Use of isomangiferin in the preparation of a drug for preventing and treating depression

Abstract

The application belongs to the field of medicine and relates to application of an isoflavone compound, isoformononetin (IFM), in preparation of a medicine or health care product for preventing and treating depression; in mouse hippocampal neuron cells (HT22) or mouse microglial cells (BV2), IFM can reduce neuroinflammation caused by corticosterone (CORT) or lipopolysaccharide (LPS) and increase the expression level of brain-derived neurotrophic factor (BDNF); in a chronic mild unpredictable stress mouse depression model, IFM can relieve depression-like behavior of the mouse and reduce the inflammation level of brain tissue, indicating that IFM has a good effect of relieving depression; IFM degrades PDE4D protein by directly combining with a PDE4D target protein. The isoflavone compound IFM has the advantages of outstanding effect and high safety and is expected to develop into an effective medicine for treating and preventing depression.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to the application of isomygin in the preparation of drugs for the prevention and treatment of depression. Background Technology

[0002] Depression is a common and serious central nervous system disorder that has a profound and significant impact on people's daily lives, work abilities, and overall health. According to the World Health Organization's projections, depression, as a common mental disorder, will become one of the major global disease burdens by 2030. Its etiology is complex, involving multiple pathogenic factors. The pathogenesis mainly includes neurotransmitter imbalance, neuroinflammatory responses, hypothalamic-pituitary-adrenal (HPA) axis dysfunction, neurotrophic factor deficiency, gut microbiota dysbiosis, and oxidative stress. In clinical practice, although several antidepressants have been developed and put into use, such as selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs), and tricyclic antidepressants (TCAs), their clinical application still faces significant limitations. Statistics show that only about one-third of patients achieve significant therapeutic effects from existing medications, and these drugs are often accompanied by significant side effects such as nausea, vomiting, sleep disturbances, and sexual dysfunction, which not only affect patient adherence but also limit overall treatment effectiveness. Currently, the main challenges in the treatment of depression include: slow onset of action (usually requiring 4-6 weeks to show efficacy), unsatisfactory bioavailability, and significant individual variability in efficacy. Therefore, the search for new targets and treatment strategies, especially the screening of highly effective and low-toxicity antidepressant active ingredients from natural products, has become a research hotspot.

[0003] Given the complex and multifactorial pathological characteristics of depression, traditional Chinese medicine, with its multi-pathway pharmacological effects, has demonstrated significant advantages in its treatment. Recent studies have found that many natural products possess anti-inflammatory and antioxidant activities, inhibiting the release of inflammatory factors and reducing oxidative stress damage, thereby protecting neurons. They indirectly exert antidepressant effects by regulating the gut microbiota-gut-brain axis function, influencing the HPA axis and inflammatory factor levels; regulating the levels of monoamine neurotransmitters (such as 5-HT, NE, and DA), and inhibiting monoamine oxidase activity, thereby improving neurotransmission function; regulating HPA axis function, reducing plasma cortisol levels, and alleviating stress responses; and promoting the expression of brain-derived neurotrophic factor (BDNF), enhancing hippocampal neurogenesis and synaptic plasticity, and improving neuroplasticity damage. This multi-pathway synergistic effect gives natural products a unique advantage in the treatment of depression, simultaneously improving multiple symptoms such as depressive mood, sleep disorders, and appetite abnormalities.

[0004] Dang Gui Bu Xue Tang (DBD) is a traditional Chinese medicine formula composed of Angelica sinensis and Astragalus membranaceus in a 5:1 ratio. It has beneficial effects on diabetes, diabetic nephropathy, early retinopathy, atherosclerosis, and peripheral neuropathy. However, its bioactive components and potential molecular mechanisms of antidepressant effects remain unexplored. Our team used ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to quantitatively analyze 21 components absorbed in the brain of rats after administration of Dang Gui Bu Xue Tang. The results showed that isoformononetin (IFM) was the most abundant substance in the brain of rats after administration of Dang Gui Bu Xue Tang. Isoflavones, specifically isoformononetin, are methoxyisoflavones found in Astragalus membranaceus; their effects on depression and related mechanisms remain unclear.

[0005] Isoformononetin. Summary of the Invention

[0006] This invention, through a depression cell model and a mouse depression model, discovered a novel use of isomycin in antidepressants by directly binding to the PDE4D protein.

[0007] The use of isomyrhodin or its pharmaceutically acceptable salts or esters in the preparation of medicaments for the prevention or treatment of depression, wherein the structural formula of isomyrhodin is shown below: .

[0008] The use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of depression, characterized in that the pharmaceutical composition comprises isomoglossin or a pharmaceutically acceptable salt or ester thereof and pharmaceutically acceptable excipients.

[0009] The application is characterized in that the dosage form of isomycetin is an oral dosage form and a non-oral dosage form.

[0010] The application is characterized in that the oral dosage form is a tablet, capsule, powder, or granule; and the non-oral dosage form is an injection.

[0011] The use of isomygium nitrate or its pharmaceutically acceptable salts or esters in the preparation of drugs that improve inflammation of brain tissue.

[0012] The dosage of the isoflavone compound isomoglycin described in this invention can be adjusted by comprehensively considering factors such as the administration method, the severity of the disease, the patient's age, and whether there is a history of the disease.

[0013] When this invention is used to prepare a medicine for the prevention and treatment of depression, the excipients and preparation methods can be of any pharmaceutically acceptable form.

[0014] Research Approach: This invention evaluates the antidepressant effects of isomandibularin in vitro using a CORT-induced HT22 cell injury model and an LPS-induced BV2 cell neuroinflammation model. The cytotoxicity of isomandibularin on HT22 and BV2 cells, and its alleviating effect on CORT-induced HT22 cell death, were analyzed using CCK-8 assays. The effect of isomandibularin on the expression levels of inflammation-related genes in the neuroinflammation model was analyzed using RT-qPCR. The effect of isomandibularin on the expression level of brain-derived neurotrophic factor (BDNF) was analyzed using RT-qPCR and Western blot.

[0015] To further evaluate the antidepressant effect of the drug in vivo, this invention first used C57BL / 6J mice to construct an acute toxicity model by gavage administration of high doses of isomycin to assess the safety of the drug. After 14 days, the heart, liver, spleen, lungs and kidneys of the mice were collected for H&E staining.

[0016] A chronic unpredictable mild stress (CUMS)-induced chronic depression animal model was constructed to demonstrate the pharmacological activity of isomyrhodin in alleviating depression. In the CUMS animal model, 4-6 week old C57BL / 6J mice were subjected to various daily stimuli, including reversed day / night cycles, wet cages, restraint, tail clamping, dirty cages, fasting, water deprivation, and odor stimulation. Drug administration began after 2 weeks, with mice divided into a solvent group, a fluoxetine group, and an isomyrhodin group. Behavioral tests were performed after 4 weeks of administration. Blood samples were collected for biochemical analysis, and brain tissue was subjected to H&E staining and Nissl staining. The 5-HT content in the blood of the mice was also measured.

[0017] To further explore the antidepressant mechanism of isomyrhodin, we combined multi-source databases and network pharmacology methods to identify the protein bound to isomyrhodin. We then used docking, DARTS, CETSA, and BLI assays to confirm the binding of isomyrhodin to PDE4D, and Western blotting was used to detect the effect of isomyrhodin on PDE4D protein.

[0018] Beneficial Effects: This invention, based on a corticosterone (CORT)-induced mouse hippocampal neuron (HT22) damage model, screened the antidepressant activity of brain-inducing components in the classic formula Dang Gui Bu Xue Tang (Angelica Blood-Nourishing Decoction). It was found that isomandrin could restore CORT-induced decreased HT22 cell viability, indicating that isomandrin possesses antidepressant activity. Our laboratory conducted in-depth research on the antidepressant effect of isomandrin, and after examining drug safety, further verified its alleviating effect on a mouse depression model induced by chronic unpredictable mild stress (CUMS) in vivo animal models. To date, no relevant domestic or international literature has reported on the antidepressant effect of isomandrin.

[0019] Details are as follows: 1. Antidepressant activity: In vivo pharmacodynamic studies showed that isomyrhodin reversed CUMS-induced decreases in sucrose preference, increased immobility during tail suspension, increased immobility during swimming, and decreased open field exploration ability in mice, indicating that isomyrhodin can alleviate depressive-like behaviors in CUMS mice.

[0020] The results of brain tissue pathological section staining showed that isomyrhodin alleviated hippocampal neuronal damage in mice and increased the number of Nissl bodies, indicating that isomyrhodin can improve neuronal damage in depressed mice.

[0021] Mechanism studies showed that isomyrhodin directly binds to PDE4D protein, reducing PDE4D protein expression, indicating that isomyrhodin can exert its antidepressant effect by directly binding to PDE4D protein and reducing its expression level.

[0022] 2. No toxic side effects: The IC50 of isomyrhodin in both HT22 and BV2 cells was greater than 100 μM, indicating that isomyrhodin has no significant cytotoxic effect. In acute toxicity animal models, no significant damage was observed in the heart, liver, spleen, lungs, and kidneys of mice in all groups, and no signs of poisoning or death occurred, indicating that the drug has high safety. This not only provides a basis for the in vivo pharmacodynamic evaluation of the compound, but also lays the foundation for the development of new clinical applications for this type of drug.

[0023] 3. Relief effect on neuroinflammation: Isorhynchin can restore CORT-induced HT22 cell death, indicating that isorhynchin can alleviate CORT-induced HT22 cell damage.

[0024] Isorhynchin can reverse the CORT-induced increase in the expression of pro-inflammatory genes and the decrease in the expression of anti-inflammatory genes in CORT-induced HT22 cells and LPS-induced BV2 cells, indicating that isorhynchin has a relieving effect on neuroinflammation.

[0025] Therefore, the present invention provides isomygium extract, which has good market prospects and clinical value in antidepressant treatment, both in terms of safety and efficacy. Attached Figure Description

[0026] Figure 1 IC50 results of different concentrations of Isoformononetin administered to HT22(A) and BV2(B) cells; Figure 2 Cell viability results of HT22 (A) and BV2 (B) cells after administration of different concentrations of Isoformononetin in the presence of CORT and LPS; Figure 3 Isoformononetin improved LPS-induced inflammation levels in BV2 cells, where A was IL-6, B was IL-10, and C was IL-1β. Figure 4 Effects of Isoformononetin on BDNF protein expression in CORT- and LPS-induced HT22(A) and BV2(B) cells; Figure 5 CUMS-induced C57 mice were administered Vehicle, Fluoxetine, Isoformononetin (10 mg / kg), and Isoformononetin (20 mg / kg) for 6 weeks and 4 weeks respectively. OFT experiment diagrams are shown, where A is the mouse movement trajectory, B is the total distance the mouse moved, C is the distance the mouse entered the central region, D is the time the mouse was in the central region, and E is the number of times the mouse entered the central region. Figure 6 CUMS-induced C57 mice were administered Vehicle, Fluoxetine, Isoformononetin (10 mg / kg), and Isoformononetin (20 mg / kg) for 6 weeks, respectively, and then subjected to SPT (A), FST (B), and TST (C) experiments for 4 weeks. Figure 7 HE staining images of brain tissue from C57 mice induced by CUMS for 6 weeks, and administered with Vehicle, Fluoxetine, Isoformononetin (10 mg / kg), and Isoformononetin (20 mg / kg) for 4 weeks, respectively. Figure 8 Nissl staining images of brain tissue from C57 mice induced by CUMS for 6 weeks, and administered with Vehicle, Fluoxetine, Isoformononetin (10 mg / kg), and Isoformononetin (20 mg / kg) for 4 weeks, respectively. Figure 9 Effects of Isoformononetin on mouse serum 5-HT; Figure 10 Isoformononetin improved the level of inflammation in mouse brain tissue induced by CUMS, where A is IL-6, B is IL-10, C is IL-1β, and D is TNF-α; Figure 11 Effects of Isoformononetin on BDNF protein expression (A) and BDNF mRNA levels (B) in mouse brain tissue; Figure 12Images indicating direct binding of Isoformononetin to PDE4D protein, including docking (A), DARTS (B), CETSA (C), and BLI (D). Figure 13 The effect of isoformononetin on the expression level of PDE4D protein, where A represents HT22 and B represents BV2. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, but the description is for explanation and not limitation of the present invention; unless otherwise specified, the experimental methods in the following embodiments involve common commercially available raw materials and reagents that can be purchased from the market.

[0028] Example 1: In vitro study of the effect of Isoformononetin on improving depression 1. Experimental Methods 1.1 Detection of cell viability The cells were divided into three groups: a blank group (containing only culture medium and no cells), a control group (normally cultured HT22,BV2 cells with a drug concentration of 0 μM), and drug-treated groups (normal HT22,BV2 cells with different concentrations of drug added: 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM, respectively), with 6 replicates per group. HT22,BV2 cells in the logarithmic growth phase were digested with trypsin, counted using an automated cell counter, and diluted to a cell density of 6 × 10⁴ cells / mL. 100 μL of the drug was seeded into each well of a 96-well plate. The cells were incubated in a cell culture incubator at 37°C with 5% CO₂ for 24 h. The cell supernatant was discarded, and 100 μL of the corresponding concentration of drug-containing culture medium was added to each well (the blank group and control group only received culture medium), and the cells were incubated for another 24 h. Under light-protected conditions, add 10 μL of CCK-8 to each well, wrap the 96-well plate with aluminum foil, shake on a shaker for 10 min, and then place it in an incubator. After 2 h, measure the OD450 using a microplate reader. Plot the IC50 of isomerin in HT22 and BV2 cells using GraphPad Prism 8.0.2 software.

[0029] 1.2 qRT-PCR detection of inflammatory factor levels Total RNA was extracted using an RNA extraction kit, and the obtained RNA was reverse transcribed using a HiScript® II Q Select RTSuperMix kit to obtain cDNA. The mRNA of inflammatory factors IL-6, IL-1β, IL-10, TNF-α and β-actin was detected using gene-specific quantitative primers and a SYBR Green PCR kit.

[0030] 1.3 Western blot detection of protein expression levels For Western blot analysis, cells were lysed using protein lysis buffer, and the supernatant was collected for BCA quantification. The quantified protein was then boiled in hot water for 10 min. Equal volumes of immune complexes or lysates were separated in an 8-12% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked at room temperature by soaking in 5% defatted PBST buffer (PBS containing 0.2% Tween 20) for 2 hours, followed by overnight immunoblotting at 4°C with a diluted primary antibody. After incubation with peroxidase-labeled secondary antibody for 2 hours, the immunoblot was electrochemiluminescently analyzed on a ChemiDoc XRS+ imaging system using ECL reagent according to the manufacturer's instructions, and imaged using ImageLab software. β-actin was used as an internal control.

[0031] 2. Experimental Results The results showed that the IC50 of Isoformononetin in hippocampal neurons of HT22 mice and microglia of BV2 mice was greater than 100 μM (115.4 μM and 112 μM, respectively), indicating that Isoformononetin had no significant toxicity to HT22 and BV2 cells. Figure 1 Different concentrations (1 μM, 3 μM, 10 μM) of Isoformononetin had no significant effect on cell viability in CORT- and LPS-induced HT22 and BV2 cells. Figure 2 ), and Isoformononetin can reduce the expression of CORT and LPS-induced inflammation-related genes ( Figure 3 ), increasing the protein expression level of brain-derived neurotrophic factor (BDNF) Figure 4 ).

[0032] Example 2: In vivo study of the effect of Isoformononetin on improving depression 1. Experimental Methods 1.1 Establishment of a chronic unpredictable mild stress (CUMS) mouse model Mice were randomly divided into six groups (n=10–12 per group): control group (Ctrl), model group (CUMS), positive control group (CUMS + Fluoxetine (10 mg / kg), ig), Isoformononetin 10 mg / kg group (CUMS + Isoformononetin (10 mg / kg), ig), and Isoformononetin 20 mg / kg group (CUMS + Isoformononetin (20 mg / kg), ig). Fluoxetine (Flu) was dissolved in dimethyl methoxide, and Isoformononetin was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na). Except for the control group, all other mice were exposed to the following unpredictable stressors daily in randomized order for 6 weeks: (1) ice bath (5 minutes), (2) moist bedding (500 ml of 22°C water poured into the bedding), (3) water deprivation (24 hours), (4) circadian rhythm reversal (24 hours), (5) restraint (2 hours), (6) odor stimulation (24 hours), (7) fasting (24 hours), (8) cage tilted at 45 degrees (24 hours), (9) tail clamping (5 minutes). Behavioral tests (including forced swimming test, tail suspension test, and sucrose preference test) were performed in week 7. After the behavioral assessment, the mice were euthanized, and serum and brain tissue samples were collected for further histological evaluation and metabolic analysis.

[0033] 1.2 Behavioral Testing 1.2.1 Sugar Water Preference Experiment The Sucrose Preference Test (SPT) was used to assess anhedonia behavior. In the sucrose preference test, mice were provided with two standard drinking bottles: one containing 1% sucrose and the other containing tap water. Mice drank from these bottles for 24 hours. Before starting the sucrose preference test, all mice were fasted and deprived of water for 12 hours. The positions of the two bottles were switched every 6 hours to avoid favoritism. At the end of the sucrose preference test, sucrose and water intake were measured, and sucrose preference was expressed as sucrose intake / (sucrose intake as a percentage of total intake) plus water intake.

[0034] 1.2.2 Open Mine Experiment The open mine experiment was used to assess exploratory drive and anxiety-like behaviors in mice. Each mouse was placed individually in the center of an open area and given six minutes of free exploration time, during which their activities were recorded by an overhead camera. Analytical metrics included total distance traveled, distance traveled in the central area, time spent in the central area, and number of times the mouse entered the central area. After the experiment, the area was disinfected with 75% ethanol to eliminate olfactory interference.

[0035] 1.2.3 Suspended Tail Experiment The tail suspension test is used to assess despair in animals. A mouse's tail is taped upwards, approximately 50 cm above the ground, and left suspended for 6 minutes. When the mouse stops struggling to escape and passively and stillly droops its tail, it is considered to be in a state of despair. The experimenters assess the test using a time sampling method without knowing the experimental groups. In the last 5 minutes of the test, the time spent struggling (i.e., remaining still) is calculated, with the first minute considered an adaptation period.

[0036] 1.2.4 Forced Swimming Experiment The forced swimming test has also been used to assess behavioral despair. In this experiment, rats are allowed to swim for 6 minutes. Rats are placed in a polycarbonate cylinder (35 cm high, 20 cm in diameter), filled two-thirds full with water at a temperature of 25 °C, for a training period of 6 minutes. The time the rats remain still is measured. Stillness is defined as the absence of any active movement other than that required to maintain buoyancy. The time spent floating (stillness) is then calculated in the last 5 minutes of the test, with the first minute considered an adaptation period.

[0037] 1.3 Hematoxylin-eosin staining (H&E staining) Brain tissue samples were collected, fixed in 10% formalin solution, decalcified, dehydrated, cleared, and finally embedded in paraffin. 5-micron-thick tissue sections were prepared using a microtome. The sections were dewaxed with xylene, rinsed with a series of ethanol solutions, stained with hematoxylin and eosin (HE), and their morphological and structural changes were observed under an optical microscope.

[0038] 1.4 Nissl staining Nissl staining is used to determine neuronal damage in the hippocampus. The specific procedure is as follows: Following the instructions, methylene blue or other staining solutions are used to stain the dewaxed hippocampal sections for 10 minutes. After dehydration, clearing, and mounting, the sections are observed and imaged under a microscope.

[0039] 1.5 Plasma Sample Collection After anesthetizing mice, blood was collected from the eyeballs in 1.5 ml EP tubes (with heparin sodium anticoagulant added). After standing for 30 min, the tubes were centrifuged at 4000 rpm for 10 min at 4℃. The supernatant clear plasma was gently transferred and stored at -80℃ for subsequent biochemical index detection.

[0040] 2. Experimental Results 2.1 Isoformononetin significantly alleviated CUMS-induced depressive-like behavior in mice. Multiple methods were used to detect depressive-like behaviors, including the open field test, forced swimming test, tail suspension test, and sucrose preference test. For all behavioral tests, mice were transferred to the testing room for at least one hour prior to the experiment. Animals were tested in a randomized order, and the equipment was thoroughly cleaned with 75% ethanol to reduce odor.

[0041] Open mine experiments showed that Isoformononetin significantly restored CUMS-induced reductions in motor function and exploration interest in mice. Figure 5 The sucrose preference test showed that Isoformononetin significantly restored CUMS-induced anhedonia in mice, and the forced swimming and tail suspension tests showed that Isoformononetin significantly alleviated CUMS-induced increases in behavioral despair in mice. Figure 6 ).

[0042] 2.2 Isoformononetin can protect against CUMS-induced neuronal damage and morphology in mice. Mouse brain tissue was stained with hematoxylin and eosin (HE) and Nissl stain, respectively. The results showed that in the control group, hippocampal neurons were neatly and tightly arranged, with intact morphology and structure, and normal intercellular spaces. However, in the chronic unpredictable mild stress (CUMS) group, hippocampal neurons showed significant defects, characterized by sparse and disordered cell arrangement, deeply stained nuclei, cytoplasmic vacuolation, and significant changes in cell morphology and structure. Notably, these signs of hippocampal damage in CUMS mice were alleviated after treatment with fluoxetine (Flu) and isoformononetin. Figure 7 Nissl staining showed that the mean optical density of Nissl bodies in the CA1 and DG regions of the hippocampus in the CUMS group was reduced, with neuronal loss, breakage, irregular arrangement, and unclear Nissl body laminar structure. However, Flu and Isoformononetin restored this damage. Figure 8 ).

[0043] 2.3 Isoformononetin can significantly restore serotonin (5-HT) levels. Using the 5-HT ELISA kit instructions, the 5-HT content of collected mouse serum was measured. The results showed that Isoformononetin could significantly restore 5-HT levels. Figure 9 This indicates that Isoformononetin can improve depressive-like symptoms in mice.

[0044] 2.4 Isoformononetin can restore the expression of inflammation-related genes in mice. Mouse brain tissue was collected, RNA lysis was added, and homogenization was performed. Total RNA was then extracted according to the instructions of the reagent manufacturer's RNA extraction kit. RNA was reverse transcribed into cDNA using the PrimeScript™ RT kit. The corresponding mRNA and β-actin mRNA levels were detected using gene-specific quantitative primers and the QuantiTect SYBR Green PCR kit. Target mRNA levels were normalized to the geometric mean of β-actin mRNA levels. Results showed that isoformononetin could restore the expression of CUMS-induced inflammation-related genes in mice. Figure 10 ).

[0045] 2.5 Isoformononetin can restore protein expression of BDNF in mice. Mouse brain tissue was collected, and 1×RIPA buffer containing protease inhibitors and phosphatase inhibitors was added. The tissue was homogenized, and the supernatant was collected. The experiment was performed according to the instructions provided by the Western blot reagent manufacturer. The results showed that Isoformononetin could restore the protein expression of mouse BDNF. Figure 11 ).

[0046] Example 3: Study of target proteins of Isoformononetin 1. Experimental Methods 1.1 Molecular docking simulation (Docking) Molecular docking simulations were performed using a docking algorithm based on the PDE4D protein structure (AlphaFold: Q01063) and the structure of Isoformononetin, employing the Schrödinger model.

[0047] 1.2 Target stability of drug affinity response (DARTS) Cells were collected and total protein was separated using lysis buffer. Cell lysates were centrifuged at 18000 × g at 4 °C for 10 min. The supernatant was diluted 1:10 with 10 × TNC buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM CaCl2), and treated with different concentrations of isoformononetin or DMSO as controls. After incubation at room temperature for 2 h, pronase was added at 37 °C and incubated for another 5 min. The reaction was stopped by adding protease inhibitors and SDS-PAGE loading buffer, followed by Western blotting.

[0048] 1.3 Cell thermal transfer assay (CETSA) HT22,BV2 cells were cultured in 10 cm culture dishes until 70-80% confluence. Cells were then treated with isoformononetin or DMSO and incubated for 6 h. Cells were collected, washed with PBS, and resuspended in PBS supplemented with protease inhibitors to a final volume of 5 × 10⁻⁶. 6 Cells / ml. Aliquot 100µl of each cell suspension into PCR tubes, heat at 38-46°C for 3 minutes using a heat cycler, and immediately lyse in liquid nitrogen. Centrifuge the cell lysate at 15000 × g for 15 minutes at 4°C until clear. Analyze the supernatant using Western blotting.

[0049] 1.4 PDE4D protein expression assay HT22 and BV2 cells were seeded into cell culture dishes. After the cells adhered overnight, the drug was added for treatment. The cells were collected 24 hours later, and the expression of PDE4D in the cells was detected by Western blotting according to the instructions of the protein extraction kit.

[0050] 2. Experimental Results Molecular docking simulations, DARTS, CETSA, and BLI results showed that Isoformononetin directly binds to the PDE4D protein. Figure 12 Western blot results showed that isoformononetin reduced the protein expression level of PDE4D. Figure 13 This indicates that Isoformononetin degrades PDE4D by directly binding to it, and the specific mechanism may affect the therapeutic effect of Isoformononetin on depression.

Claims

1. The use of isomoglossin or a pharmaceutically acceptable salt or ester thereof in the preparation of medicaments for the prevention or treatment of depression, wherein the structural formula of isomoglossin is shown below: 。 2. The use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of depression, characterized in that... The pharmaceutical composition comprises isomoglycan or a pharmaceutically acceptable salt or ester thereof and pharmaceutically acceptable excipients.

3. The application according to claim 1 or 2, characterized in that, Isomoglycin is available in both oral and non-oral formulations.

4. The application according to claim 3, characterized in that, The oral dosage form is tablets, capsules, powders, or granules; the non-oral dosage form is an injection.

5. The application according to claim 1 or 2, characterized in that, Isorhynchin exerts its antidepressant effect by directly binding to the PDE4D protein.

6. The use of isomygianin or its pharmaceutically acceptable salts or esters in the preparation of drugs that improve inflammation of brain tissue.

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