDNF expression in PC12 cells. Plant-derived extracellular vesicles of Lactobacillus plantarum achieve the inhibitory effect by reversing the effect of alcohol on BDNF expression in PC12 cells.

[0017] (2) This invention provides a biological agent and its application for inhibiting alcohol consumption in alcohol-dependent individuals. Using male Sprague-Dawley (SD) rats as the research subject, the study investigated the inhibitory effect of extracellular vesicles derived from Lactobacillus plantarum on alcohol consumption in alcohol-dependent rats and the related mechanism, providing a new approach for the clinical prevention and treatment of alcohol dependence. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0019] In the attached diagram:

[0020] Figure 1 The effect of L-EVs on alcohol consumption in alcohol-dependent rats; among them, Figure 1In this context, A represents the effect of intraperitoneal injection of L-EVs on the amount of alcohol consumed by rats after alcohol withdrawal. Figure 1 B in the table represents the effect of microinjection of L-EVs into the VTA region on re-drinking volume in rats after alcohol withdrawal; data are expressed as mean ± SEM, **P<0.01, ****P<0.0001, 6 rats per group.

[0021] Figure 2 This is a diagram illustrating the antagonistic effect of K252a (a BDNF receptor-specific antagonist) on L-EVs-regulated alcohol consumption in alcohol-dependent rats; where, Figure 2 In the figure, A represents the amount of alcohol consumed by rats after alcohol withdrawal by pre-injecting a small amount of K252a into the VTA before intraperitoneal injection of L-EVs. Figure 2 In the figure, B represents the amount of alcohol consumed by rats after alcohol withdrawal by pre-injecting a micro-dose of K252a into the VTA region before micro-injecting L-EVs. Figure 2 C represents the effect of microinjection of BDNF into the VTA region on alcohol consumption after alcohol withdrawal in rats. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. There were 6 rats in each group.

[0022] Figure 3 The effect of intraperitoneal injection of L-EVs on VTABDNF in alcohol-dependent rats was shown in the figure. L-EVs were injected every 24 hours during the 72-hour alcohol withdrawal period. The expression of BDNF protein in VTA was measured by Western blot. **P<0.001, ****P<0.0001, 6 rats in each group.

[0023] Figure 4 L-EVs can reverse the inhibitory effect of alcohol on BDNF expression in PC12 cells; among them, Figure 4 In the figure, c represents the effect of Western blot analysis on the regulation of BDNF expression in PC12 cells by L-EVs. Figure 4 In the figure, d represents the expression of intracellular BDNF in PC12 cells after co-incubation with L-EVs at concentrations of 10 μg / ml, 20 μg / ml, and 30 μg / ml. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001, n=3. Detailed Implementation

[0024] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0025] The extracellular vesicles of Lactobacillus plantarum described in this invention are obtained by extracting the extracellular vesicles of Lactobacillus plantarum using differential centrifugation, and then identified using transmission electron microscopy (TEM) and a nanoparticle analysis system (NTA).

[0026] Preparation of Lactic Acid Bacteria Extracellular Vesicles: *Lactobacillus plantarum* was cultured to the stationary phase (i.e., activated *Lactobacillus plantarum* was anaerobically cultured in sterile MRS broth at 37℃ for 24 h). The cells were removed by centrifugation at 3000×g for 15 min, followed by continuous centrifugation at 10000×g for 60 min to remove large bacterial fragments and intact organelles. Subsequently, the supernatant was ultracentrifuged at 200,000×g for 60 min, the supernatant was removed, and the precipitate was retained. The EVs were resuspended in PBS. The sample was passed through a 0.22 μm pinhole filter to obtain purified L-EVs. Protein quantification of L-EVs was performed using the BCA method. Identification was performed using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The samples were then frozen at -80℃ for later use.

[0027] L-EVs morphology: Through NTA and TEM images, it was found that the extracellular vesicle samples have a double lipid membrane structure, are spherical in shape, and are about 20-200 nm in size.

[0028] L-EVs active ingredients: Lactobacillus plantarum EVs possess a plasma membrane and carry nucleic acids, lipids, proteins, and bacterial metabolites, which can affect multiple cellular pathways in the host. Based on current understanding of their biogenesis, extracellular vesicles can be broadly classified into two categories: exosomes and microvesicles. Exosomes, with a diameter of 30-100 nm, are intraluminal vesicles (ILVs) formed by budding inward from the endosome membrane during the maturation of multivesicular endosomes (MVEs). They are intermediates within the endosome system and are secreted after MVEs fuse with the cell surface. Microvesicles range in diameter from 50 nm to 1000 nm, but can be larger in tumors (up to 10 μm). They are produced by budding and fission of the plasma membrane and subsequent vesicle release into the extracellular space.

[0029] Identification: Metabolomics analysis using LC-MS was used to identify the constituent molecules of *Lactobacillus plantarum* EVs, revealing 1519 compounds. Fatty acids detected in whole *Lactobacillus plantarum* cells included C12:0, C12:1, C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, and C20:119,20. Proteomics analysis of *Lactobacillus plantarum* EVs showed that many proteins were located in the bacterial cell membrane and cytoplasm, while a few were located in the cell wall or extracellular environment. Small RNA analysis of L-EVs revealed that the amount of RNA in RNase-treated EVs was similar to that in untreated EVs, indicating that *Lactobacillus plantarum* EVs have an RNA coating that protects them from RNase degradation. RNA-seq (transcriptome sequencing) was then performed to comprehensively analyze the nucleotide sequence and RNA chain length of L-EVs. Finally, L-EVs were identified as containing small RNAs and ribosomal proteins.

[0030] Example 1: This example establishes a chronic intermittent active alcohol consumption model in male SD rats.

[0031] Experimental Methods: SD rats were randomly divided into an alcohol group (n=12) and a water control group (n=6). The control group received two bottles of free access to water, while the alcohol group received one bottle of drinking water and the other a bottle of 20% alcohol solution. The amount of alcohol and water consumed by the rats was recorded daily, and their body weight was measured weekly. After 28 days, the rats were randomly divided into a 0h withdrawal group and a 72h withdrawal group. After withdrawal, the rats were again given 20% alcohol, and the amount of alcohol consumed within 6 hours was recorded. Six rats were in each group.

[0032] Experimental results: During the modeling period, there were no statistically significant differences in body weight (428.3±31.57mL) and total 24-hour fluid intake (52.72±9.784mL) between the alcohol-drinking group and the water-drinking control group (430.0±22.14mL) and total fluid intake (56.08±9.253mL) (P>0.05). After 28 days of modeling, the alcohol consumption (4.45±1.13g / kg / 24h) and alcohol preference (37.75±8.27%) of the alcohol-drinking group reached stable levels, and the rat alcohol dependence model was successfully established.

[0033] Example 2: This example is to verify the effect of L-EVs on alcohol consumption in alcohol-dependent rats.

[0034] Experimental Methods: Male SD rats were randomly divided into an intraperitoneal injection group (n=36) and a VTA (ventral tegmental area) injection group (n=36). After IA2BC training for 21 days, stereotactic brain localization was performed on the VTA area of ​​the rats in the VTA injection group, a cannula was implanted and fixed, and alcohol consumption continued for one week, followed by a 72-hour abstinence period. During the 72-hour abstinence period, the intraperitoneal injection group was randomly divided into a control group, a PBS group, and an L-EVs group (1. Injection dose: 5 μg / 0.5 mL or 30 μg / 0.5 mL); the VTA injection group was randomly divided into a Sham group, a PBS group, and an L-EVs group (2. Injection dose: 1 μg / μL or 2 μg / μL). The amount of alcohol consumed by the rats within 6 hours in each experimental group was recorded, with 6 rats in each group.

[0035] Experimental results: Figure 1 The effect of L-EVs on alcohol consumption in alcohol-dependent rats; among them, Figure 1 In this context, A represents the effect of intraperitoneal injection of L-EVs on the amount of alcohol consumed by rats after alcohol withdrawal. Figure 1 B in the table represents the effect of microinjection of L-EVs into the VTA region on re-drinking volume in rats after alcohol withdrawal; data are expressed as mean ± SEM, **P<0.01, ****P<0.0001, 6 rats per group.

[0036] The amount of alcohol consumed again in rats after 72 hours of abstinence was significantly increased (P<0.001). Intraperitoneal injection of L-EVs (15 μg / 0.5 mL or 30 μg / 0.5 mL) significantly reduced the amount of alcohol consumed again in alcohol-dependent rats after 72 hours of abstinence (P<0.005 or P<0.0001). Microinjection of L-EVs (1 μg / μL or 2 μg / μL) into the VTA area also significantly reduced the amount of alcohol consumed again in alcohol-dependent rats after 72 hours of abstinence (P<0.01 or P<0.0001).

[0037] Example 3: This example verifies the antagonistic effect of K252a (BDNF receptor-specific antagonist) on the regulation of alcohol consumption in L-EVs-dependent rats.

[0038] Experimental methods: An alcohol consumption model was successfully established in SD rats. As described above, the VTA region of the brain was stereotactically localized and a cannula was implanted. After resuming alcohol consumption, the rats were given a 72-hour abstinence period. Rats were randomly divided into four groups: Sham group, L-EVs(ip)+K252a(VTA) group, K252a(VTA) group, L-EVs(ip) group, K252a(VTA)+L-EVs(VTA) group, and L-EVs(VTA) group (injection dosage: 15 μg / 0.5 mL L-EVs(ip), 40 μM / μL K252a(VTA), 1 μg / μL L-EVs(VTA)). The same amount of solvent was injected into the control group. The injection rate of the reagent was 0.5 μL / min. After injecting 0.5 μL into each side of the VTA area, the needle was left in place for at least 2 minutes to facilitate drug diffusion. Rats were given alcohol 30 minutes after drug injection, and the amount of alcohol consumed by the rats was measured 72 hours after withdrawal. There were 6 rats in each group.

[0039] Experimental results: Figure 2 This is a diagram illustrating the antagonistic effect of K252a (a BDNF receptor-specific antagonist) on L-EVs-regulated alcohol consumption in alcohol-dependent rats; where, Figure 2 In the figure, A represents the amount of alcohol consumed by rats after alcohol withdrawal by pre-injecting a small amount of K252a into the VTA before intraperitoneal injection of L-EVs. Figure 2 In the figure, B represents the amount of alcohol consumed by rats after alcohol withdrawal by pre-injecting a micro-dose of K252a into the VTA region before micro-injecting L-EVs. Figure 2 In the figure, C represents the effect of microinjection of BDNF into the VTA region on alcohol consumption after withdrawal in rats. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each group consisted of 6 rats. K252a antagonized the inhibitory effect of L-EVs on alcohol consumption in rats (P<0.01). Microinjection of BDNF into the VTA reduced alcohol consumption after withdrawal in alcohol-dependent rats to a similar degree as L-EVs (P<0.001). BDNF expression in the VTA region was low 72 h after withdrawal (P<0.001); intraperitoneal injection of L-EVs (15 μg / 0.5 mL or 30 μg / 0.5 mL) increased BDNF expression in the VTA region (P<0.01 or P<0.0001). In in vitro cell culture experiments, L-EVs increased BDNF expression in PC12 cells (P<0.05); alcohol decreased BDNF expression in PC12 cells, and L-EVs reversed the inhibitory effect of alcohol on BDNF expression in PC12 cells (P<0.0001).

[0040] Example 4: This example shows the effect of intraperitoneal injection of L-EVs on VTA BDNF in alcohol-dependent rats.

[0041] Experimental Methods: 72-hour alcohol abstinence: As previously described, a cannula was successfully implanted in the brain region, and rats were given a 72-hour abstinence period. They were randomly divided into Sham, BDNF, PBS, and L-EVS groups. Alcohol consumption was measured in rats after 72 hours of abstinence. L-EVs were injected during the 72-hour alcohol abstinence period: After successful modeling in SD rats, a 72-hour abstinence period was observed. L-EVs were injected intraperitoneally every 24 hours during this period. BDNF protein expression in the VTA was measured using Western blotting.

[0042] Experimental results: Figure 3 The effect of intraperitoneal injection of L-EVs on VTA BDNF in alcohol-dependent rats was shown in the figure. L-EVs were injected during a 72-hour alcohol withdrawal period, and BDNF protein expression in the VTA was measured using Western blot. The results showed that intraperitoneal injection of L-EVs increased BDNF expression in the VTA, and L-EVs may regulate alcohol consumption in alcohol-dependent rats by modulating BDNF expression in the VTA. **P<0.001, ****P<0.0001, 6 rats per group.

[0043] Example 5: This example verifies the regulatory effect of L-EVs on intracellular BDNF expression in in vitro cultured cells.

[0044] Experimental Methods: First, the optimal alcohol incubation time was selected using a CCK-8 assay kit. Healthy PC12 cells were seeded into 96-well plates and cultured for 24 hours. After discarding the supernatant, 100 μL of DMEM complete medium containing different concentrations of alcohol was added to each well. Alcohol concentrations were 0, 10, 50, 100, 150, and 200 mM, with five wells for each concentration and one blank control group. After 24, 48, and 72 hours of culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 hours. The absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group). Western blotting analysis was used to select the optimal alcohol concentration for PC12 cell incubation at 50 mM, 100 mM, and 150 mM. Secondly, Western blotting analysis was used to determine the regulatory effect of different concentrations of L-EVs co-incubated with alcohol on intracellular BDNF expression in PC12 cells. Each experiment was repeated three times.

[0045] Experimental results: Figure 4 L-EVs can reverse the inhibitory effect of alcohol on BDNF expression in PC12 cells; among them, Figure 4 In the figure, c represents the effect of Western blot analysis on the regulation of BDNF expression in PC12 cells. Figure 4 In the figure, 'd' represents the expression of intracellular BDNF in PC12 cells after co-incubation with ethanol at concentrations of 10 μg / ml, 20 μg / ml, and 30 μg / ml. The results indicate that BDNF is involved in the regulation of alcohol consumption behavior in IA2BC rats by L-EVs. Furthermore, co-incubation of PC12 cells with L-EVs directly interferes with BDNF expression in PC12 cells, suggesting that L-EVs may directly act on cells to induce intracellular responses. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001, n=3.

[0046] Example 6: This example is to verify the efficacy of a pharmaceutical composition for inhibiting alcohol consumption in alcohol-dependent individuals provided by the present invention. The composition includes the extracellular vesicles of *Lactobacillus plantarum* as described in the present invention, and also includes N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catechyl]methylglycine.

[0047] Experimental Methods: A group of rats was used to examine the efficacy of the drug composition. An equal amount of single-dose N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catyryl]methylglycine or an equal amount of *Lactobacillus plantarum* extracellular vesicles served as a positive control, and an equal amount of water served as a negative control. Rats were treated with the drug composition or N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catyryl]methylglycine or *Lactobacillus plantarum* extracellular vesicles or water for 3 days. The rats were then exposed to ethanol deprivation (restricted drinking) for 2 weeks, during which time they were only allowed to drink water for 3 hours daily. After the restricted drinking period, rats were re-exposed to the drug composition or N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catechyl]methylglycine or water, and then re-exposed to ethanol for 13 days. At the end of the experiment, the dose of the drug composition was gradually reduced (5-2.5-0.5 mg / kg) to check whether the observed effects were dose-dependent.

[0048] Experimental Results: Rats treated with the drug composition were exposed to ethanol deprivation (limited drinking) for 13 days. Statistical analysis showed that during the limited drinking period, the number of times the drug composition was used to treat rats was significantly reduced (8 times), and there was no dose dependence. Rats in the positive control group (single dose of N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catechyl]methylglycine) also had a reduced number of times of ethanol intake (15 times), and there was no dose dependence. Rats in the positive control group (equal amount of Lactobacillus plantarum extracellular vesicles) also had a reduced number of times of ethanol intake (14 times), and there was no dose dependence. However, rats in the negative control group did not have a reduced number of times of ethanol intake (28 times), and there was a dose dependence. This indicates that the pharmaceutical composition comprising *Lactobacillus plantarum* extracellular vesicles and N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-cathyyl]methylglycine has a more significant effect on inhibiting alcohol than the single inhibitor N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-cathyyl]methylglycine or *Lactobacillus plantarum* extracellular vesicles. This suggests that *Lactobacillus plantarum* extracellular vesicles and N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-cathyyl]methylglycine have a synergistic effect on inhibiting alcohol uptake.

[0049] In summary, *Lactobacillus plantarum* extracellular vesicles regulate alcohol consumption in alcohol-dependent rats by modulating BDNF expression. In in vitro cell culture experiments, *Lactobacillus plantarum* extracellular vesicles increased BDNF expression in PC12 cells, while alcohol decreased BDNF expression in PC12 cells. *Lactobacillus plantarum* extracellular vesicles exert their inhibitory effect by reversing the alcohol-induced BDNF expression in PC12 cells. This invention provides a biological agent for inhibiting alcohol consumption in alcohol-dependent individuals and its application. Using male Sprague-Dawley (SD) rats as the research subject, this study investigated the inhibitory effect and related mechanisms of *Lactobacillus plantarum*-derived extracellular vesicles (L-EVs) on alcohol consumption in alcohol-dependent rats, providing new insights for the clinical prevention and treatment of alcohol dependence.

[0050] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A biological agent for inhibiting alcohol consumption in individuals with alcohol dependence, characterized in that, The biological agent is Lactobacillus plantarum extracellular vesicles.

2. The application of a biological agent in the preparation of a drug to inhibit alcohol consumption in individuals with alcohol dependence, characterized in that, The biological agent is the Lactobacillus plantarum extracellular vesicle as described in claim 1.

3. The application according to claim 2, characterized in that, K252a has an antagonistic effect on the regulation of alcohol consumption in alcohol-dependent individuals by extracellular vesicles of Lactobacillus plantarum. K252a is a specific antagonist of brain-derived neurotrophic factor receptor.

4. The application according to claim 2, characterized in that, The extracellular vesicles of *Lactobacillus plantarum* can increase the expression of BDNF in the ventral tegmental area of ​​the midbrain.

5. The application according to claim 4, characterized in that, The concentration of the extracellular vesicles of *Lactobacillus plantarum* is 15 μg / 0.5 mL or 30 μg / 0.5 mL.

6. The application according to claim 2, characterized in that, The extracellular vesicles of *Lactobacillus plantarum* can enhance the expression of BDNF in PC12 cells.

7. The application according to claim 6, characterized in that, The concentration of the extracellular vesicles of *Lactobacillus plantarum* is 0 μg / ml, 20 μg / ml, or 30 μg / ml.

8. A pharmaceutical composition for inhibiting alcohol consumption in individuals with alcohol dependence, characterized in that, The pharmaceutical composition comprises the Lactobacillus plantarum extracellular vesicles as described in claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further includes N-methyl-N-[[(1R,2S)-1,2,3,4-tetrahydro-6-methoxy-1-phenyl-2-catechyl]methylglycine.

10. An inhibitor to suppress alcohol consumption in individuals with alcohol dependence, characterized in that, The inhibitor includes the Lactobacillus plantarum extracellular vesicles as described in claim 1.

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