Use of free fatty acid receptor 4 agonists for the preparation of a medicament for the treatment and / or prevention of depression and pharmaceutical compositions

Abstract

The application discloses application of a free fatty acid receptor 4 agonist in preparation of a medicine for treating and / or preventing depression; the free fatty acid receptor 4 agonist is an agonist capable of specifically agonizing free fatty acid receptor 4 expressed by colon goblet cells. The application also provides a medicine composition containing a pharmaceutically effective dose of the free fatty acid receptor 4 agonist and a pharmaceutically acceptable carrier, excipient or diluent. The application unexpectedly discovers and verifies that FFAR4 specifically expressed by colon goblet cells is a key target point for mediating the anti-depression effect of the intestine-brain axis. Colon goblet cells specifically highly express FFAR4, regulate the secretion of various bioactive substances and thus protect the intestinal tract. Depression stress causes a significant decrease in the secretion of TFF3 protective factors by colon goblet cells. Agonizing the FFAR4 receptor, in particular, using FFAR4 agonists such as crocin, crocetin, TUG-891, GW9508 and the like, can effectively protect the function of goblet cells, promote the release of beneficial factors and thus relieve depression-like behaviors.

Description

Technical Field

[0001] This invention relates to the fields of disease treatment and pharmaceuticals, specifically to the use of a free fatty acid receptor 4 agonist in the preparation of a medicament for treating and / or preventing depression, and a pharmaceutical composition thereof. Background Technology

[0002] Depression is a common and serious mental disorder with a high global prevalence. Its core symptoms include persistent low mood, loss of interest, and cognitive impairment; in severe cases, it can lead to suicide. Currently, first-line antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), typically require several weeks of continuous use to become effective and suffer from issues such as insufficient response rates and significant side effects. Although drugs like ketamine have shown potential for rapid antidepressant effects, their addictive nature and psychological side effects limit their widespread use.

[0003] In recent years, the role of the gut-brain axis in mood regulation has attracted much attention. The gut is not only a digestive organ, but its vast neural network and endocrine cells constitute a "second brain." Colonic goblet cells are specific secretory cells of the intestinal mucosa. Besides secreting mucus to form a physical barrier, they also secrete various bioactive substances, such as peptides from the Muc and trefoil families. Our research found that colonic trefoil factor 3 expression levels were significantly decreased in depressed individuals and animal models of depression, while exogenous supplementation of TFF3 produced antidepressant-like behavioral effects. However, the key upstream targets regulating colonic goblet cell function and the secretion of its protective factors remain a technological gap in this field. Free fatty acid receptor 4 (FFAR4), also known as G protein-coupled receptor 120, is a receptor that responds to long-chain free fatty acids and plays an important role in regulating inflammation, metabolism, and hormone secretion. FFAR4 is expressed in various tissues, but its specific high expression in colonic goblet cells and its function in mental illness have not yet been elucidated. Summary of the Invention

[0004] Purpose of the invention: The first purpose of this invention is to provide the use of a free fatty acid receptor 4 agonist in the preparation of a medicament for the treatment and / or prevention of depression, and the second purpose is to provide a pharmaceutical composition.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of a free fatty acid receptor 4 agonist in the preparation of a medicament for the treatment and / or prevention of depression, wherein the free fatty acid receptor 4 agonist is an agonist capable of specifically activating the free fatty acid receptor 4 expressed by colonic goblet cells.

[0007] The inventors have for the first time discovered and demonstrated that functional defects of the free fatty acid receptor FFAR4 in colonic goblet cells play a crucial role in the development and progression of depression. Therefore, this invention provides the application of free fatty acid receptor 4 agonists in the preparation of drugs for the treatment and / or prevention of depression. These agonists specifically activate the free fatty acid receptor 4 expressed by colonic goblet cells. Colonic goblet cells and their molecular target FFAR4 represent a novel approach to antidepressant treatment mechanisms and drugs that have not yet been targeted. Therefore, the agonist provided by this invention is particularly suitable for patients with depression who have not responded to existing antidepressant methods and drug treatments.

[0008] Further, the free fatty acid receptor 4 agonist includes one of the following: saffron, crocin, DHA, EPA, ALA, linoleic acid, arachidonic acid, palm oil, myristic acid, palmitic acid-9-hydroxystearic acid, FAHFAs, Griffolias impricifolia extract, TUG-891, GW9508, TUG-770, TUG-469, TUG-1202, TUG-1197, Griffolic simplicifolia extract derivative, Icosabutate, Compound A, Merck Compound 18, TUG-1197 & TUG-1199, MAR701, Compound 40, DFL23916, AZ13581837, and Metabolex-36.

[0009] Furthermore, the free fatty acid receptor 4 agonist includes crocin, crocinic acid, TUG-891, or GW9508.

[0010] Furthermore, the drug is an oral targeted formulation or a localized formulation; the oral targeted formulation includes tablets, capsules, or suspensions; the localized formulation includes enemas, foams, or gels.

[0011] The drug described in this invention acts in the colon, meaning it is administered via a route of administration that allows the drug to be delivered to the colon. Specifically, this can be done orally or locally, allowing the drug to reach the colon and act on the goblet cells. For antidepressant methods and drugs targeting intestinal target tissues such as colonic goblet cells, targeting the drug to the colon and ensuring it reaches the colon to achieve an effective concentration for FFAR4 activation is crucial for exerting a therapeutic effect. This also reduces drug distribution in non-target tissues and lowers potential side effects. When using the drug of this invention, consideration must be given to whether it can reach the colon via the selected route of administration and whether it can reach the effective concentration required to activate FFAR4 in the colonic tissue. In this invention, the drug can be prepared in dosage forms suitable for oral or local administration. For example, oral administration can be in the form of oral formulations such as capsules, tablets, or suspensions, while local administration can be in the form of sustained-release formulations for colon-targeted release, such as enemas, foams, or gels. In addition, the above-mentioned drugs can be formulated into colon-targeted delivery systems. For example, active ingredients with FFAR4 activation function, such as crocin, FFAR4 specific agonists, FFAR4-targeting active peptides or nucleic acid molecules, can be combined with colon tissue-specific delivery carriers, such as colonic mucosa-targeting polymers and gut microbiota-responsive carriers, to form a composite preparation that can specifically accumulate in the colon and target and bind to colonic goblet cells, thereby further improving activation efficiency and therapeutic specificity.

[0012] Furthermore, the drug is a fast-acting antidepressant; the fast onset time is within 24 hours.

[0013] Most antidepressants in the art typically take one to several weeks to exert their antidepressant effect. For example, commonly used serotonin reuptake inhibitors (SSRIs) generally take 2-3 weeks to become effective, while dual serotonin and norepinephrine reuptake inhibitors typically take about one week. However, the antidepressant or pharmaceutical composition provided by this invention has a significantly faster onset of action than existing drugs, with an onset time of less than one week, preferably less than three days, more preferably less than one day, and as fast as 12 hours.

[0014] Preferably, the drug takes effect in the colon after administration to activate free fatty acid receptor 4 and exert a rapid antidepressant effect.

[0015] The present invention also provides a pharmaceutical composition comprising an effective dose of any of the above-mentioned agonists and a pharmaceutically acceptable carrier, excipient or diluent.

[0016] Although the active ingredient of the pharmaceutical compositions applicable to the present invention can be administered directly in the form of a raw material compound, it is preferred to combine the active ingredient (optionally in the form of a physiologically acceptable salt) with one or more pharmaceutically conventional excipients, including adjuvants, excipients, carriers, buffers, diluents, etc., to prepare a standard pharmaceutical composition for use.

[0017] Preferred routes of administration include oral administration, which can be formulated into tablets, capsules, suspensions, emulsions, etc.; intravenous administration, which can be formulated into injections, lyophilized powder injections, etc.; and colonic administration, which can be formulated into colon-targeted sustained-release capsules, enemas, colonic mucosal patches, etc. These administration methods ensure that the drug effectively reaches the colon and acts on colonic FFAR4. The pharmaceutical compositions of the present invention can be prepared by those skilled in the art using standard methods and conventional techniques suitable for the desired formulation. Furthermore, the pharmaceutical compositions of the present invention can also be prepared in a form suitable for administration via a sustained-release system. Examples of suitable sustained-release systems include a colon-targeted solid hydrophobic polymer semi-permeable matrix containing the active ingredient (FFAR4 activator) of the present invention. This matrix can be processed into molded articles, such as colon-targeted sustained-release microspheres, matrix formulations, coated sustained-release particles, or microcapsules, capable of slowly releasing the active ingredient at the colon, prolonging its sustained activation of FFAR4, and maintaining stable antidepressant efficacy. All of the above administration methods ensure that the drug effectively reaches the colon, specifically acts on colonic goblet cells FFAR4, and exerts an antidepressant effect.

[0018] The pharmaceutical compositions of the present invention (whose active ingredient is an FFAR4 activator for activating colonic goblet cells FFAR4 to exert an antidepressant effect) can be prepared in solid or liquid form, suitable for oral, intravenous injection, or other routes of administration: solid formulations include powders, tablets, pills, capsules, pouches, suppositories, and dispersible granules, wherein the solid carrier can also serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulation material; liquid formulations include solutions, suspensions, and emulsions, which, in addition to the active ingredient, may also contain excipients such as colorants, flavoring agents, stabilizers, buffers, artificial or natural sweeteners, dispersants, thickeners, and solubilizers, and also cover solid formulations that need to be converted into an oral liquid form before use, while aqueous suspensions suitable for oral use can be prepared by dispersing finely pulverized active ingredients in water containing known viscous substances such as natural or synthetic gums, resins, methylcellulose, and sodium carboxymethylcellulose. All of the above formulations can ensure that the active ingredient effectively reaches the colon and specifically acts on colonic goblet cells FFAR4.

[0019] The therapeutic effective dose refers to the amount of the active ingredient in this invention used to activate colonic goblet cells FFAR4 to exert an antidepressant effect, which can effectively alleviate symptoms of depression or related conditions. Its therapeutic efficacy (e.g., ED50, median effective dose) and toxicity (e.g., LD50, median lethal dose) can be determined by standard pharmacological procedures in cell cultures or laboratory animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed by the LD50 / ED50 ratio. The dose given should be carefully adjusted according to the individual's age, weight, condition, and the selected route of administration (e.g., oral, intraperitoneal injection), dosage form, administration regimen, and expected treatment outcome. The exact dose should be determined by the physician.

[0020] Beneficial Effects: Compared with existing technologies, this invention has significant advantages: This invention unexpectedly discovered and verified that FFAR4, specifically expressed by colonic goblet cells, is a key target mediating the antidepressant effects of the gut-brain axis. Colonic goblet cells specifically and highly express FFAR4, regulating the secretion of various bioactive substances to protect the intestine. Depressive stress leads to a significant decrease in the secretion of the protective factor TFF3 by colonic goblet cells. Activating FFAR4 receptors, especially using FFAR4 agonists such as crocin, crocinic acid, TUG-891, and GW9508, can effectively protect goblet cell function, promote the release of beneficial factors, and thus alleviate depressive-like behaviors. Attached Figure Description

[0021] Figure 1 The t-SNE diagram of single-cell RNA sequencing shows that Muc2 and Ffar4 are specifically highly expressed in colon goblet cells.

[0022] Figure 2 Crocin significantly reversed the low expression of the colonic Tff3 gene induced by a depression model;

[0023] Among them, A is a volcano diagram of differentially expressed genes between the control group and the depression model group, showing a significant decrease in Tff3 gene expression in the colonic goblet cells of depressed mice; B is a volcano diagram of differentially expressed genes between the depression model group and the crocin-treated group, showing a significant increase in Tff3 expression after crocin treatment.

[0024] Figure 3 Exogenous supplementation with Tff3 significantly improved depressive-like behavior in mice with chronic social frustration.

[0025] In this graph, A is a statistical chart of social scores in mice in the social experiment; B is a statistical chart of immobility duration in mice in the tail suspension experiment; C is a statistical chart of immobility duration in mice in the forced swimming experiment; and D is a statistical chart of the degree of anhedonia in mice in the sucrose preference experiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and *p<0.05 indicate that the data differences are statistically significant.

[0026] CTRL group, normal control group;

[0027] SS represents the depression model group;

[0028] SS+Tff3 represents the depression model plus Tff3 administration group.

[0029] Figure 4 Crocin, a metabolite, is a highly potent agonist of FFAR4, while crocin does not directly activate FFAR4.

[0030] Figure 5 Crocin exerts its antidepressant effect through Tff3 mediation;

[0031] In the figure, A is a statistical graph of social scores of mice in the social experiment; B is a statistical graph of immobility duration of mice in the tail suspension experiment; C is a statistical graph of immobility duration of mice in the forced swimming experiment; and D is a statistical graph of the degree of anhedonia in mice in the sucrose preference experiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and *p<0.05 indicate that the data differences are statistically significant.

[0032] Figure 6 A schematic diagram showing the hydrolysis of crocin into crocin by intestinal flora β-glucuronidase;

[0033] Figure 7 To illustrate the activation of colonic goblet cells by oral administration of crocin to crocin acid, in situ hybridization signal maps of Ffar4-mRNA and Muc2-mRNA, immunofluorescence signal map of c-Fos protein, and colocalization map were obtained.

[0034] Figure 8 The study investigated the antidepressant effects of two FFAR4 agonists, TUG-891HE and GW9508, after colonic instillation. C represents the normal control group, SS represents the depression model group, TUG-891 represents the group receiving TUG-891 after depression modeling, and GW9508 represents the group receiving GW9508 after depression modeling. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *p<0.05 indicate statistically significant differences. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions and specific operation processes of the embodiments of this application are now described in detail with reference to the accompanying drawings. However, the content of this application is not limited to the following examples. Reagents or instruments used without a specified manufacturer are considered to be conventional products available on the market.

[0036] In this invention, "treatment" refers to any of the following processes or outcomes: improving, alleviating, reducing, or preventing the occurrence and progression of symptoms related to depression; improving the evolution and final effect of symptoms related to depression; restoring bodily functions impaired due to a specific disease or condition to normal; or promoting improvement in one or more clinically detectable parameters related to the disease. Treatment aims include preventing related symptoms, or optimizing medical, physiological, and clinical indicators, physical therapy and occupational therapy parameters, assessment indicators for healthcare professionals or patients, and parameters related to "quality of life" or activities of daily living recognized in the art. Beneficial or desired clinical outcomes in this invention include, but are not limited to: reducing the severity of symptoms; narrowing the scope of the disease, condition, or related status; stabilizing the state of the disease or condition (i.e., preventing its deterioration); delaying the onset of the disease or condition or slowing its progression; improving or alleviating the severity of the disease or condition; partially or completely reducing the impact of the disease (whether detectable or not); or enhancing the body's tolerance and adaptation to the disease or condition. Furthermore, the term "treatment" in this invention may refer to administering medication or performing medical procedures on a patient, with the core aim of preventing disease occurrence, curing symptoms, improving the patient's clinical condition (including shortening the course of the disease and reducing the severity of the disease), subjectively improving the patient's quality of life, slowing the progression of adverse physiological conditions, or achieving other beneficial clinical outcomes.

[0037] This invention discloses a method for screening potential therapeutic agents for depression using an animal model, with the core mechanism of action being the activation of FFAR4 in colonic goblet cells. Specifically, the method involves administering the test substance to a depressed animal model, followed by observing the depression-related symptoms and the activation level of colonic goblet cells, including but not limited to the binding activity of the test substance to FFAR4 and the amount of Tff3 secreted, and comparing these observations with a control group. If the depression-related symptoms in the animal model are significantly improved and the FFAR4 activation level is significantly higher than that in the control group, it indicates that the test substance is a potential therapeutic agent for depression. This screening method may further include preliminary... The screened potential substances were subjected to specific validation, such as testing their specific activation effect on FFAR4 in colonic goblet cells, their regulatory effect on downstream calcium signaling and Tff3 secretion of FFAR4, or after optimizing the dosage and administration method, they were administered again to animal models of depression to verify their effect on improving depressive symptoms and the persistence of FFAR4 activation. If, compared with the negative control group, the activation level of FFAR4 (such as specific binding rate, downstream signal activation intensity) and the amount of Tff3 secretion were significantly increased in the test group and accompanied by improvement of depressive symptoms, it indicates that the test substance is a potential substance that exerts its antidepressant effect by targeting and activating FFAR4.

[0038] Example 1: Materials and Methods

[0039] Animal materials

[0040] All animal procedures were performed in accordance with the National Institutes of Health's guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of China Pharmaceutical University. Male C57BL / 6J mice (8 weeks old, weighing 20-25 g) and male retired CD-1 mice (6-7 months old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Tff3 - / - The mice were purchased from Jiangsu Jinzhihe Biotechnology Co., Ltd. Each CD-1 mouse was housed individually, while the other mice were housed in groups of five per cage. All animals were housed in the SPF animal facility of the Animal Experiment Center of China Pharmaceutical University, with a temperature of 23-25℃, humidity of 40-50%, and a 12-hour light / 12-hour dark day-night cycle. Food and water were readily available.

[0041] Mouse colon single-cell RNA sequencing

[0042] Crypt isolation and cell dissociation: Mouse colon cells were rinsed with ice-cold PBS, cut into 2 mm pieces, and shaken with dissociation buffer for 15 minutes. The crypt-rich suspension was digested into single cells using an enzymatic digestion mixture. The resulting cell suspension was subjected to cytokine splitting and removal of dead cells, followed by scRNA-seq cell capture and sequencing. Single-cell capture was performed using a 10x Genomics GemCode Single-Cell Instrument and a Chromium Next GEM Single Cell 3' HT Kit v3.1, and high-throughput sequencing was performed using an Illumina sequencing platform. After quality control, an integrated expression matrix was obtained. t-SNE visualization and dimensionality reduction results were obtained through cluster analysis, and differentially expressed genes were analyzed in subpopulations. The purpose of this experiment was to confirm the gene expression status of colonic goblet cells.

[0043] Chronic Social Defeat Stress (CSDS) model

[0044] Aggressive CD1 mice were selected based on the aggressive behavior of retired male CD1 mice towards C57BL / 6J mice. This model lasted for 10 days. C57BL / 6J mice were exposed daily to new CD1-aggressive mice. A perforated plastic partition was placed in the middle of the cage. After social frustration, the CD1-aggressive mice and C57BL / 6J mice were placed on either side of the partition. Daily exposure included 5-10 minutes of physical contact stress and 24 hours of olfactory-visual psychological stress. Wounds in C57BL / 6J mice were monitored daily, and appropriate measures were taken to prevent infection. Control group mice were housed in pairs and received no stress. The aim of this experiment was to obtain mice in a depressed state for behavioral testing and efficacy evaluation. Social interaction tests were used 24 hours after the last social frustration to assess whether the mice were depressed.

[0045] Open Field Test (OFT)

[0046] Mice were placed in a 42 cm × 42 cm × 42 cm square area and allowed free exploration during the testing period. Each mouse's movement was recorded for 6 minutes. Any-maze software was used to analyze the time spent in the central area and the number of times the mouse entered the central area in the last 4 minutes. After each mouse's test, the open space box was cleaned with 75% alcohol to eliminate odor and avoid affecting subsequent mice. The purpose of this experiment was to assess mouse depression and drug efficacy by measuring the percentage of time spent in the central area.

[0047] Social Interaction Test (SI)

[0048] Social interaction tests were conducted in an opaque white social box measuring 42 cm × 42 cm × 42 cm, with a metal cage placed in the center of one side. The test area surrounding the metal cage was defined as the social zone. The test consisted of two phases: in the first phase, the activity of C57BL / 6J mice was recorded for 150 seconds in an empty metal cage; in the second phase, the activity of C57BL / 6J mice was recorded for 150 seconds in the metal cage with CD-1 present. CD-1 was performed on completely unfamiliar but aggressive mice. Any-maze software was used to analyze the time mice spent in the social zone. The purpose of this experiment was to assess whether mice were depressed and the efficacy of the medication using the social coefficient (SI) (the ratio of the time spent in the second social zone to the time spent in the first social zone). Mice with an SI < 1 were considered to be depression-sensitive mice.

[0049] Tail Suspension Test (TST)

[0050] The tail suspension test was conducted in a tail suspension box (55 cm high, 15 cm wide, and 12 cm deep). C57BL / 6J mice were secured in the box by attaching medical tape to the tip of their tails 0.5–1 cm above the tip. The distance between the mouse's nose and the bottom of the box was approximately 20 cm. The test lasted for 6 minutes and was video recorded. During the test, the mice could not see each other. Two single-blind researchers recorded the immobility time of the mice in the last 4 minutes of the video recording. Immobility was defined as complete immobility or only minor limb movements. The purpose of this experiment was to assess the presence of depression and drug efficacy in mice by measuring the immobility time in the tail suspension test.

[0051] Forced Swimming Test (FST)

[0052] The forced swimming test was conducted in a transparent 5 L glass beaker (27.5 cm high, 17.8 cm in diameter), filled with approximately 18 cm of water (22-25°C), ensuring the mouse's tail did not touch the bottom. The experiment lasted 6 minutes and was video-recorded, with mice unable to see each other during the test. The video recording, taken by two single-blind researchers, documented the duration of immobility in the water for the last 4 minutes. Immobility was defined as ceasing to struggle, floating motionless, or making small limb movements to keep the head above water. The purpose of this experiment was to assess the immobility time in mice during the forced swimming test to evaluate depression and drug efficacy.

[0053] Sucrose Preference Test (SPT)

[0054] Mice were housed individually and acclimatized to water and 1% sucrose for 24 hours. During the acclimatization process, the water bottle positions were changed every 12 hours to prevent repeated drinking from the same bottle. Water was then withheld for 24 hours. For the sucrose preference test, mice were allowed free access to water and 1% sucrose. The positions of the two water bottles were changed every 12 hours, and the amount of water consumed from each bottle over 24 hours was measured. The mouse sucrose preference index was calculated as: (sucrose intake / (water intake + sucrose intake)) * 100%. The purpose of this experiment was to assess the mouse's depression and drug efficacy using the preference index in the sucrose preference test.

[0055] In situ hybridization

[0056] Mice were anesthetized with isoflurane and their hearts were perfused with physiological saline and 4% paraformaldehyde. Vagal ganglia were removed and fixed in 4% PFA at 4°C for 2 hours. Tissues were graded-dehydrated in PBS containing 15% and 30% sucrose until settling. Tissues were embedded in OCT gel and immediately stored at -80°C. Before sectioning, tissue blocks were equilibrated at -20°C for at least 30 minutes (Leica, CM1950). 20 μm sections were collected and attached to glass slides. After washing sections with 0.1 mol / L PBS, the sections were baked at 60°C for 30 minutes, then post-fixed in pre-cooled 4% paraformaldehyde for 15 minutes. The sections were then dehydrated sequentially in 50%, 75%, 100%, and 100% ethanol. RNAscope hydrogen peroxide (ACD, #322335) was added, and the sections were incubated at room temperature for 10 minutes. After washing with distilled water, the sections were immersed in 1× co-detection target retrieval solution (ACD, #323165) preheated to 98–102°C for 15 minutes. The sections were immediately transferred to distilled water for cooling and washing. Primary antibody c-Fos diluted with co-detection antibody dilution buffer was incubated overnight at 4°C. After washing with PBS-T, the sections were incubated in 4% paraformaldehyde at room temperature for 30 minutes. After washing with PBS-T, RNAscope protease Plus (ACD, #322331) was added, and the sections were incubated in a hybridization oven at 40°C for 2 hours. After washing with washing buffer, AMP 1 (ACD, #323101) and AMP were added sequentially. AMP2 (ACD, #323102) and AMP3 (ACD, #323103) were incubated at 40℃ for 30 minutes, 30 minutes, and 15 minutes respectively, washing with wash buffer each time. Then, Muc2 and FFAR4 probes were added, and the slides were incubated with fluorescent dye (ACD, #323273) and HRP blocker (ACD, #323107) at 40℃ for 15 minutes, 30 minutes, and 15 minutes respectively, washing with wash buffer each time. Finally, secondary antibody diluted with co-detection antibody dilution buffer was added and incubated at room temperature for 30 minutes. After washing with PBS, DAPI was added and incubated at room temperature for 5 minutes. After washing with PBS, a small amount of anti-fluorescence quenching mounting medium was added to the slides, and covers with a coverslip. Immunofluorescence images were acquired using an FV3000 (Olympus). The purpose of this experiment was to locate, qualitatively and quantitatively determine the distribution and co-localization of target gene signals in colon tissue or cells.

[0057] Statistical analysis

[0058] Experimental data are expressed as mean ± SEM, and the results were analyzed using GraphPad Prism8 software. One-way ANOVA or two-way ANOVA was used for analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and p-values ​​< 0.05 were considered statistically significant. Social interaction tests were analyzed using ANY-maze.

[0059] Example 2: Based on single-cell RNA sequencing, Ffar4 was found to be specifically highly expressed in colonic goblet cells.

[0060] Single-cell RNA sequencing analysis was performed on mouse colon tissue samples. First, the tissue was prepared into a single-cell suspension through collagenase digestion and mechanical dissociation. Single-cell capture, library construction, and high-throughput sequencing were then performed using the 10x Genomics platform. After standardized data processing and dimensionality reduction clustering, a cell atlas (e.g., t-SNE visualization technology) was used to construct the cell atlas. Figure 1 As shown in the figure, a distinct and well-defined cell cluster was observed. This cell cluster simultaneously and specifically highly expresses the mucin gene Muc2 and the free fatty acid receptor gene Ffar4 at the transcriptomic level. Muc2 is a classic functional marker of goblet cells, while the expression pattern of Ffar4 highly overlaps with that of Muc2, and both are located in this cell population.

[0061] Example 3: Use of crocin in upregulating Tff3 expression in colonic goblet cells

[0062] By establishing a mouse model of depression and performing single-cell RNA sequencing analysis on colon tissue, it was found that the expression level of the Tff3 gene in the colonic goblet cells of the depressed model group mice was significantly decreased (e.g., Figure 2 As shown in A), after intervention with crocin administration, the expression of this gene was significantly restored (as shown in A). Figure 2 As shown in Figure B), changes in Tff3 expression are closely related to the function of colonic goblet cells in a depressive state. This result suggests that crocin can improve depression-related colonic dysfunction by regulating the expression of the Tff3 gene in colonic goblet cells.

[0063] Example 4: Tff3 supplementation improves depressive-like behavior

[0064] By exogenously supplementing Tff3 into a depressed mouse model and assessing behavioral changes, the significant antidepressant effect of Tff3 was confirmed. Compared with the depressed model group, the Tff3-supplemented experimental group showed a significant improvement in social index scores in the social interaction test, and the difference was statistically significant (e.g., ...). Figure 3As shown in A). The duration of immobility in the Tff3 supplementation group mice was significantly shortened in both the tail suspension test and the forced swimming test, indicating that their despair-like behavior was effectively improved (e.g., Figure 3 (As shown in B and C). Meanwhile, in the sucrose preference experiment, the mice in the Tff3-supplemented group showed a significantly enhanced preference for sucrose solution, indicating that the anhedonia state was reversed (as shown in B and C). Figure 3 (As shown in D). The results of the four independent behavioral experiments above consistently demonstrate that exogenous supplementation of Tff3 can comprehensively and significantly improve the core behavioral symptoms of a depressed mouse model, providing direct and strong functional evidence for the development of novel antidepressants that regulate Tff3 secretion from colonic goblet cells.

[0065] Example 5: Application of crocin, a metabolite of crocin, as an FFAR4-specific agonist in the preparation of drugs regulating colonic goblet cell function.

[0066] This invention demonstrates through in vitro pharmacological experiments that crocin, a metabolite of saffron, is a specific agonist of free fatty acid receptor 4. For example... Figure 4 As shown, in cell systems expressing FFAR4, crocin can activate the FFAR4-mediated signaling pathway more strongly than the endogenous ligand EPA, and has no significant activating effect on wild-type cells within the same concentration range, indicating that crocin is a highly effective agonist of the FFAR4 receptor, while crocin cannot activate FFAR4. This finding reveals for the first time that crocin can directly act on FFAR4, providing a molecular pharmacological basis for its regulation of colonic goblet cell function and promotion of Tff3 secretion through the FFAR4 receptor.

[0067] Example 6: Screening Model for Tff3-Dependent Antidepressants and Pharmaceutical Applications of Crocin

[0068] By establishing Tff3 - / - In a mouse model, it was confirmed that the antidepressant effect of crocin depends on the complete expression of the Tff3 gene. In wild-type depressed mice, oral administration of crocin significantly improved social indices (e.g., social performance). Figure 5 As shown in A), shorten the immobility time in the tail suspension test and forced swimming test (e.g. Figure 5 (as shown in B and C), and restore the preference for sugar water (as shown in B and C). Figure 5 As shown in D), it has a clear antidepressant behavioral effect. However, in a Tff3 gene knockout depressed mouse model, the same dose of crocin failed to improve any of the above behavioral indicators, and its effect was not statistically different from the untreated group (e.g., Figure 5 As shown in AD), this demonstrates that the antidepressant effect of crocin is mediated through the Tff3 pathway.

[0069] Example 7: Crocin was hydrolyzed into crocin by intestinal flora β-glucuronidase.

[0070] In embodiments of the present invention, the specific metabolic pathway by which crocin is converted into crocin acid is elucidated, revealing the essence of its function. For example... Figure 6 As shown, crocin contains a specific ester bond (i.e., a glycosidic ester bond) in its structure. This structure undergoes hydrolysis in vivo (e.g., catalyzed by β-glucosidase from gut microbiota) to convert it into crocin acid. This conversion process is chemically well-defined and direct, leading to the exposure of the active aglycone of crocin acid, which enables it to bind to specific biological targets (such as transcription factors or key proteins in inflammatory signaling pathways) and produce pharmacological activity. Therefore, when crocin is administered, it is converted in situ into crocin acid in vivo via the aforementioned enzymatic hydrolysis pathway, and crocin acid mediates the subsequent core biological effects. This embodiment demonstrates from a metabolic mechanism perspective that crocin, as a precursor to crocin acid, has its active metabolite, crocin acid, as the key to achieving its final efficacy.

[0071] Example 8: In vivo verification and application of oral administration of crocin to activate colonic goblet cells

[0072] like Figure 7 As shown, using in situ hybridization and immunofluorescence co-staining techniques, FFAR4 gene mRNA signal, Muc2 gene mRNA signal, and c-Fos protein signal (a cell activation marker) were observed simultaneously in the colonic goblet cell region of mice orally administered crocin. The spatial distribution of these three signals highly overlapped, indicating that crocin metabolites can specifically act on colonic goblet cells, activate goblet cells through the FFAR4 receptor, regulate Tff3 secretion, and thus achieve an antidepressant effect.

[0073] Example 9

[0074] Antidepressant effect after colonic instillation of two FFAR4 agonists

[0075] like Figure 8 As shown, the pharmacodynamics of two FFAR4 agonists, TUG-891 and GW9508 (1 mg / ml), administered via colonic instillation were evaluated. In a chronic social frustration stress model in normal mice, treatment with the three drugs improved the social index, immobility time in tail suspension and forced swimming, and sucrose preference in the social avoidance test, indicating that colonic instillation of these two FFAR4 agonists can exert an antidepressant effect.

[0076] Based on the above embodiments, this invention demonstrates that: 1) FFAR4 is specifically highly expressed in colonic goblet cells; 2) the natural FFAR4 agonists crocin / crocetin can effectively target FFAR4 and activate colonic goblet cells, promoting their secretion of protective factors such as TFF3; 3) oral administration of crocin can produce a rapid and significant antidepressant behavioral effect in a depressed animal model by regulating TFF3 secretion; colonic instillation of TUG-891 and GW9508, respectively, can produce a rapid antidepressant behavioral effect in a depressed animal model. Therefore, targeting colonic goblet cells (FFAR4) provides a novel strategy for the treatment and / or prevention of depression, and crocin / crocetin is a candidate drug with great development potential. In addition, other agonists targeting colonic goblet cells (FFAR4) and regulating TFF3 secretion can also produce antidepressant effects.

Claims

1. The use of free fatty acid receptor 4 agonists in the preparation of medicaments for the treatment and / or prevention of depression, characterized in that, The free fatty acid receptor 4 agonist is an agonist that can specifically activate the free fatty acid receptor 4 expressed by colonic goblet cells.

2. The application according to claim 1, characterized in that, The free fatty acid receptor 4 agonists include one of the following: crocin, crocinic acid, DHA, EPA, ALA, linoleic acid, arachidonic acid, palm oil, myristic acid, palmitic acid-9-hydroxystearic acid, FAHFAs, Griffolia simplicifolia extract, TUG-891, GW9508, TUG-770, TUG-469, TUG-1202, TUG-1197, Griffolic simplicifolia extract derivatives, Icosabutate, Compound A, Merck Compound 18, TUG-1197 & TUG-1199, MAR701, Compound 40, DFL23916, AZ13581837, and Metabolex-36.

3. The application according to claim 1, characterized in that, The free fatty acid receptor 4 agonists include crocin, crocinic acid, TUG-891, or GW9508.

4. The application according to claim 1, characterized in that, The drug is an oral targeted dosage form or a local dosage form; the oral targeted dosage form includes tablets, capsules or suspensions; the local dosage form includes enemas, foams or gels.

5. The application according to claim 1, characterized in that, The drug is a fast-acting antidepressant; the fast onset time is within 24 hours.

6. The application according to claim 1, characterized in that, The drug takes effect in the colon after administration, activating free fatty acid receptor 4 to exert a rapid antidepressant effect.

7. A pharmaceutical composition, characterized in that, The drug contains an effective dose of any of the agonists as described in claims 1-6 and a pharmaceutically acceptable carrier, excipient, or diluent.

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