A bama pig model for preclinical study of weight loss drugs and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a Bama pig model for preclinical research of a weight loss drug COST and application. Healthy Bama pigs are first adaptively fed with ordinary feed, and then fed with high-fat feed to model, and the Bama pigs are detected for obesity-related indexes at a fixed time period every week; the high-fat feed comprises ordinary feed, beef tallow, artificial butter, sucrose, casein, experimental animal premix, microcrystalline cellulose, calcium bicarbonate, gypenoside, peppermint leaf, maslinic acid, ursolic acid, atractylodes, nutmeg. The application can quickly construct a Bama pig model with stable phenotype, and provides a reliable model for preclinical research of a new drug for treating obesity, and provides guarantee for research on the mechanism of obesity, screening, development and mechanism research of a new drug for treating obesity and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a Bama pig model for preclinical research of a weight loss drug COST and application thereof. BACKGROUND

[0002] According to the definition of disease by the World Health Organization (WHO), obesity refers to excessive accumulation of fat in the body, which is an abnormal state that can damage health, and was officially listed as one of the endocrinology diseases in 1997. Obesity is associated with an increased risk of premature death and also increases the incidence of various disease complications, such as type 2 diabetes, sleep apnea syndrome, atherosclerosis, non-alcoholic fatty liver disease, etc. In addition, obesity also increases the risk of various cancers and has a negative impact on the psychology, mood and cognitive ability of patients. At present, the global obesity rate is continuously rising, and obesity has become a global public health challenge. Therefore, modern medicine urgently needs to strengthen scientific research on obesity and strive to find more effective and harmless weight loss drugs and treatment methods.

[0003] In the screening and preclinical research stage of new drugs, mouse models are relatively common mammalian models, but the physiological differences between mice and humans lead to limitations in using mice to simulate diseases. For example, when studying metabolic diseases such as diabetes, due to the differences in metabolic levels such as sugar metabolism and lipid metabolism between mice and humans, even if the mice are induced to have the corresponding phenotype by drugs or genetic means, the disease occurrence and development process and pathophysiological mechanism are not completely consistent with humans, which leads to the fact that the research results cannot be directly applied to humans.

[0004] In preclinical drug research, due to the differences in drug metabolism enzyme systems, drug transporters, etc. between mice and humans, the metabolism process and pharmacokinetic characteristics of drugs in humans and mice are different. Therefore, some drugs that are effective in mouse models may not work well or have different adverse reactions in human clinical trials, affecting the success rate of drug development and the accuracy of safety assessment. Therefore, an animal model with less physiological difference from humans is needed to provide reliable support for further preclinical research of new drugs.

[0005] Bama pigs have a relatively long lifespan, and the occurrence and development process of some chronic diseases is closer to humans in terms of time scale. The organs of Bama pigs are more similar to humans in size, structure and physiological function, and the nutritional needs and intake patterns are similar to humans. The absorption and metabolic response to different nutritional components are also relatively close, which can more accurately simulate the physiological conditions of humans. By controlling the intake amount and composition of Bama pig feed, the occurrence mechanism of obesity that is close to the physiological conditions of humans can be better simulated, and more reliable data support can be provided for research.

[0006] CN110862988A discloses an sgRNA, a CREBRF point mutation type Bama mini-pig constructed by the sgRNA and an application, but the modeling cost is high through gene editing, and there is a long-term safety problem in screening drugs using an animal model of gene editing, and the edited gene may have some unknown and potential effects, such as causing tumors and immune system abnormalities, which may be ignored in the drug screening process but exposed in subsequent clinical trials or clinical applications.

[0007] CN114831769A discloses a method for establishing a new obstructive sleep apnea syndrome animal model pig, but does not disclose the feeding time problem. Since obesity is a complex chronic disease and is related to other diseases to some extent, other diseases may be introduced in the modeling process, affecting the results of drug research.

[0008] In summary, there is still a lack of a standardized Bama pig model construction method for successfully constructing a Bama pig model with stable obesity phenotype in a short time in the prior art. SUMMARY

[0009] The technical problem to be solved by the present application is to provide a Bama pig model for preclinical research of weight loss drugs, which has a short construction time and stable obesity phenotype, and provides a reliable animal model for preclinical research of new drugs for treating obesity, and provides protection for the research of disease characteristics and pathogenesis of obesity, screening, development and mechanism research of new drugs for treating obesity, etc.

[0010] To achieve the above-mentioned purpose, the first aspect of the present application provides a construction method of a Bama pig model, which is as follows:

[0011] Healthy Bama pigs are selected and divided into control group Bama pigs and model group Bama pigs;

[0012] The control group Bama pigs are fed with ordinary feed;

[0013] The model group Bama pigs are first fed with ordinary feed, and then fed with high-fat feed, and the obesity-related indicators of the control group Bama pigs and the model group Bama pigs are detected at a fixed time period every week, and the Bama pig model is obtained when the obesity degree of the model group Bama pigs is greater than 20%;

[0014] The calculation formula of the obesity degree is: (the body weight of the model group Bama pigs-the average body weight of the control group Bama pigs) / the average body weight of the control group Bama pigs) x 100%;

[0015] The high-fat feed comprises the following components in mass fraction: 49-50 parts of the common feed, 9-10 parts of beef tallow, 10-11 parts of margarine, 14-15 parts of sucrose, 9-10 parts of casein, 2-2.5 parts of experimental animal premix, 1-1.5 parts of microcrystalline cellulose, and 2-2.5 parts of calcium bicarbonate.

[0016] Preferably, the high-fat feed further comprises the following components in mass fraction: 0.1-0.3 parts of gypenoside, 0.5-1.0 parts of mint leaves, 0.2-0.3 parts of maslinic acid, 0.1-0.15 parts of ursolic acid, 1-2 parts of atractylodes, and 0.1-0.15 parts of nutmeg. The preferred gypenoside, atractylodes, and nutmeg of the present application can promote the absorption of the nutritional components in the high-fat feed by the Bama pigs through reasonable collocation. Since there is a large amount of oil in the high-fat feed, the Bama pigs fed with the high-fat feed for a long time will have the condition of indigestion and reduced absorption efficiency. The preferred gypenoside, atractylodes, and nutmeg of the present application are more suitable for the constitution of the Bama pigs and have a good effect on promoting the digestion and absorption of the Bama pigs. The addition of the mint leaves, maslinic acid, and ursolic acid improves the taste of the feed. The maslinic acid and ursolic acid can also promote the secretion of digestive juice of the Bama pigs, further promoting the digestion and absorption of the Bama pigs.

[0017] Preferably, the common feed comprises the following components in mass fraction: 25-26 parts of corn, 3-4 parts of fish meal, 8-9 parts of soybean meal, 9-10 parts of rice bran, 18-20 parts of wheat bran, 30-32 parts of alfalfa meal, 2-3 parts of calcium bicarbonate, 0.3-0.4 parts of iodized salt, 0.03-0.04 parts of trace element additive, and 0.03-0.032 parts of vitamin additive.

[0018] Preferably, the trace element additive is composed of the following components in mass fraction: 7.7-12 parts of copper, 41-61 parts of iron, 22-33 parts of zinc, 14.1-21 parts of manganese, 0.17-0.33 parts of iodine, 0.12-0.23 parts of selenium, 0.07-0.13 parts of cobalt, and 9.46-14.19 parts of water.

[0019] The vitamin additive is composed of the following components in mass fraction: 19-19.5 parts of vitamin A, 0.4-0.45 parts of vitamin D3, 16.75-16.8 parts of vitamin E, 5-5.1 parts of vitamin K3, 1-2.1 parts of vitamin B1, 16-16.5 parts of vitamin B2, 10-10.1 parts of vitamin C, 6-6.05 parts of vitamin B6, 0.03-0.031 parts of vitamin B12, 35-35.5 parts of nicotinic acid, 25-25.5 parts of calcium pantothenate, 0.5-0.51 parts of folic acid, 0.1-0.11 parts of biotin, 50-50.5 parts of methionine, and 50-50.5 parts of lysine.

[0020] The experimental animal premix is composed of the vitamin additive 1-1.05 parts by mass, the trace element additive 1-1.05 parts by mass, salt 10-10.5 parts, and corn flour 88-89 parts.

[0021] Preferably, the healthy Bama pigs are 4-6 month old ordinary grade Bama pigs with a weight of 20-30 kg.

[0022] Preferably, the model group Bama pigs are first fed with ordinary feed for 1.0-1.1 weeks, and then fed with high-fat feed until the obesity degree of the model group Bama pigs is greater than 20%.

[0023] The feeding is daily administration of ordinary feed or high-fat feed, and the weight of the ordinary feed or high-fat feed used for feeding is 3.0-3.01% of the body weight of the Bama pigs in the week.

[0024] Preferably, the preparation method of the ordinary feed and / or high-fat feed comprises the following steps: weighing the required raw materials, crushing the raw materials, uniformly mixing, adding the mixed powder feed into a feed granulator, preparing feed granules, cooling, and packaging into finished feed.

[0025] Another aspect of the present application provides a Bama pig model for preclinical research of a weight loss drug COST.

[0026] Preferably, the application is to use the Bama pig model of any one of claims 1-6 as a model group, and to use the Bama pigs fed with ordinary feed as a control group.

[0027] The model group is randomly divided into a model control group, a positive drug orlistat group, and a COST group.

[0028] The model control group, the positive drug orlistat group, and the COST group are subjected to drug intervention, and the stage of the drug intervention is a weight loss period.

[0029] During the weight loss period, the physiological state of the Bama pigs is observed daily, the body weight is weighed and the body size is measured every week, blood biochemical detection is performed every month, the indexes of the blood biochemical detection include fasting blood glucose, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, triglyceride, cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol, and the weight loss effect is judged in combination with the physiological state, the body weight, the body size, and the serum biochemical indexes after the weight loss period.

[0030] After the weight loss period, the Bama pigs are sacrificed, the back fat thickness and the subcutaneous fat layer thickness are measured, and then the samples are taken.

[0031] The taking of the samples includes taking out the viscera, fat tissue, and cecal contents.

[0032] The viscera is subjected to weighing, embedding, and cryopreservation treatment.

[0033] The organs include: heart, liver, kidney, duodenum, ileum, colon;

[0034] The fat tissues include: perirenal fat, abdominal subcutaneous fat, omental fat;

[0035] The fat tissues are subjected to free fatty acid detection and analysis by using a fatty acid analysis method based on a GC-MS / MS platform, so as to study the molecular level mechanism of COST in lipid metabolism and synthesis.

[0036] Preferably, the free fatty acid detection and analysis specifically comprises the following steps:

[0037] S1, sample pretreatment;

[0038] S2, analyzing sample derivatives by using a GC-EI-MS system, wherein a chromatographic column used by the GC is a DB-5MS capillary column with a specification of 30m*0.25mm*0.25um; high-purity helium is used as a carrier gas; and a heating program is as follows:

[0039] starting from 40 DEG C and lasting for 2 minutes, increasing to 200 DEG C at a rate of 30 DEG C / min and lasting for 1 minute, then increasing to 240 DEG C at a rate of 10 DEG C / min and lasting for 1 minute, finally increasing to 285 DEG C at a rate of 5 DEG C / min and lasting for 3 minutes; a flow rate is 1.0mL / min; an injection port temperature is 230 DEG C; an injection volume is 1.0uL;

[0040] the GC-EI-MS system is an Agilent 8890-5977B GC-MS system, and after GC detection, parameters of the EI-MS are set as follows: temperature: 230 DEG C; ionization voltage: 70eV; transmission line temperature: 240 DEG C; quadrupole temperature: 150 DEG C; solvent delay: 4 minutes; scanning mode: SIM;

[0041] S3, statistical analysis.

[0042] Preferably, the sample pretreatment in step S1 specifically comprises the following steps:

[0043] After the sample stored in an ultralow temperature is taken out and thawed on ice, the sample is ground into powder, methanol, methyl tert-butyl ether and 36% phosphoric acid are added for extraction; the supernatant is removed by centrifugation, dried, 15% boron trifluoride methanol solution is added, and the mixture is kept in a 60-65 DEG C oven for 30-40 minutes; after cooling to room temperature, n-hexane solution and saturated sodium chloride solution are added, mixed, centrifuged, and the n-hexane layer solution is removed for machine analysis.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] (1) The application provides a method for constructing a Bama pig model, which feeds self-made high-fat feed, reasonably matches the proportion of lipids and sugars, and adds natural ingredients such as mint leaves and hawthorn acid to promote the appetite and gastrointestinal digestion and absorption capacity of the Bama pig. Each Bama pig individual maintains a high weight state after modeling is completed, the current week and last week weight gain of the individuals in the group is relatively stable, and the Bama pig model with stable obesity phenotype can be successfully constructed within 12 weeks, the modeling time of the Bama pig obesity model is shortened, and the modeling efficiency is improved.

[0046] (2) The application provides a gas chromatography-mass spectrometry experimental method for detecting free fatty acids of Bama pigs, which can analyze the composition and content of free fatty acid metabolites of fat tissues of Bama pigs in each group, so as to help further explore the influence of drugs on specific Bama pig fat differential metabolites, and help preclinical research of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The body weight (A) and weight gain (B) of each group of Bama pigs after drug administration (n=3, mean±SEM);

[0048] Figure 2 The appearance of each group of Bama pigs after drug administration;

[0049] Figure 3 The body length (A), neck circumference (B), chest circumference (C), abdominal circumference (D) and hip circumference (E) of each group of Bama pigs after drug administration (n=3, mean±SEM) Note: & indicates that compared with the Control group, && P<0.01, &&& P<0.001, &&&& P<0.0001;

[0050] Figure 4 The obesity degree (A), back fat (B) and abdominal subcutaneous fat thickness (C) of each group of Bama pigs after drug administration (n=3, mean±SEM) Note: & indicates that compared with the Control group, & P<0.05, &&& P<0.001;

[0051] Figure 5 The PCA score plot of the mass spectrometry data of each group of Bama pigs;

[0052] Figure 6 The overall clustering plot of fatty acid metabolites of each group of Bama pigs;

[0053] Figure 7 The change levels of serum total cholesterol (A), triglycerides (B), low-density lipoprotein cholesterol (C), high-density lipoprotein cholesterol (D) and non-esterified fatty acids of each group of Bama pigs (n=3, mean±SEM);

[0054] Figure 8The values ​​of glucose (A), aspartate aminotransferase (B), alanine aminotransferase (C), low-density lipoprotein cholesterol (D), cholesterol (E), and triglycerides (F) in the liver tissue of Bama pigs (n=3, mean±SEM);

[0055] Figure 9 HE staining 100X (A) and Oil Red O staining 200X (B) of livers from Bama pigs after drug administration (n=3);

[0056] Figure 10 HE staining of Bama pig hearts at 200X (A) and HE staining of kidneys at 100X (B) (n=3);

[0057] Figure 11 HE staining of subcutaneous adipose tissue of Bama pigs (A) and perirenal lamina adipose tissue (B) (n=3, 100X);

[0058] Figure 12 HE staining of the ileum (A) and colon (B) of Bama pigs after drug administration (n=3, 100X);

[0059] Figure 13 Fasting blood glucose (A), fasting insulin (B), and insulin resistance index (C) of Bama pigs in each group after drug administration (n=3, mean±SEM);

[0060] Figure 14 The levels of AST(A) and ALT(B) in the serum of Bama pigs in each group after drug administration (n=3, mean±SEM). Detailed Implementation

[0061] The present invention will be further illustrated below with reference to embodiments. It should be noted that the following embodiments are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used in the embodiments are all commercially available.

[0062] This experiment used GraphPad Prism 9 software for data analysis. T-tests were performed to compare two groups, and one-way ANOVA was used for comparisons of more than two groups. All experimental data are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant compared to the Model group. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0063] Example 1

[0064] A Bama pig model, specifically constructed using the following steps:

[0065] Select healthy 20-30kg 5-month-old ordinary level Bama pigs 12, randomly select 3 as control group Bama pigs, the rest 9 are model group Bama pigs;

[0066] The control group Bama pigs are fed with ordinary feed, and the daily feed weight is 3% of the pig's body weight this week.

[0067] The model group Bama pigs are first fed with ordinary feed for 1 week, and then fed with high-fat feed, and the daily feed weight is 3% of the pig's body weight this week.

[0068] The control group Bama pigs and the model group Bama pigs are weighed, measured, and 2mL of blood is taken from the anterior vena cava of the Bama pig every week at a fixed time period, and the supernatant is used for blood lipid detection.

[0069] When the obesity degree of the model group Bama pigs is greater than 20%, the Bama pig model is obtained;

[0070] The obesity degree calculation formula is: (model group Bama pig weight-control group Bama pig average weight) / control group Bama pig average weight) x 100%;

[0071] Prepare ordinary feed, the specific steps are as follows:

[0072] Prepare corn 25.238 parts, fish meal 4 parts, soybean meal 8 parts, rice bran 10 parts, wheat bran 20 parts, alfalfa powder 30 parts, calcium bicarbonate 2.4 parts, iodized salt 0.3 parts, trace element additive 0.03 parts, vitamin additive 0.032 parts, and mix them evenly in a mixing machine after being fully ground. Add the mixed powder feed to a feed granulator to make ordinary feed granules, and finally cool and package into finished feed.

[0073] Prepare high-fat feed, the specific steps are as follows:

[0074] Prepare the components as shown in Table 1, mix them evenly in a mixing machine after being fully ground, add the mixed powder feed to a feed granulator to make high-fat feed granules, and finally cool and package into finished feed.

[0075] Among them, the trace element additive consists of copper 7.7-12 parts, iron 41-61 parts, zinc 22-33 parts, manganese 14.1-21 parts, iodine 0.17-0.33 parts, selenium 0.12-0.23 parts, cobalt 0.07-0.13 parts, and water 9.46-14.19 parts.

[0076] The vitamin additive consists of, by weight, 19 parts of vitamin A; 0.4 parts of vitamin D3; 16.75 parts of vitamin E; 5 parts of vitamin K3; 2 parts of vitamin B1; 16 parts of vitamin B2; 10 parts of vitamin C; 6 parts of vitamin B6; 0.03 parts of vitamin B12; 35 parts of niacin; 25 parts of calcium pantothenate; 0.5 parts of folic acid; 0.1 parts of biotin; 50 parts of methionine; and 50 parts of lysine.

[0077] The experimental animal premix consists of 1 part vitamin additive, 1 part trace element additive, 10 parts salt, and 88 parts corn flour by weight.

[0078] Comparative Example 1

[0079] A Bama pig model, essentially the same as in Example 1, except that nutmeg in the high-fat diet was replaced with gypenosides. The specific components of the high-fat diet used in Comparative Example 1 are shown in Table 1.

[0080] Comparative Example 2

[0081] A Bama pig model, essentially the same as in Example 1, except that the gypenosides in the high-fat diet were replaced with Atractylodes macrocephala. The specific components of the high-fat diet used in Comparative Example 2 are shown in Table 1.

[0082] Comparative Example 3

[0083] A Bama pig model was used, which was basically the same as in Example 1, except that the Atractylodes macrocephala in the high-fat diet was replaced with Astragalus membranaceus. The specific composition of the high-fat diet used in Comparative Example 3 is shown in Table 1.

[0084] Comparative Example 4

[0085] A Bama pig model was used, which was basically the same as that in Example 1, except that the peppermint leaves in the high-fat feed were replaced with patchouli leaves, hawthorn acid was replaced with malic acid, and ursolic acid was replaced with citric acid. The specific composition of the high-fat feed used in Comparative Example 4 is shown in Table 1.

[0086] Table 1. Composition of High-Fat Feed

[0087] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Beef tallow (mass parts) 10 10 10 10 10 Artificial butter 10 10 10 10 10 Sucrose 8 8 8 8 8 Casein 10 10 10 10 10 Experimental animal premix 2 2 2 2 2 Microcrystalline cellulose 1.5 1.5 1.5 1.5 1.5 Calcium bicarbonate 2.4 2.4 2.4 2.4 2.4 Gypenoside 0.2 0.3 / 0.3 0.2 Peppermint leaf 0.7 0.7 0.7 0.7 / Maslic acid 0.25 0.25 0.25 0.25 / Ursolic acid 0.1 0.1 0.1 0.1 / Atractylodes 1 1 1.2 / 1 Myristica 0.1 / 0.1 0.1 0.1 Common feed 49.5 49.5 49.5 49.5 49.5 Astragalus / / / 1 / Malic acid / / / / 0.25 Citric acid / / / / 0.1 Agastache / / / / 0.7

[0088] Experimental Example 1: Obesity Detection in Bama Pigs

[0089] The weight of Bama pigs in Example 1 and Comparative Examples 1-4 was measured weekly, and their obesity degree was calculated. The formula for calculating obesity degree is: (actual weight of model group - average weight of control group) / average weight of control group) × 100%. The time required to reach an obesity degree of 20% is shown in the table below.

[0090] Table 2. Results of Obesity Detection in Bama Pigs

[0091]

[0092]

[0093] As shown in the above table, the embodiment 1 of the present application reached the standard of 20% obesity degree after feeding high-fat feed for 10.5 weeks, while the comparative examples 1-4 were 13-14 weeks. The lack of myristic in the comparative example 1, the lack of gypenoside in the comparative example 2, and the lack of atractylodes in the comparative example 3 affected the absorption of nutrients in the feed by the Bama pigs, thereby reducing the weight gain rate of the Bama pigs. Atractylodes, myristic, and gypenoside synergize with each other and are indispensable to each other, and can play a role in regulating the stomach and intestines and promoting digestion of the Bama pigs, thereby promoting the digestion and absorption of nutrients in the feed by the Bama pigs. The high-fat feed used in the comparative example 4 lacks peppermint leaves, maslinic acid, and ursolic acid, which affects the taste of the feed. Replacing peppermint leaves with houttuynia cordata leaf, maslinic acid with malic acid, and ursolic acid with citric acid cannot promote the secretion of digestive juice of the Bama pigs, thereby leading to loss of appetite and reducing the weight gain rate of the Bama pigs in the comparative example 4. In summary, the high-fat feed of the present application has good palatability, and the synergistic effect of atractylodes, myristic, and gypenoside promotes the digestion and absorption of the Bama pigs, thereby achieving the effect of rapid weight gain of the Bama pigs.

[0094] Test Example Two, Application of Bama Pig Model in Preclinical Research of Weight Loss Drug COST

[0095] Experimental method: The control group Bama pigs and model group Bama pigs of Example 1 were used for preclinical research test of weight loss drug COST.

[0096] The model group Bama pigs were randomly divided into: a model control group (3), a positive drug orlistat group (3), and a COST group (3);

[0097] The model control group, the positive drug orlistat group, and the COST group were given drug intervention, and the stage of drug intervention was the weight loss period. During the weight loss period, the physiological state of the Bama pigs was observed daily, and body weight and body size indicators were measured every week. Blood biochemical tests were performed every month, and the indicators of the blood biochemical tests included fasting blood glucose, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, triglyceride, cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. After the end of the weight loss period, the weight loss effect was judged in combination with the physiological state, body weight, body size, and serum biochemical indicators;

[0098] After the end of the weight loss period, the Bama pigs are sacrificed, the back fat thickness and subcutaneous fat layer thickness are measured, and then the samples are taken, including removing the organs, fat tissue and cecal contents, the organs including: heart, liver, kidney, duodenum, ileum, colon; the fat tissue including: perirenal fat, abdominal subcutaneous fat, omental fat; the organs are weighed, embedded and frozen; the fat tissue is analyzed by a GC-MS / MS platform-based fatty acid analysis method, and the free fatty acid detection and analysis of the fat tissue are performed;

[0099] The free fatty acid detection and analysis includes the following steps:

[0100] S1, after taking out the ultra-low temperature preserved sample, thawing on ice, grinding to powder, adding methanol, methyl tert-butyl ether and 36% phosphoric acid for extraction; centrifuging and removing the supernatant, blowing dry, adding 15% boron trifluoride methanol solution; keeping in a 60-65℃ oven for 30-40min; after cooling to room temperature, adding n-hexane solution and saturated sodium chloride solution, mixing, centrifuging, and removing the n-hexane layer solution for machine analysis;

[0101] S2, using a GC-EI-MS system to analyze the sample derivative, the GC uses a DB-5MS capillary column with a specification of 30m x 0.25mm x 0.25μm; the carrier gas is high-purity helium; the heating program is as follows:

[0102] Starting from 40℃ for 2min, increasing to 200℃ at 30℃ / min, lasting for 1min, then increasing to 240℃ at 10℃ / min, lasting for 1min, finally increasing to 285℃ at 5℃ / min, lasting for 3min; flow rate: 1.0mL / min; injection port temperature: 230℃; injection volume: 1.0μL;

[0103] The GC-EI-MS system is an Agilent 8890-5977B GC-MS system, after the GC detection is completed, the parameters of the EI-MS are set as follows: temperature: 230℃; ionization voltage: 70eV; transfer line temperature: 240℃; quadrupole temperature: 150℃; solvent delay: 4min; scan mode: SIM;

[0104] S3, statistical analysis.

[0105] The specific test process is as follows:

[0106] 1. Experimental animal grouping and drug administration

[0107] The model group of Example 1 was randomly divided into three groups: model control group (Model group), positive drug orlistat group (Orlistat group) and chitooligosaccharide group (COST group), each group of 3 obese Bama pigs (2 females and 1 male). After grouping, Orlistat group and COST group were given orlistat and chitooligosaccharide intervention, respectively. The dosage was determined according to the standard weight animal from adult to small pig conversion coefficient Rab = 1.370. The Control group and model group were given corresponding starch feeding. This stage is called the weight loss period. Because the oral administration of animal models in the weight loss period is easy to cause drug waste, therefore, the drug needs to be mixed with 1 / 10 of the daily feed and completely consumed, and then the remaining 9 / 10 of the feed is fed, to ensure that the drug is fully consumed. The length of the drug administration stage is 12 weeks.

[0108] 2. Observation index and sampling

[0109] During the entire experiment, the physiological state of Bama pigs needs to be observed daily, including observing whether there are conditions such as lethargy, apathy, dry and dull skin, loose stool, pale mouth, etc. Bama pigs are weighed and measured for body size indicators once a week after fasting. Bama pigs are taken from the anterior vena cava for blood sampling and supernatant is taken for blood biochemical detection every month, and the detection indexes include fasting blood glucose (FBG), glutamic oxalate transaminase (AST), glutamic pyruvic transaminase (ALT), triglyceride (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Finally, the success of the obesity model and the weight loss effect are judged in combination with the general condition of Bama pigs, body weight, body size, and serum biochemical indexes.

[0110] After the 12-week drug administration period, Bama pigs were subjected to 16-hour fasting without water, so that they were in a relatively fasting state. Before sampling, the last body weight measurement, body size data collection and intravenous blood sampling were performed. Then the Bama pigs were anesthetized with isoflurane and then bled to death, and then the back fat thickness and subcutaneous fat layer thickness of each Bama pig were measured. After the measurement was completed, the organs of the Bama pigs were completely removed, and the organs were weighed, embedded and frozen. The organs that need to be sampled include: heart, liver, kidney, duodenum, ileum, colon. The fat tissues that need to be sampled include: board oil (perirenal fat), abdominal subcutaneous fat, omental fat. In addition, Bama pig feces (cecal contents) also need to be frozen for subsequent experimental research.

[0111] (1) Daily behavior and state observation results of Bama pigs

[0112] During the whole experiment, the daily behavior and state of each group of Bama pigs were closely observed. It was found that the Control group of piglets had good mental state, good appetite, liked to wag their tails, had self-cleaning behavior, the color of the nasal disc was light white, the hair was shiny, and the stool was formed; the mental state of the Model group of piglets was general, liked to lie down and not to move, had a weak appetite in the later stage, the eyes only moved with the feeders when feeding, moved slowly, the nose was dry, the color of the nasal disc was reddish with ecchymosis, the skin was loose, the hair was not shiny, there were a large number of fat spots on the back skin, and the stool was dark in color; the mental state of the Orlistat group of piglets was good, liked to move, liked to wag their tails, and was often irritable, the nose was moist, the color of the nasal disc was slightly white, the hair was slightly dry, there were fat spots on the back skin, but less than the Model group of piglets, the stool was slightly white, and there were oil stools; the mental state of the COST group of piglets was good, liked to wag their tails, had a waiting feeding behavior, the nose was moist, the color of the nasal disc was light white, the hair was slightly dry, there were fat spots on the back skin, but still less than the Model group of piglets, the stool was formed, and the feces slightly contained oil. From the appearance characteristics, the Model group of piglets had a bloated body shape, the abdominal skin was close to the ground when standing upright, and the hind limbs were difficult to stand; the Control group of piglets had a relatively small body size and a uniform body shape; the Orlistat group and the COST group of piglets had similar body sizes, and could stand and walk upright without difficulty, as shown in Figure 2 .

[0113] (2) Bama pig weight and weight gain results table

[0114] In order to investigate the weight loss effect of chitosan oligosaccharide on obese Bama pigs, the body weight of each group of Bama pigs before sampling was recorded, and the data analysis of body weight and weight gain was performed. The results are shown in Figure 1 , Table 3.

[0115] Table 3 shows the results of body weight and weight gain of each group of Bama pigs after administration

[0116]

[0117] From the above table, compared with the Control group, the Model group fed with high-fat feed had a significant increase in body weight and a large weight gain; while the Orlistat group and the COST group fed with the same high-fat feed had a significant decrease in body weight compared with the Model group, and the body weight increased slowly. It shows that Orlistat and chitosan oligosaccharide can reduce the body weight of Bama pigs to a certain extent, thereby improving the signs of obesity.

[0118] 3. Measurement of body size indicators

[0119] The method for evaluating body fat distribution in human is also applicable to animal models. The indexes for analyzing the whole body fat composition in clinic are height and weight, sometimes including waist circumference and skinfold thickness. In this experiment, the multi-circumference of Bama pigs was measured, including body length, neck circumference, chest circumference, abdominal circumference and waist circumference. The measurement methods are as follows.

[0120] Body length: the straight-line distance from the midpoint of the line connecting the two ear roots of the Bama pig to the first scale wheel at the tail root along the back ridge of the Bama pig was measured with a tape measure, which was the body length.

[0121] Neck circumference: the circumference of the neck of the Bama pig was measured with a tape measure at the position of the back edge of the ear, and the tightness was appropriate when the tape was naturally close to the fur.

[0122] Chest circumference: the length of one circle around the body with the posterior margin of the scapula of the Bama pig as the boundary was measured with a tape measure, which was the chest circumference.

[0123] Abdominal circumference: the distance around the abdomen of the Bama pig through the lowest point of the abdomen was measured with a tape measure, which was the abdominal circumference.

[0124] Waist circumference: the circumference around the body close to the root of the hind leg of the Bama pig was measured, which was the waist circumference.

[0125] Body size data is an important indicator for measuring the body shape of small pigs. In this experiment, the body length, neck circumference, chest circumference, abdominal circumference and waist circumference of Bama pigs were measured. The body length was the longitudinal height, and the other four body size indicators were the transverse circumference.

[0126] The measurement results are shown in Figure 3 Table 4.

[0127] Table 4. Body size information of Bama pigs in each group after administration (n=3, mean±SEM)

[0128]

[0129]

[0130] From the above table, the body length of the Control group Bama pigs was smaller than that of the rest of the Bama pigs fed with high-fat feed, while there was no significant difference between the Model group, Orlistat group and COST group fed with high-fat diet, indicating that drug intervention would not inhibit the growth of the body length of Bama pigs, and the body length was more related to dietary factors. In the neck circumference index, the neck circumference of the Control group was significantly smaller than that of the Model group, and there was a significant difference between the two. The neck circumference of the Orlistat group and the COST group was also significantly smaller than that of the Model group, and there was a significant difference between the two, while there was no significant difference between the Orlistat group and the COST group. Compared with the Control group, the chest circumference of the Model group Bama pigs increased significantly, while the chest circumference of the Orlistat group and the COST group decreased significantly compared with the Model group. In the abdominal circumference index, compared with the Control group, the abdominal circumference of the Model group Bama pigs increased significantly, while the abdominal circumference of the Orlistat group and the COST group Bama pigs also decreased significantly compared with the Model group. The data analysis of the waist circumference index showed that the waist circumference of the Control group Bama pigs was significantly smaller than that of the Model group Bama pigs, and the waist circumference of the Orlistat group and the COST group was significantly smaller than that of the Model group. The above data all showed that feeding high-fat feed could accelerate the obesity of Bama pigs, and chitosan could significantly improve the degree of obesity caused by high-fat feed.

[0131] 4. Measurement of subcutaneous fat thickness

[0132] Back fat thickness: After the Bama pigs were sacrificed, the back subcutaneous fat thickness of the Bama pigs at the positions of about 4 cm away from the back median line at the positions of scapula, last rib and lumbosacral joint was measured using a vernier caliper. Then the average of the three positions was calculated, which was called "BFT" index. BFT was important for measuring the back subcutaneous fat content.

[0133] Abdominal subcutaneous fat thickness: After the pigs were sacrificed, the subcutaneous fat thickness of the Bama pigs at the lowest part of the abdomen was measured using a vernier caliper. In the obese animal model, obesity degree is often used as an index to measure whether the model is successfully modeled and whether the weight loss effect is achieved. If the obesity degree is greater than 20%, it indicates obesity.

[0134] Table 5 Results of obesity degree and fat thickness of Bama pigs in each group after administration (n=2-3, mean±SEM)

[0135]

[0136]

[0137] As shown in the above table, after 12 weeks of drug intervention, the obesity degree of the Orlistat group and the COST group decreased and was lower than 20%, while the obesity degree of the Model group continued to rise and exceeded 30%. As shown in FIG. B of Figure 4 As shown in FIG. B of Figure 4 As shown in FIG. C of

[0138] 5. Biochemical index determination

[0139] Blood collection method: The Bama pig was laid down in a supine position, the front chest fossa was exposed, the needle was disinfected with 75% ethanol, a sterile 10 mL syringe was used to vertically puncture the anterior venous sinus, and 2 mL of blood was slowly drawn.

[0140] The drawn venous blood was slowly injected into the blood collection tube to avoid hemolysis caused by blood cell rupture, and then the blood in the tube was immediately centrifuged at 3500 rpm for 15 min to obtain the upper serum for blood index detection.

[0141] Blood index: The levels of TG, TC, HDL-C, LDL-C, FFA, ALT, AST, GLU in the serum sample were determined by using the ordinary kit according to the corresponding instruction manual, and the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α), fasting insulin level (Insulin) and endotoxin level (LPS) in the serum sample were determined by using the ELISA kit.

[0142] Liver / fat tissue index: 0.100 g of liver tissue or fat tissue was weighed using a one-thousandth analytical balance in a 1.5 mL EP tube, 0.9 mL of normal saline and 2 grinding beads were added to the tube, and finally the prepared tube was subjected to liver tissue or fat tissue homogenization in a full-automatic homogenizer. After homogenization, the tube with the tissue was placed in a 4°C, 2500 r / min high-speed centrifuge for 10 min. After centrifugation, the supernatant was taken and used to determine the levels of IL-1β, IL-6, IL-10, TNF-α in the liver of Bama pigs (GLU, AST, ALT, TG, TC, LDL-C) and fat tissue using a general kit and an ELISA kit.

[0143] The experimental results are shown in Figure 7 、 8 , 13, 14 and Tables 6-9.

[0144] Table 6 Results of blood glucose-related indicators of Bama pigs in each group after administration (n = 3, mean ± SEM)

[0145] FBG (mmol / L) Insulin (mU / L) Insulin resistance index Control group 4.017±0.04807 11.65±1.6560 2.086±0.3162* Model group 5.290±0.53260 18.90±2.4240 4.523±0.9273 Orlistat group 4.497±0.29160 13.85±1.5350 2.762±0.3510 COST group 4.280±0.27430 10.15±1.5930* 1.966±0.4052*

[0146] Table 7 Results of serum AST and ALT indicators of Bama pigs after administration (n = 3, mean ± SEM)

[0147] AST (U / L) ALT (U / L) Control group 20.90±2.2000* 25.07±1.9630* Model group 27.30±0.8963 32.83±1.7900 Orlistat group 26.10±0.5568 25.30±1.0150* COST group 21.20±1.7210* 24.40±1.9010*

[0148] Table 8 Results of changes in serum lipid levels of Bama pigs after administration (n = 3, mean ± SEM)

[0149]

[0150] Table 9 Results of liver biochemical indicators of Bama pigs after administration (n = 3, mean ± SEM)

[0151]

[0152]

[0153] 6. Pathological section and staining

[0154] HE staining: Place the embedding box containing the tissue in a 4% paraformaldehyde solution for 24 hours, then place the fixed embedding box in a full-automatic dehydrator for stepwise dehydration; after dehydration, paraffin embedding is performed. The embedded paraffin tissue is pre-cooled and then sliced using a microtome, and the glass slide with the tissue is stained with hematoxylin and eosin. The staining process and time are based on the laboratory's previous experience. Finally, the stained slices are stored in a slide box for subsequent microscopic observation.

[0155] Oil red O staining: The embedding box containing the tissue was placed in a 4% paraformaldehyde solution for 24 hours, and then the embedding box was sequentially placed in a 15% sucrose solution for 14 hours, a 20% sucrose solution for 14 hours, and a 30% sucrose solution for 14 hours for step-by-step dehydration. After dehydration, the tissue was embedded with OCT embedding agent. After embedding, it was stored in a -20°C refrigerator. Then, a frozen section machine was used to make a section with a thickness of 8 μm. The section was stained with oil red O staining solution. Finally, the stained section was stored in a section box for subsequent microscopic observation.

[0156] To further understand the deposition of lipid droplets in the liver tissue of obese Bama pigs, the liver paraffin sections of the Bama pigs in each group were subjected to HE staining, and the frozen sections were subjected to oil red O staining. The HE staining of the liver of the Bama pigs is shown in Figure 9 (A). The liver cells of the Bama pigs in the Control group were arranged regularly, and the cytoplasm was uniform. The liver cells of the Bama pigs in the Model group contained lipid droplets and vacuoles of different sizes in the cytoplasm. After the intervention of COST, the lipid vacuoles of the Bama pigs in the COST group were significantly reduced compared with those of the Bama pigs in the Model group, and the arrangement of the liver cells was obviously improved. The oil red O staining of the liver of the Bama pigs is shown in Figure 9 (B). Compared with the Bama pigs in the Control group, the liver of the Bama pigs in the Model group contained a large number of red lipid droplets, while the lipid droplets in the liver of the Bama pigs in the COST group were significantly reduced. In combination with the results of HE staining and oil red O staining, COST can reduce the fatty degeneration of the liver of obese Bama pigs and reduce the accumulation of lipid droplets in the liver.

[0157] In Figure 10 (A), the myocardial cells of the four groups of Bama pigs were in normal morphology, and the myocardial cells did not appear hypertrophy. In Figure 10 (B), the kidney cells of the four groups of Bama pigs were in normal morphology, the glomerulus and renal corpuscle were clearly visible, and the nuclear membrane was clear. According to the HE staining and in combination with the appearance of the kidney of each group of Bama pigs, it was judged that the kidney tissue was in a normal state. Therefore, according to the pathological sections, it was preliminarily concluded that COST had no obvious toxic side effects on the heart and kidney tissues of Bama pigs.

[0158] Adipose tissue is one of the important indicators for measuring obesity. In this study, the subcutaneous fat and board oil (perirenal fat) of the Bama pigs in each group were subjected to HE staining. As shown in Figure 11 (A), the subcutaneous fat cells of the Bama pigs in the Control group were uniform in size, and the number of cells was relatively large. The subcutaneous fat cells of the Bama pigs in the Model group were large in size, and the nuclear membrane was not clear. After the administration of COST, the number of cells was obviously increased, and the size of the fat cells was reduced. In the perirenal board oil fat of the Bama pigs in each group, the fat cells of the Bama pigs in the Control group were uniform in size, and the number of cells was relatively large. The fat cells of the Bama pigs in the Model group were large in size, and the nuclear membrane was not clear. After the administration of COST, the number of cells was obviously increased, and the size of the fat cells was reduced. Figure 11In (B), the Model group of Bama pigs had large cell volume and less number of plate oil tissue, and the nuclear membrane was not clear. However, the Control group and the COST group of Bama pigs could significantly improve the state of the plate oil cells of the Model group of Bama pigs.

[0159] In order to observe the morphological structure of the intestinal tract of Bama pigs, HE staining was performed on the ileum and colon tissues of Bama pigs. As shown in (A), the intestinal villi and muscle layer of the Model group of Bama pigs were damaged, the barrier function was weakened, and the intestinal crypt depth was shallower. Compared with the Model group, the muscle layer of the Control group and the COST group was relatively complete, and the crypt depth was deeper. In the HE staining of the colon of Bama pigs Figure 12 (A), the fat cells of the Model group of Bama pigs had infiltrated the intestinal muscle layer, the goblet cells that secreted mucus were abnormal in shape, and the number was reduced. Compared with the Model group, the number of goblet cells in the Control group and the COST group was significantly increased, and more mucus could be secreted, which was helpful to reduce the damage of fecal balls to the intestinal mucosa. This indicates that chitooligosaccharide can reduce the damage of high-fat diet to the intestinal tract of Bama pigs and is beneficial to intestinal metabolism. Figure 12 (B), the fat cells of the Model group of Bama pigs had infiltrated the intestinal muscle layer, the goblet cells that secreted mucus were abnormal in shape, and the number was reduced. Compared with the Model group, the number of goblet cells in the Control group and the COST group was significantly increased, and more mucus could be secreted, which was helpful to reduce the damage of fecal balls to the intestinal mucosa. This indicates that chitooligosaccharide can reduce the damage of high-fat diet to the intestinal tract of Bama pigs and is beneficial to intestinal metabolism.

[0160] In summary, feeding chitooligosaccharide can reduce the body weight and weight gain of obese Bama pigs, reduce blood lipid, reduce blood sugar, improve inflammation in serum and adipose tissue, improve liver function, and reshape the intestinal structure of piglets, which has certain weight loss activity and lays a foundation for further mechanism exploration.

[0161] 7. Detection of Bama pig fat free fatty acid composition

[0162] Fatty acids (FA) exist in different forms in organisms and participate in lipid metabolism as key compounds. Fatty acid detection uses mass spectrometry scanning mode to select ion monitoring mode (SIM). After the sample molecules are ionized, only the pre-selected target ions can pass through the quadrupole to the detector for detection, while other ions cannot pass through the quadrupole to the detector. Selective ion monitoring mode can effectively remove matrix interference, improve sensitivity, and better complete quantitative and qualitative analysis of Bama pig free fatty acid composition. Based on the GC-MS / MS platform, the fatty acid analysis method was used to analyze the free fatty acid composition of each group of Bama piglets, and the molecular level mechanism of COST in lipid metabolism and synthesis was further explored.

[0163] In this experiment, 48 kinds of free fatty acids in Bama pig fat tissue were detected, and the composition information is as follows.

[0164] Table 10 Information table of 48 kinds of fatty acids

[0165]

[0166]

[0167]

[0168] Four groups of samples, a total of 12 samples, were selected for detection. The sample information is shown in the table below.

[0169] Table 11 Sample information table

[0170] Species Tissue Group Serial number Pig Subcutaneous adipose tissue Control 722-4 Pig Subcutaneous adipose tissue Control 722-8 Pig Subcutaneous adipose tissue Control 1209-15 Pig Subcutaneous adipose tissue Model 722-5 Pig Subcutaneous adipose tissue Model 722-6 Pig Subcutaneous adipose tissue Model 1209-13 Pig Subcutaneous adipose tissue Orlistat 1213-1 Pig Subcutaneous adipose tissue Orlistat 722-10 Pig Subcutaneous adipose tissue Orlistat 709-1 Pig Subcutaneous adipose tissue COST 715-1 Pig Subcutaneous adipose tissue COST 722-7 Pig Subcutaneous adipose tissue COST 1209-14

[0171] The specific steps of the free fatty acid detection method are as follows:

[0172] 7.1 Sample pretreatment

[0173] (1) Take the sample out of the -80°C refrigerator and thaw on ice (all subsequent operations require being performed on ice)

[0174] (2) Take the biological sample stored in the ultra-low temperature out and grind it with a grinder (30 Hz, 1 min to a powder.

[0175] (3) Accurately weigh 50 mg of the ground sample into a new EP tube, add 150 μL of methanol solution, 200 μL of methyl tert-butyl ether solution, and 50 μL of 36% phosphoric acid solution for extraction.

[0176] (4) Vortex for 3 min, and centrifuge at 4°C, 12000 r / min for 5 min.

[0177] (5) Take 200 μL of the supernatant and dry it with a nitrogen evaporator, and add 300 μL of 15% boron trifluoride methanol solution.

[0178] (6) Vortex for 3 min, and keep in a 60°C oven for 30 min.

[0179] (7) Cool to room temperature, accurately add 500 μL of n-hexane solution, and 200 μL of saturated sodium chloride solution.

[0180] (8) Vortex for 3 min, and after centrifugation at 4°C, 12000 r / min for 5 min, take 100 μL of the n-hexane layer solution for machine analysis.

[0181] 7.2 Chromatography mass spectrometry collection and analysis

[0182] The sample derivatives were analyzed using a GC-EI-MS system (GC, Agilent 8890, https: / / Agilent.com.cn / ; MS, 5977B System, https: / / Agilent.com.cn / ). The analysis conditions were as follows: GC: column, DB-5MS capillary column (30 m x 0.25 mm x 0.25 μm, Agilent); carrier gas, high-purity helium (purity > 99.999%); heating program starting from 40 °C (2 min), increasing by 30 °C / min to 200 °C (1 min), then increasing by 10 °C / min to 240 °C (1 min), and finally increasing by 5 °C / min to 285 °C (3 min); flow rate: 1.0 mL / min; injection port temperature: 230 °C; injection volume: 1.0 μL

[0183] Agilent 8890-5977B GC-MS system, temperature 230 °C; ionization voltage: 70 eV; transfer line temperature: 240 °C; quadrupole temperature: 150 °C; solvent delay: 4 min; scan mode: SIM.

[0184] 7.3 Statistical analysis

[0185] (1) Principal component analysis (PCA)

[0186] Unsupervised principal component analysis (PCA) was performed using the statistical function prcomp in www.r-project.org. The data needed to be unit-variance scaled before unsupervised PCA was performed.

[0187] (2) Hierarchical cluster analysis and Pearson correlation coefficient

[0188] A tree map was drawn according to the hierarchical cluster analysis (HCA) of samples and metabolites, while the Pearson correlation coefficient (PCC) between samples was calculated by the cor function in R and only presented in the form of a heat map. Both the hierarchical cluster analysis and the Pearson correlation coefficient were performed by the R package pheatmap. For the hierarchical cluster analysis, the normalized signal intensity (unit-variance scaling) of metabolites was visualized in the form of a color gradient. 7.4 Results of detection of different fatty acid contents in the fatty tissue of Bama pigs

[0189] The main components of free fatty acids in the fat tissues of each group of Bama pigs were detected, and the results were grouped and statistically analyzed, and the results are shown in the following table. By comparing the results, it was found that for C6-0, C11-0, C15-0, C16-2, C17-0, C22-6n3, C18-1n9c, C18-1n9t, C19-0, C23-0, C20-3n3, C22-1n9 components, their contents in the Model group Bama pigs were significantly higher than those in the Control group, which can be speculated that high-fat diet-induced obesity in Bama pigs may interfere with the normal lipid metabolism process by increasing the synthesis of these fatty acid components. Similarly, there were significant differences in some fatty acids between the COST administration group and the Model group. Specifically, for C6-0, C11-0, C12-0, C15-0, C16-2, C17-0, C22-6n3, C18-1n9c, C18-1n9t, C19-0, C23-0, C20-1(cis-11), C20-3n3, C20-4n6, C21-0, C22-1n9 components, the contents of Bama pigs in the COST group were significantly lower than those in the Model group, and the contents of these components in the Model group were significantly higher than those in the Control group. This shows that COST administration can reduce the content of free fatty acids that cause obesity and thus alleviate the fat accumulation state induced by high-fat diet.

[0190] Table 12: Content of each component of free fatty acids in fat tissue of Bama pigs (n=3, mean ± SD)

[0191]

[0192] 7.5 Principal component analysis of Bama pig fat samples

[0193] By the method of multivariate statistical analysis, high-dimensional complex data can be reduced in dimension, and the original data can be saved as much as possible. This method helps to accurately establish a mathematical model, thereby inducing the metabolic profile characteristics of the sample. Principal component analysis (PCA) is a multivariate data statistical analysis method of unsupervised pattern recognition, which converts a set of variables that may be correlated into a set of linearly uncorrelated variables through orthogonal transformation. The converted set of variables is called principal components. The results are shown in Figure 5 . Figure 5 PC1 represents the first principal component, PC2 represents the second principal component, and PC3 represents the third principal component. The percentage represents the explanation rate of the principal component to the data set. Each point in the figure represents a sample, and samples in the same group are represented by the same color. Group is the grouping.

[0194] The overall metabolic differences between the groups of Bama pig fat tissues and the metabolic differences within the groups of Bama pig fat tissues were understood by performing PCA analysis on the fat tissue samples of each group. The PCA results can show the metabolic group trends between each group and suggest the metabolic group situation within each group of fat tissues. The QC is a quality control sample. The results are shown in Figure 5 As shown in the principal component analysis (PCA) score plot, the principal components showed a significant deviation in the fat samples of the Control group and the Model group, and there was a significant metabolic difference between the two groups. This indicates that the lipid metabolism of the Bama pig fat tissue in the Model group was significantly disturbed. In the Bama pig samples after COST administration, the principal components of the metabolites were closer to the Control group. In summary, COST treatment can partially restore the metabolic characteristics of Bama pig fat tissue, reduce the metabolic difference between the Control group and the Model group, and suggest that COST may have a certain impact on regulating the lipid metabolism of Bama pig fat tissue.

[0195] 7.6 Cluster analysis of Bama pig fat sample metabolites

[0196] Cluster analysis is a classification multivariate statistical analysis method. This method classifies individuals or samples based on their characteristics, aiming to make individuals within the same category as homogeneous as possible and as heterogeneous as possible between categories. Through cluster analysis, the accumulation of fat metabolites in different Bama pig samples can be more intuitively observed.

[0197] In this experiment, the Bama pig fatty acid metabolite data was processed by UV (unit variance scaling), and the cluster heat map was drawn using the R program script, as shown in Figure 6 .

[0198] As can be seen from Figure 6 , for fatty acids C6-0, C11-0, C15-0, C16-2, C17-0, C22-6n3, C18-1n9c, C18-1n9t, C19-0, C23-0, C20-3n3 and C22-1n9, etc., the Bama pig samples in the Model group showed a higher content, which was significantly higher than that in the Control group. However, in the COST administration group, the cluster content of these fatty acids was significantly lower than that in the Model group. This is consistent with the results of the single sample metabolite detection in the previous section, and further confirms that high-fat diet-induced obesity in Bama pigs may interfere with normal fat metabolism by increasing the synthesis of these fatty acid components. COST administration can reduce the content of free fatty acids that cause obesity, thereby alleviating the fat accumulation state induced by high-fat diet.

[0199] Free fatty acid metabolites play an important role in obesity. In obese patients, the level of free fatty acids in the body is usually increased. These free fatty acids are the products of the hydrolysis of triglycerides in adipocytes, and can be transported to other tissues through the blood circulation and used as an energy source. However, when the level of free fatty acids is too high, it can cause problems such as inflammation of adipocytes, insulin resistance, and fat accumulation. Studies have shown that excessive free fatty acids can promote the growth and number of adipocytes, leading to hypertrophy and excessive accumulation of adipose tissue, i.e. free fatty acid metabolites may also affect the proliferation and differentiation of adipocytes, and thus affect the physiological function of adipose tissue, in which case the normal metabolic function of adipose tissue may be disrupted. In addition, excessive free fatty acids can interfere with lipid synthesis and degradation pathways, leading to lipid metabolism disorders and inflammation of adipose tissue, thereby exacerbating the development of obesity.

[0200] In summary, COST treatment plays a certain role in restoring the metabolic characteristics of the adipose tissue of Bama pigs, helping to reduce the metabolic differences between the Control and Model groups. These findings provide new clues for the study of treatment strategies for obesity-related metabolic disorders, and are worth further research and discussion.

Claims

1. A Bama pig model for preclinical study of weight loss drugs COST, characterized in that, The method for constructing the Bama pig model comprises the following steps: healthy Bama pigs are selected and divided into control group Bama pigs and model group Bama pigs; the control group Bama pigs are fed with ordinary feed; the model group Bama pigs are first fed with ordinary feed, and then fed with high-fat feed; the control group Bama pigs and the model group Bama pigs are detected for obesity-related indexes at a fixed time period every week; when the obesity degree of the model group Bama pigs is greater than 20%, the Bama pig model is obtained; the obesity degree is calculated according to the formula: (the body weight of the model group Bama pigs - the average body weight of the control group Bama pigs) / the average body weight of the control group Bama pigs) * 100%; the high-fat feed comprises the following components in mass fraction: 49-50 parts of ordinary feed, 9-10 parts of beef tallow, 10-11 parts of artificial butter, 14-15 parts of sucrose, 9-10 parts of casein, 2-2.5 parts of experimental animal premix, 1-1.5 parts of microcrystalline cellulose and 2-2.5 parts of calcium bicarbonate; the high-fat feed further comprises the following components in mass fraction: 0.1-0.3 parts of gypenoside, 0.5-1.0 parts of mint leaves, 0.2-0.3 parts of maslinic acid, 0.1-0.15 parts of ursolic acid, 1-2 parts of atractylodes, and 0.1-0.15 parts of nutmeg.

2. The Bama pig model of claim 1, wherein, the ordinary feed comprises the following components in mass fraction: 25-26 parts of corn, 3-4 parts of fish meal, 8-9 parts of soybean meal, 9-10 parts of rice bran, 18-20 parts of wheat bran, 30-32 parts of alfalfa meal, 2-3 parts of calcium bicarbonate, 0.3-0.4 parts of iodized salt, 0.03-0.04 parts of trace element additive and 0.03-0.032 parts of vitamin additive.

3. The Bama pig model of claim 2, wherein, the trace element additive is composed of the following components in mass fraction: 7.7-12 parts of copper, 41-61 parts of iron, 22-33 parts of zinc, 14.1-21 parts of manganese, 0.17-0.33 parts of iodine, 0.12-0.23 parts of selenium, 0.07-0.13 parts of cobalt and 9.46-14.19 parts of water; the vitamin additive is composed of the following components in mass fraction: 19-19.5 parts of vitamin A, 0.4-0.45 parts of vitamin D3, 16.75-16.8 parts of vitamin E, 5-5.1 parts of vitamin K3, 2-2.1 parts of vitamin B1, 16-16.5 parts of vitamin B2, 10-10.1 parts of vitamin C, 6-6.05 parts of vitamin B6, 0.03-0.031 parts of vitamin B12, 35-35.5 parts of nicotinic acid, 25-25.5 parts of calcium pantothenate, 0.5-0.51 parts of folic acid, 0.1-0.11 parts of biotin, 50-50.5 parts of methionine and 50-50.5 parts of lysine; the experimental animal premix is composed of 1-1.05 parts of the vitamin additive, 1-1.05 parts of the trace element additive, 10-10.5 parts of salt and 88-89 parts of corn flour.

4. The Bama pig model of claim 1, wherein, the healthy Bama pigs are 4-6-month-old ordinary-grade Bama pigs with a body weight of 20-30 kg.

5. The Bama pig model of claim 1, wherein, The model group of the Bama pigs is fed with common feed for 1.0-1.1 weeks, and then fed with high-fat feed until the obesity degree of the model group of the Bama pigs is greater than 20%; The feeding is to give the common feed or the high-fat feed every day, and the weight of the common feed or the high-fat feed used for feeding is 3.0-3.01% of the body weight of the Bama pigs in the week.

6. The use of the Bama pig model according to any one of claims 1-5 in the preclinical study of the weight loss drug COST.

7. Use according to claim 6, characterized in that, The model group of the Bama pigs is used as the model group, and the Bama pigs fed with common feed are used as the control group; The model group is randomly divided into a model control group, a positive drug orlistat group and a COST group; The model control group, the positive drug orlistat group and the COST group are subjected to drug intervention, and the stage of the drug intervention is a weight loss period; During the weight loss period, the physiological state of the Bama pigs is observed daily, the body weight is weighed and the body size index is measured every week, blood biochemical detection is performed every month, the index of the blood biochemical detection includes fasting blood glucose, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, triglyceride, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, and the weight loss effect is judged in combination with the physiological state, the body weight, the body size, the serum biochemical index after the weight loss period ends; After the weight loss period ends, the Bama pigs are sacrificed, the back fat thickness and the subcutaneous fat layer thickness are measured, and then the samples are taken; The samples include the removal of the viscera, the adipose tissue and the cecal contents; The viscera is subjected to weighing, embedding and cryopreservation treatment; The viscera includes the heart, the liver, the kidney, the duodenum, the ileum and the colon, and the adipose tissue includes the perirenal fat, the abdominal subcutaneous fat and the omental fat; The adipose tissue is subjected to free fatty acid detection and analysis by using a fatty acid analysis method based on a GC-MS / MS platform, so as to study the molecular level mechanism of COST in lipid metabolism and synthesis.

8. Use according to claim 7, characterized in that, The free fatty acid detection and analysis specifically include the following steps: S1, sample pretreatment; S2, analysis of sample derivatives by using a GC-EI-MS system, a chromatographic column used for GC is a DB-5MS capillary column with a specification of 30 m x 0.25 mm x 0.25 μm, high-purity helium is used as the carrier gas, and the heating program is as follows: starting from 40℃ for 2 minutes, increasing to 200℃ at a rate of 30℃ / min for 1 minute, then increasing to 240℃ at a rate of 10℃ / min for 1 minute, finally increasing to 285℃ at a rate of 5℃ / min for 3 minutes; flow rate: 1.0 mL / min; injection port temperature: 230℃; injection volume: 1.0 μL; The GC-EI-MS system is an Agilent 8890-5977B GC-MS system, and after the GC detection is completed, the parameters of the EI-MS are set as follows: temperature: 230℃; ionization voltage: 70 eV; transfer line temperature: 240℃; quadrupole temperature: 150℃; solvent delay: 4 minutes; scan mode: SIM; S3, statistical analysis. The sample pretreatment in step S1 specifically includes the following steps:

9. Use according to claim 8, characterized in that, ​ After the sample stored in ultra-low temperature is taken out, it is thawed on ice, ground into powder, and extracted by adding methanol, methyl tert-butyl ether and 36% phosphoric acid; the supernatant is removed by centrifugation, dried, and then 15% boron trifluoride methanol solution is added; the mixture is kept in a 60-65°C oven for 30-40 min; after cooling to room temperature, n-hexane solution and saturated sodium chloride solution are added, mixed, centrifuged, and the n-hexane layer solution is removed for machine analysis.

Images