CN108815706B

CN108815706B - Method for establishing mouse model of functional dyspepsia

Info

Publication number: CN108815706B
Application number: CN201810503881.7A
Authority: CN
Inventors: 王术玲; 龚彦溶
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2022-02-01
Anticipated expiration: 2038-05-23
Also published as: CN108815706A

Abstract

The invention relates to the field of medical animal model modeling, in particular to a method for establishing a mouse model with functional dyspepsia. The method for establishing the functional dyspepsia mouse model comprises the following steps: the mice were subjected to restraint, shock and fatigue swimming in sequence daily followed by random application of chronic unpredictable mild stress for 28-32 days of molding. The method has the advantages that the chronic unpredictable mild stress is randomly applied after the restraint, the electric shock and the fatigue swimming are sequentially carried out on the mouse, so that the experimental animal shows the similar functional dyspepsia symptom to the clinical patient, meanwhile, the stress intensity is moderate, the physical injury to the experimental animal cannot be caused, the organic change cannot occur, and the characteristic of the functional dyspepsia is met.

Description

Method for establishing mouse model of functional dyspepsia

Technical Field

The invention relates to the field of medical animal model modeling, in particular to a method for establishing a mouse model with functional dyspepsia.

Background

Functional dyspepsia, also known as non-ulcer dyspepsia, is a common group of clinical signs including persistent or repetitive epigastric discomfort, loss of appetite, early satiety, poststernal pain discomfort, nausea, vomiting, or other epigastric symptoms lasting for at least 3 months without evidence of local or systemic organic disease. The pathogenesis of functional dyspepsia is not clear at present due to complex etiology, but is generally considered to be related to abnormal gastrointestinal motility regulating mechanism, gastrointestinal hormone secretion disorder, abnormal secretion of gastric acid, increased visceral sensitivity, brain-intestine axis disorder, pathogen infection and psychological factors, wherein the gastrointestinal motility disorder and the psychological factors are hot spots of research in a plurality of induced factors. Research shows that the incidence rate of functional dyspepsia is positively correlated with the psychological stress degree of a patient, more and more clinical data show that psychological factors play an important role in the induction and development process of functional dyspepsia, and the functional dyspepsia belongs to functional psychosomatic diseases with physical and psychological traumas.

At present, a tail clamping irritation method is mostly adopted in a functional dyspepsia animal model, and the principle of tail clamping modeling is to irritate a mouse by using a tail clamp to fight against other mice, so that the rat is violent and irritable for a long time, and dyspepsia symptoms are further caused. However, most of patients with functional dyspepsia in clinic have long-term depressed mood and depression and are low in albinism, and the patients show depression symptoms to a certain extent, but not irritability and irritability, so that the tail clamping method has the defects that the molding process is not completely consistent with the disease process of the clinical patients, and the tail of the rat is injured by clamping the tail for a long time, so that other problems such as infection and the like are caused.

Disclosure of Invention

The invention provides a method for establishing a functional dyspepsia mouse model, which can lead the performance of a modeled animal to be consistent with the functional dyspepsia symptom of a clinical patient, and simultaneously has moderate stress intensity, does not cause physical damage to an experimental animal and does not change the organic quality.

The invention is realized by the following steps:

a method of establishing a mouse model of functional dyspepsia comprising the steps of:

the mice were subjected to restraint, shock and fatigue swimming in sequence daily followed by random application of chronic unpredictable mild stress for 28-32 days of molding.

The invention has the beneficial effects that: according to the invention, the chronic unpredictable mild stress is randomly applied after the restraint, the electric shock and the fatigue swimming are sequentially carried out on the mouse, so that the experimental animal shows the similar functional dyspepsia symptom to the clinical patient, and meanwhile, the stress intensity is moderate, the physical injury to the experimental animal is avoided, and the organic change is avoided, so that the characteristic of the functional dyspepsia is met.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 is a diagram showing defecation of a mouse in example 1;

FIG. 2 is a diagram showing the state of mouse hair in example 1;

FIG. 3 is a graph showing the body weight change of mice during molding in example 1;

FIG. 4 is a graph showing the change of daily average food intake of mice during molding in example 1;

FIG. 5 is a graph showing the variation of average daily water intake of mice during molding in example 1;

FIG. 6 is a graph showing the change in percentage of sugar water consumption during molding in example 1;

FIG. 7 is a graph showing the change in the horizontal exercise score of the mouse during the molding in example 1;

FIG. 8 is the change in the score of vertical movement of the mouse during the molding in example 1;

FIG. 9 is a micrograph (100X) of a pathological section of a mouse colon in example 1;

FIG. 10 is a microphotograph (100X) of a pathological section of a mouse stomach tissue in example 1;

FIG. 11 is a microphotograph (100X) of a pathological section of liver tissue of a mouse in example 1;

FIG. 12 is a (%) showing the gastric remnant rates of mice of the normal group and the model group in example 1;

FIG. 13 is a comparison of serum motilin (ng/L) in mice of example 1;

FIG. 14 shows a comparison of substance p (ng/L) in the serum of mice in example 1;

FIG. 15 is a comparison of the serum 5-hydroxytryptamine levels (ng/mL) in example 1;

FIG. 16 is a comparison of the 5-hydroxytryptamine content (ng/mg) in brain tissue of example 1;

FIG. 17 is a graph showing the change in percentage of sugar water consumption of mice during molding in comparative example 1;

FIG. 18 is a graph showing the change in the horizontal motor score of the mouse during the molding in comparative example 1;

FIG. 19 is a graph showing the change in the score of vertical movement of a mouse during molding in comparative example 1;

FIG. 20 is a graph showing defecation of mice in comparative example 2 and comparative example 3;

FIG. 21 is a (%) showing the residual ratio of the stomach of the mice in the normal group and the model group in comparative example 3;

FIG. 22 is a comparison of serum motilin (ng/L) in mice of comparative example 3;

FIG. 23 shows a comparison of substance p (ng/L) in the mouse serum of comparative example 3;

FIG. 24 is a comparison of the 5-hydroxytryptamine levels in serum (ng/mL) of comparative example 2;

FIG. 25 is a comparison of the 5-hydroxytryptamine content (ng/mg) in brain tissue of comparative example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The method for establishing a mouse model of functional dyspepsia according to the present invention will be described in detail below.

The traditional Chinese medicine considers that the cause of functional dyspepsia is mainly incoordination between liver and stomach, namely long-term depression of liver qi, which affects digestive dysfunction of stomach, so that the pathological changes of liver (mainly characterized by liver depression) in the traditional Chinese medicine and digestive tract symptoms (mainly stomach digestive dysfunction and organic change can not be caused) are reflected during modeling. Methods that can be used to replicate the "liver depression" model are: chronic restraint, fatigue swimming, electric shock, chronic unpredictable mild stress, tail suspension, sequestration, noise stimulation, and the like, which are mostly used alone or in random combination. The stress mode is singly used or combined randomly, the mice do not have obvious symptoms of stomach digestive dysfunction, and the stress degree is not enough to cause the condition that the experimental animals have 'wood depression earth' (liver depression affects the stomach digestive function).

The embodiment of the invention provides a method for establishing a functional dyspepsia mouse model, which comprises the following steps:

s1, selecting and pretreating animals;

selecting 20 SPF Kunmin mice, weighing about 20g each, and adaptively feeding the mice for 5-7 days before molding. Specifically, adaptive feeding is that water and feed are sufficiently supplied and mice are freely drunk during feeding. The mouse is ensured to adapt to the surrounding experimental environment, the mind of uneasiness and anxiety caused by environmental stress to the mouse is avoided, and the accuracy of the modeling result is ensured.

After the adaptive feeding is finished, the mice are divided into two groups, one group is a normal group, the other group is a modeling group, and each group of mice comprises 10 mice. The normal group is used for comparison, and then the molding effect of the molding group is determined.

However, the selected mice are not limited to 20 mice, and can be increased or decreased according to the requirements, the grouping is not limited to the sharing, and the normal group and the modeling group can have a slight difference. The 20 described above are divided into two groups, and each group 10 is only one of the preferred embodiments.

S2, establishing an animal model;

after the adaptive feeding is finished, modeling is started, specifically, the mice are subjected to restraint, electric shock and fatigue swimming in turn every day, and then chronic unpredictable mild stress is randomly applied for continuous modeling for 30 days.

Specifically, the restraint, the electric shock and the fatigue swimming are sequentially carried out, namely, after the mouse is restrained in the restraint tube, the mouse is shocked for the first time, then the restraint is continuously carried out, after the mouse is shocked for the second time, the restraint is released, and then the fatigue swimming is carried out.

The first shocked mouse is shocked for 3-5 minutes on the tail part of the mouse, the duration of restraint is 5.5-6.5 hours, and the second shocked mouse is shocked for 3-5 minutes on the tail part of the mouse.

The fatigue swimming is characterized in that 45-65 minutes after the restraint is released, the mice are controlled to swim in water at 15-19 ℃, and the standard that the nose of the mouse is submerged in the water for 2-4 seconds and cannot be lifted is taken as the fatigue standard.

The restraint, electric shock and fatigue swimming are fixed stimulation, unpredictable mild stress is random chronic stimulation, and the combination of the two modes can further promote the modeling effect of the mouse. Specifically, chronic restraint for a long time is to limit the degree and range of physical activity of the animal to cause psychological and behavioral changes such as impatience, struggle without help, anxiety, depression, despair and the like, which have similarities with the formation of human chronic psychosomatic diseases, and the psychosomatic changes can be promoted by adding pain stimuli (electric shock) on the basis of restraint. In addition, animals can have escape behaviors in a severe environment, behavior despair occurs when the animals cannot escape (behavioral death), when mice are forced to swim in a limited space (forced swimming), the mice firstly spell swimming and try to escape, and when the escape cannot be realized, the mice are in a floating and motionless state. The three stress methods are connected in series and applied to a mouse, so that the experimental animal can be quickly caused to have the state of liver qi stagnation to further influence the digestion function. Meanwhile, considering that the experimental animals can resist the stress by using fixed stress stimulation every day, a random chronic unpredictable mild stress is applied on the basis of the three kinds of fixed stimulation, and a triple-combined stress method is created. By using the triple-compound stress method for molding, the experimental animal shows the similar functional dyspepsia symptom to that of a clinical patient, and meanwhile, the stress intensity is moderate, so that the physical injury of the experimental animal is avoided, and the organic change is avoided, so that the characteristic of functional dyspepsia is met.

Further, the chronic unpredictable mild stress comprises 7 stress modes, wherein the 7 stress modes comprise any one of tail lifting rotation for 45-75 seconds, water bath swimming for 2-4min at 40-45 ℃, wet cage feeding for 22-26h, noise stimulation for 1.8-2.2h, tail hanging for 8-12min, squirrel cage tilting, 22-26h and ice bath for 2-3min at 10-15 ℃.

Further, randomly applying the chronic unpredictable mild stress was one of the stress patterns described in 7 per application, and each of the stress patterns had to be applied repeatedly over 5 days.

In the molding process, the weight, the daily average food intake and the daily average water intake of the mouse are measured every 2-3 days, and a sugar water preference experiment and an open field experiment are performed regularly to observe the mental state and defecation condition of the mouse. After the molding is finished, detecting the residual rate of the stomach, preparing pathological sections from liver tissues, stomach tissues and colon tissues for histomorphology observation, collecting serum and brain tissues, and measuring the content of motilin, substance p and 5-hydroxytryptamine. The mode is adopted to judge the molding effect.

Through the mode, the experimental animal can quickly show the similar functional dyspepsia symptom to that of a clinical patient, and meanwhile, the stress intensity is moderate, so that the physical injury of the experimental animal can not be caused, and the organic change can not occur.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The present example provides a method for establishing a mouse model of functional dyspepsia, comprising the following steps:

s1, selecting and pretreating animals;

SPF-grade Kunming 20 mice, each weighing about 20g, were selected and were acclimatized for 7 days prior to molding. During the feeding process, water and feed are sufficiently supplied, and the mice drink the feed freely.

After the adaptive feeding is finished, the mice are divided into two groups, one group is a normal group, the other group is a modeling group, and each group of mice comprises 10 mice.

S2, establishing an animal model;

after the adaptive feeding is finished, the molding is started, the restraint, the electric shock and the forced swimming are fixedly given every day in sequence, and the three stimulations are performed according to the following operations: after the mouse is successfully bound by the binding tube, the tail of the mouse is electrically shocked for 5min, the binding is continued for 6h, then the mouse is shocked for 5min again, the binding is released, and after 1 h, the over-fatigue swimming is carried out in water at the temperature of 17 ℃, and the standard that the nose of the mouse is immersed in the water for 3 seconds and cannot be lifted is taken as fatigue. A chronic unpredictable mild stress is additionally arranged every day, and the stress method is randomly selected from the following 7 types: extracting tail and rotating for 1 min; ② heat stimulation (swimming in water bath at 43 ℃ for 3 min); wet cage breeding for 24 h; fourthly, stimulating for 2 hours by noise; suspending tail for 10 min; sixthly, inclining the mouse cage for 24 hours; seventhly, swimming with ice water (ice bath at 15 ℃ for 3min), wherein each stress is not reused within 5 days. The molding was continued for 30 days.

In the molding process, the weight, the daily average food intake and the daily average water intake of the mouse are measured every 2 to 3 days, and a sugar water preference experiment and an open field experiment are performed regularly to observe the mental state and defecation condition of the mouse. After the molding is finished, detecting the residual rate of the stomach, preparing pathological sections from liver tissues, stomach tissues and colon tissues for histomorphology observation, collecting serum and brain tissues, and measuring the content of motilin, substance p and 5-hydroxytryptamine.

And (3) molding results:

1. mouse body shape and defecation

At the beginning of molding, mice in each group had similar body types and defecation. After 30 days of molding by the triple-combined stress method, compared with the normal group, the mice in the model group had decreased appetite, slowly increased weight, listlessness, loose stools without forming shapes (see fig. 1), dry and unsmooth hair (see fig. 2). Wherein A is a normal group and B is a model group.

2 weight Change in mice

From the weight change, it was observed that the normal group mice gained weight faster, and the growth rate became slower from day 24, and the model group (model) mice gained weight slowly from day 18 and had statistical significance (p < 0.01) compared to the normal group, suggesting that long-term modeling slowed the weight gain of the mice (see fig. 3).

3 Change in food intake and Water intake of mice

The mice in the model group in the pre-molding period (0-12 days) have higher daily food intake than the mice in the normal group because the mice have higher energy consumption due to daily stress stimulation (such as forced swimming) and have higher food intake. In the middle stage of molding (12-24 days), the mice in the model group are in an excitable state and show dysphoria and biting of cages. The mice in the post-model (24-30 days) model group exhibited states of stress and anxiety, listlessness, decreased food and water intake (see fig. 4 and fig. 5).

4 determination of percentage sugar Water consumption

The statistical results show that the percentage of sugar water consumption of the mice in the early and middle groups of the model group has no significant difference, but the sugar water consumption of the mice in the model group gradually decreases along with the model making, and the percentage of sugar water consumption is significantly reduced (p < 0.05) compared with the normal group by the 28 th day, which indicates that the mice in the model group have a depression-like state (see figure 6).

5 Change in autonomic Activity in mice

As can be seen from the open field experimental results of FIG. 7 and FIG. 8, the triple-compound stress method significantly reduced the level of autonomic activity (p < 0.05) in the model group mice, suggesting a depressive-like state in the model group mice.

6 histological section observation of liver, stomach and colon

Referring to fig. 9 and 10, the results of the stomach and colon tissue sections show that no necrosis and inflammatory cell infiltration are observed in the normal group and the model group, which indicates that the stomach and colon of the model group mice are not organically changed after modeling. Referring to fig. 11, it can be seen from the liver tissue section that the liver cells in the liver lobules of the model group mice slightly swell, the cytoplasm is lightly stained, the cells are obviously enlarged, and the liver cells of the normal group mice are normal. Wherein A is a normal group and B is a model group.

7 gastric residual rate test results

As can be seen from FIG. 12, the gastric residual rate of the normal group mice is 23.7%, and the gastric residual rate of the model group mice is 44.3%, which indicates that the gastric residual rate of the mice after molding is obviously increased, and has statistical significance (p is less than 0.01) compared with the normal group, and the gastric motility of the model group mice is obviously reduced by the molding stimulation.

8ELISA method for measuring content of motilin and substance p in serum

Referring to FIGS. 13 and 14, the results show that the serum content of motilin and substance p is significantly reduced (p < 0.01) in the model group mice compared to the normal group, indicating that the gastric motility is reduced in the model group mice.

9ELISA method for measuring 5-hydroxytryptamine content in brain tissue and serum

Referring to FIGS. 15 and 16, the measurement of 5-hydroxytryptamine levels in both groups of mice showed that the 5-hydroxytryptamine levels in serum and brain tissues of the model group of mice were significantly lower than those of the normal group (p < 0.01).

The detection proves that the mouse has the characteristics of liver depression, and is manifested by listlessness, dry hair, no smoothness and no luster, obvious reduction of sugar water consumption and obvious reduction of autonomous activity; and meanwhile, gastric digestive dysfunction is manifested by reduced food and water intake, slow weight increase, loose stool without shaping, obviously increased gastric residual rate, reduced gastrointestinal hormone (motilin and substance p) level directly related to gastric motility, obviously reduced 5-hydroxytryptamine content in brain and serum, and no organic lesion in digestive tract organs proved by tissue section, which prompts successful model building of a functional dyspepsia mouse model.

Examples 2 to 3

Examples 2-3 provide molding methods that operate in accordance with the molding method provided in example 1, with the difference that the conditions are different.

Example 2

The mice are bred adaptively for 5 days, the first time of electric shock is to shock the tail of the mice for 3 minutes, the duration of restraint is 5.5 hours, and the second time of electric shock is to shock the tail of the mice for 3 minutes.

The fatigue swimming was performed 45 minutes after the restraint was released, and the mice were controlled to swim in water at 19 ℃ with the nose submerged in water for 2 to 4 seconds and were not lifted as a criterion of fatigue. And molding for 28 days.

Example 3

The mice were bred adaptively for 6 days, the first shocked mice were shocked for 7 minutes on the tail, the duration of restraint was 6.5 hours, and the second shocked mice were shocked for 7 minutes on the tail.

The fatigue swimming is a standard in which the mouse is controlled to swim in water at 15 ℃ 65 minutes after the restraint is released, and the nose of the mouse is submerged in the water for 2 to 4 seconds and cannot be lifted. And molding for 32 days.

Examples 2 and 3 were also successfully molded, and the results were the same as those of example 1, and will not be described in detail.

Comparative example 1: the molding was performed according to the molding method provided in example 1, except that the stress applied to the mice was only irritative to the tail.

Comparative example 2: the molding was performed according to the molding method provided in example 1, except that the stress applied to the mice was only chronic unpredictable mild stress.

Comparative example 3: the molding was performed according to the molding method provided in example 1, except that the stress applied to the mice was only a chronic unpredictable mild stress after the fatigue swimming.

And (4) conclusion: the mice of comparative example 1 were apparently free from the marked "liver depression" characteristic (sugar water consumption and autonomic activity change were not obvious, see fig. 17, fig. 18 and fig. 19), while the mice of comparative example 2 and comparative example 3 were not apparently free from the symptoms of digestive dysfunction in the stomach (for example, no symptoms of loose stool were found, see fig. 20; no difference in the gastric residual rate was found in the mice, see fig. 21; serum and brain tissue were collected, and the contents of motilin, substance p, and 5-hydroxytryptamine were determined to be substantially unchanged, see fig. 22, fig. 23, fig. 24 and fig. 25), indicating that the three molding processes were unsuccessful.

In conclusion, the invention discovers that the experimental mouse has the characteristic of liver depression by randomly applying chronic unpredictable mild stress after sequentially performing restriction, electric shock and fatigue swimming on the mouse, and the characteristic is manifested by listlessness, unsmooth and lusterless hair, obviously reduced sugar water consumption and obviously reduced autonomous activity; and meanwhile, gastric digestive dysfunction is manifested by reduced food and water intake, slow weight increase, loose stool without shaping, obviously increased gastric residual rate, reduced gastrointestinal hormone (motilin and substance p) level directly related to gastric motility, obviously reduced 5-hydroxytryptamine content in brain and serum, and no organic lesion in digestive tract organs proved by tissue section, which prompts successful model building of a functional dyspepsia mouse model. The experimental animal shows the similar functional dyspepsia symptom with clinical patient, and simultaneously the stress intensity is moderate, can not cause physical injury to the experimental animal, can not appear organic change, therefore accords with the characteristic of functional dyspepsia.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of establishing a mouse model of functional dyspepsia comprising the steps of:

the method comprises the steps of sequentially carrying out restraint, electric shock and fatigue swimming on a mouse every day, then randomly applying chronic unpredictable mild stress for 28-32 days, wherein the sequential execution of restraint, electric shock and fatigue swimming is to firstly shock the tail of the mouse for 3-5 minutes after the mouse is restrained in a restraint tube, then continuously restrain for 5.5-6.5 hours, then shock the tail of the mouse for 3-5 minutes, then release the restraint for 45-65 minutes, control the mouse to swim in water at 15-19 ℃, and take the condition that the nose of the mouse is immersed in the water for 2-4 seconds and cannot be lifted as the standard of fatigue;

the chronic unpredictable mild stress comprises any one of 7 stress modes, each stress mode cannot be repeatedly applied within 5 days, and the 7 stress modes comprise any one of tail lifting rotation for 45-75 seconds, water bath swimming for 2-4min at 40-45 ℃, wet cage feeding for 22-26h, noise stimulation for 1.8-2.2h, tail hanging for 8-12min, squirrel cage inclination for 22-26h and ice bath for 2-3min at 10-15 ℃.

2. The method of claim 1, wherein the mice are acclimatized for 5-7 days prior to molding.

3. The method of establishing a functional dyspepsia mouse model according to claim 2, wherein the adaptive feeding is that water and feed are sufficiently supplied during feeding and the mouse is freely drunk.

4. The method of claim 2, wherein the mice are divided into two groups after the adaptive feeding, one group is a normal group, the other group is a model group, and each group comprises 10 mice.

5. The method for establishing a mouse model of functional dyspepsia according to claim 1, wherein the body weight, daily average food intake and daily average water intake of a mouse are measured every 2 to 3 days during modeling, and a sugar water preference test and an open field test are periodically performed to observe the mental state and defecation of the mouse.