Liver-stomach disharmony chronic gastritis animal model and construction method and application thereof

Abstract

The invention discloses a liver-stomach disharmony chronic gastritis animal model and a construction method and application thereof.The construction method includes the step that experimental animals are treated with improper diet, a tail clamping stimulation method and a chronic unexpected stress method.By means of the construction method, the chronic gastritis animal model with the liver-stomach disharmony syndrome can be obtained; the method is easy to operate and high in repeatability, and a unique method and thought are provided for construction of the liver-stomach disharmony chronic gastritis animal model.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to an animal model of chronic gastritis caused by liver-stomach disharmony, and a construction method and application thereof. Background Art

[0002] Chronic gastritis (CG) is a persistent inflammation of the gastric mucosa with no specific clinical manifestations and is the most common gastrointestinal disease. The persistence and recurrence of chronic non-atrophic gastritis (CNAG) may lead to chronic atrophic gastritis (CAG), which is characterized by atrophy of the gastric mucosa, often accompanied by metaplasia or dysplasia. As the first step in the malignant transformation of chronic gastritis, CAG is considered to be a precancerous lesion of gastric cancer.

[0003] As one of the most important and oldest complementary medicine methods in the world, TCM has formed a unique etiology and diagnostic theory since ancient times. According to TCM theory, syndrome refers to the classification pattern of symptoms and signs of patients at a specific stage in the course of a specific disease, and is considered to be the most important unit for evaluating pathogenesis. TCM practitioners have been applying the core principle of syndrome differentiation and treatment in clinical practice to identify the patient's physical condition and the pathogenesis of the disease for more than 2,500 years. Today, TCM continues to use the traditional "four diagnostic methods", namely, observation, auscultation, smelling, questioning, and palpation to comprehensively collect information on patients' symptoms and signs. Physicians can then conduct a comprehensive assessment of the above clinical information based on TCM theory and clinical experience to reveal the etiology (syndrome) of the disease, thereby guiding personalized herbal prescriptions. Although great leaps have been made in the past few decades in elucidating the pathogenesis of CAG, modern medicine still has certain deficiencies in the treatment of CNAG and CAG. Due to the heavy burden of recurrence, chronicity, and malignant transformation of CG, a considerable number of patients have now focused their attention on TCM treatment.

[0004] According to TCM theory, chronic gastritis includes different syndromes, and liver-stomach disharmony syndrome is one of the most common syndromes. It is a syndrome caused by abnormal flow of liver qi, characterized by stomach bloating, chest tightness, and sighing. As far as current research is concerned, no animal model of chronic non-atrophic gastritis with liver-stomach disharmony has been reported. Therefore, establishing an animal model of chronic gastritis with liver-stomach disharmony is essential for further exploring the TCM syndromes of chronic gastritis.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an animal model of chronic gastritis due to liver-stomach disharmony, and a construction method and application thereof. The animal model of chronic gastritis due to liver-stomach disharmony obtained by the construction method of the present invention can be applied in the disease research of chronic gastritis.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a method for constructing an animal model of chronic gastritis caused by liver-stomach disharmony, which comprises subjecting experimental animals to an irregular diet, tail clamping stimulation method, and chronic unexpected stress method;

[0009] Improper diet includes: giving irritating food to experimental animals to form a gastric ulcer model;

[0010] The tail clamping stimulation method includes: repeatedly clamping the tail of the experimental animal with a clamp at a low intensity, angering the experimental animals and causing them to fight each other, causing liver depression and qi stagnation in the experimental animals;

[0011] The chronic unpredictable stress method includes: subjecting experimental animals to a random chronic unpredictable mild stress every day. Chronic unpredictable mild stress includes noise exposure, wet cage tilting, fasting, day and night reversal, and temperature change.

[0012] In some embodiments, the above-mentioned experimental animals include rats.

[0013] In some embodiments, the experimental animals are subjected to dietary restriction, tail clamping and chronic unexpected stress for 1-3 months.

[0014] In some embodiments, the tail clamp stimulation method includes: clamping the proximal third of the tail of the experimental animal with a clamp to irritate the experimental animals and make them fight each other; wherein each stimulation lasts for 30 minutes, and the position of the clamp is changed every 10 minutes; twice a day.

[0015] In some embodiments, the noise exposure in chronic unpredictable mild stress comprises subjecting the experimental animals to noise stimulation for 1 hour.

[0016] In some embodiments, the wet cage tilting during chronic unpredictable mild stress comprises wet-caging the experimental animals for 24 hours and tilting the cages of the experimental animals for 24 hours.

[0017] In some embodiments, fasting during chronic unpredictable mild stress is fasting the experimental animals for 24 hours.

[0018] In some embodiments, the temperature change during chronic unpredictable mild stress includes subjecting the experimental animals to swimming in 15°C cold water or 43°C water bath for 3 minutes.

[0019] In a second aspect, the present invention provides an animal model of chronic gastritis caused by liver-stomach disharmony obtained by the above-mentioned construction method.

[0020] In a third aspect, the present invention also provides application of the above-mentioned animal model of chronic gastritis due to liver-stomach disharmony in the study of chronic gastritis due to liver-stomach disharmony.

[0021] The present invention has the following beneficial effects:

[0022] The present invention can obtain an animal model of chronic gastritis with liver-stomach disharmony by subjecting experimental animals to irregular diet, tail clamping stimulation method, and chronic unexpected stress method. The method is simple to operate and has strong repeatability, and provides a unique method and idea for constructing an animal model of chronic gastritis with liver-stomach disharmony. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 This is a schematic diagram of the treatment of improper diet in Example 1;

[0025] Figure 2 This is a schematic diagram of the treatment of tail clamping stimulation in Example 1;

[0026] Figure 3 The figure is a graph showing the changes in body weight of rats in the model group and the control group in Example 1;

[0027] Figure 4 The figure is a graph of water consumption of rats in the model group and the control group in Example 1;

[0028] Figure 5 The figure is the movement trajectory diagram of rats in the model group and the control group in Example 1;

[0029] Figure 6 The open field scores of the rats in the model group and the control group in Example 1;

[0030] Figure 7 This is a comparison chart of sugar water consumption of rats in the model group and the control group in Example 1;

[0031] Figure 8 H&E staining of rat liver tissues in the model group and the control group in Example 1;

[0032] Fig. 9 H&E staining of gastric tissue of rats in the model group and the control group in Example 1;

[0033] Fig.10 This is a comparison of the stomach and small intestine propulsion rates of rats in the model group and the control group in Example 1;

[0034] Fig.11This is a comparison chart of activated partial thromboplastin time, thrombin time, and fibrinogen levels in the plasma of rats in the model group and the control group in Example 1;

[0035] Fig.12 This is a comparison chart of the levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, and alkaline phosphatase in the plasma of rats in the model group and the control group in Example 1;

[0036] Fig.13 The figure is a graph showing the changes in body weight of rats in the positive drug group, model group and control group in Example 1;

[0037] Fig.14 The figure is the movement trajectory diagram of rats in the positive drug group, model group and control group in Example 1;

[0038] Fig.15 The open field scores of the rats in the positive drug group, model group and control group in Example 1;

[0039] Fig.16 H&E staining of rat liver tissues in the positive drug group, model group and control group in Example 1;

[0040] Fig.17 H&E staining of rat stomach tissue in the positive drug group, model group and control group in Example 1;

[0041] Fig.18 This is a comparison chart of the levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin and alkaline phosphatase in the plasma of rats in the positive drug group, model group and control group in Example 1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1

[0045] This example provides a method for constructing an animal model of chronic gastritis caused by liver-stomach disharmony, which is as follows:

[0046] 1. Experimental Animals

[0047] 60 SPF grade SD rats.

[0048] 2. Experimental reagents

[0049] According to the Chinese Pharmacopoeia 2005 edition, the conventional dosage of Chaihu Shugan Powder is selected: 9g of dried tangerine peel, 5g of roasted licorice root, 9g of chuanxiong, 9g of bitter orange, 9g of cyperus rotundus, 15g of white peony root, and 9g of bupleurum. The medicine is administered in the form of mixed formula granules according to the above dosage ratio.

[0050] Before the experiment, dissolve the calculated amount in distilled water, mix well with a vortex mixer, and prepare 200 ml of 2.5 g / ml Chaihu Shugan San solution. Refrigerate until use and take out and return to room temperature 2 hours before use.

[0051] The basis for the concentration of Chaihu Shugan San solution: The dosage of Chaihu Shugan San for rats should be 6.7 times the oral dosage for adults (4g / kg), that is, 25.2g / kg. This embodiment adopts a dosage of 25g / kg. Since the oral dosage for rats is 10ml / kg, the concentration of Chaihu Shugan San solution is calculated to be 2.5g / ml.

[0052] 3. Experimental methods

[0053] (1) Animal grouping

[0054] The model was constructed by dividing the rats into two groups: normal group and model group, with 12 rats in each group.

[0055] The Chinese medicine counter-evidence was divided into 3 groups: normal group, model group, and positive drug group, with 12 mice in each group.

[0056] (2) Modeling

[0057] The experimental animals in the normal group received normal diet and water from the first day;

[0058] The model group and the positive drug group were treated with irregular diet + tail clamping stimulation + chronic unexpected stress from the first day. The specific treatment methods are as follows:

[0059] Improper diet: The model group and the positive drug group were given fried, spicy and other foods to create a gastric ulcer model. Figure 1 As shown;

[0060] The rats in the model group and the positive drug group were given a certain amount of tail clamping stimulation every day: a long-tail sponge clamp wrapped in gauze was clamped at the proximal third of the rat's tail to make it furious. During the tail clamping stimulation, the rats stood upright against each other and screamed, then hugged each other and bit and wrestled. The stimulation time should last for 30 minutes, and the position of the clamp was changed once every 10 minutes to avoid damage caused by ischemia. This was done twice a day to make the rats suffer from liver depression and qi stagnation. Figure 2 As shown;

[0061] The rats in the model group and the positive drug group were subjected to a random chronic unpredictable mild stress every day. The arrangement of chronic unpredictable mild stress is shown in Table 1:

[0062] Table 1 Chronic unexpected mild stress

[0063]

[0064] The model group and the positive drug group were treated with the above modeling treatment for 30 days, thereby creating a liver-stomach disharmony model.

[0065] The positive drug group was given Chaihu Shugan San decoction every day on the basis of the model group, and the normal group was given only an equal amount of distilled water for gavage. After the last administration, the rats were fasted and deprived of water for 12 hours, and blood was collected from the abdominal aorta of the anesthetized rats to separate serum. The stomach was removed and cut open along the greater curvature of the stomach, the stomach contents were cleaned, and the ulcer condition was recorded by taking photos.

[0066] The mental state, overall activity, appetite, hair gloss, and stool characteristics of each group of rats were observed, and the tongue, nose and lips, auricle, and claws were carefully observed. The body weight of the rats was measured once a week.

[0067] (3) Post-experimental processing

[0068] 1) After administration, the rats were fasted for 1 day (but not water).

[0069] 2) At 9:00 am on the second day, all rats were gavaged with drugs.

[0070] 3) After 20 minutes, give ink semi-solid paste, 3 ml per mouse.

[0071] 4) After 20 minutes, 4 ml / kg of chloral hydrate was injected intraperitoneally to kill the animals.

[0072] 5) Determine the gastric emptying rate and intestinal propulsion rate of SD rats.

[0073] 4. Detection of the modeling effect of chronic gastritis rat model with liver-stomach disharmony

[0074] (1) Open field test

[0075] This experiment is used to evaluate the autonomous activities of rats, with horizontal and vertical activity scores as observation indicators. Each group of rats was placed one by one in the center grid of the equipment (size 100×100×80cm, made of black acrylic board, and the bottom surface was evenly divided into 25 grids). The rats moved freely in the open box for 3 minutes, and the activities were observed and recorded throughout the process. The horizontal and vertical activity scores of the rats within 3 minutes were counted. The horizontal activity scoring standard is: each time the rat horizontally steps out of a grid along the bottom of the open box, and all four paws enter the grid, it is scored as 1 point; the vertical activity scoring standard is: the rat's front paws are off the ground and the hind paws are upright, each time it is scored as 1 point. Before each rat is tested, the feces of the previous rat are cleaned up, and then the open box is thoroughly cleaned with alcohol gauze. After the alcohol is completely evaporated, the rat is put in for testing to eliminate the influence of odor on the rat's behavior.

[0076] (2) Sugar water preference

[0077] ① Train rats to adapt to sugary drinking water in a quiet environment, and place 2 identical drinking water bottles in each cage. In the first 24 hours, the two bottles contain 1% sucrose water of equal weight; in the second 24 hours, one bottle contains 1% sucrose water and the other bottle contains the same weight of purified water. Then fast and deprive rats of water for 23 hours; ② Give rats 2 identical drinking water bottles at the same time, one bottle contains 1% sucrose water and the other bottle contains the same weight of purified water, and measure the consumption after 1 hour. Measurement indicators: sugar water consumption, sugar water preference (sugar water consumption / total liquid consumption × 100%).

[0078] (3) H&E staining of pathological sections

[0079] The rat liver, stomach and colon tissues preserved in 4% paraformaldehyde were removed, embedded in paraffin, cut into appropriate sizes and then prepared for conventional HE staining sections.

[0080] (4) Biochemical testing:

[0081] Mindray biochemical analyzer was used to detect various biochemical indicators.

[0082] (5) Statistical methods

[0083] Graphpad Prism 8.0 statistical software was used for data analysis. One-way analysis of variance (independent sample t-test) was performed between the two groups. The test level was P < 0.05, which was considered statistically significant (compared with the model control group, *P < 0.05, **P < 0.01, ***P < 0.001).

[0084] 5. Modeling effect of rat model of chronic gastritis caused by liver-stomach disharmony

[0085] (1) Body shape and defecation status of rats

[0086] like Figure 3 and Figure 4 As shown, 30 days after modeling by irregular diet + tail clamping stimulation + chronic unexpected stress method, compared with the normal control group, the modeling group had slow weight gain, listlessness, dry and yellow stools, and dry, rough and dull hair.

[0087] (2) Scores of rats’ spontaneous activity

[0088] The results of the open field test are as follows Figure 5 and Figure 6 As shown, it can be seen that irregular diet + tail clamping stimulation method + chronic unexpected stress method significantly reduced the level of spontaneous activity of rats in the model group, indicating that the rats in the model control group were in a depressive-like state.

[0089] (3) Sugar water consumption experiment

[0090] like Figure 7 As shown, the sugar water consumption of the model group was significantly higher than that of the control group, indicating that the model rats had emotional changes.

[0091] (4) Observation of liver and stomach tissues

[0092] From liver tissue sections ( Figure 8 ) It can be seen that the hepatocytes in the liver lobules of the rats in the model control group underwent watery degeneration of hepatocytes, fatty degeneration, and paravascular inflammatory cell infiltration, indicating that the liver tissue section model group underwent organic changes, which has a certain significance for evaluating whether the model of functional dyspepsia of liver-stomach disharmony type is successful. In the normal control group, only some of the hepatocytes of the rats had loose cytoplasm, and the rest were normal.

[0093] Gastric tissue sections ( Fig. 9 ), the normal control group had a relatively intact mucosal epithelium, with no obvious epithelial cell shedding; the number of glands in the lamina propria was abundant, with chief cells and parietal cells, and no obvious glandular damage; no obvious inflammatory cell infiltration was observed. In the model group, the mucosal epithelium was relatively intact, with no obvious epithelial cell shedding; some glands were loosely arranged, with individual glandular epithelial cells necrotic (dark arrows), nuclear condensation, and scattered inflammatory cell infiltration (light arrows); scattered eosinophil infiltration (light arrows) was observed at the bottom of the lamina propria.

[0094] (5) Gastrointestinal propulsion rate

[0095] like Fig.10 As shown in the data, the gastric residual rate in the model group was lower than that in the normal control group, indicating that the gastric motility was faster; the small intestinal propulsion rate was also lower than that in the normal group, but there was no significant difference.

[0096] (6) Coagulation function

[0097] Liver qi stagnation leads to qi stagnation and blood stasis, coagulation function test results ( Fig.11 ) showed that the activated partial thromboplastin time (APTT) and thrombin time (TT) in the plasma of the model group decreased, while the fibrinogen (FIB) level increased, indicating that the rats in the model group showed significant blood stasis.

[0098] (7) Biochemical indicators

[0099] Elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important signs of liver damage. Liver blood tests are the most common blood tests that can be used to evaluate liver function or liver damage. ALT and AST are mainly distributed in liver cells. If the liver is damaged or damaged, the transaminases in the liver cells enter the blood, and the ALT and AST levels in the blood increase, indicating a sign of liver disease.

[0100] Elevated total bilirubin (T-bil) is seen in toxic or viral hepatitis, hemolytic jaundice, pernicious anemia, etc.

[0101] Alkaline phosphatase (ALP) is widely distributed in human bones, liver, intestines, placenta and other tissues. It is mainly used for the examination of obstructive jaundice, primary liver cancer, secondary liver cancer and cholestatic hepatitis. When suffering from these diseases, liver cells overproduce ALP, which enters the blood through lymphatic tract and hepatic sinusoids. At the same time, due to the obstruction of bile excretion in the intrahepatic bile duct, it refluxes into the blood, causing a significant increase in serum alkaline phosphatase.

[0102] like Fig.12 As shown, ALT, AST, T-bil, and ALP in the model group were significantly increased, indicating that the liver function of mice in the model group was abnormal.

[0103] The above experimental results show that the rats in the model group had slow weight gain, listlessness, dry and yellow stools, dry and dull hair, depression, organic changes in liver tissue, slower gastrointestinal digestion, blood stasis, and abnormal liver function, all of which are consistent with the symptoms of chronic gastritis caused by liver-stomach disharmony.

[0104] 6. Traditional Chinese medicine proves the modeling effect

[0105] Chaihu Shugan San has the effects of soothing the liver and regulating qi, promoting blood circulation and relieving pain. It is mainly used to treat liver qi stagnation syndrome. It is often used clinically to treat chronic hepatitis, chronic gastritis, intercostal neuralgia and other patients with liver qi stagnation. Therefore, Chaihu Shugan San was used as a positive drug to further verify whether the construction of the liver-stomach disharmony type gastritis model was successful.

[0106] (1) Body shape of rats

[0107] After 30 days of modeling through irregular diet + tail clamping stimulation + chronic unexpected stress method, the positive drug group was gavaged with Chaihu Shugan powder for one week after the modeling was successful. Fig.13 As shown, compared with the normal control group, the model group had slow weight gain, listlessness, dry and yellow stool, and dry and dull hair. The positive drug group gained weight compared with the model group after one week of administration.

[0108] (2) Scores of rats’ spontaneous activity

[0109] From the open field test results ( Fig.14 and Fig.15 ) can be seen that irregular diet + tail clamping stimulation method + chronic unexpected stress method significantly reduced the level of spontaneous activity of rats in the model group, while Chaihu Shugan San could significantly enhance the level of spontaneous activity of rats.

[0110] (3) Observation of liver and stomach tissues

[0111] From liver tissue sections ( Fig.16) It can be seen that the hepatocytes in the liver lobules of the mice in the model control group underwent watery degeneration of hepatocytes, fatty degeneration, and paravascular inflammatory cell infiltration, indicating that the liver tissue section model group underwent organic changes, which has a certain significance for evaluating whether the model of functional dyspepsia of liver-stomach disharmony type is successful. In the normal control group, only some of the hepatocytes in the liver cells of the mice had loose cytoplasm, and the rest were normal.

[0112] Stomach tissue sections ( Fig.17 ), the normal control group had an intact gastric epithelial structure in the mucosal layer, clear gastric pit structure, tightly arranged epithelial cells, abundant gastric glands in the lamina propria, and normal morphology and structure of chief cells and parietal cells, with no obvious inflammation. The model group had an incomplete epithelial structure in the mucosal layer, with many epithelial cells falling off (black arrows); local calcification foci were visible in the lamina propria (gray arrows); edema was visible in the local submucosal layer, with loosely arranged connective tissue (white arrows), accompanied by a small amount of lymphocyte and mast cell infiltration (dark arrows). The positive drug group had an incomplete epithelial structure in the gastric mucosal layer, with many epithelial cells falling off (black arrows); abundant gastric glands in the lamina propria, with normal morphology and structure of chief cells and parietal cells, with no obvious inflammation; edema was visible in the local submucosal layer, with loosely arranged connective tissue (white arrows). This shows that Chaihu Shugan San can alleviate the inflammation of gastritis of liver-stomach disharmony type.

[0113] (4) Biochemical indicators

[0114] from Fig.18 It can be seen that ALT, AST, T-bil and ALP in the model group were significantly increased, while after treatment with Chaihu Shugan San, these indicators were significantly adjusted back, which shows that Chaihu Shugan San can alleviate liver damage in the liver-stomach disharmony type gastritis model.

[0115] The above counter-experimental results prove that the liver-stomach disharmony type gastritis model constructed in this example is successfully constructed.

[0116] The success rate statistics of the liver-stomach disharmony type gastritis model obtained by the construction method of this embodiment are shown in Table 2:

[0117] Table 2 Data on the success rate of constructing the liver-stomach disharmony type gastritis model

[0118]

[0119]

[0120] It can be seen from the results in Table 2 that the success rate of the liver-stomach disharmony type gastritis model obtained by the construction method of the present invention is relatively high, reaching more than 66.7% overall, and the symptoms of liver-stomach disharmony in rats still exist one week after stopping the modeling.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of chronic gastritis due to liver-stomach disharmony, characterized in that: Subjecting experimental animals to unhealthy diets, tail clamping stimulation, and chronic unexpected stress; The improper diet includes: giving irritating food to the experimental animals to form a gastric ulcer model; The tail clamping stimulation method includes: repeatedly clamping the tail of the experimental animal with a clamp at a low intensity to anger the experimental animals and make them fight each other, causing liver depression and qi stagnation in the experimental animals; The chronic unpredictable stress method includes: subjecting the experimental animals to a random chronic unpredictable mild stress every day, wherein the chronic unpredictable mild stress includes noise exposure, wet cage tilting, fasting, day and night reversal and temperature change.

2. The construction method according to claim 1, characterized in that: The experimental animals include rats.

3. The construction method according to claim 2, characterized in that: The experimental animals are subjected to irregular diet, tail clamping stimulation and chronic unexpected stress for 1 to 3 months.

4. The construction method according to claim 3, characterized in that: The tail clamping stimulation method includes: clamping the proximal third of the tail of the experimental animal with a clamp to irritate the experimental animals and make them fight each other; wherein, each stimulation lasts for 30 minutes, and the position of the clamp is changed every 10 minutes; twice a day.

5. The construction method according to claim 4, characterized in that: The noise exposure in the chronic unpredictable mild stress includes subjecting the experimental animals to noise stimulation for 1 hour.

6. The construction method according to claim 5, characterized in that: The wet cage tilting in the chronic unpredictable mild stress includes keeping the experimental animals in wet cages for 24 hours and tilting the cages of the experimental animals for 24 hours.

7. The construction method according to claim 6, characterized in that: The fasting in the chronic unpredictable mild stress is to fast the experimental animals for 24 hours.

8. The construction method according to claim 7, characterized in that: The temperature change in the chronic unpredictable mild stress includes the experimental animals swimming in 15°C cold water or 43°C water bath for 3 minutes.

9. An animal model of chronic gastritis caused by liver-stomach disharmony constructed by the construction method according to any one of claims 1 to 8.

10. Use of the animal model of chronic gastritis due to liver-stomach disharmony as claimed in claim 9 in the study of chronic gastritis due to liver-stomach disharmony.

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