A method for constructing a phlegm-dampness constitution animal model and application thereof in evaluating intervention effect of regulating body constitution

Abstract

The application discloses a method for constructing an animal model of phlegm-dampness constitution and application of the method in evaluating intervention effect of body regulation, compared with a method for copying phlegm-dampness constitution by feeding mice with high-fat feed for a long time to cause metabolic disorder, the method provided by the application significantly improves the modeling effect by transplanting intestinal flora of a phlegm-dampness constitution subject into a pseudo-sterile mouse. Further, on the basis of high-fat feed combined with intestinal flora transplantation, the application surprisingly finds that the step of adding 0.5% dextran sodium sulfate in drinking water can further significantly improve the modeling rate of the phlegm-dampness constitution mouse animal model. The phlegm-dampness constitution mouse animal model obtained by the constructing method provided by the application can be used not only for evaluating intervention effect of body regulation, but also for studying pathogenesis of phlegm-dampness constitution and for screening and preparing drugs for intervention of body regulation of phlegm-dampness constitution.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and relates to a construction method of an animal model and application thereof, in particular to a construction method of an animal model of phlegm-damp constitution and application thereof in evaluation of therapeutic effect of constitution intervention. BACKGROUND

[0002] Phlegm-damp constitution is a constitution state caused by stagnation of water and fluid, and is mainly characterized by sticky and heavy turbidity, mainly manifested as symptoms such as abdominal fullness and softness, sticky feeling in the mouth, much phlegm and chest tightness, sticky sweating, much oil on the forehead, thick and greasy tongue fur, and heavy and uncomfortable body (Guo Wenqi, Wan Jiyi, Yao Haiqiang; Explore the Microscopic Mechanism of Spleen Dysfunction in Phlegm-damp Constitution from Intestinal Barrier Dysfunction; Journal of Beijing University of Chinese Medicine, 2022).

[0003] Intestinal flora is known as the "second genome" of the human body, and can exert a wide range of regulatory effects on host physiological functions. Changes in the structure of intestinal flora and imbalance of homeostasis are important mechanisms for the occurrence of many diseases, and are closely related to the occurrence of metabolic diseases such as obesity and metabolic syndrome in phlegm-damp constitution. The interaction between intestinal flora and host intestinal epithelium plays an important role in regulating the metabolic process of the body (Wang Xiaolu, Yao Haiqiang, Wan Jiyi; Explore the Mechanism of Qi-tonifying Therapy in Clinical Constitution Intervention of Phlegm-damp Constitution Based on the Interaction between Intestinal Epithelium and Intestinal Flora; Chinese Journal of Chinese Medicine, 2023).

[0004] Animal models are essential tools for modern medical research on the occurrence, development and outcome of diseases, as well as the evaluation of drug efficacy and mechanism. With the development of experimental animal science and the advancement of the modernization process of traditional Chinese medicine, animal models have gradually become a common method and important means for the study of traditional Chinese medicine theory (Shenyang, Yang Xue, Wang Lina, Yin Zifei; Research Progress of Animal Models of Traditional Chinese Medicine "Six Exogenous Pathogens"; Modern Journal of Integrated Traditional Chinese and Western Medicine, 2019).

[0005] There are few construction methods of phlegm-damp constitution animal models, and currently the main method is to feed high-fat feed for a long time to cause metabolic disorders and thus replicate the characteristics of phlegm-damp constitution. However, the model formation rate of this method needs to be improved. SUMMARY

[0006] The first object of the present application is to overcome the shortcomings of the prior art and provide a construction method of a phlegm-damp constitution animal model. The second object of the present application is to provide the application of the construction method of the phlegm-damp constitution animal model in the evaluation of therapeutic effect of constitution intervention.

[0007] The above objects of the present application are achieved by the following technical solutions:

[0008] A construction method of a phlegm-damp constitution mouse animal model, comprising feeding high-fat feed, and transplanting intestinal flora of a phlegm-damp constitution subject into a pseudo-sterile mouse by gavage before feeding the high-fat feed.

[0009] Preferably, the phlegm-dampness constitution mice are fed with high-fat diet and given drinking water added with 0.5% dextran sodium sulfate.

[0010] More preferably, the method comprises the following steps:

[0011] Step S1, administering broad-spectrum antibiotics to the experimental mice to eliminate the main intestinal flora in the mice and construct pseudo-sterile mice;

[0012] Step S2, transplanting the intestinal flora of the phlegm-dampness constitution subject into the pseudo-sterile mice by gavage;

[0013] Step S3, feeding the phlegm-dampness constitution mice with high-fat diet and giving them drinking water added with 0.5% dextran sodium sulfate;

[0014] In the method, the intestinal flora of the phlegm-dampness constitution subject is transplanted into the pseudo-sterile mice, and at the same time, the intestinal flora of the normal constitution subject is transplanted into the pseudo-sterile mice by gavage to construct normal constitution control mice, and the normal constitution control mice drink normal drinking water without 0.5% dextran sodium sulfate;

[0015] Step S4, taking the body weight of the phlegm-dampness constitution mice being 20% higher than that of the normal constitution control mice as a success marker.

[0016] More preferably, the method for administering broad-spectrum antibiotics in Step S1 is to give drinking water containing 1 g / L ampicillin, 1 g / L neomycin, 0.25 g / L metronidazole and 0.5 g / L vancomycin.

[0017] More preferably, the broad-spectrum antibiotics are administered for 7 days in Step S1.

[0018] More preferably, the pseudo-sterile mice are rested for 3 days before the intestinal flora transplantation.

[0019] More preferably, the duration of Step S3 is 6 weeks.

[0020] The phlegm-dampness constitution mice animal model obtained by any of the above construction methods is used in the evaluation of the efficacy of a body constitution intervention.

[0021] The phlegm-dampness constitution mice animal model obtained by the above construction method is used in the study of the pathogenesis of phlegm-dampness constitution.

[0022] The phlegm-dampness constitution mice animal model obtained by the above construction method is used in the screening of drugs for treating phlegm-dampness constitution.

[0023] Beneficial effects:

[0024] Compared with the method of feeding mice with high-fat feed for a long time to cause metabolic disorders to replicate phlegm-dampness constitution, the step of transplanting the intestinal flora of the phlegm-dampness constitution subject into the pseudo-sterile mouse in the method provided by the application significantly improves the modeling effect. Further, on the basis of high-fat feed combined with intestinal flora transplantation, the application unexpectedly finds that the step of adding 0.5% dextran sodium sulfate in the drinking water can further significantly improve the modeling rate of the phlegm-dampness mouse animal model. Those skilled in the art know that dextran sodium sulfate is commonly used for modeling of an animal model of colitis, and 2-3% dextran sodium sulfate is commonly used for gavage to construct an animal model of chronic colitis, and 3-5% dextran sodium sulfate is commonly used for gavage to construct an animal model of acute colitis. The application finds that an ultra-low dose of dextran sodium sulfate not only does not cause colon damage in mice, but also can promote the formation of pathological characteristics of phlegm-dampness constitution, and produces unexpected technical effects. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The body weight of each group of mice (n=30); Note: compared with the FMT-BC group at the same time, * represents a significant difference (P<0.05); compared with the FMT-PD group at the same time, # represents a significant difference (P<0.05);

[0026] Figure 2 The oil red O staining results of the skin of each group of mice (×100);

[0027] Figure 3 The food preference of each group of mice (n=30); Note: compared with the FMT-BC group, * represents a significant difference (P<0.05); compared with the FMT-PD group, # represents a significant difference (P<0.05);

[0028] Figure 4 The OGTT and area under the curve of each group of mice (n=30); Note: compared with the FMT-BC group at the same time, * represents a significant difference (P<0.05); compared with the FMT-PD group at the same time, # represents a significant difference (P<0.05)

[0029] Figure 5 The ITT and area under the curve of each group of mice (n=30); Note: compared with the FMT-BC group at the same time, * represents a significant difference (P<0.05); compared with the FMT-PD group at the same time, # represents a significant difference (P<0.05)

[0030] Figure 6 The four blood lipids of each group of mice (n=30); Note: compared with the FMT-BC group, * represents a significant difference (P<0.05); compared with the FMT-PD group, # represents a significant difference (P<0.05);

[0031] Figure 7 Fat mass and fat coefficient of each group of mice (n=30); Note: * indicates significant difference (P<0.05) compared with FMT-BC group; # indicates significant difference (P<0.05) compared with FMT-PD group;

[0032] Figure 8 H&E staining results of colon tissue of each group of mice (×200). DETAILED DESCRIPTION

[0033] The substantial content of the present application will be specifically described below in combination with examples, but the protection scope of the present application is not limited thereto.

[0034] I. Experimental materials

[0035] 1. Research object

[0036] 1.1 Case source

[0037] Phlegm-dampness constitution and moderate constitution subjects were recruited, and the specific sources included: Beijing University of Chinese Medicine Guowutang Outpatient Department and Beijing University of Chinese Medicine Third Affiliated Hospital.

[0038] 1.2 Constitution determination standard

[0039] The determination of phlegm-dampness constitution and moderate constitution was in reference to the standard of "TCM Constitution Classification and Determination" issued by Chinese Medicine Association in 2009 (number: ZZYXH / T157-2009). The recruited subjects first filled in the "TCM Constitution Scale" to preliminarily determine the constitution type, and finally determined the constitution type by artificial recognition to exclude the influence of subjective factors. (Background information supplement: Phlegm-dampness constitution is a long-term stable bias state, characterized by obesity, abdominal obesity, more facial skin oil secretion, more phlegm, chest tightness, preference for fatty food, white and greasy tongue fur, and slippery pulse. Phlegm-dampness constitution is closely related to the occurrence of various metabolic diseases, and is a high-risk population for metabolic diseases. Moderate constitution refers to good congenital endowment and proper postnatal care, and is a constitution type characterized by moderate body shape, ruddy complexion, abundant energy, and strong and robust function state of viscera. Here, moderate constitution is taken as a healthy control group.)

[0040] 1.3 Inclusion criteria

[0041] (1) Inclusion criteria, i.e. after scale screening and artificial constitution identification, the constitution was established as moderate constitution and phlegm-dampness constitution;

[0042] (2) Age 18 to 60 years old, age calculated according to the difference between the survey date and the birthday date;

[0043] (3) Gender is not limited;

[0044] (4) The subjects are informed and sign the informed consent form.

[0045] 1.4 Exclusion criteria

[0046] (1) Those with obvious mixed constitution;

[0047] (2) Breastfeeding or pregnant women;

[0048] (3) Those with metabolic diseases, gastrointestinal diseases, cardiovascular and cerebrovascular diseases or other clearly diagnosed diseases;

[0049] (4) Subjects who have a history of taking antibiotics, gastrointestinal motility drugs, and microecological modulators within one month before sampling;

[0050] (5) Participated in other research projects within the past 3 months.

[0051] 1.5 Baseline control

[0052] There were no significant differences in the basic information of the subjects such as age and gender.

[0053] 1.6 Study grouping

[0054] A total of 20 cases of phlegm-dampness constitution (PD) and 20 cases of balanced constitution (BC) were recruited.

[0055] 1.7 Quality control

[0056] The artificial constitution identification was reviewed by professionals to ensure accuracy. The data entry was checked for the second time.

[0057] 2. Materials and reagents

[0058] The modeling agent DSS (MW 36000-50000) was purchased from MPBiomedicals Company in the United States, and the rest of the reagents were purchased from the National Pharmaceutical Group. The ordinary maintenance feed, low-fat feed, and high-fat feed (D12492) for mice were purchased from Sperofree (Beijing) Biotechnology Co., Ltd. The ACCU-CHEK Performa blood glucose meter and matching test paper were purchased from Roche Company in Germany. The serum total cholesterol (TC) detection kit, triglycerides (TG) detection kit, high-density lipoprotein cholesterol (HDL-C) detection kit, and low-density lipoprotein cholesterol (LDL-C) detection kit were purchased from Nanjing Jiancheng Biological Engineering Institute.

[0059] 3. Experimental animals

[0060] 180 healthy C57BL / 6 male mice, 8 weeks old, weighing 20-23 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The feeding environment was: SPF level, the room temperature was always maintained at 20-25°C, the relative humidity was controlled at 50%-60%, and the light and darkness were alternated for 12 hours per day. After 1 week of pre-adaptation, the mice were randomly divided into the following three groups: FMT-BC group, FMT-PD group and FMT-PD+DSS group, 60 mice in each group.

[0061] II. Experimental methods

[0062] 1. Collection of human fecal samples

[0063] On the sampling day, the subjects collected their morning feces. The subjects independently collected the feces in a disposable feces collection tube. The intestinal condition should be stable and good at the time of collection. If the sampling is delayed due to unexpected situations, such as sudden diarrhea or menstruation, the sampling should be delayed. The specific collection method is as follows: first, rinse the toilet bowl thoroughly, then lay several layers of toilet paper, take the middle section of the feces (without mixing with urine), use a disposable sampling spoon to collect 5g of fresh feces naturally excreted, and each subject's feces is divided into a sterile feces sampling tube, immediately frozen in liquid nitrogen, and the sample is returned to the laboratory within 2 hours, and stored in a -80°C freezer.

[0064] 2. Construction of a pseudo-sterile mouse model

[0065] Broad-spectrum antibiotics (1 g / L ampicillin, 1 g / L neomycin, 0.25 g / L metronidazole and 0.5 g / L vancomycin, free drinking water) were added to the drinking water of the experimental mice for 7 days to eliminate the main intestinal flora in the mice and construct a pseudo-sterile mouse model.

[0066] 3. Fecal microbiota transplantation (FMT) experiment

[0067] 20 fecal samples of the phlegm-damp constitution and 20 fecal samples of the phlegm-damp constitution were prepared into intestinal flora suspensions, i.e. the fecal bacteria suspension was prepared according to the ratio of 250 mg feces / 2 mL PBS, centrifuged at 800 rpm for 5 minutes, and the supernatant was taken as the flora inoculum after discarding the fecal residue, which was used for FMT. After the pseudo-sterile mouse model was successfully constructed, the FMT was started after 3 days of rest. The specific method was as follows:

[0068] FMT was performed by gavage. The Pinghe BC group was inoculated with the BC flora, and the Pinghe PD group and the FMT-PD+DSS group were inoculated with the PD flora. The operation was performed at a fixed time every day, and fresh flora inoculum was used each time, 200 μL per mouse. The flora sample from each subject in each group was transplanted into 3 mice, and the experimental period was 6 weeks. The three groups of mice were given high-fat feed. The FMT-BC and FMT-PD groups were given normal drinking water throughout the experiment, and the FMT-PD+DSS group was given 0.5% (0.5 g / 100 mL) dextran sodium sulfate (DSS) in drinking water throughout the experiment.

[0069] The body weight of the mice was recorded daily during the experiment. The body weight of the mice in the FMT-PD or FMT-PD+DSS group was 20% higher than that of the mice in the FMT-BC group, which was taken as the success marker of modeling. The food preference test was performed one day before the mice were taken. After the experiment, the mice in each group were taken from the orbital blood, centrifuged at 4000 rpm for 10 minutes to obtain the upper serum, and stored in a -80°C refrigerator. The mice were sacrificed and the samples were taken.

[0070] 4. Mouse skin histological determination

[0071] The square skin tissue on the back of the mouse was taken, and oil red staining was determined by skin frozen section. The oil secretion function of the mouse sebaceous gland was observed under an optical microscope.

[0072] 5. Mouse food preference determination

[0073] The mice were subjected to food preference determination, and low-fat feed and high-fat feed were placed at the same time. The food intake of the mice in each group within 3 hours was counted to determine the food preference.

[0074] 6. Oral glucose tolerance test (OGTT)

[0075] After 5 weeks of FMT intervention, the mice were fasted for 12 hours, and a glucose solution was administered at a dose of 2 g / kg by gavage. Tail blood was collected, and the blood glucose values of the mice at 0, 15, 30, 60, and 120 minutes after gavage were determined. The OGTT changes were evaluated by calculating the area under the curve.

[0076] 7. Insulin tolerance test (ITT)

[0077] After 6 weeks of FMT intervention, the mice were fasted for 4 hours, and insulin was injected intraperitoneally at a dose of 0.75 U / kg. Tail blood was collected, and the blood glucose values of the mice at 0, 15, 30, 60, and 120 minutes after gavage were determined. The ITT changes were evaluated by calculating the area under the curve.

[0078] 8. Mouse blood lipid index determination

[0079] Take the mouse serum, according to the experimental steps of the kit instructions to detect the content of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) in serum of each group of mice.

[0080] 9, the determination of mouse fat coefficient

[0081] Take the mouse perirenal fat and epididymal fat, weigh and calculate the fat coefficient: fat coefficient = (perirenal fat mass + epididymal fat mass) / mouse body mass.

[0082] 10, the determination of mouse colon and its length

[0083] Take the mouse colon from the anus to the ileocecal colon, observe the surface of the colon and measure its length.

[0084] 11, mouse colon H&E staining

[0085] Take part of the mouse colon, fix it in 4% paraformaldehyde, perform H&E staining with paraffin section, and then observe it with optical microscope.

[0086] 12, data processing

[0087] The experimental results conform to normal distribution, the data are expressed as mean ± standard deviation, single factor analysis of variance is performed by using SPSS20.0 statistical software, and P<0.05 is considered to have statistical significance.

[0088] III. Experimental results

[0089] 1, the evaluation of FMT mouse body mass and modeling rate

[0090] The body mass of three groups of mice is recorded throughout the experiment, and the results are shown in Figure 1 The results show that, since the fourth week of modeling, the body mass of FMT-PD group mice is significantly improved compared with that of FMT-BC group mice (P<0.05), and the gap gradually increases with time; compared with FMT-PD group mice, the body mass of FMT-PD+DSS group mice increases more significantly (P<0.05). Taking 20% higher than the body mass of FMT-BC group mice as the modeling success mark, the modeling rate of FMT-PD group mice reaches 81% at the end of 6 weeks; the modeling rate of FMT-PD+DSS group mice reaches 93% at the end of 6 weeks. It is shown that through FMT animal model, the increase of body mass of phlegm-damp constitution group can be observed in a short period (6 weeks), and adding DSS will shorten the modeling period and improve the modeling efficiency to a certain extent.

[0091] 2, the evaluation of FMT mouse skin oil secretion

[0092] Oil Red O is a lipid-soluble dye that turns lipid droplets in tissues orange-red. Oil Red O staining was used to observe sebum secretion in the sebaceous glands of FMT mice. Figure 2 As shown. Compared with the FMT-BC group mice, the FMT-PD group mice showed a large number of clusters of red lipid droplets in the cytoplasm of sebaceous gland cells. The orange-red lipid droplets around the sebaceous acini of the FMT-PD+DSS group mice merged into sheets, indicating more vigorous sebum secretion, which is consistent with the clinical characteristics of people with phlegm-dampness constitution who have more sebum secretion.

[0093] 3. Assessment of feeding preferences in FMT mice

[0094] Figure 3 The study presented the dietary preferences of FMT mice in different groups for low-fat and high-fat diets. Results showed no significant difference in the amount of low-fat and high-fat diets consumed by FMT-BC mice within 3 hours, while FMT-PD and FMT-PD+DSS mice consumed significantly more high-fat diets than low-fat diets (P<0.05), demonstrating a clear preference for high-fat foods, consistent with the dietary characteristics of those with phlegm-dampness constitution who prefer rich and sweet foods.

[0095] 4. Assessment of oral glucose tolerance in FMT mice

[0096] The oral glucose tolerance test (OGTT) is a glucose load test used to assess the body's tolerance and regulation of blood glucose. Figure 4 The results of OGTT (Oral Glucose Tolerance Test) in three groups of FMT mice after 5 weeks of intervention are presented. It can be seen that compared to the FMT-BC group, the FMT-PD and FMT-PD+DSS groups showed a rapid and significant increase in blood glucose, while the decrease was relatively slow. The area under the curve (AUC) statistical results indicate that the FMT-PD and FMT-PD+DSS groups were significantly higher than the FMT-BC group, with the FMT-PD+DSS group showing a more significant increase (P<0.05). The FMT animal model confirms that individuals with phlegm-dampness constitution are at risk of abnormal glucose metabolism even in the pre-disease stage.

[0097] 5. Assessment of insulin tolerance in FMT mice

[0098] Insulin tolerance tests can assess the sensitivity of mice to insulin. For example... Figure 5 As shown, after 6 weeks of intervention, compared with the FMT-BC group, the glucose decline trend after insulin injection was slower in the FMT-PD group and the FMT-PD+DSS group. Further statistical analysis revealed that the AUC was significantly higher in the FMT-PD group than in the FMT-BC group, and the increase was even more pronounced in the FMT-PD+DSS group (P<0.05). This indicates that the FMT animal model can observe insulin resistance in the "pathological state" of phlegm-dampness constitution in a short period.

[0099] 6. Assessment of blood lipid levels in FMT mice

[0100] The results of the measurement of four lipid indicators in three groups of FMT mice are as follows: Figure 6 As shown in the figure, compared with the FMT-BC group, the serum TC, TG, LDL-C, and HDL-C levels in mice in the FMT-PD and FMT-PD+DSS groups were significantly increased (P<0.05), indicating dyslipidemia; the lipid dyslipidemia was more severe in the FMT-PD+DSS group. These results demonstrate that the FMT mouse model can effectively prove, at the animal experimental level, that individuals with phlegm-dampness constitution are at risk of developing dyslipidemia even in a disease-free state.

[0101] 7. Assessment of Fat Index in FMT Mice

[0102] In addition to blood lipid levels, the distribution of fat in FMT mice was also investigated. Figure 7 As shown, the mass of perirenal and periepididymal fat in the FMT-PD group was significantly higher than that in the FMT-BC group (P<0.05), and similar results were observed in the FMT-PD+DSS group (P<0.05). The fat coefficient more clearly indicates the level of abdominal fat. The results showed that compared to the FMT-BC group, the fat coefficients of the FMT-PD and FMT-PD+DSS groups were significantly increased (P<0.05). Therefore, the abnormal fat coefficient in the FMT animal model is consistent with the clinical characteristics of fat accumulation and central obesity in individuals with phlegm-dampness constitution.

[0103] 8. Evaluation of colon morphology and length in FMT mice

[0104] Observation of the colons of mice revealed that the colonic mucosa of the three FMT groups was smooth, the intestinal wall color was normal, and no congestion or ulceration was observed on the surface. Furthermore, as shown in Table 1, there was no significant difference in colon length among the FMT-BC group, FMT-PD group, and FMT-PD+DSS group (P>0.05). Therefore, it is evident that the addition of 0.5% DSS did not affect the morphology and physiological characteristics of the mouse colon.

[0105] Table 1. Colon length results for each group of mice (n=30)

[0106]

[0107] 9. Evaluation of colon tissue sections from FMT mice

[0108] like Figure 8As shown in the results of H&E staining of the mouse colon tissue sections under a light microscope, the epithelial cells of the colon mucosa tissue of the three groups of FMT mice were columnar, regular in shape, closely arranged, and had many goblet cells, all of which were in a normal physiological state (P>0.05). It was indicated that the addition of 0.5% DSS did not affect the structure of the colon tissue of the mice and did not induce a significant intestinal inflammatory response.

[0109] The above experimental results show that, compared with the method of feeding mice with high-fat feed for a long time to cause metabolic disorders of the mice and thereby replicate the phlegm-damp constitution, the step of transplanting the intestinal flora of the phlegm-damp constitution subject into a pseudo-sterile mouse in the method provided by the application significantly improves the modeling effect. Further, on the basis of the high-fat feed combined with the intestinal flora transplantation, the application unexpectedly finds that the step of adding 0.5% dextran sulfate sodium in the drinking water can further significantly improve the modeling rate of the phlegm-damp constitution mouse animal model. The phlegm-damp constitution mouse animal model obtained by the construction method provided by the application can not only be used for evaluating the efficacy of body regulation intervention, but also be used for studying the pathogenesis of phlegm-damp constitution and for screening drugs for treating phlegm-damp constitution.

[0110] The above examples serve to specifically introduce the essential content of the application, but those skilled in the art should know that the protection scope of the application should not be limited to the specific examples.

Claims

1. A method for constructing a mouse animal model of phlegm constitution, characterized in that, Comprising the following steps: Step S1 Pseudobiotic mice were constructed by administering broad-spectrum antibiotics to experimental mice to eliminate the major intestinal flora in the mice. Step S2 The intestinal flora of the phlegm-damp constitution subject is transplanted into the pseudo-sterile mouse by gavage. Step S3 , after transplantation, high-fat diet was given, and 0.5% dextran sulfate sodium was added to the drinking water; Wherein, the intestinal flora of phlegm-dampness constitution subject is transplanted into the pseudo-sterile mice, at the same time, the intestinal flora of mild constitution subject is transplanted into the pseudo-sterile mice by gavage to construct the mild constitution control mice, and the mild constitution control mice drink the conventional drinking water without adding 0.5% dextran sodium sulfate; Step S4 The success of construction was marked by the body weight of the phlegm-dampness constitution mice being 20% higher than that of the normal constitution control mice.

2. The method of construction of claim 1, wherein: Procedure S1 The method for administering broad spectrum antibiotics in the middle was to administer drinking water containing 1 g / L ampicillin, 1 g / L neomycin, 0.25 g / L metronidazole, and 0.5 g / L vancomycin.

3. The method of construction of claim 2, wherein: Step S1 Broad-spectrum antibiotics were given for 7 days.

4. The method of construction of claim 1, wherein: The pseudo-sterile mice are rested for 3 days before intestinal flora transplantation.

5. The method of construction of claim 1, wherein: Step S3 The duration was 6 weeks.

6. The application of the phlegm-dampness constitution mouse animal model obtained by the construction method of any one of claims 1-5 in the evaluation of the efficacy of body regulation intervention, wherein the evaluation of the efficacy of body regulation intervention refers to the evaluation of the efficacy of body regulation intervention on the phlegm-dampness constitution subject.

7. The application of the phlegm-dampness constitution mouse animal model obtained by the construction method of any one of claims 1-5 in the study of the pathogenesis of phlegm-dampness constitution.

8. The application of the phlegm-dampness constitution mouse animal model obtained by the construction method of any one of claims 1-5 in the screening of drugs for the preparation of phlegm-dampness constitution body regulation intervention.

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