CN111758669B

CN111758669B - Construction method and application of animal model with yang-deficiency constipation

Info

Publication number: CN111758669B
Application number: CN202010685318.3A
Authority: CN
Inventors: 刘晓节; 赵迪; 赵思俊; 李震宇; 张丽增; 秦雪梅
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2021-09-28
Anticipated expiration: 2040-07-16
Also published as: CN111758669A

Abstract

The invention belongs to the technical field of animal model construction, and provides a construction method and application of a yang-deficiency constipation animal model in order to solve the problems of high mortality, poor reproducibility and the like of the yang-deficiency constipation animal model. Performing intragastric administration on experimental animals for 1-8 weeks by using 1-8% v/v acetic acid solution and 5-50% g/v activated carbon-containing ice-water mixture, wherein the specific administration method comprises the following steps: the first dosage of the acetic acid solution is 0.5-10.0 mL/mouse, then 0.5-10 mL/100g of the acetic acid solution is administered, and after 0.5-8 hours, 0.5-10 mL/100g of the ice-water mixture containing the activated carbon is administered by intragastric administration. The invention provides an animal model of yang-deficiency type constipation with high stability, easy popularization, low mortality, good reproducibility and economic applicability, and provides experimental basis for prescription screening for treating yang-deficiency type constipation, preclinical research and development of medicines, function evaluation, mechanism research and the like, and research and development of functional foods and health-care foods.

Description

Construction method and application of animal model with yang-deficiency constipation

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a yang-deficiency constipation animal model.

Background

Constipation is a common disorder of the digestive tract, and clinically manifested as dry and hard stools, difficult or incomplete defecation, and decreased weight and frequency of stools. According to epidemiological investigation, the incidence rate of constipation in our country is 3% -17%. With the development of modern society and the change of life style and dietary structure, the incidence rate of chronic constipation is on the rise, and has the characteristics of universality, repeatability, age correlation and the like. The non-lethal nature of constipation, however, often results in therapeutic neglect. But constipation is a great hazard, and can lead to the reduction of the life quality of patients, such as neurasthenia, insomnia, dysphoria, halitosis and the like; secondary anorectal diseases, colon cancer, cardiovascular diseases, senile dementia and other diseases are also increased.

The western medicine considers that the constipation is only a clinical symptom and does not belong to independent diseases. In traditional Chinese medicine, constipation is used as an independent disease, the advantages of syndrome differentiation and treatment are exerted, and individualized treatment is carried out on patients.

The traditional Chinese medicine has more than 2000 years of history of treating constipation, and a great deal of theoretical experience is accumulated in practice grope. The book Su Wen ㆍ treatise on the other five zang organs: the sou men is also the five internal organs, even though secret in the heart, lung, liver, spleen and stomach, kidney, etc., it is closely related to spleen and kidney. The theory of su wen ㆍ jue (su wen ㆍ): "faint due to taiyin" can cause abdominal distension, difficulty in eating, vomiting and inability to lie down. "indicate the relationship between constipation and spleen. Su Wen ㆍ Ci malaria records that "kidney malaria is unrestrained, pain in the loins and back, turning, difficulty in urination and defecation, eyes , cold hands and feet", clearly indicates that constipation is related to kidney yang deficiency.

In conclusion, constipation is closely related to the spleen and kidney. Therefore, the yang-deficiency constipation animal model not only accords with the yang-deficiency performance in the theory of traditional Chinese medicine on the syndrome type, but also accords with the scope of difficult defecation and constipation on the disease, and can better simulate the clinical performance of the patients with yang-deficiency constipation in clinic.

As a scientific research guide, animal experiments can make up for the deficiency of clinical research. Therefore, an animal model with good objectivity, recognition and repeatability plays an important role in understanding the pathogenesis, pathophysiological processes and drug development of diseases. At present, although there is experimental research on traditional Chinese medicine syndrome type animal models of yang-deficiency constipation in China, high mortality and poor reproducibility are important defects of the model research.

Disclosure of Invention

The invention provides a construction method and application of a yang-deficiency constipation animal model, aiming at solving the problems of high mortality rate, poor reproducibility and the like of the current yang-deficiency constipation animal model.

The invention is realized by the following technical scheme: a construction method of an animal model of yang-deficiency constipation comprises the steps of performing intragastric lavage on an experimental animal for 1-8 weeks by using 1-8% v/v acetic acid solution and an ice-water mixture containing 5-50% g/v activated carbon, wherein the specific administration method comprises the following steps: the first dosage of the acetic acid solution is 0.5-10.0 mL/piece, then the acetic acid solution is administered according to 0.5-10 mL/100g, after 0.5-8 hours, the ice-water mixture containing the active carbon is administered according to 0.5-10 mL/100g per piece, the intragastric administration time of the acetic acid solution and the active carbon ice-water mixed solution is 1-8 weeks, namely 7-56 days, and the intragastric administration is carried out for 1-7 times per week;

when the experimental animal shows 5 items of the following (1) (2) (3) (4) (5) (6) (7) major symptoms and 3 items of the following (8) (9) (10) (11) minor symptoms, the construction of the rat animal model with yang-deficiency constipation is successful;

the main symptoms are: (1) the anal temperature decreases; (2) the number of the defecation grains is reduced within 12 hours; (3) the defecation time is prolonged; (4) a decrease in the rate of small bowel advancement; (5) the water content of the excrement is reduced; (6) in colon pathological tissue: epithelial cells fall off or local submucosa edema, connective tissues are arranged loosely, cytoplasm is loose and lightly stained, but the intestinal glands of the inherent layer are rich, more goblet cells can be seen, and obvious inflammation is not seen; (7) decreased mucus secretion from the colon;

the secondary symptoms are: (8) the hair color of the rat becomes grey-dark and dull; (9) weight loss; (10) decreased autonomic activity; (11) a decline in thymus index; (12) the food intake is reduced.

The experimental animal is an SPF-grade healthy adult animal, and the adult animal is any one of an SD rat, a Wistar rat, a C57BL/6 mouse, a BALB/C mouse or a Kunming mouse.

The acetic acid concentration is controlled by pH. The pathological specimen is mainly the colon part.

The invention has the following beneficial effects: the existing constipation model with yang deficiency has the defects of unstable model making, high fatality rate and the like. The invention provides the yang-deficiency constipation animal model which is high in stability, easy to popularize, low in mortality rate and economical and applicable, and provides an experimental basis for the preliminary clinical experimental research and the research and development of foods and medicines with the function of improving the yang-deficiency constipation. Provides experimental basis for prescription screening for treating yang deficiency type constipation, preclinical research and development, function evaluation, mechanism research and the like of medicines, and research and development of functional food and health food.

The invention improves the model on the basis of the previous research, and the new model can greatly reduce the death condition of experimental animals and can obviously improve the replication rate of the model. The invention not only can deepen the understanding of the yang deficiency constipation disease, but also can provide a high-level animal model for the research and development of transformed medicine and new drugs.

Drawings

FIG. 1 is a graph showing the anal temperature of a rat after modeling in an example of the present invention;

FIG. 2 is a graph showing the small intestine propulsion rate of each group of rats after modeling in the example of the present invention;

FIG. 3 is an optical microscope photograph of HE staining of colon tissue specimens wherein (a) is a control group (. times.200 times), (b) is a 3% acetic acid group (. times.200 times), (c) is a 5% acetic acid group (. times.200 times) in an example of the present invention;

FIG. 4 is an optical microscope photograph of the AB-PAS staining of a colon tissue specimen in an example of the present invention, wherein a is a control group (. times.200 times), b is a 3% acetic acid group (. times.200 times), c is a 5% acetic acid group (. times.200 times), and d is the amount of mucus in each group;

FIG. 5 is a diagram of stool morphology of various groups of rats after modeling in an example of the present invention;

FIG. 6 is a graph showing the weight gain of rats after modeling according to the example of the present invention;

FIG. 7 is a graph of thymus index of a modeled rat in an example of the present invention;

FIG. 8 is a survival curve diagram of a rat animal model with acetic acid-induced yang-deficiency constipation modeled for 35 days in example 1 of the present invention;

FIG. 9 is a graph showing the survival rate of rats molded with white vinegar for 35 days in comparative example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific embodiments. It should be understood that the embodiments described herein are merely illustrative of the present invention and that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.

Example 1: establishment of acetic acid induced yang deficiency type constipation rat animal model

Preparation of the reagent: 3% (v/v) acetic acid solution: adding edible glacial acetic acid solution (CH)₃COOH, tianyu food manufacturers (korea)) was added to distilled water at room temperature (about 25 c) and stirred with a glass rod to be well mixed. When the pH value of the solution is 2.53, the acid addition is stopped.

5% (v/v) acetic acid solution: adding edible glacial acetic acid solution (CH)₃COOH, tianyu food manufacturers (korea)) was added to distilled water at room temperature (about 25 c) and stirred with a glass rod to be well mixed. When the pH value of the solution is 2.42, the acid addition is stopped.

Activated carbon ice water mixture: 200g of activated carbon powder (Fuchen chemical Co., Ltd.) was added to 1000 mL of ice water to prepare an ice water mixture containing 20% of activated carbon. The activated carbon is uniformly dispersed in the solution by stirring before use.

Experimental animals: SPF-grade male Sprague-Dawley (SD) rats weighing 180-200 g were provided by the laboratory animal technology of Wintolite, Beijing (Certification number: SCXK (Kyoto) 2016-. During the feeding period, food and water are taken freely, 12-hour light/dark alternation is adopted, and the feeding environment is proper in temperature and humidity.

Animal grouping: SD rats are 50, 10 are randomly selected as a control group and 40 are selected as a model group according to a digital random table. Model groups rats were randomly subdivided into 3% (v/v) acetic acid model groups and 5% (v/v) acetic acid model groups of 20 rats each.

The molding method comprises the following steps: and (3) irrigating the stomach with 1-8% (v/v) acetic acid solution and 5-50% (g/v) activated carbon-containing ice-water mixture for 1-8 weeks (7-56 days), and evaluating the established yang-deficiency constipation animal model by adopting an evaluation method combining major symptoms and minor symptoms. The construction of the rat animal model for yang-deficiency constipation was successful when the experimental animals exhibited 5 of the following major symptoms (1) (2) (3) (4) (5) (6) (7) and 3 of the following minor symptoms (8) (9) (10) (11): (1) anal temperature is reduced (2), defecation grain number is reduced by 12 hours, (3) defecation time is prolonged, (4) small intestine propulsion rate is reduced, (5) water content of excrement is reduced, (6) epithelial cells in colon pathological tissues drop off or local submucosa edema is seen, connective tissues are arranged loosely, but inherent layer intestinal glands are rich, more goblet cells are seen, obvious inflammation is not seen, (7) mucus secretion of colon is reduced, (8) rat hair is grayed and dark, weight is reduced, (9) self-activity ability is reduced, (10) thymus index is reduced, (12) food intake is reduced.

The method comprises the following specific steps:

A. the rats in the 3% acetic acid model group were fed a first dose of 5 mL/3% (v/v) acetic acid solution while eating a normal diet, and the rats were gavaged with a 3% (v/v) acetic acid solution at a dose of 1.5 mL/100g the following day, and after 2 hours, each of them was gavaged with 2.5 mL/100g of an activated carbon-ice water mixture containing 20% (g/v) activated carbon, and were fed 3 times per week; for 35 consecutive days.

B. The rats in the 5% acetic acid model group were fed a first 5.0 mL/5% (v/v) acetic acid solution simultaneously with the normal diet, and were gavaged with a 5% (v/v) acetic acid solution at a dose of 1.5 mL/100g the following day, and after 2 hours, each was gavaged with 2.5 mL/100g of an ice-water mixture containing 20% (g/v) activated carbon 3 times a week; for 35 consecutive days.

C. The rats in the control group were given distilled water in an amount equal to that of the model group while eating a normal diet, 3 times per week; for 35 consecutive days.

Evaluation of establishment of rat animal model: the rat model of constipation due to yang deficiency established in the above examples was subjected to the evaluation test of the following items.

First, evaluation of chief complaints

1. And (3) anal temperature measurement: after the molding is finished, the anal temperatures of all mice are measured by an electronic thermometer.

2. Number of defecation granules in 12 hours: on

days

0, 7, 14, 21, 28, 35, all rats were placed in metabolic cages to collect the number of feces per rat over 12 hours; normal group rats were observed contemporaneously.

3. Measurement of first-particle black feces excretion time: after the molding was completed, all rats were gavaged with home-made ink (1 mL/100 g), placed in a metabolic cage, and the time from gavage of the ink to the first removal of black feces was observed and recorded.

4. Determination of small intestine propulsion rate: after fasting of the rats for 12 hours, the rats were gavaged with self-prepared ink (1 mL/100 g), and after 30 min, the rats were sacrificed, the entire gastrointestinal tract was removed by laparotomy, and the distance (cm) from the front end of the ink to the pyloric sphincter and the distance (cm) from the pyloric sphincter to the end of the small intestine were measured. The small intestine propulsion rate was calculated as follows.

Small intestine propulsion rate = distance from ink front to pyloric sphincter (cm)/pyloric sphincter to small intestine end (cm) × 100%

5. And (3) measuring the water content of the excrement: after the model building is finished, the rat is placed in a metabolism cage, and fresh excrement of the rat is collected and weighed as wet weight. The feces were dried in an oven at 60 ℃ for 24 hours, and the dry weight of the feces was determined. The fecal water content was calculated as follows.

Stool water content = (stool wet weight-stool dry weight)/stool wet weight × 100%

6. And (3) observing colon pathological tissue morphology: all rats were sacrificed after fasting for 12 hours without water deprivation, and colon tissue specimens (colon tissue was left at a distance of about 2cm from the cecum) were fixed with 10% (v/v) formaldehyde for 24 hours before HE staining. The pathological changes of the mucosa, the lamina propria and the muscular layer and whether inflammation occurs are observed by using an optical microscope.

7. Secretion of mucus in colonic tissue: all rats were sacrificed after fasting for 12 hours without water deprivation, and colon tissue specimens (colon tissue was left at about 2cm from the cecum) were stained with AB-PAS after 10% (v/v) formaldehyde was fixed for 24 hours. The average number of positive cells for analysis of mucus was counted using Image Pro Plus 6.0 software.

Second, observation and determination of secondary symptoms:

1. general observation of yang deficiency signs: after the modeling is finished, all rats are observed for yang deficiency characteristics such as mental state, abdominal distension degree, hair glossiness, body contracture degree, diet condition, excrement form and the like.

2. Weight gain change: rat body weights were measured at 9 am, three times per rat, and recorded as an average. Three body weights were recorded a week.

3. Determination of autonomic activity capacity: on

days

0, 7, 14, 21, 28, 35, all rats were placed in an open field laboratory setting of 100 cm x 100 cm, 60cm high. The test was performed for 5 minutes, acclimated for the first 1 minute, and the number of erections and crossing of the rats was recorded for the last 4 minutes.

4. Thymus index: after the model is made, the weights of all rats are counted before the experimental animals are sacrificed, and the thymus is taken out after the sacrifice and the weight of the thymus is recorded. The organ index of the thymus was calculated as follows.

Organ index of thymus = weight of thymus/body weight.

Thirdly, a statistical method: the experimental data in the examples were analyzed using One-way ANOVA in ANOVA, and the results are expressed as mean. + -. standard deviation (` x. + -. SD).

Fourth, experimental results

(I) results of the experiment of the chief complaints

1. Anal temperature: after the molding is finished, compared with a blank group, the anal temperature of the 3% acetic acid group and the anal temperature of the 5% acetic acid group are both reduced remarkably, and the difference has statistical significance (P is less than 0.01). The results are shown in FIG. 1. The anal temperature is close to the deep blood temperature, so that the real body temperature of the rat can be reflected. The result shows that the acetic acid and the activated carbon ice water can obviously reduce the anal temperature of the rat and show the symptom of yang deficiency.

2. Number of 12-hour defecation grains: the results are shown in Table 1. On day 21 of molding, the 12-hour faecal pellet count was significantly reduced in the 5% acetic acid group compared to the blank group, with a statistically significant difference (P < 0.05). On day 28 of molding, the 12-hour faecal pellet count was significantly reduced in the 3% acetic acid group compared to the blank group, with a statistically significant difference (P < 0.05). The results show that after the molding is finished, the constipation group obviously reduces the number of the defecation granules within 12 hours, and the constipation symptom is obvious.

TABLE 1 number of 12-hour defecation grains for each group of rats: (

± SD）

Note: p < 0.05 compared to blank group.

3. Feces transport time: the results are shown in table 2, where the fecal transit times were significantly longer in the 3% and 5% acetate groups compared to the blank group at 14 days of molding, with the difference being statistically significant (P < 0.05). The prolonged fecal transport time of the model group shows that constipation occurs obviously in rats with constipation due to prolonged defecation interval time.

TABLE 2 fecal transit time for each group of rats: (

± SD）

Note: p < 0.001, P < 0.01, P < 0.05 compared to the blank group.

4. Small intestine propulsion rate: as shown in fig. 2, the small intestine propulsion rates of the 3% acetic acid group and the 5% acetic acid group were significantly decreased compared to the control group, and the difference was statistically significant (P < 0.05). The rate of intestinal transit is one of the important indicators of constipation. The intestinal transit rate was decreased in the constipated rats compared to the blank group, indicating insufficient gastrointestinal motility in the constipated rats.

5. Results for stool water content: the results are shown in Table 3. After the molding is finished, compared with a blank group, the moisture content of the feces of the 3% acetic acid group and the feces of the 5% acetic acid group are obviously reduced, and the difference has statistical significance (P is less than 0.05). The moisture content of the feces reflects the degree of dryness of the feces. Compared with the blank group, the water content of the feces of the model group is obviously reduced, which shows that after the model group is made, the water content in the intestinal tract of the rat is reduced, and the difficulty of defecation is increased.

TABLE 3 fecal water content of various groups of rats: (

± SD）

Note: p < 0.01, P < 0.05 compared to blank group.

6. And (3) observing colon pathological tissue morphology: the results are shown in FIG. 3. After the model building is finished, the colon mucosal epithelium, the lamina propria, the muscular layer and the submucosa glands of the rats in the blank group have clear and regular structures and are completely arranged, more goblet cells can be seen, and no obvious inflammation is seen. The colon mucosa layer of the 3% acetic acid group has a complete intestinal epithelial structure, epithelial cells have normal morphological structure and are arranged closely, the inherent layer intestinal gland is rich, more goblet cells can be seen, and no obvious inflammation is seen. Mucosal layers in the 5% acetate group were mostly exfoliated by intestinal epithelial cells (black arrows); edema is visible in the local submucosa, with loose arrangement of connective tissue (black arrows); myofibroedema and cytopenia were seen in the local muscle layer, but more goblet cells were seen without significant inflammation. The yang-deficiency constipation model belongs to functional constipation, although pathological tissues of colon have epithelial cell shedding and local mucosal edema symptoms, the number of goblet cells is normal, and inflammation is not seen, which indicates that the yang-deficiency constipation model does not show obvious organic injury.

7. Secretion of mucus in colonic tissue: after the molding is finished, the average number of mucus secreting positive cells of the 3% acetic acid group and the 5% acetic acid group is remarkably reduced compared with that of the blank group, and the difference is statistically significant (P < 0.01). The results are shown in FIG. 4. Colonic mucus is a high molecular weight epithelial glycoprotein, and an immune barrier formed by the colonic mucus can resist the erosion of microorganisms and insoluble substances and provide protection for surface mucosa. The average number of mucus secreting positive cells in the model component is significantly reduced, so that the mucus layer on the mucosal surface is thinned or disappeared, the probability that colon epithelial cells of constipation rats are invaded by bacteria is increased, and the colon immune barrier is damaged.

Secondary, secondary symptom results

1. General observation of yang deficiency signs: the rats in the control group have bright hair color, ruddy paw tails, normal appetite, formed excrement, spindle shape, no abnormal characteristics and no death. The 3% acetic acid group rats had decreased body hair brightness, pale paw, decreased appetite, decreased activity, and different degrees of mental depression. The 5% acetic acid rats had decreased body hair brightness, withered and hard, pale paw, decreased appetite, hard feces, less lying and movement, and were listened to different degrees. Stool morphology is shown in figure 5. The results show that the constipation rats show obvious yang deficiency characteristics.

2. Weight gain variation curve: from the aspect of weight increment change, after modeling, the weight of rats in the 3% model group and the 5% model group is obviously reduced compared with that of the control group, but the weight is not statistically significant. The results are shown in FIG. 6. The results show that the modeling factor has no significant effect on the body weight of the constipation rats.

3. Autonomic activity capacity: after 21 days of molding, the number of erections and crossing grids was significantly reduced in the 3% and 5% acetic acid model groups compared to the blank group, and the difference was statistically significant (P < 0.05). The results are shown in Table 4. The results of the study showed that the rats with constipation had reduced ability to move autonomously.

Table 4 number of erections and crossing of the rats in each group in the open field experiment (

± SD）

Note: p < 0.001, P < 0.01, P < 0.05 compared to the blank group.

4. Thymus index: after the molding is finished, the thymus index of the 3% acetic acid group and the thymus index of the 5% acetic acid group are obviously reduced compared with that of the blank group, and the difference has statistical significance (P < 0.05). The results are shown in FIG. 7. The thymus, an important central immune organ, contributes to the differentiation and maturation of T cells. The thymus index of the model group rats is low, which indicates that the astriction can cause the thymus atrophy of the rats and the immunity is reduced.

The measurement results of the main symptoms and the secondary symptoms of the experimental rats show that the yang-deficiency type constipation model constructed by acetic acid and activated carbon ice water can show obvious yang-deficiency symptoms and constipation symptoms. The successful construction of the model of constipation caused by yang deficiency is shown. In addition, no experimental animal died in example 1 of the present invention, and the survival curve is detailed in FIG. 8. In order to highlight and further verify the advantage of low fatality rate of the yang-deficiency constipation model constructed by the invention, a white vinegar-induced constipation rat model is used as a comparison experiment, and the construction method of the acetic acid-induced yang-deficiency constipation rat model is evaluated under the condition of consistent molding time.

Experimental example 1: establishment of white vinegar-induced yang-deficiency constipation rat animal model

Preparation of the reagent: commercial edible white vinegar (Shanxi mature vinegar factory, batch number: 20140513, total acidity greater than or equal to 6.0 degree), activated carbon powder (Fuchen chemical reagent Co., Ltd.)

Experimental animals: same as example 1

Animal grouping: 30 healthy male SD rats were randomly divided into a blank control group (n = 10) and a white vinegar model group (n = 20).

The molding method comprises the following steps: 5mL of Shanxi white vinegar is administrated to the rats for first time, and 3 mL of vinegar is administrated to the rats for later time according to the weight of 200 g. On the next day after vinegar administration, animals were administered 16.67 mol/L of activated charcoal ice water at 0 ℃ to the animals at a rate of 5 mL/200g body weight. And alternately molding the white vinegar and the activated carbon ice water in single or double days. The molding time was 35 days in total.

And (3) evaluating the survival rate: the survival of the experimental rats during the experiment was recorded and a survival curve was constructed. The results are shown in FIG. 9. The results show that the acetic acid-induced constipation rate in the rat model is only 50% in 35 days. In fig. 8, it is evident that the survival rate of the rat model constructed by the method of the present invention is 100% in 35 days of modeling, and none of the rats died, and it is evident that the mortality rate of the method of the present invention is very low and the reproducibility is good.

Claims

1. A construction method of an animal model of yang-deficiency constipation is characterized by comprising the following steps: performing intragastric lavage on experimental animals by using 1% -8% v/v acetic acid solution and 5% -50% g/v activated carbon-containing ice-water mixture, wherein the specific administration method comprises the following steps: the first dosage of the acetic acid solution is 0.5-10.0 mL/piece, then the acetic acid solution is administered according to 0.5-10 mL/100g, after 0.5-8 hours, the ice-water mixture containing the activated carbon is administered according to 0.5-10 mL/100g per piece, the gastric lavage time of the ice-water mixture of the acetic acid solution and the activated carbon is 1-8 weeks, namely 7-56 days, and the gastric lavage is performed for 1-7 times per week;

the main symptoms are: (1) the anal temperature decreases; (2) the number of the defecation grains is reduced within 12 hours; (3) the defecation time is prolonged; (4) a decrease in the rate of small bowel advancement; (5) the water content of the excrement is reduced; (6) epithelial cells in colon pathological tissues drop or local submucosal edema is seen, connective tissues are arranged loosely, but the intestinal glands in the inherent layer are rich, more goblet cells can be seen, and obvious inflammation is not seen; (7) decreased mucus secretion from the colon;

2. The method for constructing an animal model of yang-deficiency constipation according to claim 1, wherein: the experimental animal is an SPF-grade healthy adult animal, and the adult animal is an SD rat or a Wistar rat.

3. The method for constructing an animal model of yang-deficiency constipation according to claim 1, wherein: the acetic acid solution is 3% v/v acetic acid solution and 5% v/v acetic acid solution, and the activated carbon in the activated carbon ice-water mixture is 20% g/v.

4. The method for constructing an animal model of yang-deficiency constipation according to claim 3, wherein: the acetic acid solution is 3% v/v acetic acid solution, the activated carbon in the activated carbon ice-water mixture is 20% g/v, and the specific administration method comprises the following steps: the rats are fed with 5 mL/3% v/v acetic acid solution of the first dose while eating normal diet, and are intragastrically fed with 3% v/v acetic acid solution of 1.5 mL/100g dose from the next day, and after 2 hours, each intragastrically fed with 2.5 mL/100g activated carbon ice-water mixture containing 20% g/v activated carbon is fed 3 times per week; for 35 consecutive days.

5. The method for constructing an animal model of yang-deficiency constipation according to claim 3, wherein: the acetic acid solution is a 5% v/v acetic acid solution, the activated carbon in the activated carbon ice-water mixture is 20% g/v, and the specific administration method comprises the following steps: rats were fed a first 5.0 mL/5% v/v acetic acid solution while on a normal diet, gavage the 5% v/v acetic acid solution at a dose of 1.5 mL/100g the following day, 2 hours later, each was gavaged with 2.5 mL/100g of an ice-water mixture containing 20% g/v activated charcoal, 3 times a week; for 35 consecutive days.

6. The method for constructing an animal model of yang-deficiency constipation according to claim 1, wherein: epithelial cells in pathological tissues of colon drop or local submucosal edema is seen, connective tissues are arranged loosely, muscle fiber edema is seen in local muscle layers, cytoplasm is loose and lightly stained, but intestinal glands in the lamina propria are abundant, more goblet cells are seen, and obvious inflammation is not seen.