Use of a dipeptide for the prevention and treatment of depression

Abstract

The application discloses use of a dipeptide or a salt or other derivatives thereof in preparation of a medicine for preventing and treating depression and / or depression-like behavior, wherein the dipeptide is gamma-L-glutamyl-L-tyrosine. The ingredient has an improvement effect on depression-like behavior of a mouse depression-like animal model established by lipopolysaccharide injection, has extremely high anti-depression potential, and exerts an anti-depression effect by being combined with target proteins p-GSK-3beta and CyclinD1.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of a dipeptide in the prevention and treatment of depression. Background Technology

[0002] Sophora flavescens seeds are the seeds of the legume Sophora flavescens Ait., which contain two non-alkaloid components in relatively high amounts: γ-L-glutamyl-L-tyrosine and guava acid.

[0003] Depression, also known as major depressive disorder (MDD), is one of the most common mental disorders, clinically characterized by a significant and persistent depressed mood. It often has a prolonged course, with recurrent episodes; most episodes resolve, but some may leave residual symptoms or become chronic, leading to severe impairment of social functioning. The pathogenesis of depression is not yet fully understood, but numerous studies have shown that inflammatory factors invade the central nervous system, leading to neurological dysfunction, and neuroinflammation is closely related to the development of depression. Lipopolysaccharide (LPS) can induce a neuroinflammatory state in the hippocampus, increase pro-inflammatory cytokines in the brain, and induce depression-like behavior. The LPS intraperitoneal injection-induced depression-like mouse model is currently a widely accepted animal model that can effectively explore antidepressant effects.

[0004] There are no reports in the existing technology regarding the antidepressant effects of γ-L-glutamyl-L-tyrosine, a monomeric component of Sophora flavescens extract. Summary of the Invention

[0005] Based on this, the present invention provides the use of a dipeptide or its salt or other derivative in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0006] According to another aspect of the invention, there is a use of a pharmaceutical composition comprising a dipeptide or a salt or other derivative thereof in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0007] According to another aspect of the invention, there is a use of a pharmaceutical preparation comprising a dipeptide or a salt or other derivative thereof in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0008] Furthermore, the depression and / or depression-like behavior is lipopolysaccharide-induced depression and / or depression-like behavior.

[0009] Furthermore, the depression and / or depression-like behavior are lipopolysaccharide-induced depression and / or depression-like behavior.

[0010] Furthermore, this depression and / or depression-like behavior is characterized by a significant increase in the expression levels of p-GSK-3β and CyclinD1 proteins and a significant decrease in the expression levels of β-catenin and Wnt1 proteins in mammals.

[0011] Furthermore, this depression and / or depression-like behavior is characterized by a significant increase in the level of cell apoptosis in the hippocampus of the mammalian brain.

[0012] Furthermore, the depression and / or depressive-like behavior includes one or more of the following conditions (symptoms or circumstances): reduced distance and speed of movement in the middle zone of the open field in the open field test, reduced open-arm dwell time in the elevated cross maze test, and increased immobility time in the forced swimming test.

[0013] Furthermore, the dipeptide exerts its effect by binding to the CyclinD1 protein.

[0014] Furthermore, the dipeptide exerts its effect by binding to the p-GSK-3β protein.

[0015] Furthermore, the dipeptide is obtained by extraction and isolation from plants, synthesis by transpeptidase, or from commercially available sources.

[0016] The beneficial effects of this invention are:

[0017] γ-L-glutamyl-L-tyrosine ameliorates depressive-like behavior in a mouse model of depression induced by lipopolysaccharide injection, and exerts its antidepressant effect by binding to target proteins p-GSK-3β and Cyclin D1. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0019] Figure 1 These are macroscopic and microscopic diagrams of the interaction between γ-L-glutamyl-L-tyrosine and CyclinD1 protein. A represents the macroscopic diagram, and B represents the microscopic diagram.

[0020] Figure 2The diagrams show the macroscopic and microscopic views of the interaction between γ-L-glutamyl-L-tyrosine and p-GSK-3β protein. A represents the macroscopic view, and B represents the microscopic view.

[0021] Figure 3 The effect of γ-L-glutamyl-L-tyrosine on the movement trajectory of mice in the open field experiment. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0024] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0025] To a certain extent, the terms “comprising,” “including,” and “having, has, with,” or variations thereof, are used in the specific implementation and / or claims, and these terms are intended to include in a manner similar to the term “comprising.”

[0026] As described in the background section, there are no reports in the prior art regarding the antidepressant effect of γ-L-glutamyl-L-tyrosine, a monomeric component of Sophora flavescens extract. To address the above problem, this invention provides the use of a dipeptide or its salt or other derivative in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0027] According to another aspect of the invention, there is a use of a pharmaceutical composition comprising a dipeptide or a salt or other derivative thereof in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0028] According to another aspect of the invention, there is a use of a pharmaceutical preparation comprising a dipeptide or a salt or other derivative thereof in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

[0029] In a preferred embodiment, the depression and / or depression-like behavior is lipopolysaccharide-induced depression and / or depression-like behavior.

[0030] In a preferred embodiment, the depression and / or depression-like behavior is lipopolysaccharide-induced depression and / or depression-like behavior.

[0031] In a preferred embodiment, the depression and / or depression-like behavior is characterized by a significant increase in the expression levels of p-GSK-3β and CyclinD1 proteins and a significant decrease in the expression levels of β-catenin and Wnt1 proteins in mammals.

[0032] In this invention, after intraperitoneal injection of LPS into mice, the expression levels of p-GSK-3β and Cyclin D1 proteins in the model group mice were significantly increased, while the expression levels of β-catenin and Wnt1 proteins were significantly decreased. This indicates that LPS injection leads to abnormalities in the Wnt / β-catenin signaling pathway. The dipeptide (γ-L-glutamyl-L-tyrosine) can significantly reduce the content of Cyclin D1 and p-GSK-3β proteins in the depression model group, while increasing the expression levels of β-catenin and Wnt1 proteins (using Western blot experiments). The depressive-like behavior of mice was significantly improved, and the expression levels of the bound proteins tended to normalize. This suggests that the antidepressant effect of Sophora flavescens extract is achieved by activating the Wnt / β-catenin signaling pathway and inhibiting neuroinflammation.

[0033] In a preferred embodiment, the depression and / or depression-like behavior is depression and / or depression-like behavior characterized by a significant increase in the level of cell apoptosis in the hippocampus of the mammalian brain.

[0034] In this invention, after intraperitoneal injection of LPS into mice, the number of TUNEL-positive cells in the model group mice was significantly increased compared with the control group. Compared with the model group, the number of TUNEL-positive cells in the dipeptide (γ-L-glutamyl-L-tyrosine) group mice was significantly reduced (as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay of apoptotic cells).

[0035] In a preferred embodiment, the depression and / or depressive-like behavior includes one or more conditions (symptoms or circumstances) selected from the following: reduced distance and speed of movement in the open field mid-zone of an open field test, reduced open-arm dwell time in an elevated cruciate maze test, and increased immobility time in a forced swimming test. Furthermore, in practice, the conditions (symptoms or circumstances) are not limited to those listed above.

[0036] In a preferred embodiment, the dipeptide functions by binding to the CyclinD1 protein.

[0037] In a preferred embodiment, the dipeptide exerts its effect by binding to the p-GSK-3β protein.

[0038] In a preferred embodiment, the dipeptide is obtained by extraction and isolation from plants, synthesis by transpeptidase, or from commercially available sources. For example, refer to the method described in patent application number 2021114897173.

[0039] Example

[0040] 1. Materials and Methods

[0041] 1.1 Animals

[0042] Forty-eight SPF-grade male C57BL / 6 mice, weighing 18-20g, were purchased from Vital River Co., Ltd. They were housed in an SPF-grade animal facility at a temperature of 20℃-24℃, relative humidity of 50-60%, and a light-dark cycle of 12L / 12D (7:00-19:00). Sufficient food and water were provided, the facility was kept quiet, and regular cleaning and disinfection were carried out. After a one-week acclimatization period, the mice had free access to food and water.

[0043] 1.2 Medicines and Reagents

[0044] γ-L-glutamyl-L-tyrosine was prepared in the laboratory with a purity >98%; fluoxetine hydrochloride dispersible tablets (Prozac) were purchased from Eli Lilly Suzhou Co., Ltd.; LPS was purchased from Sigma (product number L2880).

[0045] 1.3 Animal grouping

[0046] Forty-eight male mice were divided into six groups: control group, model group, fluoxetine group (2.5 mg / kg), low-dose γ-L-glutamyl-L-tyrosine group (1.88 mg / kg), medium-dose γ-L-glutamyl-L-tyrosine group (3.76 mg / kg), and high-dose γ-L-glutamyl-L-tyrosine group (7.52 mg / kg).

[0047] 1.4 Modeling and Drug Administration

[0048] Mice were pretreated with γ-L-glutamyl-L-tyrosine by gavage for 7 days. The fluoxetine group was administered 2.5 mg / kg fluoxetine hydrochloride by gavage for 7 days, while the control and model groups were administered the corresponding doses of physiological saline by gavage for 7 days. On day 7, the model group, fluoxetine group, and each dose group of γ-L-glutamyl-L-tyrosine were administered LPS via intraperitoneal injection (1.5 mg / kg).

[0049] 1.5 Behavioral Testing

[0050] 1.5.1 Open Field Experiment

[0051] Behavioral tests were performed 24 hours after drug administration. The open field test apparatus was 60cm × 60cm in size, with the central 30cm area defined as the central zone and the other areas as the peripheral zone. At the start of the experiment, mice were placed in the peripheral zone of the open field, and their free movement trajectory in the open field was recorded using a camera for 5 minutes. The movement time and distance in the central zone of the open field were also recorded.

[0052] 1.5.2 Elevated Cross Maze Experiment

[0053] Before the experiment, the mice were placed in an elevated cross maze room for 30 minutes to acclimatize. At the start of the experiment, the mice were placed facing the open arms in the center of the maze. The mice were recorded by camera as they moved freely in the maze for 5 minutes, and the time spent in the closed and open arms was recorded.

[0054] 1.5.3 Forced Swimming Test

[0055] The forced swimming device was a cylindrical plastic bucket, 20 cm in diameter and 50 cm in height. The water depth in the bucket was about 30 cm, and the water temperature was maintained at about 24 ℃. Each mouse swam for 6 minutes without pre-selection. The immobility time of the mice was recorded for the last 4 minutes, with immobility defined as the mouse floating on the surface without struggling.

[0056] 1.6 Molecular docking

[0057] The structural formula of γ-L-glutamyl-L-tyrosine was downloaded from PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The ligand molecule energy was minimized using Chem3D software and output in mol² format. The PDB format structural file of the receptor protein (PDB ID: 2OW3) was then downloaded from the PDB database (http: / / www.rcsb.org / ). The protein was pre-processed using PyMOL software. AutoDockTools 1.5.6 software was used to convert the mol² format of γ-L-glutamyl-L-tyrosine and the PDB file of the receptor protein to PDBQT format, and the active pocket was located. The search conformation range was set. Finally, the Vina script was run to calculate the molecular binding energy and display the molecular docking results. A stable docking was indicated when the Vina binding energy ≤ −5.0 kcal / mol and the RMSD value < 2.00. A stable docking was indicated when the Vina binding energy ≤ −7.0 kcal / mol. When the concentration is kcal / mol, it indicates that the ligand molecule binds very strongly to the receptor protein. Finally, the ligand-receptor complex generated by molecular docking was visualized in 3D using PyMOL software.

[0058] 1.7 Statistical Analysis

[0059] SPSS 16.0 statistical software was used for data analysis. Experimental data are presented as mean ± standard error (σ). The mean ± standard deviation (±s) indicates the mean between groups. The t-test was used to compare the means between groups, and P < 0.05 was considered statistically significant.

[0060] 2 Results

[0061] 2.1 Docking analysis of γ-L-glutamyl-L-tyrosine with target protein molecules

[0062] We selected target sites Cyclin D1 and p-GSK-3β and performed molecular docking with γ-L-glutamyl-L-tyrosine to reveal the affinity of γ-L-glutamyl-L-tyrosine for target proteins in the signaling pathway.

[0063] like Figure 1As shown, using the Vina script to dock γ-L-glutamyl-L-tyrosine with Cyclin D1 protein, the resulting binding energy was -5.1 kcal / mol, indicating that the ligand molecule γ-L-glutamyl-L-tyrosine can stably bind to Cyclin D1. Subsequent analysis of the interaction between the ligand and receptor protein complex using PyMOL revealed that γ-L-glutamyl-L-tyrosine forms a hydrogen bond with ASN151 of the receptor protein Cyclin D1 at a distance of 2.9 Å, and two hydrogen bonds with ARG87 at distances of 3.2 Å and 3.1 Å, respectively; it also exhibits hydrophobic interaction with LEU91 and forms a salt bridge with ARG87, indicating that γ-L-glutamyl-L-tyrosine is mainly stably bound to the pocket of the receptor protein through hydrogen bonds.

[0064] like Figure 2 As shown, using the Vina script to dock γ-L-glutamyl-L-tyrosine with the p-GSK-3β protein, the resulting binding energy was -6.2 kcal / mol, indicating that the ligand molecule γ-L-glutamyl-L-tyrosine can stably bind to p-GSK-3β. Subsequent analysis of the interaction between the ligand and receptor protein complex using PyMOL revealed that γ-L-glutamyl-L-tyrosine forms hydrogen bonds with GLU97, PHE201, and PHE67 of the receptor protein p-GSK-3β; it also exhibits hydrophobic interactions with VAL70, ALA83, and LYS85, and forms a salt bridge with LYS. This indicates that γ-L-glutamyl-L-tyrosine is stably bound to the receptor protein pocket mainly through hydrogen bonds and hydrophobic interactions.

[0065] 2.2 Effects of γ-L-glutamyl-L-tyrosine on depressive-like behavior

[0066] 2.2.1 Effects of γ-L-glutamyl-L-tyrosine on mouse open field test

[0067] like Figure 3 As shown, compared with the control group, the model group showed a significant reduction in movement trajectories in the middle area of ​​the open field; compared with the model group, the fluoxetine group, the medium-dose group of γ-L-glutamyl-L-tyrosine, and the high-dose group of mice all showed a significant increase in movement trajectories in the middle area of ​​the open field.

[0068] Compared with the control group, the open field distance and speed of mice in the model group were significantly decreased (P<0.01). Compared with the model group, the open field distance and speed of mice in the fluoxetine group were significantly increased (P<0.05); the open field distance and speed of mice in the medium-dose and high-dose γ-L-glutamyl-L-tyrosine groups were significantly increased (P<0.05) and significantly increased (P<0.05 and P<0.01), respectively. The results are shown in Table 1.

[0069] Table 1. Effects of γ-L-glutamyl-L-tyrosine on movement distance and speed in the middle zone of the open field in mouse open field experiments. ±s, n=8)

[0070]

[0071] Note: Compared with the control group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01.

[0072] 2.2.2 Effects of γ-L-glutamyl-L-tyrosine on the elevated cruciate maze test in mice

[0073] Compared with the control group, the open-arm dwell time in the elevated cruciate maze was significantly decreased in the model group mice (P<0.01). Compared with the model group, the open-arm dwell time in the elevated cruciate maze was significantly increased in the fluoxetine group mice (P<0.05); the open-arm dwell time in the elevated cruciate maze was also significantly increased in the medium-dose and high-dose γ-L-glutamyl-L-tyrosine groups (P<0.05 and P<0.01, respectively). The results are shown in Table 2.

[0074] Table 2. Effects of γ-L-glutamyl-L-tyrosine on open-arm dwell time in mouse elevated cruciate maze ( ±s, n=8)

[0075]

[0076] Note: Compared with the control group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0077] 2.2.3 Effect of γ-L-glutamyl-L-tyrosine on immobility time in forced swimming test in mice

[0078] Compared with the control group, the forced swimming immobility time of mice in the model group was significantly increased (P<0.01). Compared with the model group, the forced swimming immobility time of mice in the fluoxetine group was significantly decreased (P<0.05); the forced swimming immobility time of mice in the medium-dose and high-dose γ-L-glutamyl-L-tyrosine groups was also significantly decreased (P<0.05 and P<0.01, respectively). The results are shown in Table 3.

[0079] Table 3. Effects of γ-L-glutamyl-L-tyrosine on immobility time in forced swimming test in mice. ±s, n=8)

[0080]

[0081] Note: Compared with the control group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0082] 3. Discussion

[0083] Depression is a common mental illness characterized by depressed mood, anhedonia, and suicidal tendencies in severe cases. This experiment induced a mouse model of depression by intraperitoneal injection of LPS and measured changes in depression-related behaviors. Open field tests showed that, compared to the normal group, the model group mice exhibited significantly reduced distance and speed in the central open field, significantly shorter open-arm dwell time in the elevated cruciate maze, and significantly increased immobility time in the forced swimming test. These results indicate that LPS-injected mice exhibited significant depression-like behaviors, demonstrating the successful establishment of a mouse model of depression. After gavage administration of fluoxetine to LPS-injected mice, various behavioral indicators were improved.

[0084] This invention confirms the antidepressant effect of γ-L-glutamyl-L-tyrosine, the main active ingredient in Sophora flavescens extract. First, molecular docking technology was used to connect two key targets, p-GSK-3β and CyclinD1, to γ-L-glutamyl-L-tyrosine. It was found that p-GSK-3β and CyclinD1 have good binding affinity to γ-L-glutamyl-L-tyrosine. CyclinD1 protein binds stably to γ-L-glutamyl-L-tyrosine mainly through hydrogen bonds, while p-GSK-3β protein binds stably to γ-L-glutamyl-L-tyrosine mainly through hydrogen bonds and hydrophobic interactions. Molecular docking results indicate that γ-L-glutamyl-L-tyrosine has a good binding effect with the target protein, suggesting that this component has extremely high antidepressant potential. After administration of γ-L-glutamyl-L-tyrosine, behavioral abnormalities in model mice were improved. Compared with the model group, γ-L-glutamyl-L-tyrosine increased the distance and speed of movement in the open field mid-zone of the mouse open field test, increased the open-arm dwell time in the elevated cruciate maze test, and reduced the immobility time in the forced swimming test. These results indicate that γ-L-glutamyl-L-tyrosine has a good antidepressant effect.

[0085] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. Use of a dipeptide as the sole active ingredient in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

2. Use of a pharmaceutical preparation comprising a dipeptide as the sole active ingredient in the preparation of a medicament for the prevention and treatment of depression and / or depressive-like behavior, wherein the dipeptide is γ-L-glutamyl-L-tyrosine.

3. The use according to claim 1 or 2, characterized in that, The depression and / or depression-like behavior mentioned are lipopolysaccharide-induced depression and / or depression-like behavior.

4. The use according to claim 1 or 2, characterized in that, The depression and / or depression-like behavior referred to are depression and / or depression-like behavior induced by intraperitoneal injection of lipopolysaccharide.

5. The use according to claim 1 or 2, characterized in that, The depression and / or depression-like behavior described herein refers to depression and / or depression-like behavior in mammals characterized by a significant increase in the expression levels of p-GSK-3β and CyclinD1 proteins and a significant decrease in the expression levels of β-catenin and Wnt1 proteins.

6. The use according to claim 5, characterized in that, The depression and / or depression-like behavior described herein refers to depression and / or depression-like behavior characterized by a significant increase in the level of cell apoptosis in the hippocampus of the mammalian brain.

7. The use according to claim 1 or 2, characterized in that, The depression and / or depression-like behavior includes one or more of the following conditions: reduced distance and speed of movement in the middle zone of the open field in the open field test, reduced open-arm dwell time in the elevated cross maze test, and increased immobility time in the forced swimming test.

8. The use according to claim 1 or 2, characterized in that, The dipeptide functions by binding to the CyclinD1 protein.

9. The use according to claim 1 or 2, characterized in that, The dipeptide exerts its effect by binding to the p-GSK-3β protein.

10. The use according to claim 1 or 2, characterized in that, The dipeptide is obtained by extraction and isolation from plants, or by synthesis via transpeptidase, or by commercial purchase.

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