Application of indolepropionic acid in preparation of medicine for treating depression

Abstract

The invention discloses application of indolepropionic acid or pharmaceutical salt thereof in preparation of a medicine for preventing and / or treating depression. The indole-3-propionic acid provided by the invention is used as an auxiliary medicine for treating depression, and the indole-3-propionic acid can relieve depression-like behaviors of depression mice and improve hippocampal neuron damage of the depression mice, so that the indole-3-propionic acid can be used as a medicine for treating depression.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of indolepropionic acid in preparing a medicine for treating depression. Background Art

[0002] Depression is a common mental disorder syndrome, with clinical manifestations of persistent low mood, slow reaction, and decreased willpower. In severe cases, suicidal tendencies may even occur. According to the definition of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), if an individual has 5 or more symptoms listed in DSM-5 (such as sadness, despair, decreased activity, insomnia, etc.) for 2 consecutive weeks, accompanied by low mood or anhedonia, he or she can be diagnosed with depression. Monoamine neurotransmitters, neuroendocrine, neurotrophic, immune, oxidative stress, and the gut-brain axis may all be involved in the pathophysiological process of depression.

[0003] As the research continues to deepen, researchers have found that the levels of some active metabolites in tryptophan metabolism are closely related to the occurrence and treatment of depression. Some metabolic enzymes in tryptophan metabolism, such as monoamine oxidase, have become one of the therapeutic targets of antidepressants. More and more scientific research evidence shows that changes in the tryptophan metabolic pathway play an important role in the pathogenesis and treatment of depression. Tryptophan is mainly metabolized through three pathways: "tryptophan-kynurenine", "tryptophan-5-hydroxytryptamine" and "tryptophan-indole". According to research reports, disorders in tryptophan metabolism and changes in neurotransmitters such as 5-hydroxytryptamine are closely related to depression.

[0004] Indolepropionic acid, also known as indole-3-propionic acid (IPA), is one of the products of the bacterial tryptophan indole metabolic pathway in the lower intestine. Studies have reported its immunosuppressive and anti-inflammatory biological activities in the intestinal microenvironment, and found that oral IPA can improve autism symptoms in mice. However, there are few studies on the role of IPA in depression, and the relevant mechanisms of IPA regulating depressive-like behaviors have not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an application of indolepropionic acid in preparing a medicine for treating depression.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a use of indolepropionic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating depression.

[0008] The indolepropionic acid is indole-3-propionic acid (IPA), with a CAS number of 830-96-6. The compound can effectively alleviate the depressive-like behavior of depressed mice and improve the damage of hippocampal neurons in depressed mice.

[0009] The pharmaceutically acceptable salts refer to the following salts formed by indolepropionic acid and the corresponding base: sodium salt, calcium salt, potassium salt, zinc salt or meglumine salt, etc.

[0010] The pharmaceutically acceptable salt refers to a salt formed by the reaction of indolepropionic acid with a pharmaceutically acceptable inorganic acid or organic acid; wherein the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, carbonic acid, nitric acid or sulfuric acid; and the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, stearic acid, nicotinic acid, isonicotinic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acids.

[0011] Depression is a common mental disorder syndrome, which is mainly manifested clinically as persistent low mood, slow reaction, decreased will activity, and even suicidal tendencies in severe cases. The indolepropionic acid can improve the depression-like behaviors of depressed mice, such as anhedonia, decreased will activity, and despair.

[0012] In the application, indolepropionic acid or its pharmaceutically acceptable salt is used as the only active ingredient.

[0013] The second aspect of the present invention also provides a pharmaceutical preparation, which is made of indolepropionic acid or a pharmaceutically acceptable salt thereof and medically acceptable excipients.

[0014] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano preparation and the like.

[0015] The third aspect of the present invention provides a use of indolepropionic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating mental illness and / or a drug for a disease associated with chronic stress.

[0016] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0017] The present invention provides the use of indole-3-propionic acid as an auxiliary drug for treating depression. Indole-3-propionic acid can alleviate the depressive-like behavior of depressed mice and improve the damage of hippocampal neurons in depressed mice. Therefore, indole-3-propionic acid can be used as a drug for treating depression.

[0018] Indolepropionic acid is an intestinal flora-derived metabolite of tryptophan, an essential amino acid for the human body. It can regulate human physiological functions by exerting antioxidant and anti-inflammatory effects. As a metabolite of intestinal bacteria, it has good safety and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the test results of sugar water preference (Mean±SD, n=6) in the sugar water preference experiment of mice in each experimental group.

[0020] Figure 2 It is a schematic diagram of the test results of the central area distance (Mean±SD, n=6) of mice in each experimental group in the open field test.

[0021] Figure 3 It is a schematic diagram of the test results of the immobility time (Mean±SD, n=6) of mice in each experimental group in the tail suspension test.

[0022] Figure 4 It is a schematic diagram of the test results of the floating immobility time (Mean±SD, n=6) of mice in each experimental group in the forced swimming test.

[0023] Figure 5 This is a schematic diagram of the Nissl staining results (×40) of the ipsilateral hippocampal CA1 region of mice.

[0024] Figure 6 It is a schematic diagram of the effects of different concentrations of IPA on the survival rate of HT22 cells (Mean±SD, n=6).

[0025] Figure 7 It is a schematic diagram of the effects of different concentrations of IPA on the survival rate of cells induced by 5 mM Glu injury (Mean±SD, n=6). DETAILED DESCRIPTION

[0026] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0027] Example 1

[0028] Indole-3-propionic acid reduces depressive-like behavior in mice:

[0029] 1. Establishment of chronic depression model and animal grouping

[0030] The indolepropionic acid used in the examples is indole-3-propionic acid (IPA), with a CAS number of 830-96-6.

[0031] The experimental animals were male C57BL / 6 mice, a total of 18 mice, all weighing 20-22g. After one week of adaptation, the animals were randomly divided into 3 groups, 6 mice in each group, namely blank control group (Ct), depression model group (CUMS), and indole-3-propionic acid (IPA) group.

[0032] The internationally recognized CUMS (chronic unpredictable mild stimulation) method was used to establish a chronic depression model (Picard K, Bisht K, Poggini S, et al. Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice. Brain Behav Immun. 2021; 97: 423-439. doi: 10.1016 / j.bbi.2021.07.022). The blank control group did not receive any stimulation except for behavioral evaluation. The depression model group and the indole-3-propionic acid (IPA) group received chronic stress stimulation for 6 weeks, once a day, and each stimulation was not repeated within two consecutive days. The stress factors used in the experiment included: wet bedding for 20 hours, tilting the cage at 45 degrees for 24 hours, restraint for 4 hours, swimming in 4℃ cold water for 5 minutes, baking at 40℃ for 5 minutes, shaking the cage for 15 minutes, depriving water for 24 hours, and depriving food for 24 hours.

[0033] Blank control group: The mice did not receive CUMS stimulation, and were gavaged with control solvent (CMC-Na solution) daily from 4 weeks after modeling until the end of the experiment.

[0034] Depression model group: The mice received CUMS stimulation and were gavaged with a control solvent (CMC-Na solution) once a day from 4 weeks after modeling until the end of the experiment.

[0035] Indole-3-propionic acid group: 4 weeks after modeling, the rats were gavaged with IPA CMC-NA suspension at a dose of 50 mg / kg once a day until the end of the experiment.

[0036] Preparation of CMC-NA suspension of indole-3-propionic acid:

[0037] Sodium carboxymethylcellulose (CMC-Na) powder was added to pure water to make the final concentration of CMC-Na 5% (w / v). Indole-3-propionic acid was added to the above CMC-Na solution and mixed to prepare a CMC-NA suspension of indole-3-propionic acid with a concentration of 6.25 mg / mL.

[0038] 2. Behavioral evaluation methods

[0039] (1) Sugar water preference experiment: Before the experiment, mice were trained with sugar water for a total of 3 days during the one-week adaptive feeding period. The mice were housed in single cages, and 2 bottles of 1% sugar water were placed in each cage for 1 day. Then, 1 bottle of 1% sugar water and another bottle of pure water were placed for 2 days. The left and right positions of 1% sugar water and pure water were changed on the second day. Then, the mice were subjected to sugar water preference experiments on day 0 (week 0) and day 42 (week 6) of modeling. Water was deprived before the experiment. During the experiment, mice were given a bottle of 1% sugar water and a bottle of pure water at the same time. The detection time was 24 hours. The volume of sugar water and pure water before and after the experiment was measured, and the drinking volume and total drinking volume of the two were calculated respectively. The percentage of sugar water drinking volume to total drinking volume was the percentage of sugar water preference.

[0040] (2) Open field test (OFT): Use an open field test box with a size of 50cm×50cm×40cm, adjust the camera to shoot perpendicular to the ground, and the field of view covers the edge of the open field box. Ensure that the light in the open field box is uniform and there are no obvious light and dark corners. After wiping the bottom of the open field box with 75% ethanol, place a mouse in the center of the box from the same position and direction each time, and start the test. Record the activity of the mouse within 5 minutes. After the end, remove the mouse, clean its excrement, and wipe the open field box with 75% ethanol before the next round of testing. Use behavioral analysis software to evenly divide the open field into 16 grids and divide it into the central area, peripheral area, and wall area. The mouse's activity distance in the central area during this period is used as an evaluation indicator to evaluate the mouse's spontaneous activity behavior and exploratory behavior in an unfamiliar environment.

[0041] (3) Tail suspension test (TST): The tail suspension test was performed on the 43rd day. The tail suspension test was conducted from 15:00 to 17:00. Before the start of the experiment, the mice were transferred to the experimental site 1 hour in advance to adapt to the environment. A cotton thread was tied about 2 cm at the end of the mouse's tail, and the mouse was suspended on a stand so that the mouse was about 25 cm from the ground. Every two mice were separated by an opaque baffle to avoid mutual interference. The camera recorded the behavior of the mice for 6 minutes, and the behavioral analysis software Visutrack was used to analyze the immobility time of the mice after 4 minutes. In the tail suspension test, the mouse gave up struggling, the body hung in the air, and only showed necessary movements to maintain balance, which was considered immobile. After the tail suspension test, the mouse was put back into the cage.

[0042] (4) Forced swimming test (FST): FST was conducted from 15:00 to 17:00. Before the experiment began, mice pre-swimmed for 5 minutes 24 hours in advance to adapt to the environment. A cylindrical barrel with a diameter of 20 cm and a height of 50 cm was filled with warm water at a temperature of about 25°C and a water depth of about 35 cm to prevent the mice from escaping or supporting their limbs on the barrel wall and bottom. Every two animals were separated by an opaque baffle. After the mice were placed in the water, the camera recorded the behavior of the mice for 6 minutes, and the behavioral analysis software Visutrack was used to analyze the immobility time of the mice after 4 minutes. The immobility standard was that the mice stopped struggling in the water and were floating, with only slight limb movement to keep their heads above the water. After each mouse finished swimming, it was wiped dry and replaced with new clean water to continue the forced swimming experiment.

[0043] (5) Statistical analysis: GraphPad Prism 8 (GraphPad Software, USA) was used for statistics and graphing.

[0044] 3. Results display

[0045] (1) The results of the sugar water preference experiment are as follows Figure 1 As shown, Figure 1 It is a schematic diagram of the test results of sugar water preference (Mean±SD, n=6) in the sugar water preference experiment of mice in each experimental group. *** P<0.001, compared with the blank control group, # P<0.05, compared with the model group. After 6 weeks of stimulation in the depression model group and 2 weeks of gavage treatment in the IPA group, the percentage of sugar water preference in the depression model group was reduced compared with the blank control group (P<0.001); the percentage of sugar water preference in the IPA group was increased compared with the depression model group (P<0.05). This indicates that gavage of IPA can improve the anhedonia state of depression model mice.

[0046] (2) The results of the open field experiment are as follows Figure 2 As shown, Figure 2 It is a schematic diagram of the test results of the central area distance (Mean±SD, n=6) of mice in each experimental group in the open field test. *** P<0.001, compared with the blank control group, #P<0.05, compared with the model group. Compared with the blank control group, the 6-week stimulation reduced the central area distance of the depression model group mice (P<0.001); compared with the depression model group, the central area distance of the indole-3-propionic acid (IPA) group mice increased (P<0.05). The results showed that IPA improved the reduction of the central area distance of mice induced by CUMS.

[0047] (3) The results of the tail suspension test are as follows Figure 3 As shown, Figure 3 It is a schematic diagram of the test results of the immobility time (Mean±SD, n=6) of mice in each experimental group in the tail suspension test. ** P<0.01, compared with the blank control group, # P<0.05, compared with the model group. Compared with the blank control group, the 6-week stimulation prolonged the immobility time of the mice in the depression model group (P<0.01); compared with the depression model group, the immobility time of the mice in the indole-3-propionic acid (IPA) group was shortened (P<0.05). The results showed that IPA improved the prolongation of the immobility time of the mice induced by CUMS.

[0048] (4) The results of the forced swimming test are as follows Figure 4 As shown, Figure 4 It is a schematic diagram of the test results of the floating immobility time (Mean±SD, n=6) of mice in each experimental group in the forced swimming test. ** P<0.01, compared with the blank control group, ## P<0.01, compared with the model group. Compared with the blank control group, the immobility time of mice in the depression model group was significantly prolonged (P<0.01, respectively); compared with the depression model group, the immobility time of mice in the indole-3-propionic acid (IPA) group was shortened (P<0.01). This indicates that IPA can improve the behavior of prolonged forced swimming immobility time in mice induced by CUMS.

[0049] Example 2

[0050] Effects of indole-3-propionic acid on hippocampal neurons in depressed mice:

[0051] Nissl staining of mouse hippocampal sample slices: after behavioral evaluation, one mouse was taken from each group, anesthetized with 20% urethane (0.4 mL / 100 g) by intraperitoneal injection, the chest was quickly opened and the heart was completely exposed, the needle was inserted into the left ventricle, the right atrial appendage was cut open, the abdominal aortic vein was clamped, and 30 mL PBS was injected to quickly pressurize and perfuse the blood until the fluid outflowing from the right atrial appendage was basically clear, and the perfusion was stopped. At this time, the mouse lungs, eyeballs and claws were observed to be white. Then 30 mL of 4% paraformaldehyde was quickly perfused under pressure. At this time, the mouse upper limb head muscle stiffness was used as a sign, the perfusion was stopped, and the brain was removed by decapitation. After the brain tissue was sampled through the coronal plane, it was fixed in 4% paraformaldehyde for 48 hours as soon as possible. After fixation, the tissue was dehydrated in ethanol from low to high concentrations, 50% ethanol (2h) → 75% ethanol (2h) → 85% ethanol (2h) → 95% ethanol (1h) → 95% ethanol (2h) → anhydrous ethanol (1h) → anhydrous ethanol (1.5h). Then it was transparent in xylene, 1h in the first tank and 1h in the second tank. It was embedded in paraffin with a melting point of 52-60℃, and the transparent brain tissue was transferred to the melted paraffin for wax immersion, the first tank (1h), the second tank (2h). The paraffin around the brain tissue was retained for about 2mm, and the excess paraffin was trimmed with a blade, and the tissue was sliced ​​to a thickness of 5μm. The temperature of the slide was 45℃, and the sliced ​​slides were placed in a 60℃ baking machine for 2h. Paraffin sections were dewaxed in xylene, 5 min each in the first, second and third cylinders, followed by 5 min each in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol, and rinsed with tap water for 1 min. The sections were placed in a 1% toluidine blue aqueous solution and stained in a 56°C incubator for 20 min, and then washed with distilled water. After differentiation in 70% ethanol for about 1 min, they were differentiated in 95% ethanol and controlled under a microscope, with the Nissl bodies showing clearly. Dehydrated quickly in anhydrous ethanol. Xylene was transparent and the sections were sealed.

[0052] The results are as follows Figure 5 As shown, Figure 5 Figure 1 is a schematic diagram of the Nissau staining results (×40) in the ipsilateral hippocampal CA1 region of mice. A is a schematic diagram of the results of the Ct group; B is a schematic diagram of the results of the CUMS group; and C is a schematic diagram of the results of the IPA group. Compared with the blank control group, the pyramidal cells in the hippocampal CA1 region of the depression model group were disordered, the intercellular space was enlarged, the Nissau bodies in the cell bodies were reduced or completely disappeared, and the cells were pyknotic; compared with the depression model group, the pyramidal cells in the hippocampal CA1 region of the indole-3-propionic acid (IPA) group were neatly arranged, and the Nissau bodies in the cell bodies were clearly visible, which was close to the blank control group. This suggests that IPA has antidepressant activity against CUMS-induced depressed mice.

[0053] Example 3

[0054] Effects of indole-3-propionic acid on the viability of HT22 cells (mouse hippocampal neurons) induced by glutamate

[0055] 1. Cell Culture

[0056] DMEM medium was supplemented with 10% fetal bovine serum and 1% double antibody for culturing HT22 cells. The cells were placed in a constant temperature incubator at 37°C and containing 5% carbon dioxide.

[0057] 2. CCK8 assay to determine cell viability

[0058] CCK-8 was used to detect the cytotoxicity of IPA and the protective effect of IPA on glutamate-induced oxidative stress injury in HT22 cells. 4 Cells were inoculated at a density of 100 μL / mL in a 96-well plate, 100 μL of cell suspension was added to each well, and the plate was cultured in a constant temperature incubator for 24 h, and the culture medium was discarded for subsequent operations.

[0059] (1) Evaluation of indole-3-propionic acid cytotoxicity

[0060] 100 μL of culture medium containing IPA or DMSO was added, with the concentrations being normal culture medium, 0 μM (the culture medium only contained the solvent DMSO), 50 μM, 500 μM, 1 mM, 2.5 mM, 5 mM, and 10 mM. Six replicate wells were prepared for each concentration and cultured in a constant temperature incubator for 24 h.

[0061] (2) Protective effect of indole-3-propionic acid on glutamate-induced cellular oxidative stress damage

[0062] Glutamate (Glu) is an important excitatory neurotransmitter. Its normal function in the brain is to regulate signal transmission between neurons, but excessive glutamate can cause excitotoxicity and damage nerve cells.

[0063] 100 μL of complete medium containing 5 mM glutamate and a series of concentrations of indole-3-propionic acid was added to the wells for stimulation for 12 h, with 6 replicate wells for each concentration.

[0064] Remove the culture medium from the wells, add 100 μL of culture medium containing 10% CCK-8, incubate in a constant temperature incubator for 1 to 4 hours, and measure the OD value at a wavelength of 450 nm. The wells without cells and culture medium containing only 10% CCK-8 are used as negative control wells, and the wells without any stimulation of cells are used as positive control wells to calculate the cell survival rate.

[0065] Cell survival rate = (OD drug-containing well-OD negative control well) / (OD positive control well-OD negative control well)

[0066] (3) Statistical analysis: GraphPad Prism 8 (GraphPad Software, USA) was used for statistics and graphing.

[0067] 3. Experimental results

[0068] (1) Indole-3-propionic acid cytotoxicity results Figure 6 As shown, Figure 6 The figure is a schematic diagram of the effect of different concentrations of IPA on the survival rate of HT22 cells (Mean±SD, n=6). It can be seen from the figure that except for 10mM (P<0.0001) IPA stimulation of cells for 24h, which significantly reduced the survival rate of cells, the other concentrations had no obvious toxicity to the cells.

[0069] (2) The protective effect of indole-3-propionic acid on glutamate-induced oxidative stress injury Figure 7 As shown, Figure 7 The figure is a schematic diagram of the effect of different concentrations of IPA on the survival rate of cells induced by 5mM Glu injury (Mean±SD, n=6). It can be seen from the figure that 1mM (P=0.0229) and 2.5mM (P=0.0013) IPA can significantly reduce the cell injury induced by 5mM glutamate and improve the cell survival rate.

[0070] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Use of indolepropionic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating depression.

2. The use according to claim 1, characterized in that: In the application, indolepropionic acid or its pharmaceutically acceptable salt is used as the only active ingredient.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt refers to the following salts formed by indolepropionic acid and the corresponding base: sodium salt, calcium salt, potassium salt, zinc salt or meglumine salt.

4. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt refers to a salt formed by the reaction of indolepropionic acid with a pharmaceutically acceptable inorganic acid or organic acid.

5. The use according to claim 4, characterized in that: The inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, carbonic acid, nitric acid or sulfuric acid.

6. The use according to claim 4, characterized in that: The organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalene disulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, stearic acid, nicotinic acid, isonicotinic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

7. A pharmaceutical preparation, characterized in that The invention is prepared from indolepropionic acid or its pharmaceutically acceptable salt and medically acceptable auxiliary materials.

8. The pharmaceutical preparation according to claim 7, characterized in that The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, and nano preparation.

9. Use of indolepropionic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating mental illness and / or a drug for treating a disease associated with chronic stress.

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