CN114159563A

CN114159563A - Anti-depression medicine and screening method thereof

Info

Publication number: CN114159563A
Application number: CN202111540219.7A
Authority: CN
Inventors: 宋永贵; 艾志福; 苏丹; 汪婷婷
Original assignee: Jiangxi University of Traditional Chinese Medicine
Current assignee: Jiangxi University of Traditional Chinese Medicine
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-03-11

Abstract

The invention discloses an anti-depression drug and a screening method thereof, and relates to the field of medicines, wherein the drug takes inflammatory factor regulation intestinal IDO1 as a target spot, and through inhibiting proinflammatory factors in intestinal tracts, IDO1 in the intestinal tracts is further reduced, and tryptophan metabolism is regulated to play an anti-depression role. Whether the animal body has intestinal inflammation or not is detected, the candidate drug is administrated to the animal model, whether the candidate drug has an antidepressant effect or not is detected, the target point is further identified, the target point is a novel therapeutic drug design target point, and screening of antidepressant drugs based on the components for inhibiting the intestinal inflammation and/or IDO1 expression is expected to be promoted.

Description

Anti-depression medicine and screening method thereof

Technical Field

The invention relates to the field of medicines, in particular to an anti-depression medicine and a screening method thereof.

Background

Depression is a common mental disorder disease, and its clinical manifestations include low mood, insomnia, poor appetite and poor concentration. Severe depression may even increase the risk of suicide. According to the world health organization data, more than 3.2 million people worldwide suffer from depression. Research analysis has predicted that depression will become a major disease burden in both moderate and high income countries by 2030. Therefore, the deep exploration of the pathogenesis of depression is imminent.

A wide variety of antidepressant drugs are currently on the market, mainly comprising monoamine oxidase inhibitors, tricyclic drugs and selective serotonin reuptake inhibitors. However, the medicines have the problems of long effective time, strong side effect, different drug sensitivity of people and the like. The reason for this analysis may be due to the complex pathogenesis of the disease and the poor specificity of the target for which the drug is directed. The key point for solving the problems lies in finding out an exact medicine target point on the basis of disclosing a depression mechanism.

The pathophysiological mechanisms of depression are complex, and the central mental disorder presented by patients involves pathological changes of multiple systems and involves metabolic and microstructural abnormalities in vivo. The association of intestinal inflammation with depression is of increasing concern. Clinical studies indicate that IBD (inflammatory bowel disease) patients are more likely to be diagnosed with depression within 9 years before diagnosis is confirmed; persons reporting gastrointestinal symptoms prior to the appearance of depressive symptoms have a 40% higher likelihood of having IBD than persons without depressive symptoms. In recent years, research shows that patients with UC (ulcerative colitis) are often accompanied by various mental health problems such as anxiety, depression and the like. Intestinal inflammation is a cause of depression to some extent, but the related mechanisms are unclear, and the action mechanism of intestinal inflammation in depression cannot be studied from peripheral inflammation change. Therefore, the mechanism of intestinal inflammation and depression is required to be deeply explored, and a novel anti-depression drug is searched by taking the mechanism as a target.

Disclosure of Invention

The invention aims to solve at least one technical problem in the prior art and provides an anti-depression drug and a screening method thereof.

The technical solution of the invention is as follows:

an application of a medicine for inhibiting intestinal inflammation in preparing antidepressant is disclosed.

The invention also discloses an antidepressant drug which takes the intestinal inflammation factor to regulate intestinal IDO1 as a target spot.

Preferably, the medicament for inhibiting the intestinal inflammation is one or more of mesalamine, olsalazine, balsalazide and sulfasalazine.

Preferably, it is capable of inhibiting inflammatory factors in the intestine and downregulating IDO1 in the intestine.

Preferably, the antidepressant drug is mesalazine or scutellaria baicalensis-coptis chinensis extract.

The invention also discloses a screening method of the antidepressant, which is to establish an animal chronic depression model, detect whether intestinal inflammation exists in the animal body, administer the candidate drug to the model and detect whether the candidate drug has the antidepressant effect.

Further, the method also comprises the following steps:

detecting whether the drug to be selected can reduce inflammatory factors in animal intestinal tracts;

detecting whether the drug to be selected down-regulates the expression of IDO1 in the intestinal tract of the animal;

detecting whether the drug to be selected regulates the tryptophan metabolism in the intestinal tract of the animal; thereby identifying the action target of the antidepressant in the animal body.

Further, in the first step, the judgment condition of whether to play an antidepressant role is as follows: expression of IDO1 in colon tissues of animals was detected by immunoblotting.

Further, the level of the inflammatory factor was measured by ELISA.

The invention has the beneficial effects that: the anti-depression drug takes inflammatory factors to regulate intestinal IDO1 as a target spot, and regulates IDO1 in the intestinal tract by inhibiting proinflammatory factors in the intestinal tract, so that tryptophan metabolism is regulated, the conversion of tryptophan in the intestinal tract to kynurenine is inhibited, and the conversion of tryptophan to 5-HT level is promoted, thereby playing the role of anti-depression. The invention is a new therapeutic drug design target and is expected to promote the screening of antidepressant drugs based on the components for inhibiting intestinal inflammation and/or inhibiting IDO1 expression.

Drawings

FIG. 1 shows the results of a sugar water preference test in test group 1 (mesalazine) according to the present invention;

FIG. 2 shows the results of forced swimming in test group 1 (mesalazine) according to the present invention;

FIG. 3 shows the results of the inflammatory factor ELISA in test group 1 (mesalamine) according to the present invention;

FIG. 4 is a graph showing the results of the expression level of intestinal IDO1 in test group 1 (mesalazine) according to the present invention;

FIG. 5 shows the results of tryptophan metabolism in test group 1 (mesalamine) according to the present invention;

FIG. 6 shows the results of a sugar water preference test in Experimental group 2 (Scutellariae radix-Coptidis rhizoma) according to the present invention;

FIG. 7 shows the results of forced swimming in Experimental group 2 (Scutellariae radix-Coptidis rhizoma) according to the present invention;

FIG. 8 shows the results of the inflammatory factor ELISA in Experimental group 2 (Scutellariae radix-Coptidis rhizoma) according to the present invention;

FIG. 9 shows the results of expression of IDO1 in intestine in test group 2 (Scutellariae radix-Coptidis rhizoma) according to the present invention;

FIG. 10 is a graph showing the results of tryptophan metabolism in Experimental group 2 (Scutellariae radix-Coptidis rhizoma) according to the present invention;

in the figure, "+" indicates p <0.05, "+" indicates p <0.01, "+" indicates p <0.001, and p <0.05 has statistical significance, in comparison with the model group.

Detailed Description

This section will describe in detail specific embodiments of the invention, which should not be construed as limiting the scope of the invention.

The embodiment is an anti-depression drug taking inflammation factors to regulate intestinal IDO1 as an action target, and whether the anti-depression effect is to regulate intestinal IDO1 as the action target is verified through the intestinal inflammation level, IDO1 expression and tryptophan metabolism.

Specific examples mesalamine is used as a drug for treating anti-depression, and is not particularly limited thereto. The animal depression behavior is obviously improved, reduced or disappeared in an animal behavior despair depression model experiment, and further detection shows that the intestinal inflammation of experimental animals is reduced, the intestinal IDO1 expression is reduced, and the tryptophan metabolism is recovered to be normal. The specific action target test method of the medicine is as follows:

it should be noted that IDO1 refers to the tryptophan metabolism rate-limiting enzyme, indoleamine 2, 3-bis-oxidase 1.

ELISA: enzyme linked immunosorbent assay.

CUMS: chronic unpredictable mild stress.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The method comprises the following steps: establishment and administration of mouse depression model

The experimental animals were male, 7-week-old C57 mice. All mice were free to ingest water, using a day-night cycle of 12h (8: 00-20: 00 at night, 20: 00-8: 00 at daytime.)

Experimental group 1: the mesalazine is prepared into a concentration of 30mg/ml, and the mesalazine is specifically mesalazine sustained release granules (Aidisha) produced by Shanghai Haifeng pharmaceutical Co.

Experimental group 2: preparing Scutellariae radix-Coptidis rhizoma extract into 2 g/mL^-1The suspension of (4) was administered by intragastric administration at a rate of 0.1mL/10 g. Wherein the preparation of the scutellaria baicalensis-coptis chinensis extract comprises the following steps: taking decoction pieces of scutellaria baicalensis and coptis chinensis, crushing, drying, and then weighing 50g of scutellaria baicalensis and coptis chinensis respectively as raw materials, wherein the raw materials are as follows: adding water at a ratio of 1: 12 (g: m L), soaking for 30min, decocting for 2 hr, and filtering while hot. Extracting the residue with 10 times of water for 2 hr, and filtering while it is hot. Mixing the extractive solutions for 2 times, concentrating to obtain soft extract with relative density of 1.02 (at 80 deg.C), and freeze drying to obtain Scutellariae radix-Coptidis rhizoma extract.

The mice are randomly divided into a model group, a blank group, an experimental group 1 and an experimental group 2, 8 mice in each group need to establish a depression model except the blank group of mice by orally taking corresponding medicines. The model was established by chronic unpredictable stimulation of mice. In the experiment, 14 stress sources are adopted, and 14 stress modes are respectively 14 modes of changing the light property, adjusting the circadian rhythm, forbidding water, fasting, swimming with ice water at 4 ℃, swimming at high temperature of 45 ℃, inclining a squirrel cage at 45 ℃, using a moist pad, horizontally shaking, lightly clamping the tail, binding, white noise, intermittent (flashing) stimulation and hanging the tail. The specific stress pattern is shown in table 1. The CUMS group mice were randomly given 2 stresses at random times daily, with each stress source occurring 2 times in a discrete fashion.

TABLE 1 CUMS Depression modeling stress stimulus test Schedule

After the model building is completed, the drug administration is carried out on each model building group. The administration was performed according to the scheduled groups, respectively, with molding and three weeks of administration.

(1) Blank group (no modeling required): as a blank control, gavage was continued for 15 days without any treatment except that gavage was given daily with 0.2mL/20g of physiological saline.

(2) Model group: as a negative control, after the mice are successfully molded, the mice are continuously gavaged with 0.2mL/20g of physiological saline every day for 21 days.

(3) Mesalazine group: as an experimental group 1, after the mice were successfully molded, the mice were administered with mesalamine in an amount of 0.2mL/20 g/day and a concentration of 30mg/mL, and the gavage was continued for 21 days.

(4) The scutellaria-coptis drug pair group: as an experimental group 2, after the mice are successfully modeled, the mice are continuously gavaged for 21 days by feeding 0.1mL/10g of scutellaria baicalensis-coptis chinensis drug pair per day.

Step two: mouse syrup experiment

The sugar water preference experiment is used for evaluating the preference of the sugar water of the mice and can reflect the anhedonia degree of the mice. A total of two sugar water preference experiments were performed throughout the animal experiments. Before modeling, sucrose preference coefficients of all mice are measured through a sugar water preference experiment, and the consistency of the states of the mice subjected to the experiment is ensured. At the end of the experiment, a sugar water preference experiment was performed as part of animal behaviours to assess the extent of anhedonia in mice. The sugar water preference experiment comprises two parts, namely an adaptive training part and a testing part. In training, mice were placed in two 1% (w/v) bottles of sucrose solution per cage for the first 24h, one of them was replaced with pure water immediately after 24h, and after the adaptation was completed, the mice were fasted and water was deprived for 24h, after which the sugar water preference factor was measured. In the test, mice were only able to select two bottles weighed beforehand, one bottle of 1% (w/v) sucrose solution and the other bottle of pure water, fasted, after 24h, the two bottles were taken away and weighed, and the total liquid consumption, sugar water consumption and pure water consumption of the mice were recorded. The calculation mode of the sweet water preference coefficient is as follows: the sugar water preference coefficient (%) is sugar water consumption/(sugar water consumption + pure water consumption) × 100%.

Prior to the experiment, all mice measured a baseline of sugar water bias, with no significant difference between the groups of mice at the baseline (fig. 1A and fig. 6A). After molding, as shown in fig. 1B and fig. 6B, both the sugar water preference coefficients of the CUMS group were significantly decreased (P <0.01), while both the mesalazine group and the scutellaria-coptis drug were more preferable to the sugar water than the model group (P < 0.01).

Step three: forced swimming experiment of mouse

The forced swimming experiment instrument is a transparent round barrel filled with purified water, the diameter of the barrel is 12cm, and the water depth is 25 cm. The pure water temperature is 23-25 ℃. The camera is arranged right in front of the instrument and is flush with the water surface. After the mice were placed in water, their activity videos were collected for 6min in water. Analysis software was used to analyze the quiescence time of the mice within 5min after the video. Immobility behavior is defined as the behavior of a mouse floating, not struggling, or relying only on occasional swings to maintain floating immobility.

The results of the experiment are expressed as mean ± standard error, statistically processed by t-test.

The results of the forced swimming test are shown in fig. 2 and 7. The immobility time was significantly greater in the CUMS group than in the blank group (P < 0.05). The immobility time of both the mesalazine group and the scutellaria-coptis group was significantly less than that of the CUMS group (model group).

The animal experiment models prove that the oral administration of mesalazine can increase the sweet water preference of mice and shorten the total immobility time of the mice in the forced swimming process, and the mesalazine has exact curative effect on depression.

Step four: determination experiment of inflammatory factors in mouse serum and colon tissues

ELISA assay the levels of inflammatory factors TNF-alpha and IFN-gamma in the blank, model, mesalazine group serum and colon tissue were determined and the results are shown in FIG. 3 and FIG. 8.

As can be seen from FIG. 3 and FIG. 8, compared with the blank group, the levels of proinflammatory factors TNF-alpha and IFN-gamma in the serum and colon tissues of the model group are significantly increased (P <0.05), which indicates that CUMS modeling can cause the proinflammatory cytokines of the mouse body and local colon tissues to change, the levels of TNF-alpha and IFN-gamma in the serum and colon (P <0.05) are significantly reduced in both the mesalamine group and the Scutellariae-rhizoma Coptidis group, which indicates that mesalamine and the Scutellariae-rhizoma Coptidis group have significant influence on the proinflammatory factors of depressed mice in the treatment of the group, and the levels of the proinflammatory factors in the serum and colon tissues can be significantly reduced. The invention establishes a chronic depression model, detects that the proinflammatory factors of intestinal tracts in vivo are obviously increased, and can treat depression by adopting a medicament for inhibiting intestinal inflammation.

Step five: detection of IDO1 expression in colonic tissue by immunoblotting

The colon tissue of each group of mice after the lysis and grinding was lysed with RIPA lysate (beijing solibao technologies ltd) on ice for half an hour and centrifuged for 15 minutes at 12000r/min in a 4 ℃ centrifuge. Protein concentration was measured by BCA kit (Thermo Scientific), protein denaturation followed by electrophoresis and membrane transfer, and protein quantification was performed after incubation with IDO1 antibody, and the results are shown in fig. 4 and 9.

As can be seen from fig. 4 and fig. 9, compared with the blank group, the expression of IDO1 in colon tissue of the model group is significantly increased (P <0.05), which indicates that the CUMS modeling can cause the change of IDO1 in colon tissue of mice, and both the mesalazine group and the scutellaria-coptis group significantly reduce the expression of IDO1 in colon (P <0.05), which indicates that both the mesalazine and the scutellaria-coptis group have significant effects on IDO1 of depressed mice, and both can significantly reduce the expression in colon tissue.

Step six: UPLC-MS/MS tryptophan targeted metabolic analysis

Preparing a brain tissue test solution, a colon tissue test solution and a serum test solution of each group of mice, adding the internal standard solution, mixing uniformly, and testing the samples.

Adding an internal standard solution with the same volume into mixed standard solutions with different concentrations (4000, 2000, 1000, 500, 200, 100, 5, 2 and 1ng/mL), adding the internal standard solution into an EP tube, then adding 100ul of each blank mixed homogenate solution under each condition, uniformly mixing to obtain a calibration standard solution, determining the peak area of the calibration standard solution to be A, then adding 100ul of each blank mixed homogenate solution under each condition, not adding the mixed standard solution and the internal standard solution, and determining the peak area B of the calibration standard solution to be used as a blank matrix background effect. Since endogenous neurotransmitters and their precursor compounds are present in the tissue, their blank value should be subtracted from each calibration point. The calibration curve was established by least squares linear regression analysis of the analyte peak areas (y, A-B) with the corresponding concentrations (x) to give a standard curve.

Statistical analysis of experimental data was performed by GraphPad Prism 7(GraphPad software, San Diego, CA, USA) software, all statistical data being expressed as mean ± SEM, comparing more than three groups of data using one-way ANOVA, comparing two groups of data using t-test, the difference in P <0.05 being statistically significant. The results are shown in FIGS. 5 and 10.

As can be seen from fig. 5A and 10A, the TRP content in the serum of the mice in the model group was significantly reduced (p <0.001) compared to the blank group; a significant increase in colonic tissue TRP levels (p < 0.01); the brain tissue TRP level of the mouse in the model group is obviously increased (p < 0.05); whereas the mesalazine group can significantly reverse this. As shown in FIGS. 5B and 10B, the serum 5-HT levels in the model group mice were significantly reduced (p <0.05) compared to the blank group; the colon 5-HT content of the mouse in the model group is obviously reduced (p < 0.01); the 5-HT content of the mouse brain in the model group is obviously reduced (p < 0.01); the mesalazine group and the scutellaria-coptis drug group have obvious influence on the 5-HT of a depressed mouse, and can obviously increase the expression of colon, brain tissues and serum. As shown in fig. 5C and fig. 10C, the serum KYN content of the model group mice was significantly reduced (p <0.001) compared to the blank group; the colon KYN content of the mice in the model group is obviously increased (p is less than 0.05); the content of KYN in the mouse brain of the model group is obviously increased (p is less than 0.01); the mesalazine group and the scutellaria-coptis drug group can obviously reverse the situation.

Therefore, it can be shown from the above tests that the CUMS molding process not only causes depression-like behavior, but also causes peripheral inflammation such as intestinal inflammation. The mesalazine and the scutellaria baicalensis-coptis medicine pair group are used as a medicine for treating intestinal inflammation, and depression can be treated while the intestinal inflammation is reduced. Indicating that there is a certain correlation between intestinal inflammation and depression. The medicine taking the inflammatory factor to regulate the intestinal IDO1 as the action target can effectively treat the depression, and the main reasons are as follows: the medicine can inhibit interferon IFN-gamma, inhibit IDO1 expression, influence tryptophan metabolism, reduce kynurenine activity, and increase 5-HT level, thereby treating depression. Therefore, IDO1 overexpression caused by intestinal inflammation can further regulate tryptophan metabolism to cause depression, and IDO1 expression caused by intestinal inflammation can be used as an action target of an anti-depression drug and is applied to screening drugs with potential anti-depression efficacy.

The above additional technical features can be freely combined and used in superposition by those skilled in the art without conflict.

The above description is only a preferred embodiment of the present invention, and the technical solutions that achieve the objects of the present invention by substantially the same means are within the protection scope of the present invention.

Claims

1. An application of a medicine for inhibiting intestinal inflammation in preparing antidepressant is disclosed.

2. The use according to claim 1, wherein the agent that inhibits intestinal inflammation is one or more of mesalamine, olsalazine, balsalazide, sulfasalazine.

3. An antidepressant drug, characterized in that the intestinal inflammation factors regulating the intestinal IDO1 are taken as targets.

4. The antidepressant drug according to claim 3, characterized in that it is capable of inhibiting inflammatory factors in the intestine and of downregulating the expression of IDO1 in the intestine.

5. The antidepressant drug according to claim 3, characterized in that it is mesalazine or an extract of scutellaria baicalensis-coptis chinensis.

6. A screening method of an antidepressant drug is characterized in that: whether intestinal inflammation exists in an animal body is detected by establishing an animal chronic depression model, the model is administrated with a candidate medicament, and whether the candidate medicament has an anti-depression effect is detected.

7. The screening method according to claim 6, further comprising the steps of:

8. The screening method according to claim 7, wherein in the first step, the judgment condition of whether or not the compound plays an antidepressant role is as follows: expression of IDO1 in colon tissues of animals was detected by immunoblotting.

9. The screening method according to claim 7, wherein the level of the inflammatory factor is detected by ELISA.