Application of cannabinoid receptor agonist and blood neurogenic exosome autophagy marker in preparation of drugs for treating depression or diagnosing depression

Abstract

The invention discloses an application of a cannabinoid receptor stimulant and a blood neurogenic exosome autophagy marker in preparation of a medicine for treating depression or diagnosis of depression. The cannabinoid receptor stimulant can induce LHb region neurons to generate LTD, and the autophagy marker can be used for treating depression. Therefore, possible theoretical and technical help is provided for research and development of drugs for preventing and / or delaying depression attack. Compared with the existing antidepressant drugs, the cannabinoid receptor stimulant provided by the invention has the advantage of faster onset time in preparation of drugs for relieving depression. The content of the autophagy marker Beclin-1 or LC3 in the blood neurogenic exosome is detected through western blot, regulation of the antidepressant drug on depression can be indirectly reflected, and the detection method is simple, rapid and low in cost.

Description

(1) Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of a cannabinoid receptor agonist and a blood neurogenic exosome autophagy marker in the preparation of a drug for treating depression or diagnosing depression. (2) Background Art

[0002] Major depressive disorder (MDD) is one of the most severe mental disorders, with a global lifetime prevalence as high as 10.6%. Stress is an important risk factor leading to depression, which widely exists in daily life. Chronic stress has now become the biggest inducement of depression. Although moderate stress in the short term is beneficial for survival, long-term exposure to stress can lead to disorders of physiological functions, thereby triggering depression. Depression includes a series of symptoms, such as low mood, loss of interest, anorexia, cognitive impairment, sleep disorder, and decreased responsiveness to stimuli.

[0003] The brain is the hub for regulating the stress response. Neurons regulate their own homeostasis by degrading and synthesizing specific proteins, thereby laying the foundation for the emotional homeostasis of the brain. Among the various mechanisms for regulating protein trafficking, macroautophagy (hereinafter referred to as autophagy) has received increasing attention in recent decades. Autophagy is one of the main catabolic pathways in the body and is a key process to ensure cell homeostasis. Autophagosomes engulf excess accumulated proteins, defective organelles, and pathogens in the cytoplasm and transport them to lysosomes for degradation. Previous studies have shown that autophagy is an important process for organisms to respond to stimuli. Brain autophagy is not only a process of self-digestion of cells but also specifically driven within synapses to degrade synaptic components, and can regulate synaptic function and various related physiological and pathological functions, including learning, memory, and mental diseases. More and more evidence indicates that autophagy is associated with a variety of diseases, including stress-related diseases such as depression. Autophagy genes are widely expressed in the nervous system, but most of the current mainstream research directions focus on the role of brain autophagy in neurodegenerative diseases, and there is less research on how autophagy regulates emotions under stress.

[0004] Existing drugs for treating or alleviating depression are mainly selective serotonin reuptake inhibitors (SSRIs), such as Fluoxetine, Paroxetine, etc., which improve mood by selectively inhibiting the reuptake of serotonin by neurons and increasing the concentration of serotonin in the synaptic cleft. The onset time of this type of drug is usually slow. (3) Summary of the Invention

[0005] The object of the present invention is to provide an application of a cannabinoid receptor agonist and a blood neurogenic exosome autophagy marker in the preparation of a drug for treating depression or in the diagnosis of depression. The cannabinoid receptor agonist can induce long-term inhibition of neuronal synapses and increase the level of neuronal autophagy, thereby achieving the effect of alleviating depression and providing a new drug for the treatment of depression. At the same time, an application for diagnosing depression by detecting the expression level of autophagy markers in blood neurogenic exosomes is provided. The method detects the content of Beclin-1 or LC3 in blood exosomes by Western blotting, and then assists in judging the level of neuronal autophagy and indirectly reflects the antidepressant efficacy of the cannabinoid receptor agonist.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides an application of a cannabinoid receptor agonist in the preparation of a drug for treating depression.

[0008] The cannabinoid receptor agonist refers to WIN-55,212-2 mesylate, and its molecular formula is C 28 H 30 N 2 O 6 S, and its structural formula is as follows:

[0009]

[0010] The drug of the present invention can change the plasticity of neuronal synapses, induce long-term depression (LTD) of neuronal synapses, and increase the level of neuronal autophagy, thereby alleviating depressive symptoms.

[0011] Furthermore, the drug regulates the level of neuronal autophagy, especially the level of autophagy of neurons in the lateral habenula (Lhb) region.

[0012] Furthermore, the level of neuronal autophagy includes the expression level of autophagy-related genes Beclin-1 or LC3.

[0013] Since autophagy is an important regulatory factor for neuronal synaptic plasticity, the present invention also provides an application of a blood neurogenic exosome autophagy marker in the diagnosis of depression. The method of the application is to extract neurogenic exosomes from blood and detect the content of autophagy markers in the exosomes by Western blotting. Taking the content of β-actin protein as a control, when the content of autophagy markers increases, it indicates an increase in the level of neuronal autophagy and a decrease in the depressive condition, indicating that the drug used for treating depression is effective.

[0014] Furthermore, the autophagy markers include Beclin-1 or LC3. When the content of one or both of Beclin-1 or LC3 increases, it indicates an increase in the level of neuronal autophagy.

[0015] Furthermore, the Beclin-1 refers to the cell-penetrating peptide Tat-beclin 1, also known as beclin 1, with the molecular formula C 164 H 251 N 57 O 45 .

[0016] The LC3 refers to microtubule-associated protein 1A / 1B-light chain 3 (MAP1LC3), abbreviated as LC3. The molecular formula is C 28 H 22 N 2 O 5 , and its structural formula is as follows:

[0017]

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] (1) The cannabinoid receptor agonist of the present invention can induce long-term depression (LTD) in neurons in the LHb region, thus providing possible theoretical and technical assistance for the research and development of drugs for preventing and / or delaying the onset of depression.

[0020] (2) Compared with existing antidepressant drugs, the cannabinoid receptor agonist provided by the present invention has the advantage of a faster onset time in the preparation of drugs for relieving depression.

[0021] (3) By detecting the content of Beclin-1 or LC3 in blood exosomes through Western blotting, the present invention can not only indirectly reflect the regulation of antidepressant drugs on depression, but also has the advantages of simple, rapid and low-cost detection methods. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For the relationship between the autophagy gene expression level and the severity of depressive-like behavior; among them, (a) is the experimental design; (b) is the correlation between BECN1 (Beclin-1 gene name) mRNA FPKM (fragments per kilobase of exon per million mapped reads) and the behavior score (FST immobility duration); (c) is the correlation between LC3 mRNA FPKM (fragments per kilobase million) and the behavior score (FST immobility duration);

[0023] Figure 2Schematic diagram of the effect of antidepressant drug dosage regimens treated with paroxetine on the autophagy levels in different brain regions of mice; (a) Schematic diagram of the experimental design and western blot results; (b) Schematic diagram showing that the antidepressant drug dosage regimen treated with paroxetine can lead to a decrease in P62 in the LHb; (c) Schematic diagram showing that the antidepressant drug dosage regimen treated with paroxetine can lead to an increase in Beclin-1 in the LHb.

[0024] Figure 3 Schematic diagram of the effect of antidepressant drug dosage regimens treated with ketamine on the autophagy levels in different brain regions of mice; (a) Schematic diagram of the experimental design and western blot results; (b) Schematic diagram showing that the antidepressant drug dosage regimen treated with ketamine can lead to a decrease in P62 in the LHb; (c) Schematic diagram showing that the antidepressant drug dosage regimen treated with ketamine can lead to an increase in Beclin-1 in the LHb.

[0025] Figure 4 Effect of Win-55 on CRS-induced depressive mice; (a) Virus injection site and optical fiber implantation site; (b) Electrophysiological recording results; (c) Experimental procedure; (d) Schematic diagram of the immobile time of mice in the FST; (e) Schematic diagram of the sucrose preference of mice in the SPT.

[0026] Figure 5 Effect of Win-55 on ATG gene knockout mice; (a) Virus injection site and optical fiber implantation site; (b) Electrophysiological recording results; (c) Experimental procedure; (d) Schematic diagram of the immobile time of mice in the FST. (V) Specific implementation manners

[0027] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0028] All behavioral experiments in the embodiments of the present invention were randomly grouped, and data analysis was performed using GraphPad Prism software 8. If the administration site was not in the target brain region, the data points were excluded from the analysis. For all data, the normality and homogeneity of variance were first examined. If applicable, paired or unpaired t-tests, or unpaired t-tests with Welch correction, one-way analysis of variance using Dunnett's multiple comparison test, uncorrected Fisher's LSD or Tukey's multiple comparison test, or mixed-effect analysis using Holm-Sidak's multiple comparison test were used. For data with non-Gaussian distributions, Wilcoxon test, Mann-Whitney test, Kruskal-Wallis test, and Dunn's multiple comparison test were employed. The data were expressed as mean ± S.E.M. All statistical tests were two-tailed, and statistical significance was set at p < 0.05.

[0029] Example 1: Correlation analysis of autophagy gene expression level and severity of depressive-like behavior under chronic stress Refer to Figure 1 the design of experiment a in

[0030] 1. Experimental animals:

[0031] The mice used in this study were C57BL / 6 mice, purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were housed in cages of four or five in a room with constant temperature and humidity, free access to food and water, and a 12-hour light-dark cycle (the light-on period was from 7:00 am to 7:00 pm every day). The use and operation of all animals complied with the requirements of the Laboratory Animal Management Committee of the Laboratory Animal Center of Zhejiang University regarding laboratory animal operations and animal welfare.

[0032] 2. Stress modeling:

[0033] CRS is a well-established animal model of depression.

[0034] CRS group: Referring to the method of chronic restraint stress (CRS) reported by Kyoung-Shim Kim et al. (2006), six C57BL / 6 mice aged 8 to 11 weeks were respectively placed in 50 mL centrifuge tubes (dozens of small holes with a diameter of 2 mm were engraved on the centrifuge tubes for ventilation). Chronic restraint was carried out for 2 hours per day (10:00 - 12:00) for 14 consecutive days.

[0035] Blank control group: Six C57BL / 6 mice aged 8 to 11 weeks were not given any intervention and were placed in another room.

[0036] 3. Detection of depressive-related behaviors:

[0037] On the 15th day, the forced swimming test was conducted: The forced swimming experiment was carried out under normal brightness lighting. Mice in the CRS group and the blank control group were separately placed into a cylindrical bucket filled with water (bucket height 25 cm, diameter 12 cm) for 6 minutes of swimming. The water temperature was 23 - 25 °C, and the water depth was such that the hind legs of the mice could not touch the bottom of the bucket. A video recording device was used to record the behavior of the mice for analysis. The immobile behavior during swimming (FST) was analyzed by someone who was unaware of the experimental grouping. The immobile behavior during swimming was defined as a state of remaining basically immobile or floating, except for small movements to keep the head above the water surface within 4 minutes.

[0038] Mice in the CRS group were divided into those with high FST scores (FST immobile time > 110 seconds, n = 3) and those with low FST scores (50 seconds < FST immobile time < 110 seconds, n = 3).

[0039] Blank control mice were divided into those with high FST scores (50 seconds < FST immobile time < 80 seconds, n = 3) and those with low FST scores (FST immobile time < 50 seconds, n = 3).

[0040] 4. RNA sequencing RNA-seq:

[0041] (1) mRNA FPKM and FST mouse immobile behavior analysis

[0042] On the 17th day, 12 mice in the CRS group and the blank control group were anesthetized with 1% sodium pentobarbital NEMBUTAL (100 mg / kg), and their brains were perfused with 20 mL of ice-cold phosphate buffer (PBS, pH 7.4) at 4°C. The brains were quickly collected on an ice tray and transferred to liquid nitrogen for 25 seconds. The frozen brains were placed in a stainless-steel mouse brain mold and cut into approximately 1-mm-thick coronal sections, including brain samples of the LHb (lateral habenula, from bregma -1.5 mm to -1.9 mm), vHippo (ventral hippocampus, from bregma -2.9 mm to -3.3 mm), mPFC (medial prefrontal cortex, from bregma +1.9 mm to +1.5 mm), VTA (ventral tegmental area, from bregma -2.9 mm to -3.3 mm), LH (lateral hypothalamus, from bregma -0.6 mm to -1.0 mm), and NAc (nucleus accumbens, from bregma +1.3 mm to +0.9 mm). The sections were immersed in pre-cooled PBS at 4°C, and the relevant brain regions were quickly dissected under a microscope, placed in labeled Eppendorf tubes, and immediately transferred to liquid nitrogen for subsequent mRNA sequencing services. The mRNA sequencing services for autophagy-related genes BECN1 and LC3 were provided by Hangzhou Lianchuan Biotechnology Co., Ltd. After generating the final transcriptome, StringTie and ballgown were used to estimate the expression levels of BECN1 and LC3 of all transcripts, and the mRNA expression abundance was determined by calculating the FPKM value. The results are shown in Figure 1 b and c in

[0043] Figure 1 As shown in b in

[0044] Figure 1 As shown in c in

[0045] (2) Correlation analysis

[0046] Differential gene expression analysis was completed using the DESeq2 software. Genes with p < 0.05 and an absolute fold change ≥ 1.2 were considered differentially expressed genes. The differentially expressed genes were subsequently subjected to enrichment analysis of GO functions and KEGG pathways. The Z-score value represents the upregulation or downregulation of all genes in the pathway. Bioinformatics analysis was performed using OmicStudio tools at https: / / www.omicstudio.cn / tool. KEGG analysis and mRNA expression level statistics were only compared between CRS group mice with high FST scores and blank control mice with low FST scores.

[0047] Compared with the blank control group, among the six brain regions of the lateral habenula (LHb), ventral hippocampus (vHippo), medial prefrontal cortex (mPFC), ventral tegmental area (VTA), lateral hypothalamus (LH), and nucleus accumbens (NAc) analyzed in parallel in CRS group mice, the absolute value of the autophagy enrichment score of the LHb was the highest. This indicates that the autophagy level of the LHb can indirectly reflect the severity of depressive-like behavior, that is, the severity of depression can be evaluated by detecting the autophagy level of the LHb.

[0048] Correlation analysis showed that there was a strong negative correlation between the mRNA levels of the core autophagy genes BECN1 and LC3 in the LHb of mice under chronic stress and the individual immobility time detected by the FST, indicating a direct link between the impairment of autophagy function in the LHb and the severity of depressive-like behavior, that is, the expression levels of the autophagy genes BECN1 and LC3 in the LHb region can reflect the severity of depression.

[0049] Example 2: Selection analysis of antidepressant paroxetine on brain autophagy regions

[0050] To evaluate whether antidepressant drugs would regulate the levels of autophagy-related genes (p62, Beclin-1) in specific brain circuits of chronic restraint stress (CRS) mice, the specific method is as follows:

[0051] Using the experimental animals and CRS mice with stress-induced models in Example 1, they were divided into the Pxt group and the Veh group, with 6 mice in each group.

[0052] Intraperitoneal injection for drug administration:

[0053] The Pxt group was intraperitoneally injected with paroxetine. Paroxetine was administered for 7 days after restraint stress, at a dose of 5 mg / kg. The Veh group was injected with an equal volume of normal saline.

[0054] Protein immunoblotting Western blot:

[0055] On the first day after administration, 9 stress-related brain regions were perfused and sampled using the method of Example 1, including the lateral habenula (Lhb), ventral hippocampus (vHippo), medial prefrontal cortex (mPFC), ventral tegmental area (VTA), lateral hypothalamus (LH), nucleus accumbens (NAc), lateral septum (LS), dorsal raphe nucleus (DRN), and median raphe nucleus (MRN). The corresponding brain tissues were quickly dissected under a microscope and then stored in liquid nitrogen, and total proteins were extracted. Samples were electrophoretically separated on a 10% SDS-PAGE gel with a protein loading amount of 5-15 μg per well, and then immunoblot color development analysis was performed after transfer. The antibodies used were anti-p62, anti-Beclin-1, and anti-β-actin antibody. Finally, a highly sensitive ECL reaction solution from Millipore was used for color development. The results are shown in Figure 2 .

[0056] Compared with the corresponding Veh group, the changes in the content of p62 protein in the brain regions of the Pxt group (presented as the p62 / β-actin ratio) were as follows: LHb decreased by 0.7124 ± 0.1472 times (p < 0.01), vHippo increased by 1.049 ± 0.1466 times (n.s.), mPFC increased by 1.083 ± 0.1925 times (n.s.), VTA decreased by 0.6704 ± 0.2986 times (n.s.), LH increased by 1.252 ± 0.3913 times (n.s.), NAc increased by 1.066 ± 0.2134 times (n.s.), LS decreased by 0.8885 ± 0.3660 times (n.s.), DRN decreased by 0.9832 ± 0.2907 times (n.s.), and MRN increased by 1.082 ± 0.4107 times (n.s.).

[0057] In addition, compared with the corresponding Veh group, the changes in the content of Beclin-1 protein in the brain regions (presented as the Beclin-1 / β-actin ratio) were as follows: LHb increased by 1.539 ± 0.3693 times (p < 0.01), vHippo increased by 1.097 ± 0.1404 times (n.s.), mPFC decreased by 0.9129 ± 0.2612 times (n.s.), VTA decreased by 0.9113 ± 0.2308 times (n.s.), LH increased by 1.186 ± 0.2569 times (n.s.), NAc increased by 1.042 ± 0.1559 times (n.s.), LS decreased by 0.8379 ± 0.2544 times (n.s.), DRN decreased by 0.9727 ± 0.2021 times (n.s.), and MRN decreased by 0.9990 ± 0.3460 times (n.s.).

[0058] It was observed that paroxetine specifically enhanced the protein expression level of the autophagy-related gene Beclin-1 in the LHb region and decreased the protein expression level of the autophagy-related gene p62. This indicates that the regulatory effect of paroxetine on brain autophagy occurs selectively in the LHb region.

[0059] Example 3: Selective analysis of the antidepressant ketamine on brain autophagy regions

[0060] Using the experimental animals and CRS mice with stress-induced models in Example 1, they were divided into a Ket group and a Veh group, with 6 mice in each group.

[0061] Intraperitoneal injection for drug administration:

[0062] The Ket group was intraperitoneally injected with ketamine. Ketamine was administered for 7 days after restraint stress, with a dose of 10 mg / kg. The Veh group was injected with an equal volume of normal saline.

[0063] Protein immunoblotting (Western blot):

[0064] One hour after drug administration, 9 stress-related brain regions were perfused and sampled using the method in Example 1, including the lateral habenula (Lhb), ventral hippocampus (vHippo), medial prefrontal cortex (mPFC), ventral tegmental area (VTA), lateral hypothalamus (LH), nucleus accumbens (NAc), lateral septum (LS), dorsal raphe nucleus (DRN), and median raphe nucleus (MRN). The corresponding brain tissues were quickly dissected under a microscope and then stored in liquid nitrogen, and total proteins were extracted.

[0065] Samples were electrophoretically separated on a 10% SDS-PAGE gel according to a protein loading amount of 5 - 15 μg per well, and then immunoblotting color development analysis was performed after transfer. The antibodies used were anti-p62, anti-Beclin-1, and anti-β-actin antibody. Finally, a high-sensitivity ECL reaction solution from Millipore was used for color development. The results are shown in Figure 3 .

[0066] Compared with the corresponding Veh group, the changes in the content of p62 protein in the brain regions of the Ket group (presented in the form of the p62 / β-actin ratio) were as follows: in the LHb, it decreased by 0.7525 ± 0.09661 times (p < 0.01); in the vHippo, it increased by 1.062 ± 0.2746 times (n.s.); in the mPFC, it increased by 1.020 ± 0.3323 times (n.s.); in the VTA, it increased by 1.397 ± 0.8433 times (n.s.); in the LH, it increased by 1.100 ± 0.4205 times (n.s.); in the NAc, it increased by 1.024 ± 0.4746 times (n.s.); in the LS, it increased by 1.115 ± 0.3085 times (n.s.); in the DRN, it increased by 1.097 ± 0.3551 times (n.s.); in the MRN, it decreased by 0.7779 ± 0.1965 times (n.s.).

[0067] In addition, compared with the corresponding Veh group, the changes in the content of Beclin-1 protein in the brain regions of the Ket group (presented in the form of the Beclin-1 / β-actin ratio) were as follows: in the LHb, it increased by 1.775 ± 0.6097 times (p < 0.01); in the vHippo, it increased by 1.099 ± 0.2131 times (n.s.); in the mPFC, it increased by 1.317 ± 0.3799 times (n.s.); in the VTA, it increased by 1.067 ± 0.6078 times (n.s.); in the LH, it increased by 1.086 ± 0.1667 times (n.s.); in the NAc, it decreased by 0.9391 ± 0.1849 times (n.s.); in the LS, it increased by 1.240 ± 0.1895 times (n.s.); in the DRN, it decreased by 0.9646 ± 0.1448 times (n.s.); in the MRN, it increased by 1.042 ± 0.4087 times (n.s.).

[0068] It was observed that ketamine specifically enhanced the protein expression level of the autophagy-related gene Beclin-1 in the LHb and decreased the protein expression level of the autophagy-related gene p62. This indicates that the regulatory effect of ketamine on brain autophagy also occurs selectively in the LHb region.

[0069] Example 4: Analysis of the effect of Win-55,212-2 mesylate on CRS-induced depressive mice

[0070] 1. Reagents and materials

[0071] The composition of the artificial cerebrospinal fluid perfusion fluid (ACSF) was as follows: 210 mM sucrose, 125 mM sodium chloride, 2.5 mM potassium chloride, 25 mM sodium bicarbonate, 1.25 mM sodium dihydrogen phosphate, 1 mM magnesium chloride, 1 mM calcium chloride, 25 mM glucose, and 1 mM sodium pyruvate. The solvent was ultrapure water, and it was continuously oxygenated with 95% oxygen and 5% carbon dioxide.

[0072] Composition of artificial cerebrospinal fluid working fluid (ASCF): 125 mM sodium chloride, 2.5 mM potassium chloride, 25 mM sodium bicarbonate, 1.25 mM sodium dihydrogen phosphate, 1 mM magnesium chloride, 1 mM calcium chloride, 25 mM glucose, with ultrapure water as the solvent, continuously oxygenated with 95% oxygen and 5% carbon dioxide.

[0073] Composition of intracellular solution: 127 mM K-gluconate, 13 mM potassium chloride, 4 mM Mg-ATP, 0.3 mM GTP-Na3 (trisodium 5'-guanosine triphosphate), 0.3 mM ethylene glycol tetraacetic acid (EGTA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 mM creatine phosphate sodium, with ultrapure water as the solvent, pH = 7.25.

[0074] AAV virus: Adenovirus AAV2 / 9hSyn-oChIEF, with a virus titer of approximately 10 12 virus particles per mL.

[0075] Glass electrode: A glass electrode (Sutter Instrument) was pulled using a vertical pipette puller (PC-100, Narishige), and the resistance of the glass electrode was 5 - 6 MΩ.

[0076] Optical fiber: With a diameter of 200 μm and a numerical aperture of 0.37, purchased from Inper Yingbo (Hangzhou) Technology Co., Ltd.

[0077] CRS depression model mice: Using the experimental animals in Example 1 and subjecting them to stress modeling for 14 days to obtain CRS depression model mice.

[0078] 2. Effects of WIN-55,212-2 mesylate on LTD in CRS-depressed mice

[0079] Refer to Figure 4 the schematic diagram of a in

[0080] (1) Adenovirus injection into the lateral hypothalamus (LH) region

[0081] C57BL / 6 mice aged 8 to 11 weeks were anesthetized with 1% sodium pentobarbital (100 mg / kg) and then fixed on a stereotaxic apparatus. The stereotaxic coordinates of the LH region were: ±1.00 mm lateral to the midline (ML), -0.75 mm anterior to bregma (AP), and -4.78 mm deep vertically (DV).

[0082] Using a glass electrode, 100 - 150 nL of AAV virus was injected into the LH region on each side of each mouse at a rate of 100 - 150 nL / min. After the injection, the glass electrode remained in the brain for an additional 10 minutes and then was slowly removed. The animals were allowed to recover for at least four weeks after the virus injection surgery, and then the CRS depression model was established.

[0083] (2) Preparation and recording of electrophysiological brain slices

[0084] The mice after modeling in step (1) were anesthetized with 1% pentobarbital (100 mg / kg). 20 mL of artificial cerebrospinal fluid perfusion solution was frozen to the state of ice - water mixture at - 80 °C and then used to perfuse the anesthetized mice. The lateral habenula (LHb) brain region was taken using the method of Example 1, and the LHb was coronally sliced (300 μm) using a Leica VT1200S slicer. The brain slices were immediately transferred to ASCF at 32 °C for incubation and recovered for at least 30 minutes, and then placed at room temperature (26 °C) for continued incubation for at least 1 hour.

[0085] The brain slices incubated at room temperature were moved to the recording chamber, and ACSF was continuously perfused in the recording chamber at a rate of 2 - 3 mL / min. Using the optogenetic stimulation mode, the LH - LHb synaptic terminals were stimulated at a frequency of 0.1 Hz and a light intensity of 5 mW to obtain light - evoked postsynaptic currents (eEPSC). Whole - cell patch - clamp recording of LHb neurons was performed using a glass electrode for 10 minutes, and then WIN - 55,212 - 2 mesylate (concentration 1 μM, solvent is physiological saline) was added, and recording continued for at least 40 minutes. The recording used pCLAMP 10.6 software (Axon Instruments), a MultiClamp700B amplifier to amplify neuron signals, and then Digidata 1550B for 2 kHz noise filtering and 10 kHz sampling. Neurons with impedance changes exceeding 20% during the recording process were not used for subsequent data analysis. Clampfit 10.6 (Molecular Devices) and Mini analysis Program (Synaptosoft Inc., NJ) were used to analyze the electrophysiological data, and the results are shown in Figure 4 Figure b, and the results showed that the synaptic plasticity in the LHb brain region decreased from 98.99 ± 3.151% to 70.56 ± 3.862% (p < 0.0001) and stabilized, indicating that WIN - 55,212 - 2 mesylate can induce LTD in CRS - depressed mice and relieve the depressive - like state.

[0086] 3. Effects of WIN - 55,212 - 2 mesylate on the behavior of CRS - depressed mice

[0087] Refer to Figure 4The test procedure for c is as follows:

[0088] (1) Implantation of cannula in the lateral habenula (LHb) region

[0089] C57BL / 6 mice aged 8 to 11 weeks were anesthetized with 1% sodium pentobarbital (100 mg / kg) and then fixed on a stereotaxic apparatus. The stereotaxic coordinates of the LHb region were: anteroposterior distance bregma -1.72 mm (AP), mediolateral distance ±0.46 mm (ML), and depth vertical -2.68 mm (DV).

[0090] As Figure 4 shown in c, a cannula (diameter 1.0 mm, Shenzhen Reword Biotechnology Co., Ltd.) was implanted above the LHb region of each mouse. After the cannula implantation surgery, the animals were allowed to recover for at least one week, and then the CRS-induced depression model was established for 14 days.

[0091] (2) Injection of WIN-55,212-2 mesylate

[0092] During the establishment of the CRS-induced depression model, the mice were divided into the WIN-55 group and the Vehicle group. Before restraining the mice every day, 150 - 200 nL of WIN-55,212-2 mesylate (concentration 2.5 μg / μL, solvent normal saline) was injected into the LHb brain region of the mice in the WIN-55 group through the cannula, and the same volume of normal saline was injected into the mice in the Vehicle group every day. After the drug injection, the mice were subjected to 2-hour restraint stress.

[0093] (3) Forced swimming test (FST) in mice

[0094] After the establishment of the CRS model, the immobility behavior of the mice was analyzed. The method was the same as that in Example 1, and the results are shown in Figure 4 d. It can be seen that the immobility time of the Vehicle group was 79.80 ± 30.21 s, and that of the WIN-55 group was 40.43 ± 28.86 s (p < 0.05). The shortening of the immobility time indicates the alleviation of the depressive state in mice.

[0095] (4) Sucrose preference test (SPT)

[0096] After the FST, the sucrose preference test (SPT) was conducted. After the mice were deprived of water for one day, a bottle of 1% sucrose solution and a bottle of ordinary drinking water were given to the mice, and the preference value for the sucrose solution was measured. The results are shown in Figure 4 e. The preference value for the sucrose solution in the Vehicle group was 68.98 ± 10.09%, and that in the WIN-55 group was 79.78 ± 9.435% (p < 0.01). The increase in the preference for sucrose indicates the alleviation of the depressive state in mice.

[0097] Example 5: Analysis of the effect of Win-55,212-2 methanesulfonate on the behavior of ATG gene knockout mice

[0098] Reference Figure 5 In the experimental step a, the CRS depression model mice in Example 4 were replaced with ATG gene knockout mice (the Atg7 flox / flox The mice and related manipulation methods were derived from the article published by Masaaki Komatsu et al. in Nature in 2006. The laboratory bred and donated the mice. 100-150 nL AAV2 / 9hSyn-oChIEF was injected into the LH region. AAV2 / 9hSyn-mCherry / Cre was added to the AAV virus. The virus titer was about 10 12 For every mL of virus particles, 80-100 nL of the virus was injected into the lateral habenular nucleus (LHb). Other operations were the same as in Example 4.

[0099] Figure 5 Middle b shows that WIN-55 was added at the 10th minute of electrophysiological recording. It can be seen that the synaptic plasticity in the LHb brain region decreased from 100.5±3.701% to 69.07±14.80% (p<0.0001) and stabilized, indicating that Win-55 can induce LTD in ATG gene knockout depressed mice and alleviate the depressive-like state.

[0100] Figure 5 Middle d is the immobility time of mice in FST, which was 96.00±14.00s in the Vehicle group and 31.75±7.365s in the Win-55 group (p<0.001). The shortening of the immobility time can indicate the alleviation of the depressive state of mice.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a cannabinoid receptor agonist in the preparation of a drug for treating depression.

2. The use according to claim 1, characterized in that The cannabinoid receptor agonist refers to WIN-55,212-2 mesylate.

3. The use according to claim 1, characterized in that The drug modulates neuronal autophagy levels.

4. The use according to claim 3, characterized in that The drug regulates the level of autophagy in neurons of the lateral habenular nucleus.

5. The use according to claim 4, characterized in that The drug can change the plasticity of neuronal synapses, induce long-term inhibition of neuronal synapses, and increase the level of neuronal autophagy.

6. The use according to claim 3, characterized in that The neuronal autophagy level includes the expression level of autophagy-related genes Beclin-1 or LC3.

7. Application of a blood neurogenic exosome autophagy marker in the diagnosis of depression.

8. The use according to claim 7, characterized in that The application method is to extract neurogenic exosomes from the blood, use Western blotting to detect the content of autophagy markers in the exosomes, and use the β-actin protein content as a control. When the marker content increases, the neuronal autophagy level increases.

9. The use according to claim 8, characterized in that The autophagy markers include Beclin-1 or LC3.

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