Combined medicine for treating anxiety and / or depression

Abstract

The invention belongs to the field of chemical medicines, and particularly relates to a combined medicine for treating anxiety and / or depression. The combined medicine disclosed by the invention is a combined medicine of glucosylceramide and a PPAR gamma agonist; the combined medicine improves anxiety and depression symptoms by enhancing the transcriptional activity of PPAR gamma and the target gene level of PPAR gamma, shows a synergistic effect, opens up a new direction for treating anxiety and depression, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to a combination drug for treating anxiety and / or depression. Background Technology

[0002] Anxiety and depression are prevalent mental health problems worldwide, severely impacting patients' quality of life and social functioning. Although commonly used clinical medications such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SRIs) have shown some efficacy, these medications have limitations. For example, some patients do not respond to existing medications, experiencing drowsiness, weight gain, and sexual dysfunction.

[0003] Glucosylceramide (GlcCer) is a type of glycosphingolipid widely distributed in gut microbiota and host cell membranes. Recent studies have found that GlcCer derived from gut microbiota significantly influences inflammation and neurotransmitter levels in the central nervous system by regulating intestinal immunity and intestinal barrier function. Research indicates that GlcCer can improve gut-brain axis dysfunction by reducing the release of inflammatory factors and enhancing intestinal barrier function, potentially indirectly alleviating anxiety and depressive symptoms. Furthermore, GlcCer may exert its antidepressant effects by regulating neuroinflammation and lipid metabolism in the brain, promoting neuronal survival and synaptic function.

[0004] Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor, and pioglitazone (PIOG) is its complete agonist, primarily used clinically to treat type 2 diabetes. Pioglitazone has shown antidepressant effects in clinical and animal studies, regulating the PPARγ signaling pathway and promoting brain-derived neurotrophic factor (BDNF) expression by reducing the levels of pro-inflammatory cytokines (such as IL-6 and TNF-α). However, its clinical use is severely limited due to significant side effects, such as weight gain, increased cardiovascular risk, and liver and kidney damage.

[0005] Although current medications for treating anxiety and depression have achieved some efficacy, serious adverse reactions still exist, leading to unsatisfactory results. Therefore, there is an urgent need to develop a new treatment strategy with better efficacy and fewer side effects to provide new options for patients with anxiety and depression. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides a combination drug for treating anxiety and / or depression.

[0007] A combination drug for treating anxiety and / or depression, said combination drug being glucosamine and a PPARγ agonist.

[0008] Preferably, the combined medication is a combination of glucosamine and a PPARγ agonist, administered separately or simultaneously.

[0009] Preferably, the molar ratio of the glucose ceramide to the PPARγ agonist is 100:1 to 200:1.

[0010] Preferably, the PPARγ agonist is selected from at least one of pioglitazone, pioglitazone, rosiglitazone, troglitazone, and sitiglitazone.

[0011] Preferably, the PPARγ agonist is selected from pioglitazone.

[0012] The present invention also provides the use of the above-described combination of drugs in the preparation of medicaments for treating anxiety and / or depression.

[0013] Preferably, the drug enhances PPARγ transcriptional activity.

[0014] The present invention also provides a pharmaceutical composition for treating anxiety and / or depression, which is made by adding pharmaceutically acceptable excipients or auxiliary ingredients, with glucosamine and PPARγ agonist as active ingredients; wherein the molar concentration ratio of glucosamine and PPARγ agonist is 100:1-200:1.

[0015] Preferably, the PPARγ agonist is selected from pioglitazone.

[0016] The chemical structural formula of the glucose ceramide (GlcCer) is as follows:

[0017]

[0018] The chemical structural formula of pioglitazone (PIOG) is as follows:

[0019]

[0020] This invention combines glucosamine ceramide (GlcCer) and pioglitazone (PIOG) to enhance PPARγ transcriptional activity and the level of PPARγ target genes, exhibiting a synergistic effect in improving anxiety and depressive behaviors. It provides a safe and effective treatment option, opening up new directions for the treatment of anxiety and depression.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 The combined use of GlcCer and PIOG can enhance PPARγ transcriptional activity and target gene levels. (A) Effects of different concentrations of GlcCer on PPARγ-DNA transcription in the presence and absence of PIOG; (B) Effects of GlcCer, PIOG, or GlcCer+PIOG on PPARγ-DNA transcription; (C) Effects of GlcCer, PIOG, or GlcCer+PIOG on PPARγ transcriptional activity; (D) Effects of GlcCer, PIOG, or GlcCer+PIOG on target genes Adora2a and Kcnd1.

[0024] Figure 2 The effect of chronic restraint stress on the levels of different carbon chains GlcCer in the dorsal striatum of mice.

[0025] Figure 3 To improve anxiety and depression behaviors using GlcCer, PIOG, or a combination of GlcCer and PIOG. (A) Diagram of mouse double-tube implantation and drug administration pattern. (B) Time spent in the central area of ​​mice in the open field experiment. (C) Total distance traveled by mice in the open field experiment. (D) Time spent in the bright field of mice in the black-and-white shuttle experiment. (E) Time spent in the open arm area of ​​mice in the elevated cruciate maze experiment. (F) Sugar water preference index of mice in the sugar water preference experiment. (G) Time immobile in mice in the forced swimming experiment.

[0026] Figure 4 To improve CRS-induced reduction in PPARγ target gene transcription, GlcCer, PIOG, or a combination of GlcCer and PIOG were used. (A) qRT-PCR was used to detect Adora2a mRNA levels; (B) ChIP was used to detect changes in PPARγ enrichment in the Adora2a promoter region. All data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates no significant difference. Detailed Implementation

[0027] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0028] Example 1: Combination medication for treating anxiety and / or depression

[0029] This embodiment provides a combination drug for treating anxiety and / or depression, which is a combination drug comprising 200 μM glucosamine (GlcCer purchased from Cayman, catalog number 23207) and 1 μM pioglitazone.

[0030] Example 2: Combination medication for treating anxiety and / or depression

[0031] This embodiment provides a combination drug for treating anxiety and / or depression, which is a combination drug containing 100 μM glucosamine and 1 μM pioglitazone.

[0032] The technical solution of the present invention will be further illustrated by the following experiments.

[0033] Experimental Example 1: The combined use of glucosamine ceramide (GlcCer) and pioglitazone (PIOG) can enhance PPARγ transcriptional activity and the level of PPARγ target genes.

[0034] I. Experimental Methods

[0035] 1. Cell Culture

[0036] The N2a neuroblastoma cell line was purchased from ATCC (American Type Culture Collection). N2a cells were cultured in DMEM containing 10% FBS in a humidified environment of 37°C with 5% CO2 air.

[0037] 2. Real-time quantitative PCR

[0038] RNA was extracted according to the instructions of the RNA extraction kit (Axygen). Bioscience's reverse transcription kit reverse transcribes purified and extracted RNA into cDNA. The prepared cDNA is then subjected to real-time quantitative PCR. All primers used were purchased from Shanghai Sangon Biotech Co., Ltd.

[0039] 3. PPRE-luciferase activity assay

[0040] N2a cells were seeded in 24-well plates and transfected after 24 hours of culture. A DNA mixture containing PPRE-luciferase reporter indicator (0.5 μg), PPARγ / PPARγ mutation signal (0.5 μg), and internal control indicator pRL-SV-40 (25 ng) was transfected using Lipofectamine 3000 transfection reagent. Cells were cultured for another 48 hours after transfection. The Report Assay System measures cell lysates.

[0041] 4. PPARγ transcriptional activity assay

[0042] In vitro PPARγ-PPRE binding assays were performed using a PPARγ transcription factor assay kit. Nuclear extracts were prepared from N2a cells using a nuclear extraction kit. The binding reaction between PPARγ and PPRE was performed using 3 μg of the nuclear extract at 4 °C for 16 h. The binding activity between PPARγ and PPRE was indicated by changes in optical density (OD) at 405 nm, and the enzymatic reaction was measured using an HRP-conjugated antibody in the PPRE / PPARγ / antibody complex.

[0043] II. Experimental Results

[0044] To investigate the effect of GlcCer on PPARγ transcriptional activity, the PPRE-luciferase reporter system was used for detection. The results showed that in N2a cells, glucosamine (GlcCer) increased PPARγ transcriptional activity in a dose-dependent manner, and the addition of a low dose of 100 nM pioglitazone (purchased from MedChemExpress, HY-14601) significantly increased PPARγ transcriptional activity. Figure 1 A). Exposure to 10 μM GlcCer or 100 nM PIOG alone significantly increased PPARγ transcriptional activity, while combined use of the two further enhanced transcriptional activity. Figure 1 B). Further analysis using a PPARγ transcription factor detection kit, after exposure to 10 μM GlcCer or 100 nM PIOG, or a combination of both, revealed that the combined use of 10 μM GlcCer and 100 nM PIOG significantly enhanced PPARγ transcriptional activity. Figure 1 C). qRT-PCR was used to further detect the levels of PPARγ target genes. The results showed that GlcCer or PIOG could enhance the mRNA levels of Adora2a and Kcnd1. The combination of GlcCer and PIOG significantly enhanced the mRNA levels of these two target genes compared to using GlcCer or PIOG alone. Figure 1 D, E), exhibiting a synergistic effect. The above results indicate that GlcCer or PIOG alone can enhance PPARγ transcriptional activity and target gene levels, and the combined use of both significantly enhances this effect compared to using GlcCer or PIOG alone.

[0045] Experimental Example 2: Effects of Chronic Restraint Stress on GlcCer Levels in the Dorsal Striatum

[0046] I. Experimental Methods

[0047] 1. Laboratory animals

[0048] The wild-type animals used in this invention were all male, SPF-grade, healthy, sexually mature (8-12 weeks old) C57BL / 6J mice, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 20-22g, and unmated. Housing conditions: The animals were housed in the general animal facility of the National (Chengdu) Preclinical Safety Evaluation Center for New Drugs, with a temperature of 20-25℃ and a relative humidity of 55-65%. Throughout the experiment, the animals had free access to food and water, and the housing environment met the standards of GB14925-2001, "Laboratory Animal Environment and Facilities". All animal experimental procedures involved in this invention complied with AAALAC requirements. The experimental animals were normally fed for 3-5 days before the experiment to allow the mice to become familiar with and adapt to the environment.

[0049] 2. A model of anxiety and depression induced by chronic restraint stress

[0050] Chronic Restraint Stress (CRS) model in mice: C57 mice were placed in a restraint tube modified from a 50ml BD tube, with the mouse's head facing the tail of the tube. Three small holes with a diameter of 1cm were made at the tail of the tube to ensure normal breathing. Restraint stress was applied once daily for 2 hours each time, for a total of 14 days (ensuring the procedure was performed at the same time each day). Feces were cleaned from the mice's bodies and the restraint tube after each day's experiment. Depressive-like behavioral assessments were performed the day after the experiment ended.

[0051] 3. Targeted detection of glucose ceramide (GlcCer) sphingolipids

[0052] (1) Preparation of standard curve samples

[0053] 5 mg Glucosylceramide (C18:1 / 16:0), 5 mg Glucosylceramide (C18:1 / 18:0), and 5 mg Glucosylceramide (C18:1 / 24:1) were respectively added to 5 ml of chloroform:methanol at a ratio of 1:4 (v / v) to prepare stock solutions of 1 mg / ml. The three standards were then prepared into concentration systems as shown in Table 1 to create standard curves. (Glucosylceramide (C18:1 / 16:0) was purchased from Avanti, 860539; Glucosylceramide (C18:1 / 18:0) was purchased from Avanti, 860548; Glucosylceramide (C18:1 / 24:1) was purchased from Avanti, 860549)

[0054] Table 1 Preparation of standard samples

[0055]

[0056] (2) Sphingolipid extraction

[0057] Tissue or cell samples were thawed at 4°C, and 2.5 mg of tissue was added to the extraction buffer (isopropanol:water:...).

[0058] Ethyl acetate (3:1:6, v / v / v) was added, along with an appropriate concentration of internal standard. The mixture was sonicated until no visible particles were observed, vortexed for 10 min, and centrifuged at 4°C, 6000g, for 10 min. The supernatant was transferred to a new EP tube as Solvent I. The remaining lower layer was extracted again, with 2 ml of isopropanol:water:ethyl acetate (3:1:6, v / v / v) added. The mixture was vortexed for 10 min and centrifuged at 6000g for 10 min. The supernatant was transferred to a new EP tube as Solvent II. Solvent I and Solvent II were mixed thoroughly and transferred to the same EP tube, then dried under a gentle nitrogen stream. The dried lipids were reconstituted with 200 μl of mobile phase B (containing 1 mM ammonium formate and 0.2% formic acid in methanol). If reconstitution was incomplete, the mixture was sonicated and vortexed until completely dissolved. The mixture was centrifuged at 4°C, 13000g, for 10 min. Inject 1 μl of sample and perform UPLC-MS / MS separation and detection for targeted detection of GlcCer.

[0059] (3) Chromatographic and mass spectrometric conditions

[0060] An ACQUITY UPLC I-Class (Waters) high-performance liquid chromatograph was used. Mobile phase A consisted of 0.2% formic acid and 2.0 mM ammonium formate aqueous solution; mobile phase B consisted of 0.2% formic acid and 1.0 mM ammonium formate methanol solution; flow rate: 0.5 mL / min; column: Spectra C18 Column (Waters); column temperature: 55 °C; injection volume: 1 μL. Gradient conditions are shown in Table 2.

[0061] Table 2 Mobile phase conditions

[0062]

[0063]

[0064] The mass spectrometry conditions were as follows: Scan mode was positive ion. Capillary: 3kV; Sample Cone: 30℃; Source Temperature: 140℃; Desolvation Temperature: 350℃; Cone Gas Flow: 50L / Hr; Desolvation Gas Flow: 800L / Hr; Scan range: 50kDa-1200kDa. Specific UPLC-MRM scan modes are shown in Table 3.

[0065] Table 3 Positive Ion MRM Scan Conditions

[0066]

[0067] (4) Data Analysis

[0068] Concentrations were calculated using the Quanlynx plugin for MassLynx software. GlcCer samples with similar chain lengths and abundances were fitted with standard curves using the same standard: C16:0, C18:1, C20:0, and C20:1 were fitted with the C16:0 standard curve; C18:0, C22:0, C26:0, and C26:1 were fitted with the C18:0 standard curve; and C22:1, C24:0, and C24:1 were fitted with the C24:1 standard curve. Results are expressed as mean ± standard deviation. Comparisons between two groups were analyzed using t-tests, and comparisons among four groups were analyzed using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0069] II. Experimental Results

[0070] The dorsal striatum is a brain region involved in chronic restraint stress-induced anxiety and depression. To investigate the effect of chronic restraint stress on GlcCer levels in the dorsal striatum, mice were euthanized by cervical dislocation after the last chronic restraint stress episode, and the dorsal striatum was harvested. GlcCer levels were measured using a targeted GlcCer method. The results showed that GlcCer levels in the dorsal striatum were significantly lower compared to the control group. Figure 2 This indicates that chronic restraint stress leads to a decrease in GlcCer levels in the dorsal striatum, and that administration of GlcCer to mice can improve anxiety and depressive behaviors.

[0071] Experiment Example 3: Effects of combined administration of GlcCer and PIOG to the striatum on anxiety and depression induced by chronic restraint stress.

[0072] I. Experimental Methods

[0073] 1. Targeted intracerebral injection of GlcCer or PIOG or GlcCer+PIOG into the dorsal striatum.

[0074] (1) Implantation of a cannula in the dorsal striatum region

[0075] Mice were anesthetized with 10% chloral hydrate (0.4 g / kg body weight), their heads were shaved, and they were fixed in a stereotaxic apparatus. After fixation, the head was adjusted to a horizontal position, the skin was disinfected with medical alcohol to expose the skull, and the meninges on the surface of the skull were removed with cotton balls and ophthalmic scissors. The anterior fontanelle was located and marked with a marker. The stereotaxic apparatus was adjusted, and the injection catheter (Shenzhen Ruiwode Biotechnology Co., Ltd., #62003, OD 0.48mm X ID 0.34mm) was placed and fixed. Using the anterior fontanelle as the origin, the catheter was moved directly above the injection site using the reference positioning coordinates (striatum: AP+0.8mm; ML±2mm; DV-3.5mm). When the bottom of the catheter just touched the skull, the Y coordinate was zeroed, and the injection needle was slowly moved downward to a depth of 3.5mm. The coordinate arm was released and removed, and dental powder and diluent were mixed to a paste-like consistency to fix the cannula. After the dental cement dried, a catheter cap (Shenzhen Ruiward Biotechnology Co., Ltd., #62102, OD 0.30mm) was inserted to prevent duct blockage, and the head wound was sutured. After the surgery, the mice were kept warm on an electric blanket until they woke up, and then placed in clean cages, five mice per cage. The postoperative recovery period for the mice was 7 days, and the catheter was checked for blockage the day before the formal experiment. Animals that underwent this surgery were used to study the effects of striatal injection of GlcCer or PIOG or GlcCer+PIOG on anxiety-depressive-like behaviors induced by chronic restraint stress.

[0076] (2) Targeted injection of inhibitors into the striatum

[0077] Mice that underwent bilateral dorsal striatal cannulation were administered GlcCer, PIOG, or GlcCer+PIOG at the dosages listed in Table 4, using microinjection needles and catheters. The GlcCer was administered once daily to a specific location in the dorsal striatal brain region for 14 consecutive days. GlcCer was dissolved in DMSO:PEG300:Tween80:ddH2O = 1:14:5:80 to a concentration of 200 μM. PIOG was dissolved in 5% DMSO to a concentration of 1 μM.

[0078] Table 4 Dosage Regimen

[0079]

[0080] 2. Anxiety-like behavioral indicators

[0081] (1) Open field experiment

[0082] The Open Field Test (OFT) consists of two parts: an adaptation period and a testing period. 1) Adaptation period: The day before the experiment, C57 mice were placed in a spontaneous activity detection chamber and allowed to move freely to adapt to the testing environment for 15 minutes. 2) Testing period: Mice were placed in the spontaneous activity detection chamber. No monitoring was performed for the first minute, and monitoring of the mice's activity within the chamber was conducted after one minute, for a total of 5 minutes. The monitoring indicators were the mouse's movement distance *s* (cm) and movement speed *v* (cm / s). The spontaneous activity detection chamber was divided into a central zone and a peripheral zone. Based on the *s* and *v* values ​​for different zones, the total distance and time spent in the central zone were calculated. Ethovision XT software was used to record the mice's movement throughout the experiment.

[0083] (2) Elevated Plus Maze Test (EPMT)

[0084] The elevated cross maze consists of a cross support and a camera, and includes two open arms and two closed arms. Mice are placed in the closed arms, and their movement trajectories are recorded over 5 minutes. The data are analyzed using ANY-Maze software, and the detection indicators include total movement distance (m), percentage of time spent in the open arms, and percentage of time spent in the closed arms.

[0085] (3) Light-Dark Test (LDT)

[0086] The mouse black-and-white shuttle box consisted of two identical compartments. The bright compartment was white on all sides and had a white light source, while the dark compartment was black on all sides and had a door that could be opened in the center. Mice were placed in the dark compartment, and their movement trajectories were recorded over 5 minutes and analyzed using ANY-Maze software. The key performance indicators (KPIs) included the total number of shuttles and the time spent in the bright and dark compartments (in seconds).

[0087] 3. Depressive-like behavioral indicators

[0088] (1) Sucrose preference experiment

[0089] The sucrose preference test (SPT) is an important indicator for detecting depressive-like behavior. The specific steps are as follows: 1) Adaptation period: Place bottles containing 1% sucrose solution and bottles containing pure drinking water simultaneously on the mouse cages, allowing the experimental C57 mice free access to water for 48 hours. 2) Water deprivation: Do not provide any drinking water to the experimental C57 mice for 6 hours after the adaptation period. (3) Sucrose preference test: Weigh the original 1% sucrose solution bottle and the pure drinking water bottle (g), exchange their positions on the mouse cages, and allow the mice to freely choose which water to drink. After 24 hours, weigh the 1% sucrose solution bottle and the pure drinking water bottle again (g′). Sucrose preference = sucrose (g′) / {drinking water (g′) + sucrose (g′)} × 100%

[0090] (2) Forced swimming test

[0091] The Forced Swimming Test (FST) consists of two parts: an adaptation period and a testing period. 1) Adaptation Period: The day before the experiment, C57 mice were placed in a transparent cylindrical water tank filled with purified water and allowed to move freely for 15 minutes to acclimatize to the environment. The tank dimensions were: r = 8 cm, h = 40 cm, water temperature maintained at 25 ± 2℃, and water volume two-thirds of the total capacity. 2) Testing Period: C57 mice were placed in the water tank. No monitoring was performed for the first minute, and monitoring of the mice's activity within the tank was conducted after one minute, for a total of 5 minutes. A monitoring threshold of 1.5 cm / s was set. Above this threshold, the mouse's movement was defined as moving; below this threshold, the mouse's movement was defined as not moving. A spontaneous activity detection box and Ethovision XT software were used to record the mice's movement throughout the experiment.

[0092] II. Experimental Results

[0093] This study investigated the effects of GlcCer, PIOG, and the combination of GlcCer and PIOG on anxiety and depressive behaviors. GlcCer, PIOG, and GlcCer + PIOG were used to examine their effects on chronic restraint stress-induced anxiety and depressive behaviors. Mice underwent bilateral intracerebral duct implantation in the dorsal striatum, and the model was established one week after recovery. Thirty minutes before each restraint stress event, mice were exposed to drugs in the dorsal striatum: 200 μM GlcCer, 1 μM PIOG, and 200 μM GlcCer + 1 μM PIOG, respectively. Figure 3A). The results showed that in the open field experiment, GlcCer, PIOG, or GlcCer+PIOG did not change the movement distance of mice, indicating that administration of these three drugs did not affect the spontaneous activity behavior of mice. Figure 3 C). Exposure to GlcCer or PIOG alone significantly improved the reduction in CRS-induced central zone time, while the combined use of GlcCer and PIOG enhanced this improvement. Figure 3 B). In the black-and-white shuttle experiment, exposure to GlcCer or PIOG alone significantly improved the reduction in CRS-induced bright-field entry time, while the combined use of GlcCer and PIOG enhanced this improvement. Figure 3 D). In the elevated cruciate maze experiment, exposure to GlcCer or PIOG alone significantly improved the reduction in CRS-induced entry time into the open arm, while the combined use of GlcCer and PIOG enhanced this improvement. Figure 3 E). In the sucrose preference experiment, exposure to GlcCer or PIOG alone significantly improved CRS-induced sucrose aversion, while the combined use of GlcCer and PIOG enhanced this improvement. Figure 3 F). In the forced swimming test, exposure to GlcCer or PIOG alone significantly improved the increase in immobility time induced by CRS in mice, while the combination of GlcCer and PIOG significantly reduced the immobility time induced by CRS compared to GlcCer or PIOG alone, demonstrating a synergistic effect. Figure 3 G). The above experimental results all indicate that exposure to GlcCer or PIOG alone can improve anxiety and depressive behaviors, while the combined use of GlcCer and PIOG can significantly improve anxiety and depressive-like behaviors compared to the use of GlcCer or PIOG alone.

[0094] Experiment Example 4: Effect of combined administration of GlcCer and PIOG to the striatum on the transcription of PPARγ target genes

[0095] I. Experimental Methods

[0096] 1. Chromatin Immunoprecipitation (ChIP): Fresh dorsal striatum smears from two mice were collected for this procedure. The tissue was minced, double-crosslinked with DSG for 20 minutes, double-crosslinked with 1% formaldehyde for 10 minutes, and then glycine was added for 10 minutes at room temperature. After homogenizing the tissue particles in PBS, 1 mL of lysis buffer was added. The samples were sonicated to produce fragments of 200–500 base pairs and centrifuged at 14,000 g at 4°C. The supernatant was diluted in dilution buffer and incubated overnight at 4°C with 2 mg of anti-PPARγ antibody. To monitor the specificity of the ChIP assay, the samples were also immunoprecipitated with a specific antibody isotype-matched control IgG. Dynabeads Protein G was added to the supernatant and incubated at 4°C for 2 hours. The beads were recovered and washed three times sequentially with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer. Add elution buffer to the washed beads, treat with RNase at 37°C for 2 hours, and treat with proteinase K at 65°C overnight. Purify the DNA using wash buffer and store at -20°C overnight. Use PowerUp... TM SYBR TM Quantitative PCR was performed using Green Supermix. Kcnd1 and Adora2a primers used for ChIP analysis via RT-PCR were purchased from Shanghai Sangon Biotech Co., Ltd.

[0097] 2. qRT-PCR experiment: Detect the mRNA level of the PPARγ target gene Adora2a.

[0098] II. Experimental Results

[0099] The levels of the PPARγ target gene Adora2a were detected using qRT-PCR. The results showed that CRS significantly reduced Adora2a expression. Exposure to GlcCer or PIOG alone could improve CRS-induced decreased Adora2a expression. The combined effect of GlcCer and PIOG significantly enhanced Adora2a expression compared to either GlcCer or PIOG alone. Further ChIP experiments revealed that CRS significantly reduced PPARγ enrichment in the Adora2a promoter region. Exposure to GlcCer or PIOG alone could improve the CRS-induced decrease in PPARγ enrichment in the Adora2a promoter region. The combined effect of GlcCer and PIOG significantly increased PPARγ enrichment in the Adora2a promoter region compared to either GlcCer or PIOG alone. These results indicate that the combined use of GlcCer and PIOG significantly improves CRS-induced decreased transcription of the PPARγ target gene compared to either GlcCer or PIOG alone, demonstrating a synergistic effect and thus potentially treating anxiety and depression.

[0100] In summary, the present invention combines glucosamine ceramide (GlcCer) and pioglitazone (PIOG) to enhance PPARγ transcriptional activity and the level of PPARγ target genes, demonstrating a synergistic effect in improving anxiety and depressive behaviors. This provides a new option for patients with anxiety and depression and has broad application prospects.

Claims

1. A combination medicament for the treatment of anxiety and / or depression, characterized in that: The combination drug is glucosylceramide and PPARγ agonist.

2. The combination of claim 1, wherein: The combination drug is glucosylceramide and PPARγ agonist used separately or simultaneously.

3. The combination of claim 1, wherein: The molar concentration ratio of the glucosylceramide and PPARγ agonist is 100:1-200:

1.

4. The combination of claim 1, wherein: The PPARγ agonist is at least one selected from the group consisting of pioglitazone, rosiglitazone, troglitazone, ciglitazone and sarglitazone.

5. The combination of claim 4, wherein: The PPARγ agonist is pioglitazone.

6. Use of the combination drug according to any one of claims 1-5 in the preparation of a medicament for the treatment of anxiety and / or depression.

7. Use according to claim 6, characterized in that, The medicament can enhance PPARγ transcriptional activity.

8. A pharmaceutical composition for treating anxiety and / or depression, characterized by, It is prepared with glucosylceramide and PPARγ agonist as active ingredients, and pharmaceutically acceptable adjuvant or auxiliary ingredients; the molar concentration ratio of the glucosylceramide and PPARγ agonist is 100:1-200:

1.

9. The pharmaceutical composition according to claim 8, characterized by: The PPARγ agonist is pioglitazone.

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