Application of amino acid food in construction of animal model of depression and antidepressant drugs and construction method thereof

Abstract

The present application relates to the field of biotechnology, in particular to the application of amino acid food in constructing animal model of depression and antidepressant drug and its construction method. The animal model of depression is obtained by feeding the animal with amino acid food, the protein source of the amino acid food exists in the form of hydrolysis, and the animal model of depression is constructed by feeding the animal with the amino acid food as the only food source for 3-6 weeks. The amino acid food does not affect the body weight and growth and development of the animal, and has no effect on learning and cognition, fear and anxiety, and movement ability. Unlike unpredictable chronic mild stress, glucocorticoid / corticosterone model and other stress reactions caused by stress on animals, the method is mild and effective, does not contain invasive stimulation, and the animal in the depression state can be obtained in 3-6 weeks.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of amino acid food in constructing animal models of depression and antidepressant drugs and a construction method thereof. BACKGROUND

[0002] Depression, also known as major depressive disorder or clinical depression, is a common mental disorder, with more than 320 million people worldwide suffering from depression. Depression has a negative impact on thinking and action, manifested as low mood or sadness, loss of appetite, sleep disorders, however, depression is a complex disease, and its specific cause is not clear, which may include genetic inheritance, environmental stress, drug abuse and other causes. Its prevalence and mortality are increasing year by year. Currently, the treatment drugs for depression in clinical practice are mainly serotonin, dopamine and norepinephrine reuptake inhibitors, which play an antidepressant role by increasing the concentration of neurotransmitters between nerve synapses. Since the pathological mechanism of depression has not been clearly defined, the commonly used antidepressants in clinical practice generally have low drug efficacy, slow onset, large side effects, and high relapse rate after drug withdrawal, etc. Therefore, in-depth study of the pathogenesis of depression and development of new antidepressants have become urgent scientific problems. Safety and effectiveness evaluation of antidepressants in animal models of depression is an important part of preclinical research.

[0003] Currently, preclinical research of antidepressants mainly focuses on primates and rodents. Classic animal models of depression include learned helplessness (LH), unpredictable chronic mild stress (UCMS), early life stress model, olfactory bulbectomy (OBX) model, social defeat model, chronic restraint stress model (CRS), glucocorticoid / cortisone model and genetic model, etc. However, the causes of clinical depression are caused by a variety of factors, and dietary habits and patterns are also important factors affecting the onset of depression. Studies have found that a healthy diet (such as the Mediterranean diet) is significantly associated with a reduced risk of developing depressive symptoms. Therefore, the development of a diet-induced depression animal model provides a new approach to studying the pathogenesis of depression and is crucial for the development of new antidepressants. SUMMARY

[0004] In view of the existing research and problems, the purpose of the present application is to provide the application of amino acid food in constructing animal models of depression and antidepressant drugs and a construction method thereof.

[0005] The present application is realized by the following technical solutions:

[0006] The present application protects the use of amino acid food in the construction of animal models of depression, wherein the protein source of the amino acid food is in the form of hydrolysis.

[0007] Preferably, the amino acid food further comprises carbohydrates, lipids, dietary fiber, inorganic salts, minerals and vitamins.

[0008] Further, the amino acid food further comprises other nutrients.

[0009] Preferably, the amino acid food is hydrolyzed milk powder.

[0010] The present application also protects the construction method of the above-mentioned animal model of depression, comprising the following steps:

[0011] S1, configuring amino acid food with protein source in the form of hydrolysis;

[0012] S2, using the amino acid food configured in S1 as the only food source of the animal, continuously feeding for 3-6 weeks.

[0013] Preferably, the animal in step S2 is an animal that has or has not eaten normal food.

[0014] Preferably, the feeding in step S2 is feeding to the animal body or feeding to the animal mother and then feeding through breast milk.

[0015] Preferably, the construction method further comprises step S3: verifying whether the experimental animal is in a depressive state through forced swimming test, tail suspension test and / or sugar water preference test.

[0016] Further, whether the experimental animal is in a depressive state is determined by whether there is a significant difference compared with animals fed with normal food.

[0017] The present application also protects the use of the above-mentioned animal model of depression in screening and / or verifying drugs for preventing, improving and / or treating depression.

[0018] The present application has the following beneficial effects:

[0019] The application discloses an amino acid food-induced animal depression model, which can simulate deep hydrolyzed milk powder, and early feeding of mice with the amino acid food simulates a scenario of infant feeding of deep hydrolyzed milk powder and has certain population specificity. On one hand, adult mice which have eaten normal food are fed with the amino acid food; on the other hand, female mice are fed with the amino acid food and then suckle the mice, and the depression states of the mice are monitored, so that it can be proved that the amino acid food can change the state of healthy mice, and the effect can be extended to the mice fed with the amino acid food through the breast milk to cause the generation of depression symptoms. In addition, the amino acid food does not affect the body weight and growth and development of the mice, and has no influence on learning and cognition, fear and anxiety and movement ability. Unlike unpredictable chronic mild stress, glucocorticoid / cortisone models and other stress response reactions of mice, the method is mild and effective, does not contain invasive stimulation, and can obtain mice in a depression state in 3-6 weeks. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For the tail suspension test results of the AFD-induced depression model of newborn mice (amino acid food group) and the normal food group in Example 2, the numbers marked between groups in the figure represent the P value between the two groups;

[0021] Figure 2 For the forced swimming test results of the AFD-induced depression model of newborn mice (amino acid food group) and the normal food group in Example 2, the numbers marked between groups in the figure represent the P value between the two groups;

[0022] Figure 3 For the sugar water preference test results of the AFD-induced depression model of newborn mice (amino acid food group) and the normal food group in Example 2, the numbers marked between groups in the figure represent the P value between the two groups;

[0023] Figure 4 For the results of the change of the body weight of mice with the AFD-induced depression model (amino acid food group) and the normal food group with the passage of time in Example 3;

[0024] Figure 5 For the motion trajectory graph of mice in the water maze experiment of the AFD-induced depression model (amino acid food group) and the normal food group in Example 4;

[0025] Figure 6 For the results of the time of exploration of the target area of mice in the water maze experiment of the AFD-induced depression model (amino acid food group) and the normal food group in Example 4, the numbers marked between groups in the figure represent the P value between the two groups;

[0026] Figure 7The results show the frequency of mice exploring the target area in the water maze test in the AFD-induced depression model (amino acid food group) and the normal food group in Example 4. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0027] Figure 8 The results of the Y-maze test in the AFD-induced depression model (amino acid food group) and the normal food group in Example 4. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0028] Figure 9 The results of the shock box experiment in the AFD-induced depression model (amino acid food group) and the normal food group in Example 5 are shown. The numbers marked between the groups in the figure represent the P values ​​between the two groups.

[0029] Figure 10 Figure 5 shows the movement trajectory of mice in the open field test under the AFD-induced depression model (amino acid food group) and the normal food group in Example 5;

[0030] Figure 11 The total distance traveled by mice in the open field test under the AFD-induced depression model (amino acid diet group) and the normal diet group in Example 5. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0031] Figure 12 The results of the time mice spent exploring the central area in the open field test under the AFD-induced depression model (amino acid food group) and the normal food group in Example 5. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0032] Figure 13 The results show the frequency of mice exploring the central area in the open field test under the AFD-induced depression model (amino acid food group) and the normal food group in Example 5. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0033] Figure 14 The results of the time it took for mice to fall off the rotarod in the AFD-induced depression model (amino acid food group) and the normal food group in Example 6. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0034] Figure 15 The results of the pole climbing time of mice in the AFD-induced depression model (amino acid food group) and the normal food group in Example 6. The numbers marked between the groups in the figure represent the P values ​​between the two groups;

[0035] Figure 16 The figures are the results of the gripping strength test of mice in the AFD-induced depression model (amino acid food group) and the normal food group in Example 6. The numbers marked between the groups in the figure represent the P values ​​between the two groups.

[0036] Figure 17 For the results of the tail suspension test in the AFD-induced depression model in adult mice (amino acid food group) and normal food group in Example 7, the numbers marked in the figure represent the P value between the two;

[0037] Figure 18 For the results of the forced swimming test in the AFD-induced depression model in adult mice (amino acid food group) and normal food group in Example 7, the numbers marked in the figure represent the P value between the two;

[0038] Figure 19 For the results of the sugar water preference test in the AFD-induced depression model in adult mice (amino acid food group) and normal food group in Example 7, the numbers marked in the figure represent the P value between the two. DETAILED DESCRIPTION

[0039] In order to better understand the present application, the present application will be further described below in conjunction with the examples and drawings, and the following examples are only used to illustrate the present application and not to limit it.

[0040] Example 1 Design and synthesis of an amino acid diet (AFD)

[0041] The AFD food simulates deep hydrolyzed milk powder and does not contain macromolecular antigens. The protein components contained in the food are completely hydrolyzed into amino acid form, without changing the original protein ratio, and without affecting the form and ratio of other nutritional elements. The specific formula is shown in Table 1.

[0042] Table 1 Comparison of amino acid food (AFD) and normal food (NCD) formula

[0043]

[0044]

[0045] * Customized and purchased from Nantong Trolife Feed Technology Co., Ltd., in which the eighteen kinds of complex amino acids include glycine, proline, arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, methionine, cystine, threonine, leucine, isoleucine, valine, glutamic acid, alanine, aspartic acid, and serine;

[0046] ** Purchased from Nantong Trolife Feed Technology Co., Ltd., with feed code LAD 0020.

[0047] Example 2 AFD constructs a depression model in neonatal mice

[0048] Twelve 2-day-old SPF C57 / B6j male mice (purchased from Jisui Yao Kang) were randomly divided into two groups, one group was normal food group (NCD, 6), one group was amino acid food group (AFD, 6), each group was matched with one lactating female mouse, and the corresponding food was eaten by the female mouse, and the mouse was lactated. After continuous feeding for 4 weeks, the female mice were separated, and the amino acid food group was replaced with normal food. After the mice were adapted to the environment, their depression state was monitored by forced swimming, tail suspension, and sugar water preference experiments. The specific experimental methods are as follows:

[0049] The forced swimming test (FST) instrument is a transparent 20x30cm circular barrel filled with pure water, with a diameter of 20cm and a water depth of 15cm. The temperature of the pure water is 23-25℃. A camera is installed in front of the instrument and is flush with the water surface. After the mice are placed in the water, their 6min activity video in the water is collected. The immobility time of the mice within 4min after analyzing the video is analyzed. Immobility behavior is defined as the behavior of the mouse floating, not struggling, or only relying on occasional swinging to maintain floating immobility. The longer the immobility time, the more severe the depression.

[0050] The tail suspension test (TST) uses adhesive tape to suspend the mouse's tail in a position where it cannot escape or grab the nearby surface, and a camera is installed in front to record the mouse's 6min activity video. The immobility time of the mice within 4min after analyzing the video is analyzed. This test is based on the fact that animals will develop an immobile posture when subjected to short-term unavoidable stress. The more severe the depression phenotype, the less time spent trying to escape.

[0051] The sucrose preference test (SPT) is used to evaluate the mouse's preference for sugar water, which can reflect the degree of anhedonia in mice. During the entire animal experiment, two sucrose preference tests were performed. Before modeling, the sucrose preference coefficient of all mice was measured by the sucrose preference test to ensure that the mice were in a consistent state for the experiment. After the experiment, the formal sucrose preference test was conducted, including an adaptation training part and a test part. In the training, the mice were placed in two bottles of 1% (w / v) sucrose solution per cage for the first 24h, and then one of the bottles was replaced with pure water for the next 24h. After the adaptation ended, the mice were fasted and deprived of water for 24h, and then the sucrose preference coefficient was measured. In the test, the mice could only choose two bottles that had been weighed beforehand, one bottle was 1% (w / v) sucrose solution, and the other bottle was pure water. The mice were fasted, and after 24h, the two bottles were removed and weighed. The total liquid consumption, sugar water consumption, and pure water consumption of the mice were recorded. The calculation method of the sucrose preference coefficient is as follows: Sucrose preference coefficient (%) = sugar water consumption amount / (sugar water consumption amount + pure water consumption amount) x 100%.

[0052] The monitoring results are as follows:Figures 1-3 The amino acid food group mice showed an increase in the immobility time in the FST and TST experiments and a decrease in the degree of sugar water preference in the SPT experiment after 4 weeks, indicating that the amino acid food group mice exhibited a depressive phenotype at 4 weeks.

[0053] Example 3 AFD does not affect the growth and weight gain of mice

[0054] The body weight of the mice in the AFD model constructed in Example 2 was detected, and the mice were weighed every week from 1 to 6 weeks, and the results are shown in Figure 4 The body weight of the amino acid food group mice was consistent with the body weight growth curve of the normal food group mice, indicating that the amino acid food did not affect the growth and body weight gain of the mice.

[0055] Example 4 AFD does not affect the learning and cognitive function of mice

[0056] The learning and cognitive function of the mice in the AFD model constructed in Example 2 was detected, and the learning and cognitive function of the mice was detected by water maze and Y maze experiments at 6 weeks of age, and the specific experimental methods are as follows:

[0057] The water maze experiment was completed in an opaque circular swimming pool (diameter 150 cm x height 50 cm), which was divided into four equal areas. The pool was filled with water and a non-toxic water-soluble white dye (water temperature 20±1℃), and there was a movable platform hidden under the water surface (diameter 9 cm, height 15 cm). The mouse was placed in the water with its head facing the pool wall, and the placement position was randomly selected from the east, west, south, and north four starting positions. The time (seconds) from the mouse being placed in the water to finding the underwater platform was recorded. In the first few training sessions, if the time to find the underwater platform was more than 90 seconds, the animal was guided to the platform. After the animal stayed on the platform for 10 seconds, the mouse was removed and dried. If necessary, the mouse was placed under a 150W incandescent lamp for 5 minutes, dried, and then placed back in the cage. Each animal was trained 4 times a day, with a 30-minute interval between two training sessions, and trained continuously for 5 days. After the last acquired training, the platform was removed the next day, and the mouse was placed in the water from the opposite side of the original platform quadrant. The escape latency of the mouse (i.e. the time for the mouse to find the underwater platform for the first time after entering the water) and the number of platform crossings were recorded. The time and distance in the platform quadrant were indicators of the learning ability of the mouse, and the longer the time and distance of the mouse in this quadrant, the better the spatial memory ability.

[0058] Y maze test was performed in a Y-shaped horizontal maze, which consisted of three equal-length arms (40 cm x 10 cm x 15 cm) with a 120-degree angle between each pair of arms, and each arm had a movable partition in the center. The arms and the bottom of the maze were made of opaque polyvinyl alcohol white plastic. The mice were placed at the end of any arm of the Y maze and allowed to freely explore for 8 min. The camera system recorded their behavioral changes, and the following indicators were analyzed: ① The total number of entries: the number of times the animal entered the arm of the maze (one time for the mouse to enter the arm with all four feet); ② An alternation: sequentially and continuously entering all three arms of the Y maze once, i.e., ABC, CAB, or BCA, but not ABB; ③ The number of maximum alternations: the total number of entries - 2. The spontaneous alternation behavior score = the total number of alternations / the number of maximum alternations x 100%. Between two tests, the arms of the Y maze were cleaned with diluted 10% ethanol to eliminate odors and residues. The higher the spontaneous alternation behavior score, the stronger the mouse's recognition memory ability for a new and unfamiliar environment.

[0059] The results of the water maze experiment are shown in Figures 5-7 The amino acid food group and the normal food group mice had comparable exploration time and frequency in the target area, indicating that the amino acid food did not affect the mouse's spatial learning and memory ability. The results of the Y maze experiment are shown in Figure 8 The amino acid food group and the normal food group mice had comparable spontaneous alternation behavior scores, indicating that the amino acid food did not affect the mouse's exploration memory ability for a new and unfamiliar environment.

[0060] Example 5 AFD does not affect the fear and anxiety state of mice

[0061] The mice constructed in Example 2 were subjected to fear and anxiety state detection under the AFD model. At 6 weeks of age, the mice were subjected to fear and anxiety state detection by electric shock box and open field experiment. The specific experimental methods are as follows:

[0062] In the electric shock box experiment, the mice were placed individually in the electric shock box, which included a shock generator (capable of producing various intensity shocks of 0.1-1.0 mA) associated with the grid floor of the box, a sound generator (capable of producing a wide frequency click sound or a low frequency sound), and was connected to a computer. The mice were exposed to 3s (0.6 mA) foot shock, which was randomly performed 5 times within 120 seconds, and the computer recorded the freezing time exhibited by the mice when they were in fear. The longer the mice remained motionless, the more obvious the fear state.

[0063] The open field test was performed in a square plastic box (50 x 50 x 50 cm), which was divided into a peripheral and a central area, with the central area being 20 x 20 cm in size in the center of the open field and the rest of the empty field being the peripheral area. The mice were gently taken out of the home cage and placed in the central area of the experimental box, allowing them to freely explore under dim light for 6 min. A video tracking system recorded the movement trajectory of the mice within 6 min, and a video tracking analysis system was used to analyze the total movement distance of the mice, the time and frequency of exploration and stay in the central area. The higher the time and frequency of exploration in the central area, the lower the degree of anxiety of the mice.

[0064] The results of the electric shock box experiment are shown in Figure 9 , and there was no significant difference in the immobility state exhibited by the mice in the amino acid food group and the normal food group when they were frightened; the results of the open field test are shown in Figures 10-13 , and there was no difference in the movement distance of the mice in the amino acid food group and the normal food group, and there was no significant difference in the time and frequency of exploration and stay in the central area. It is indicated that AFD does not affect the fear and anxiety state of the mice.

[0065] Example 6 AFD does not affect the exercise capacity of mice

[0066] The mice in the AFD model constructed in Example 2 were detected for exercise capacity. When the mice were 6 weeks old, the exercise capacity of the mice was detected by a rotating rod, a pole climbing and a grip meter, and the specific experimental methods were as follows:

[0067] The rotating rod test is used to evaluate the motor skills and integration of animals. The mice are placed on an accelerating rotating cylinder, and training is performed for 3 days before testing. On the 4th day, after the animals maintain balance on the rotating rod rotating at a constant speed, the speed of the rotating rod is gradually increased until the mice fall off the rotating rod. The time the mice stay on the rotating rod is recorded as an indicator of their exercise capacity. The longer the stay time, the stronger the exercise balance ability.

[0068] The pole climbing test is used to evaluate the motor coordination ability of mice. The metal rod is 5 mm in diameter and 50 cm in length, wrapped with gauze. The mice are placed at the top of the metal rod, and the total time for the mice to climb down the rod to the pole is recorded. Training is performed for 2 days before testing, and the actual test is performed on the 3rd day. The shorter the time used for climbing down, the stronger the motor coordination ability of the mice.

[0069] The grip meter detects the grip size of the mice. The grip meter is placed horizontally, and after starting the instrument, the mice are placed on the grip plate, the tail of the mouse is gently pulled back, and after the mouse holds the grip plate firmly, the instrument records the maximum grip of the mouse. Each mouse is measured 3 times, and the average value is taken as the measurement result to evaluate the muscle strength of the mice.

[0070] The results of the rotating rod test are shown inFigure 14 As shown, there was no significant difference in the fall time of the normal food group and the amino acid food group mice on the rotating stick; the pole climbing results were as follows Figure 15 As shown, there was no significant difference in the fall time of the normal food group and the amino acid food group mice on the rotating stick; the pole climbing results were as follows Figure 16 As shown, there was no significant difference in the fall time of the normal food group and the amino acid food group mice on the rotating stick; the pole climbing results were as follows

[0071] Example 7 AFD induces a depression model in adult mice

[0072] Twelve SPF C57 / B6j male mice aged 3w (purchased from Jisui Yaoke, and fed with normal food within 3w) were randomly divided into two groups, one group was a normal food group (NCD, 6), and one group was an amino acid food group (AFD, 6). After continuous feeding for 4 weeks, the mice were subjected to forced swimming, tail suspension, and sugar water preference experiments to monitor their depression state. The results were as follows Figures 17-19 As shown, the resting time of the amino acid food group mice increased in the FST and TST experiments, and the degree of sugar water preference decreased in the SPT experiment, suggesting that amino acid food can induce depression in adult mice.

[0073] The above-described embodiments merely describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. The use of amino acid food in constructing an animal model of depression is characterized by: The protein source of the amino acid food is in a hydrolyzed form, and the amino acid food is fed continuously for 3-6 weeks as the only food source for the animal; the animal is a mouse; the amino acid food also includes carbohydrates, lipids, dietary fiber, inorganic salts, minerals and vitamins.

2. The use according to claim 1, characterized in that: The amino acid food is hydrolyzed milk powder.

3. A method for constructing an animal model of depression according to claim 1, characterized in that: The following steps are involved: S1. The protein source is amino acid food in hydrolyzed form; S2: Use the amino acid food prepared in S1 as the sole food source for the animals for 3-6 weeks.

4. The method for constructing an animal model of depression according to claim 3, wherein: The animals in step S2 are animals that have eaten or have not eaten normal food.

5. The method for constructing an animal model of depression according to claim 3, wherein: The feeding in step S2 is feeding the animal itself or feeding the animal mother through breast milk.

6. The method for constructing an animal model of depression according to claim 4, characterized in that: The feeding in step S2 is feeding the animal itself or feeding the animal mother through breast milk.

7. The method for constructing an animal model of depression according to claim 3, characterized in that: The construction method further includes step S3: verifying whether the experimental animal is in a depressive state through a forced swimming test, a tail suspension test and / or a sugar water preference test.

8. The method for constructing an animal model of depression according to claim 7, characterized in that: Whether the experimental animals are in a depressed state is determined by whether there is a significant difference compared with animals fed normal food.

9. Use of the depression animal model according to any one of claims 1 to 8 in screening and / or verifying drugs for preventing, ameliorating and / or treating depression.

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