Application of indole-3-formaldehyde in preparation of antidepressant drugs

Abstract

The invention discloses an application of indole-3-formaldehyde in preparation of antidepressant drugs, and relates to the technical field of medicines.The antidepressant drugs comprise active ingredients and pharmaceutical excipients, the active ingredients comprise indole-3-formaldehyde or pharmaceutically acceptable salts thereof, and the effective dose of the active ingredients in rats is 70-200 mg / kg; indole-3-formaldehyde is adopted, so that depression-like behaviors of mice can be remarkably improved, the indole-3-formaldehyde can be used as an exogenous oral supplement to enter intestinal tracts and even can penetrate through a blood brain barrier to enter brain tissues, and the anti-depression effect is achieved from the two aspects of improving the intestinal steady state and protecting neuroinflammation. The tryptophan intestinal metabolite is applied to treatment and improvement of depression for the first time, and a theoretical basis and a novel angle are provided for subsequent research and development of antidepressant drugs applying the tryptophan intestinal metabolite.

Description

Technical Field

[0001] The present invention relates to a field, and in particular to application of indole-3-carboxaldehyde in the preparation of antidepressant drugs. Background Art

[0002] Depression is a serious affective mental disorder, with core symptoms including persistent low mood, loss of interest and decreased willpower, and is characterized by high recurrence rate, high disability rate and high suicide rate. According to statistics from the World Health Organization, there are 322 million people suffering from depression worldwide, and there are problems of repeated and chronic depression, which has become a major global public health issue. With the development of society and the increasing pressure of life, depression may become the number one burdensome disease.

[0003] The pathogenesis of depression is complex. The mainstream hypotheses include the monoamine neurotransmitter hypothesis, the intestinal microbial hypothesis, and neuroinflammation. However, clinically, only about one-third of patients are relieved by antidepressants developed based on a single hypothesis. Therefore, it is of great significance to find the intersection or key factors of various hypotheses. The monoamine neurotransmitter hypothesis occupies an important position in the pathogenesis of depression. This hypothesis believes that the reduction or absence of monoamine neurotransmitters such as 5-hydroxytryptamine (5-HT) in the central and peripheral regions may lead to depression. Basic research and clinical experiments have shown that tryptophan and its metabolites are key to the formation of 5-HT, and the indole compound metabolic pathway mediated by intestinal microorganisms is one of the main tryptophan metabolic pathways. Intestinal microorganisms metabolize some tryptophan in the intestine into indole compounds such as indole-3-carboxaldehyde (I3C). Studies have confirmed that the level of indole-3-carboxaldehyde (I3C) in patients with depression is significantly lower than that in normal people, and it can pass through the blood-brain barrier and enter the brain tissue. It also shows a significant anti-inflammatory effect.

[0004] In recent years, many studies have revealed that the neuroinflammation hypothesis can cover hypotheses such as monoamine neurotransmitters, and is an important research direction for antidepressant drugs. As an intestinal metabolite of tryptophan, indole-3-carboxaldehyde (I3C) is a precursor metabolite of 5-HT and has the characteristics of crossing the blood-brain barrier and anti-inflammatory. Therefore, whether it can exert antidepressant effects by improving neuroinflammation is worth exploring. At present, the development of depression drugs mostly only targets a single hypothesis, with obvious side effects and less than ideal treatment effects. Therefore, it is extremely important to find new ingredients that can be used to prepare depression drugs, which is of great significance for the prevention and treatment of depression. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an application of I3C in the preparation of a drug for treating depression.

[0006] The technical solution of the present invention is as follows: application of indole-3-carboxaldehyde in the preparation of antidepressant drugs.

[0007] Preferably, the drug comprises an active ingredient and a pharmaceutical excipient, and the active ingredient comprises indole-3-carboxaldehyde.

[0008] Preferably, the effective dose of the active ingredient for rats is 70-200 mg / kg.

[0009] Preferably, the active ingredient accounts for 20-80% of the total weight of the drug.

[0010] Preferably, the pharmaceutical excipients include at least one of a diluent, an antioxidant, a lubricant, a solubilizer, and a preservative.

[0011] Preferably, the dosage form of the drug is one of granules, tablets, capsules, oral liquids, injections, ointments, emulsions, and pills.

[0012] Preferably, the drug is administered orally, by enema or by injection.

[0013] The beneficial effects of the present invention are:

[0014] The present invention uses indole-3-carboxaldehyde (I3C) to significantly improve the depressive-like behavior of mice. It can be taken as an exogenous oral supplement into the intestine, and can even pass through the blood-brain barrier into the brain tissue, exerting antidepressant effects from the two aspects of improving intestinal homeostasis and protecting against neuroinflammation. The present invention is the first to use a tryptophan intestinal metabolite for the treatment and improvement of depression, providing a theoretical basis and a new perspective for the subsequent development of antidepressant drugs using this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the behavioral test results of depressed rats, where A is the open field test, B is the forced swimming test, and C is the sugar water preference test;

[0016] Figure 2 The content of I3C in the intestine and hippocampus of depressed rats;

[0017] Figure 3 This is the DCX staining of hippocampal tissue of depression model rats by I3C;

[0018] Figure 4 The effect of I3C on the number of DCX-positive cells in the hippocampus of depression model rats;

[0019] Figure 5 This is the fluorescence staining of AhR in hippocampal tissue of depression model rats by I3C;

[0020] Figure 6The results of I3C on AhR / NF-κB signaling pathway in hippocampus of depression model rats;

[0021] Figure 7 This is a bar graph showing the relationship between I3C and the AhR / NF-κB signaling pathway in the hippocampus of depression model rats;

[0022] In the figure, "*" indicates that compared with the model group, there is a significant difference of p < 0.05; "**" indicates that compared with the model group, there is a significant difference of p < 0.01; "***" indicates that compared with the model group, there is a significant difference of p < 0.001; "****" indicates that compared with the model group, there is a significant difference of p < 0.0001. DETAILED DESCRIPTION

[0023] The present invention provides a tryptophan intestinal metabolite that has a neuroprotective effect and improves depressive-like behavior, and exerts an antidepressant effect by reducing neuroinflammation and neurogenesis.

[0024] Specifically, indole-3-carboxaldehyde is used as an active ingredient in the preparation of antidepressant drugs, and the effective dose for rats is 70-200 mg / kg, specifically 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 160 mg / kg, 180 mg / kg, 190 mg / kg, or 200 mg / kg.

[0025] Preferably, the active ingredient accounts for 20-80% of the total weight of the drug.

[0026] Preferably, the pharmaceutical excipients include at least one of a diluent, an antioxidant, a lubricant, a solubilizer, and a preservative.

[0027] Preferably, the dosage form of the drug is one of granules, tablets, capsules, oral liquids, injections, ointments, emulsions, and pills.

[0028] Preferably, the drug is administered orally, by enema or by injection.

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0030] Example 1

[0031] Animal grouping: Male SPF SD rats (180±20g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. During the experiment, the temperature of the breeding environment was (24±1)℃, the humidity was (50±5)%, and the light was alternating between light and dark for 12h. The animals were allowed to drink water and eat freely. They were adaptively fed for 1 week before the experiment, and randomly divided into normal group (CON), model group (CRS), I3C low-dose group (80mg / kg), and I3C high-dose group (160mg / kg) one week later. There were 10 rats in each group. The drug was administered by gavage every morning, and the dosage was calculated according to body weight. The animal experiment was approved by the Ethics Committee of the China Academy of Chinese Medical Sciences.

[0032] Method for establishing chronic unpredictable stress model: The rats in the normal group were raised normally and did not receive any stimulation. The rats in the model group and each drug-treated group were randomly exposed to various chronic unpredictable mild stimulations every day, including 4°C ice water swimming (5 min), 45°C heat stimulation (10 min), 60 decibel ultrasound stimulation (3 h), tail clamping (2 min), day and night reversal (12 h / 12 ​​h), foot electric shock (voltage 32 V, electric shock every 10 s, each lasting 2 s, a total of 10 times), water deprivation (24 h), fasting (24 h) and empty bottle restraint (2 h), for a total of 14 days, and the stimulation method was randomly selected every day. The specific stimulation is as follows:

[0033] On the first day, the emergency stimuli were fasting (24 h) and empty bottle restraint (2 h);

[0034] On the second day, the emergency stimulus was swimming in 4°C ice water (5 min) and tail clamping (2 min);

[0035] On the third day, the emergency stimulation was water deprivation (24 h) and 45°C heat stimulation (10 min);

[0036] On the fourth day, the emergency stimulus was day-night reversal (12h / 12h) and foot shock (2s, 10 times);

[0037] On the fifth day, the emergency stimulation was 4°C ice water swimming (5 min) and 60 dB ultrasound stimulation (3 h);

[0038] On the sixth day, the emergency stimulus was 45°C heat stimulation (10 min) and tail clamping (2 min);

[0039] On the seventh day, the emergency stimulation was 60 dB ultrasound stimulation (3 h), tail clamping (2 min), and foot electric shock (2 s, 10 times);

[0040] On the eighth day, the emergency stimulus was fasting (24 h) and empty bottle restraint (2 h);

[0041] On the ninth day, the emergency stimulus was swimming in 4°C ice water (5 min) and tail clamping (2 min);

[0042] On the tenth day, the emergency stimulation was water deprivation (24 h) and 45°C heat stimulation (10 min);

[0043] On the eleventh day, the emergency stimulus was day-night reversal (12h / 12h) and foot electric shock (2s, 10 times);

[0044] On the twelfth day, the emergency stimulation was 4°C ice water swimming (5 min) and 60 dB ultrasound stimulation (3 h);

[0045] On the thirteenth day, the emergency stimulus was 45°C heat stimulation (10 min) and tail clamping (2 min);

[0046] On the fourteenth day, the acute stimulations were 60 dB ultrasound stimulation (3 h), tail clamping (2 min) and foot electric shock (2 s, 10 times).

[0047] Evaluation of the severity of depression: After modeling, behavioral tests, namely the open field test (OFT), forced swimming test (FST) and sucrose preference test (SPT), were used to evaluate the severity of depression in rats.

[0048] (1) Open field test (OFT): The bottom of the open field test apparatus (100cm×100cm×40cm) was divided into 25 equal squares, and the open field test was conducted in a relatively quiet environment (≤60 decibels) from 8:00 am to 12:00 noon. The test was conducted on the 28th day of the experiment. First, the rats were placed in the open field test apparatus for 1 minute to adapt to the environment. The number of grids crossed by the rats (at least 3 paws crossed the grid lines) and the number of upright times (both forelimbs were at least 1 cm from the ground) were observed. After adapting to the environment for 1 minute, the number of grids crossed and the number of upright times of the rats were recorded within 4 minutes. The results of the open field test are shown in Figure 1 Middle A, compared with the normal group, the number of grids crossed and the number of upright times of the rats in the model group decreased significantly, indicating that the model was successfully established; compared with the model group, the number of grids crossed and the number of upright times of the rats in the low-dose I3C group (80 mg / kg) and the high-dose I3C group (160 mg / kg) were significantly increased, indicating that both low-dose I3C and high-dose I3C have antidepressant effects.

[0049] (2) Forced swimming test (FST): The experimental apparatus was a cylindrical plexiglass container with a height of 50 cm and a diameter of 20 cm, a temperature of 25±1°C, and a water depth of 30 cm. On the 28th day after the experiment, each rat received 15 minutes of forced swimming training, and a forced swimming test was performed on the 29th day. Each rat underwent a 6-minute forced swimming test, and the immobility time was recorded in the last 4 minutes. The time when the rat kept its head suspended above the water without obvious struggle was defined as the immobility time. The results of the forced swimming test are shown in Figure 1Middle B, compared with the normal group, the immobility time of the rats in the model group was significantly increased, indicating that the model was successfully established; compared with the model group, the immobility time of the low-dose I3C group (80 mg / kg) and the high-dose I3C group (160 mg / kg) was significantly reduced, indicating that both low-dose I3C and high-dose I3C have antidepressant effects.

[0050] (3) Sugar water preference test (SPT): Before the experiment, each rat was given a 1wt% sucrose solution and daily drinking water for the sugar water preference training experiment. Two water bottles were placed in the cage of each rat (single cage). First, two bottles of 1wt% sucrose solution (150 ml each) were given within the first 24 hours. Secondly, one bottle of 1wt% sucrose solution and another bottle of daily drinking water (150 ml) were given within the second 24 hours to train all rats. After the training, all rats were fasted and deprived of water for 24 hours, and then the sugar water consumption was measured within 12 hours. During the test, the rats could freely choose 1% sucrose solution or water. A baseline measurement of the sugar water preference rate was performed on day 0, and a sugar water preference experiment was performed on day 28. The weight of the water bottles before and after the experiment was measured to calculate the sugar water preference rate of the rats. Sugar water preference rate = weight of sucrose solution consumed / (weight of sucrose solution consumed + weight of daily drinking water consumed). The results of the sugar water preference experiment are shown in Figure 1 Middle C, compared with the normal group, the sugar water preference rate of the model group rats was significantly decreased, indicating that the model was successfully established; compared with the model group, the I3C low-dose group (80 mg / kg) and I3C high-dose group (160 mg / kg) both significantly increased the sugar water preference rate, indicating that both low-dose I3C and high-dose I3C have antidepressant effects.

[0051] (4) I3C content in the intestine and hippocampus of depressed rats

[0052] After the experiment, the rats were dissected according to ethical requirements, and their colon and hippocampus tissues were taken. After grinding, lysis, protein precipitation and concentration, the content of I3C in the colon and hippocampus tissues of the rats was determined by UPLC-TQ-MS / MS technology. Figure 2 Compared with the normal group, the I3C in the colon and hippocampus tissues of rats in the model group decreased significantly. After I3C treatment, the I3C in the colon and hippocampus tissues of rats increased significantly, indicating that exogenously supplemented I3C can also pass through the blood-brain barrier and enter the brain tissue.

[0053] (5) Effect of I3C on the number of DCX-positive cells in the hippocampus of rats with depression

[0054] The rat hippocampus tissue was fixed in 4wt% paraformaldehyde for 24h, then removed for tissue dehydration, transparency, wax immersion, embedding and sectioning, and DCX was labeled and stained by immunohistochemistry. Figure 3 and Figure 4 Compared with the normal group, the number of DCX-positive cells in the hippocampus of rats in the model group decreased significantly. After I3C treatment, the number of DCX-positive cells in the hippocampus of rats increased significantly, indicating that I3C can exert antidepressant effects by promoting the occurrence of hippocampal neurogenesis.

[0055] (6) Effects of I3C on AhR receptors in hippocampal tissue of depression model rats

[0056] The AhR protein was stained by immunofluorescence technique in rat hippocampus tissue. The experimental results are shown in Figure 5 Compared with the normal group, the AhR receptor in the hippocampus of the rats in the model group was significantly activated, and I3C treatment could significantly inhibit the expression of AhR receptor.

[0057] (7) The protective effect of I3C on neuroinflammation in the hippocampus of rats with depression model at the molecular level

[0058] The hippocampal tissue of rats was taken and the expression of IL-6, IL-1β, and TNF-α was detected by Elisa technology; the effect of I3C on the AhR / NF-κB signaling pathway was analyzed by Western blot technology. Figure 6 and Figure 7 Compared with the normal group, the expression levels of IL-6, IL-1β, TNF-α and the expression levels of AhR / NF-κB related signaling pathways NLRP3, TMS1, Caspase1, and NF-κB in the hippocampal tissue of rats in the model group were significantly upregulated. After treatment with I3C, the expression of related genes was significantly inhibited, thereby improving neuroinflammation caused by depression.

[0059] Moreover, the I3C used in the present invention belongs to aldehyde substances, which are more stable than acid substances and have better bioavailability. In the metabolic pathway of tryptophan intestinal indole compounds, many indole compounds will be produced, and under different modeling methods, the content of different indole compounds varies. The chronic restraint stress depression modeling method (CRS) used in this application is different from the existing indoleacetic acid modeling method. Moreover, the pathogenesis of depression is currently uncertain, and the targets and pathways regulated by different indole compounds are different. Therefore, the present invention innovatively provides a theoretical basis for the subsequent development of antidepressant drugs for this application.

[0060] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. Application of indole-3-carboxaldehyde in the preparation of antidepressant drugs.

2. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 1, characterized in that: The medicine comprises active ingredients and pharmaceutical excipients, and the active ingredients comprise indole-3-carboxaldehyde.

3. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 1, characterized in that: The effective dose of the active ingredient in rats is 70-200 mg / kg.

4. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 2, characterized in that: The active ingredient accounts for 20-80% of the total weight of the drug.

5. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 2, characterized in that: The pharmaceutical excipients include at least one of a diluent, an antioxidant, a lubricant, a solubilizer, and a preservative.

6. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 1, characterized in that: The dosage form of the medicine is one of granules, tablets, capsules, oral liquid, injection, ointment, emulsion and pills.

7. The use of indole-3-carboxaldehyde in the preparation of antidepressant drugs according to claim 1, characterized in that: The administration route of the drug is oral administration, enema administration or injection administration.

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