Bama pig model for preclinical research of weight-reducing drug COST and application of Bama pig model

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a Bama pig model for preclinical research of a weight-reducing medicine COST and application of the Bama pig model. Healthy Bama pigs are firstly adaptively fed with common feed and then fed with high-fat feed for molding, and obesity-related indexes of the Bama pigs are detected in a fixed period of time every week; the high-fat feed comprises common feed, beef tallow, margarine, cane sugar, casein, laboratory animal premix, microcrystalline cellulose, calcium bicarbonate, gypenosides, mint leaves, maslinic acid, ursolic acid, bighead atractylodes rhizome and nutmeg. According to the invention, the Bama pig model with stable phenotype can be rapidly constructed, a reliable model is provided for preclinical research of new drugs for treating obesity, and guarantee is provided for research of obesity mechanisms and screening, development and mechanism research of the new drugs for treating obesity.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a Bama pig model and application thereof for preclinical research of a weight-loss drug COST. Background Art

[0002] According to the World Health Organization (WHO) definition of disease, obesity refers to excessive accumulation of body fat, an abnormal state that can damage health, and it was officially listed as one of the endocrine diseases in 1997. Obesity is associated with an increased risk of premature death and increases the incidence of complications of various diseases, 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 patient's psychological, emotional and cognitive abilities. At present, the global obesity rate in the world continues to rise, 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 treatments.

[0003] Mouse models are commonly used mammalian models in the screening and preclinical research stages of new drugs, but the physiological differences between mice and humans lead to limitations in using mice for disease simulation. For example, when studying metabolic diseases such as diabetes, due to differences in metabolic levels such as sugar metabolism and lipid metabolism between mice and humans, even if the corresponding phenotypes are induced in mice through drugs or genetic means, the disease development process and pathophysiological mechanisms are not completely consistent with those of humans, resulting in the research results not being directly applicable to humans.

[0004] In preclinical drug research, due to differences in drug metabolism enzyme systems and drug transporters between mice and humans, the metabolic process and pharmacokinetic properties of drugs in humans and mice are different. Therefore, some drugs that are effective in mouse models may not be effective 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 smaller physiological differences 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 of some chronic diseases are closer to humans in terms of time scale. The size, structure and physiological function of Bama pig organs are more similar to those of humans, and their nutritional needs and intake patterns are similar to those of humans. The absorption and metabolic responses to different nutrients are also similar, and they can more accurately simulate the physiological conditions of humans. By controlling the intake and composition of Bama pig feed, the occurrence mechanism of obesity that is close to human physiological conditions can be better simulated and more reliable data support can be provided for research.

[0006] CN110862988A discloses an sgRNA and a CREBRF point mutation Bama Xiang pig constructed therefrom and its application. However, the cost of modeling through gene editing is high. At the same time, there may be long-term safety issues in using gene-edited animal models to screen drugs. The edited genes may produce some unknown and potential effects, such as causing tumors, immune system abnormalities, etc. These problems may be overlooked in the drug screening process, but will be exposed in subsequent clinical trials or clinical applications.

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

[0008] In summary, the prior art still lacks a standardized Bama pig model construction method that can successfully construct a Bama pig model with a stable obesity phenotype in a short period of time. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a Bama pig model for preclinical research of the weight-loss drug COST, which has a short construction time and a stable obesity phenotype, providing a reliable animal model for preclinical research of new drugs for the treatment of obesity, and providing a guarantee for the study of the disease characteristics and pathogenesis of obesity, as well as the screening, development and mechanism research of new drugs for the treatment of obesity.

[0010] To achieve the above objectives, the first aspect of the present invention provides a method for constructing a Bama pig model, which is as follows:

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

[0012] The Bama pigs in the control group were fed with ordinary feed;

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

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

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

[0016] Preferably, the high-fat feed also includes the following components by mass: 0.1-0.3 parts of gypenosides, 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 macrocephala, and 0.1-0.15 parts of nutmeg. The preferred gypenosides, Atractylodes macrocephala, and nutmeg of the present invention can promote the absorption of nutrients in high-fat feed by Bama pigs through reasonable matching. Due to the presence of more oil in high-fat feed, long-term feeding of high-fat feed by Bama pigs will cause indigestion and reduced absorption efficiency. The preferred gypenosides, Atractylodes macrocephala, and nutmeg of the present invention are more suitable for the physique of Bama pigs, and have a better effect on promoting their digestion and absorption. At the same time, mint leaves, maslinic acid, and ursolic acid are added to improve the taste of feed. At the same time, maslinic acid and ursolic acid can also promote the secretion of digestive juices by Bama pigs, further promoting their digestion and absorption.

[0017] Preferably, the common feed comprises the following components in parts by mass: 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 additives, and 0.03-0.032 parts of vitamin additives.

[0018] Preferably, the trace element additive is composed of the following components by mass: 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 by mass: 19 to 19.5 parts of vitamin A; 0.4 to 0.45 parts of vitamin D3; 16.75 to 16.8 parts of vitamin E; 5 to 5.1 parts of vitamin K3, 2.1 parts of vitamin B12, 16 to 16.5 parts of vitamin B2, 10 to 10.1 parts of vitamin C, 6 to 6.05 parts of vitamin B6, 0.03 to 0.031 parts of vitamin B12, 35 to 35.5 parts of niacin, 25 to 25.5 parts of calcium pantothenate, 0.5 to 0.51 parts of folic acid, 0.1 to 0.11 parts of biotin, 50 to 50.5 parts of methionine, and 50 to 50.5 parts of lysine;

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

[0021] Preferably, the healthy Bama pig is a 4-6 month old ordinary grade Bama pig, weighing 20-30 kg.

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

[0023] The feeding is to give common feed or high-fat feed every day, and the weight of the common feed or high-fat feed used for feeding is 3.0-3.01% of the body weight of the Bama pig in that week.

[0024] Preferably, the method for preparing common feed and / or high-fat feed comprises the following steps: weighing the required raw materials, crushing the raw materials, mixing them evenly, adding the mixed powdered feed into a feed pelletizer to make feed pellets, cooling, and packaging them into finished feed.

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

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

[0027] The model group was randomly divided into: model control group, positive drug orlistat group and COST group;

[0028] 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;

[0029] During the weight loss period, the physiological state of the Bama pigs is observed daily, and the weight and body size indicators are measured weekly; blood biochemical tests are performed monthly, and the indicators of the blood biochemical tests include fasting blood sugar, aspartate aminotransferase, alanine aminotransferase, triglycerides, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol; after the weight loss period, the weight loss effect is judged based on the physiological state, weight, body size, and serum biochemical indicators;

[0030] After the weight loss period, the Bama pigs were killed, the back fat thickness and subcutaneous fat layer thickness were measured, and then the samples were collected;

[0031] The sampling includes taking out organs, adipose tissue and cecal contents;

[0032] The organs are weighed, embedded and cryopreserved;

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

[0034] The adipose tissue includes: perirenal fat, abdominal subcutaneous fat, and greater omentum fat;

[0035] The adipose tissue adopts a fatty acid analysis method based on a GC-MS / MS platform to detect and analyze free fatty acids in the adipose tissue, and 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. The sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with a specification of 30 m × 0.25 mm × 0.25 μm. The carrier gas was high-purity helium. The heating procedure was as follows:

[0039] Start at 40°C for 2 minutes, increase to 200°C at 30°C / min, continue for 1 minute, increase to 240°C at 10°C / min, continue for 1 minute, and finally increase to 285°C at 5°C / min, continue for 3 minutes; flow rate: 1.0mL / min; injection port temperature: 230°C; injection volume: 1.0μL;

[0040] The GC-EI-MS system was an Agilent 8890-5977B GC-MS system. After the GC detection was completed, the EI-MS parameters were set as follows: temperature: 230°C; ionization voltage: 70 eV; transfer line temperature: 240°C; quadrupole temperature: 150°C; solvent delay: 4 minutes; scan mode: SIM;

[0041] S3. Perform statistical analysis.

[0042] Preferably, the sample pre-processing in step S1 specifically includes the following steps:

[0043] After taking out the ultra-low temperature stored sample, thaw it on ice, grind it into powder, add methanol, methyl tert-butyl ether, and 36% phosphoric acid for extraction; centrifuge to remove the supernatant, blow dry, and add 15% boron trifluoride methanol solution; keep it in an oven at 60-65°C for 30-40 minutes; after cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and remove the n-hexane layer solution for analysis on the machine.

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

[0045] (1) The present invention provides a method for constructing a Bama pig model, which includes feeding a homemade high-fat feed with a reasonable ratio of lipids and carbohydrates, and adding natural ingredients such as mint leaves and hawthorn acid to promote the appetite and digestive absorption capacity of the gastrointestinal tract of the Bama pig. After the modeling is completed, each Bama pig individual maintains a high weight state, and the weight gain of individuals in the group is relatively stable between the current week and the previous week. A Bama pig model with a stable obesity phenotype can be successfully constructed within 12 weeks, shortening the modeling time of the Bama pig obesity model and improving the modeling efficiency.

[0046] (2) The present invention provides a gas chromatography-mass spectrometry experimental method for detecting free fatty acids in Bama pigs, which can analyze the composition and content of free fatty acids in the adipose tissue metabolites of each group of Bama pigs, thereby helping to further explore the effects of drugs on specific Bama pig fat differential metabolites, which is helpful for preclinical drug research. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0049] Figure 3 The body length (A), neck circumference (B), chest circumference (C), abdominal circumference (D), and hip circumference (E) of Bama pigs in each group 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 Obesity (A), back fat (B) and abdominal subcutaneous fat thickness (C) of Bama pigs in each group 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 diagram of the mass spectrometry data of each group of Bama pig samples;

[0052] Figure 6 This is the overall cluster diagram of fatty acid metabolites of Bama pigs in each group;

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

[0054] Figure 8Glucose (A), AST (B), ALT (C), LDL-C (D), cholesterol (E) and triglyceride (F) in liver tissue of Bama pigs (n=3, mean±SEM);

[0055] Fig. 9 HE staining of the liver of Bama pigs after administration 100X (A), Oil Red O staining of the liver 200X (B) (n=3);

[0056] Fig.10 HE staining of the heart of Bama pig at 200X (A) and HE staining of the kidney at 100X (B) (n=3);

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

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

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

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

[0061] The present invention is further described below in conjunction with the examples. It should be noted that the following examples are only intended to help understand the present invention, rather than to limit the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents, etc. used in the examples can be obtained through commercial channels.

[0062] This experiment was analyzed using the software GraphPad Prism 9. T-test was performed between two groups, and one-way ANOVA was performed for more than two groups. All experimental data results were expressed as mean ± SEM. Compared with the Model group, P < 0.05 was considered statistically significant. * 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, the specific construction method includes the following steps:

[0065] Twelve healthy 20-30kg 5-month-old ordinary Bama pigs were selected, 3 of which were randomly selected as the control group, and the remaining 9 were the model group.

[0066] The Bama pigs in the control group were fed with ordinary feed, and the daily feed weight was 3% of the piglets' body weight this week.

[0067] The Bama pigs in the model group were first fed with ordinary feed for 1 week and then switched to high-fat feed. The daily feed weight was 3% of the piglets' body weight this week.

[0068] At a fixed time period every week, the Bama pigs in the control group and the model group were weighed and measured, and 2 mL of blood was collected from the anterior vena cava of the Bama pigs, and the supernatant was taken for blood lipid testing.

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

[0070] The calculation formula of obesity degree is: (weight of Bama pigs in the model group - average weight of Bama pigs in the control group) / average weight of Bama pigs in the control group) × 100%;

[0071] To prepare common feed, the specific steps are as follows:

[0072] Prepare 25.238 parts of corn, 4 parts of fish meal, 8 parts of soybean meal, 10 parts of rice bran, 20 parts of wheat bran, 30 parts of alfalfa meal, 2.4 parts of calcium bicarbonate, 0.3 parts of iodized salt, 0.03 parts of trace element additives, and 0.032 parts of vitamin additives. After fully crushing, add them to a mixer and mix evenly. Add the mixed powder feed to a feed granulator to make ordinary feed pellets, and finally cool and package them into finished feed.

[0073] The preparation of high-fat feed, the specific steps are as follows:

[0074] Prepare the components shown in Table 1, grind them fully and add them into a mixer to mix evenly, add the mixed powder feed into a feed pelletizer to make high-fat feed pellets, and finally cool and package them into finished feed.

[0075] Among them, the trace element additive is composed of 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 in terms of mass.

[0076] The vitamin additive consists, by mass, of 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 of vitamin additives, 1 part of trace element additives, 10 parts of table salt, and 88 parts of corn flour, calculated by mass.

[0078] Comparative Example 1

[0079] A Bama pig model is basically the same as Example 1, the only difference being that the nutmeg in the high-fat feed is replaced with gypenosides. The specific composition of the high-fat feed used in Comparative Example 1 is shown in Table 1.

[0080] Comparative Example 2

[0081] A Bama pig model is basically the same as Example 1, the only difference being that the gypenosin in the high-fat feed is replaced with Atractylodes macrocephala. The high-fat feed ingredients used in Comparative Example 2 are specifically shown in Table 1.

[0082] Comparative Example 3

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

[0084] Comparative Example 4

[0085] A Bama pig model is basically the same as Example 1, except that the mint leaves in the high-fat feed are replaced with Patchouli leaves, the hawthorn acid is replaced with malic acid, and the ursolic acid is replaced with citric acid. The high-fat feed ingredients used in Comparative Example 4 are specifically shown in Table 1.

[0086] Table 1 High fat feed composition

[0087] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Butter (weight) 10 10 10 10 10 Margarine 10 10 10 10 10 sucrose 8 8 8 8 8 Casein 10 10 10 10 10 Laboratory 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 Gynostemma pentaphyllum saponin 0.2 0.3 / 0.3 0.2 Mint leaves 0.7 0.7 0.7 0.7 / Maslinic acid 0.25 0.25 0.25 0.25 / Ursolic acid 0.1 0.1 0.1 0.1 / Atractylodes macrocephala 1 1 1.2 / 1 nutmeg 0.1 / 0.1 0.1 0.1 Ordinary feed 49.5 49.5 49.5 49.5 49.5 Astragalus / / / 1 / Malic Acid / / / / 0.25 Citric Acid / / / / 0.1 Patchouli Leaves / / / / 0.7

[0088] Test Example 1: Detection of obesity in Bama pigs

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

[0090] Table 2 Bama pig obesity test results

[0091]

[0092]

[0093] As shown in the table above, Example 1 of the present invention reached the standard of 20% obesity after 10.5 weeks of feeding high-fat diet, while Comparative Examples 1 to 4 were all 13 to 14 weeks. Comparative Example 1 lacks nutmeg, Comparative Example 2 lacks gypenosides, and Comparative Example 3 lacks Atractylodes macrocephala. The missing components in the high-fat diets of Comparative Examples 1 to 3 affect the absorption of nutrients in the feed by Bama pigs, thereby reducing the weight gain rate of Bama pigs. Atractylodes macrocephala, nutmeg, and gypenosides work together, and none of them can be missing. They can regulate the stomach and promote digestion for Bama pigs, and promote the digestion and absorption of nutrients in the feed by Bama pigs. The high-fat diet used in Comparative Example 4 lacks mint leaves, hawthorn acid, and ursolic acid, which affects the taste of the feed. Replacing mint leaves with Patchouli leaves, hawthorn acid with malic acid, and ursolic acid with citric acid cannot promote the secretion of digestive juices in Bama pigs, thereby causing loss of appetite and reducing the weight gain rate of Bama pigs in Comparative Example 4. In summary, the high-fat feed of the present invention has good palatability and uses three natural medicinal ingredients, namely, Atractylodes macrocephala, nutmeg, and gypenoside, to synergistically enhance the effect, thereby promoting the digestion and absorption of Bama pigs and achieving the effect of rapid weight gain of Bama pigs.

[0094] Experimental Example 2: Application of Bama Pig Model in Preclinical Research of Weight Loss Drug COST

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

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

[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 the body weight and body size index were measured weekly; blood biochemical tests were performed monthly, and the indicators of the blood biochemical tests included fasting blood glucose, aspartate aminotransferase, alanine aminotransferase, triglycerides, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol; after the weight loss period, the weight loss effect was judged based on the physiological state, body weight, body size and serum biochemical indicators;

[0098] After the weight loss period, the Bama pigs were killed, and the back fat thickness and subcutaneous fat layer thickness were measured. Then, samples were taken, including organs, adipose tissue and cecal contents. The organs included: heart, liver, kidney, duodenum, ileum, colon; adipose tissue included: perirenal fat, abdominal subcutaneous fat, greater omental fat; the organs were weighed, embedded and cryopreserved; the adipose tissue was analyzed by fatty acid analysis method based on GC-MS / MS platform, and free fatty acids were detected and analyzed in adipose tissue;

[0099] Free fatty acid detection and analysis involves the following steps:

[0100] S1. Take out the sample stored at ultra-low temperature, thaw it on ice, grind it into powder, add methanol, methyl tert-butyl ether, and 36% phosphoric acid for extraction; centrifuge to remove the supernatant, blow dry, add 15% boron trifluoride methanol solution; keep it in an oven at 60-65℃ for 30-40min; after cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and remove the n-hexane layer solution for analysis;

[0101] S2. The sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with a specification of 30 m × 0.25 mm × 0.25 μm. The carrier gas was high-purity helium. The heating procedure was as follows:

[0102] Start at 40°C for 2 minutes, increase to 200°C at 30°C / min, continue for 1 minute, increase to 240°C at 10°C / min, continue for 1 minute, and finally increase to 285°C at 5°C / min, continue for 3 minutes; flow rate: 1.0mL / min; injection port temperature: 230°C; injection volume: 1.0μL;

[0103] The GC-EI-MS system was an Agilent 8890-5977B GC-MS system. After GC detection, the EI-MS parameters were set as follows: temperature: 230 °C; ionization voltage: 70 eV; transfer line temperature: 240 °C; quadrupole temperature: 150 °C; solvent delay: 4 min; scan mode: SIM;

[0104] S3. Perform statistical analysis.

[0105] The specific test process is as follows:

[0106] 1. Experimental Animal Grouping and Dosing

[0107] The Bama pig model group successfully modeled in Example 1 was randomly divided into three test groups: a model control group (Model group), a positive drug orlistat group (Orlistat group) and a chitosan oligosaccharide group (COST group), each of which consisted of three obese Bama pigs of mixed sexes (2 females and 1 male). After grouping, the Orlistat group and the COST group were given orlistat and chitosan oligosaccharide drug interventions, respectively. The dosage was determined according to the standard weight animal conversion coefficient Rab=1.370 from adults to miniature pigs. The control group and the model group were fed with corresponding starch. This stage is called the weight loss period. Since oral administration of the animal model during the weight loss period is prone to cause drug waste, it is necessary to first mix the drug with 1 / 10 of the day's feed and completely eat it, and then feed the remaining 9 / 10 of the feed to ensure that the drug is fully eaten. The duration of the drug administration phase is 12 weeks.

[0108] 2. Observation indicators and materials

[0109] During the entire experiment, the physiological state of the Bama pigs needs to be observed daily, including whether they are drowsy and lazy, indifferent to eating, dry and dull fur, loose feces, pale mouth, etc. And the Bama pigs are weighed and measured for body size indicators after fasting once a week. Blood is collected from the anterior vena cava of the Bama pigs every month and the supernatant is taken for blood biochemical testing. The test indicators include fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Finally, the general condition, weight, body size, and serum biochemical indicators of the Bama pigs are combined to judge the success of the obesity model and the weight loss effect.

[0110] After the 12-week dosing period, the Bama pigs were fasted for 16 hours but not water, so that they were in a relatively fasting state. Before sampling, the final body weight, body measurement data collection and venous blood sampling were required. After that, the Bama pigs were anesthetized with isoflurane and then bled to death. Then, the back fat thickness and subcutaneous fat layer thickness of each Bama pig were measured. After the measurement, the various 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, and colon. The adipose tissues that need to be sampled include: suet (perinephric fat), abdominal subcutaneous fat, and greater omental fat. In addition, Bama pig feces (cecal contents) also need to be frozen for subsequent experimental studies.

[0111] (1) Observation results of daily behavior and status of Bama pigs

[0112] Throughout the experimental period, the daily behaviors and status of each group of Bama pigs were closely observed. It was found that the piglets in the Control group fed with a normal diet had a good mental state, a good appetite, liked to wag their tails, had self-cleaning behavior, had pale nose discs, shiny hair, and formed stools; the piglets in the Model group had a general mental state, liked to lie down but not move, and had apathetic eating in the later period. When feeding, their eyes only moved with the breeder, moved slowly, had a dry nose, a reddish nose disc with ecchymosis, loose skin, dull hair, a large number of lipid spots on the back skin, and dark stools; the piglets in the Orlistat group had a good mental state, liked to move, liked to wag their tails, were often irritable, had a moist nose, a pale nose disc, dry hair, and lipid spots on the back skin, but less than the piglets in the Model group, with pale stools and oily stools; the piglets in the COST group had a good mental state, liked to wag their tails, had a behavior of waiting for feeding, had a moist nose, a pale nose disc, dry hair, and lipid spots on the back skin, but still less than the piglets in the Model group, with formed stools and a slight oil content in the feces. In terms of appearance, the piglets in the Model group were bloated, with their abdominal skin close to the ground when standing upright, and it was difficult for them to stand on their hind legs; the piglets in the Control group were relatively small and well-proportioned; the piglets in the Orlistat group and the COST group were similar in size, had no difficulty standing, and could walk upright. Figure 2 shown.

[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, we recorded the weight of each group of Bama pigs before sampling and analyzed the weight and weight gain data. Figure 1 , as shown in Table 3.

[0115] Table 3 Body weight and weight gain results of Bama pigs in each group after administration

[0116]

[0117] As shown in the table above, compared with the Control group, the Model group fed with high-fat diet had a significant increase in weight, and the weight gain was large; while the weight of the Orlistat group and the COST group, which also ate high-fat diet, decreased significantly compared with the Model group, and the weight gain was slow. This indicates that Orlistat and chitosan oligosaccharide can reduce the weight of Bama pigs to a certain extent, thereby improving obesity signs.

[0118] 3. Determination of body size indicators

[0119] The body fat distribution assessment method applicable to humans is also applicable to animal models. In clinical practice, the indicators commonly used to analyze the body fat composition are height and weight, and sometimes waist circumference and skinfold thickness. In this experiment, multiple circumferences of Bama pigs were measured, including body length, neck circumference, chest circumference, abdominal circumference and waist circumference. The measurement method is as follows.

[0120] Body length: Use a tape measure to measure the straight-line distance from the midpoint of the line connecting the two ears of the Bama pig to the first groove at the base of the tail along the back of the Bama pig, which is the body length.

[0121] Neck circumference: Use a tape measure to measure the circumference of the neck around the back edge of the Bama pig's ear. The tightness should be such that the tape measure naturally sticks to the fur.

[0122] Chest circumference: Use a tape measure to measure the length around the body with the back edge of the shoulder blade as the boundary as the chest circumference.

[0123] Abdominal circumference: Use a tape measure to measure the distance around the abdomen of a Bama pig through the lowest point of the abdomen.

[0124] Waist circumference: The waist circumference is the measurement around the pig's body close to the base of the Bama pig's hind legs.

[0125] Body size data is an important indicator to measure 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 is the longitudinal height, and the other four body size indicators are the lateral 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 drug administration (n=3, mean±SEM)

[0128]

[0129]

[0130] As can be seen from the above table, the body length of the Bama pigs in the Control group is smaller than that of the other groups of Bama pigs fed with a high-fat diet, while there is no significant difference between the Model group, Orlistat group and COST group, which are also fed with a high-fat diet, indicating that drug intervention will not inhibit the growth of the body length of Bama pigs, and the body length is highly correlated with dietary factors. In the neck circumference index, the neck circumference of the Control group is significantly smaller than that of the Model group, and there is a significant difference between the two. The neck circumference of the Orlistat group and the COST group is also significantly smaller than that of the Model group, and there is a significant difference with the Model group, while there is no significant difference between the Orlistat group and the COST group. Compared with the Control group, the chest circumference of the Bama pigs in the Model group increased significantly, while the chest circumference of the Orlistat group and the COST group decreased significantly relative to the Model group. In the abdominal circumference index, compared with the Control group, the abdominal circumference of the Bama pigs in the Model group increased significantly, while the abdominal circumference of the Bama pigs in the Orlistat group and the COST group also decreased significantly relative to the Model group. The data analysis of waist circumference index showed that the waist circumference of Bama pigs in the Control group was significantly smaller than that of Bama pigs in the Model group, while the waist circumference of piglets in the Orlistat group and COST group was significantly smaller than that of piglets in the Model group. The above data all indicate that feeding high-fat feed can accelerate obesity in Bama pigs, and chitosan oligosaccharides can significantly improve the degree of obesity caused by high-fat feed.

[0131] 4. Determination of subcutaneous fat thickness

[0132] Back fat thickness: After the Bama pigs were killed, the thickness of the subcutaneous fat on the back of the Bama pigs was measured using a vernier caliper at the back edge of the shoulder blade, the last rib, and the waist and sacrum junction, about 4 cm away from the midline of the back. Then the average of the three locations was calculated, which we call the "BFT" index. BFT is of great significance in measuring the subcutaneous fat content on the back.

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

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

[0135]

[0136]

[0137] As shown in the table above, after 12 weeks of drug intervention, the obesity level in the Orlistat and COST groups decreased and was below 20%, while the obesity level in the Model group continued to increase and exceeded 30%. Figure 4 As shown in Figure B, compared with the Control group, the back fat thickness of the Model group was significantly increased, and the back fat thickness of the Model group was further compared with the Orlistat group and the COST group, respectively, and the back fat thickness of the latter two groups was significantly decreased. This shows that the subcutaneous fat deposition of Bama pigs fed with chitosan oligosaccharides is relatively lower. Figure 4 As shown in Figure C, compared with the Control group, the abdominal subcutaneous fat thickness of the Model group was significantly increased, and the abdominal fat thickness of the Model group was significantly decreased when the Orlistat group and the COST group were further compared. The above results all indicate that the obesity status of Bama pigs has been improved to a certain extent after the intervention of Orlistat and Chitosan Oligosaccharide.

[0138] 5. Determination of biochemical indicators

[0139] Blood collection method: Lay the Bama pig in a supine position to expose the chest concave area, use 75% ethanol to disinfect the puncture needle, use a sterile 10mL syringe to vertically insert the needle into the anterior vena cava sinus, and slowly draw 2mL of blood.

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

[0141] Blood indicators: According to the corresponding instructions, the levels of TG, TC, HDL-C, LDL-C, FFA, ALT, AST, and GLU in serum samples were determined using common test kits, and the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α), fasting insulin levels (Insulin), and endotoxin levels (LPS) in serum samples were determined using ELISA kits.

[0142] Liver / fat tissue indicators: Use a 1 / 1000 analytical balance to weigh 0.100 g of liver tissue or fat tissue in a 1.5 mL EP tube, then add 0.9 mL of saline and 2 grinding beads to the tube, and finally homogenize the liver tissue or fat tissue in the prepared tube in an automatic homogenizer. After the homogenization, place the tube with the tissue in a high-speed centrifuge at 4°C and 2500 r / min for 10 minutes. After centrifugation, take the supernatant and use ordinary kits to measure Bama pig liver indicators (GLU, AST, ALT, TG, TC, LDL-C) and ELISA kits to measure the levels of IL-1β, IL-6, IL-10, and TNF-α in adipose tissue.

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

[0144] Table 6 Results of blood sugar 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 in 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 Changes in serum lipid levels in Bama pigs after administration (n=3, mean±SEM)

[0149]

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

[0151]

[0152]

[0153] 6. Pathological sections and staining

[0154] HE staining: Place the embedding box containing the tissue in a 4% paraformaldehyde solution for 24 hours, and then place the fixed embedding box in a fully automatic dehydrator for step-by-step dehydration; after dehydration, embed in paraffin. Precool the embedded paraffin tissue and slice it with a slicer, then stain the slide with tissue with hematoxylin and eosin dyes. The staining process and time refer to the laboratory's previous experience. Finally, store the stained slide in a slice box for subsequent microscopic observation.

[0155] Oil Red O staining: Place the embedding box containing the tissue in a 4% paraformaldehyde solution for 24 hours, then place the embedding box 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, embed the tissue with OCT embedding agent. After embedding, store in a -20°C refrigerator. Then use a frozen slicer to make slices with a thickness of 8μm. Use Oil Red O staining solution to stain the slices. Finally, store the stained slices in a slice box for subsequent microscopic observation.

[0156] To further understand the deposition of lipid droplets in the liver tissue of obese Bama pigs, the paraffin sections of the liver of each group of Bama pigs were stained with HE, and the frozen sections were stained with Oil Red O. Fig. 9 (A) The liver cells of the Bama pigs in the Control group were arranged regularly and the cytoplasm was uniform. The cytoplasm of the liver cells of the mice in the Model group contained lipid droplet vacuoles of different sizes. After COST intervention, the fat vacuoles of the Bama pigs in the COST group were significantly reduced compared with those in the Model group, and the arrangement of liver cells was significantly improved. Oil red O staining of the liver of the Bama pigs is shown in Fig. 9 (B) Compared with the Bama pigs in the Control group, the livers of the Bama pigs in the Model group showed a large number of red lipid droplets, while the lipid droplets in the livers of the Bama pigs in the COST group were significantly reduced. Combining the results of HE staining and Oil Red O staining, chitosan oligosaccharide can alleviate fatty degeneration of the liver of obese Bama pigs and reduce the accumulation of liver lipid droplets.

[0157] exist Fig.10 In (A), the cardiomyocytes of the four groups of Bama pigs were in normal morphology, and there was no hypertrophy of the cardiomyocytes. Fig.10 In (B), the kidney cells of the four groups of Bama pigs were in normal morphology, the glomeruli and renal bodies were clearly visible, and the nuclear membrane was clear. According to HE staining and the appearance of the kidneys of each group of Bama pigs, it was judged that the kidney tissue was in a normal state. Therefore, based on the pathological sections, we preliminarily concluded that chitosan oligosaccharide 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, HE staining was performed on subcutaneous fat and lard (perinephric fat) of each group of Bama pigs. Fig.11 As shown in (A), the subcutaneous adipocytes of the control group Bama pigs were uniform in size and had a large number of cells, while the subcutaneous adipocytes of the model group were larger in size and had unclear nuclear membranes. After administration of COST, the number of cells was significantly increased and the volume of adipocytes was reduced. Fig.11In (B), it can be found that the suet tissue cells of the Model group Bama pigs are large in size, small in number, and have unclear nuclear membranes, while the Control group and COST group Bama pigs can significantly improve the status of the suet cells of the Model group Bama pigs.

[0159] In order to observe the morphological structure of the intestine of Bama pigs, HE staining was performed on the ileum and colon tissues of Bama pigs. Fig.12 As shown in (A), the intestinal villi and intestinal muscle layer of the Model group Bama pigs were damaged, the barrier function was weakened, and the depth of the intestinal crypts became shallower. Compared with the Model group, the intestinal muscle layer of the Control group and the COST group was relatively intact, and the crypt depth was deeper. Fig.12 In (B), the fat cells in the colon tissue of the Model group Bama pigs have infiltrated the intestinal muscular layer, and the goblet cells that secrete mucus have abnormal morphology and decreased in number. Compared with the Model group, the number of goblet cells in the Control group and the COST group increased significantly, and they can secrete more mucus, which helps to reduce the damage of fecal balls to the intestinal mucosa. This shows that chitosan oligosaccharides can reduce the damage of high-fat diet to the intestine of Bama pigs and is beneficial to intestinal metabolism.

[0160] In summary, feeding chitosan oligosaccharides can reduce the body weight and weight gain of obese Bama pigs, reduce blood liver lipids, lower blood sugar, improve inflammation in serum and adipose tissue, improve liver function, reshape the intestinal structure of piglets, and have certain weight loss activity, laying a foundation for further mechanism exploration.

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

[0162] Fatty acid (FA) is widely present in organisms in different forms and is a key compound involved in lipid metabolism. Fatty acid detection uses mass spectrometry scanning mode selected ion monitoring mode (SIM). After the sample molecules are ionized, only the pre-selected target ions can reach the detector through the quadrupole for detection, while other ions cannot reach the detector through the quadrupole. The selected ion monitoring mode can effectively remove matrix interference, improve sensitivity, and better complete the quantitative and qualitative analysis of the free fatty acid components of Bama pigs. In this experiment, the free fatty acid components of the adipose tissue of each group of Bama miniature pigs were analyzed based on the fatty acid analysis method of the GC-MS / MS platform to further explore the molecular level mechanism of COST in lipid metabolism and synthesis.

[0163] This experiment detected 48 kinds of free fatty acids in the adipose tissue of Bama pigs. The composition information is shown in the following table.

[0164] Table 10 Information of 48 fatty acids

[0165]

[0166]

[0167]

[0168] A total of 12 samples from 4 groups were selected for testing. The sample information is shown in the table below.

[0169] Table 11 Sample information table

[0170] Species organize Grouping Serial number name pig Subcutaneous fat tissue Control 722-4 pig Subcutaneous fat tissue Control 722-8 pig Subcutaneous fat tissue Control 1209-15 pig Subcutaneous fat tissue Model 722-5 pig Subcutaneous fat tissue Model 722-6 pig Subcutaneous fat tissue Model 1209-13 pig Subcutaneous fat tissue Orlistat 1213-1 pig Subcutaneous fat tissue Orlistat 722-10 pig Subcutaneous fat tissue Orlistat 709-1 pig Subcutaneous fat tissue COST 715-1 pig Subcutaneous fat tissue COST 722-7 pig Subcutaneous fat 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 out the sample from the -80℃ freezer and thaw it on ice (all subsequent operations must be performed on ice)

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

[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 and 12,000 rpm for 5 min.

[0177] (5) Take out 200 μL of the supernatant and blow dry it on a nitrogen blower, then add 300 μL of 15% boron trifluoride methanol solution.

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

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

[0180] (8) After vortexing for 3 min, centrifugation was performed at 4 °C and 12,000 rpm for 5 min, and 100 μL of the hexane layer solution was removed for analysis.

[0181] 7.2 Chromatographic mass spectrometry acquisition 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 (30m×0.25mm×0.25μm, Agilent); carrier gas, high-purity helium (purity>99.999%); heating program started from 40°C (2 minutes), increased to 200°C (1 minute) at 30°C / min, then increased to 240°C (1 minute) at 10°C / min, and finally increased to 285°C (3 minutes) at 5°C / min; flow rate: 1.0mL / min; injection port temperature: 230°C; injection volume: 1.0μL

[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 from www.r-project.org. Before performing unsupervised PCA, the data needed to be scaled to unit variance.

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

[0188] Dendrograms were plotted based on 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 presented only as a heatmap. Both the hierarchical cluster analysis and the Pearson correlation coefficient were performed using the R package pheatmap. For the hierarchical cluster analysis, the normalized signal intensity (unit variance scaling) of the metabolites was visualized in the form of a color gradient. 7.4 Results of the detection of different fatty acid contents in adipose tissue of Bama pigs

[0189] In this experiment, the main components of free fatty acids in the adipose tissue of each group of Bama pigs were detected, and the results were grouped and statistically analyzed. The results are shown in the table below. 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, and C22-1n9, their contents in the Model group Bama pigs were significantly higher than those in the Control group. It can be inferred that high-fat diet-induced obesity in Bama pigs may interfere with the normal lipid metabolism process of Bama pigs by increasing the synthesis of these fatty acid components. Similarly, some fatty acids were significantly different between the COST group and the Model group. Specifically, the contents of 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, and C22-1n9 in Bama pig tissues in the COST group were significantly lower than those in the Model group, and most of these components were significantly higher in the Model group than in the Control group. This indicates that COST administration can reduce the content of free fatty acids that cause obesity and thus alleviate the fat accumulation state induced by a high-fat diet.

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

[0191]

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

[0193] Through multivariate statistical analysis, high-dimensional and complex data can be reduced in dimension and the original data can be preserved as much as possible. This method helps to accurately establish a mathematical model, thereby summarizing and analyzing the metabolic spectrum characteristics of the sample. Principal component analysis (PCA) is a multidimensional data statistical analysis method for unsupervised pattern recognition. It 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. Results are shown in Figure 5 . Figure 5 In the figure, PC1 represents the first principal component, PC2 represents the second principal component, PC3 represents the third principal component, and the percentage represents the explanation rate of the principal component for the data set. Each point in the figure represents a sample, and samples in the same group are represented by the same color. Group represents the grouping.

[0194] PCA analysis of the adipose tissue samples of each group of Bama pigs was performed to understand the overall metabolic differences between the adipose tissues of each group of Bama pigs and the metabolic differences of adipose tissues of Bama pigs within each group. The PCA results can show the metabolome trends between the groups and indicate the metabolome conditions within each group of adipose tissue. QC is a quality control sample. Figure 5 As shown in the figure, it can be observed through the principal component analysis (PCA) score graph that in the fat samples of the Control group and the Model group, the principal components showed a significant shift, and there were significant metabolic differences between the two groups. This shows that the lipid metabolism of the adipose tissue of Bama pigs 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 in the adipose tissue of Bama pigs and alleviate the metabolic differences between the Control group and the Model group, suggesting that COST may have a certain effect on regulating the lipid metabolism of adipose tissue of Bama pigs.

[0195] 7.6 Cluster analysis of metabolites in Bama pig fat samples

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

[0197] In this experiment, UV (unit variance scaling) processing was performed on the fatty acid metabolite data of Bama pigs, and a cluster heat map was drawn using an R program script, as shown in Figure 6 .

[0198] Depend on Figure 6 It can be seen that 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, the Bama pig samples in the Model group showed higher content, which was significantly increased compared with the Control group. However, in the COST administration group, the clustering content of these fatty acids was significantly lower than that in the Model group. This is consistent with the results of the metabolite detection of individual samples in the previous article, which further proves that high-fat diet-induced obesity in Bama pigs may interfere with the normal fat metabolism of Bama pigs 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 a high-fat diet.

[0199] Free fatty acid metabolites play an important role in obesity. In the body of obese patients, the level of free fatty acids usually increases. These free fatty acids are the products of triglyceride hydrolysis in fat cells, which 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 may cause problems such as inflammatory response of fat cells, insulin resistance and fat accumulation. Studies have shown that excessive free fatty acids can promote the enlargement and increase in the number of fat cells, leading to hypertrophy and excessive accumulation of adipose tissue, that is, free fatty acid metabolites in obesity may also affect the physiological function of adipose tissue by affecting the proliferation and differentiation of adipocytes. In this case, the normal metabolic function of adipose tissue may be damaged. In addition, excessive free fatty acids can interfere with lipid synthesis and degradation pathways, leading to lipid metabolism disorders and inflammatory responses of adipose tissue, thereby exacerbating the development of obesity.

[0200] In summary, COST treatment played a role in restoring the metabolic characteristics of adipose tissue in Bama pigs and helped to alleviate the metabolic differences between the Control group and the Model group. These findings provide new clues for studying the treatment strategies of obesity-related metabolic disorders and deserve further study and exploration.

Claims

1. A Bama pig model for preclinical research of the weight loss drug COST, characterized in that: The method for constructing the Bama pig model comprises the following steps: Healthy Bama pigs were selected and divided into control group Bama pigs and model group Bama pigs; The Bama pigs in the control group were fed with ordinary feed; The Bama pigs in the model group are first fed with ordinary feed, and then fed with high-fat feed. The Bama pigs in the control group and the Bama pigs in the model group are tested for obesity-related indicators at a fixed time period every week. When the obesity degree of the Bama pigs in the model group is greater than 20%, the Bama pig model is obtained; The calculation formula of the obesity degree is: (weight of the Bama pigs in the model group - average weight of the Bama pigs in the control group) / average weight of the Bama pigs in the control group)×100%; The high-fat feed comprises the following components in parts by mass: 49 to 50 parts of the ordinary feed, 9 to 10 parts of butter, 10 to 11 parts of margarine, 14 to 15 parts of sucrose, 9 to 10 parts of casein, 2 to 2.5 parts of experimental animal premix, 1 to 1.5 parts of microcrystalline cellulose, and 2 to 2.5 parts of calcium bicarbonate.

2. The Bama pig model according to claim 1, characterized in that: The high-fat feed also includes the following components in parts by weight: 0.1-0.3 parts of gypenoside, 0.5-1.0 parts of peppermint leaves, 0.2-0.3 parts of maslinic acid, 0.1-0.15 parts of ursolic acid, 1-2 parts of atractylodes macrocephala, and 0.1-0.15 parts of nutmeg.

3. The Bama pig model according to claim 1, characterized in that: The common feed comprises the following components in parts by weight: 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 additives, and 0.03-0.032 parts of vitamin additives.

4. The Bama pig model according to claim 3, characterized in that: The trace element additive is composed of the following components by mass: 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 by mass: 19 to 19.5 parts of vitamin A; 30.4 to 0.45 parts of vitamin D; 16.75 to 16.8 parts of vitamin E; 5 to 5.1 parts of vitamin K3, 2.1 parts of vitamin B12, 16 to 16.5 parts of vitamin B2, 10 to 10.1 parts of vitamin C, 6 to 6.05 parts of vitamin B6, 0.03 to 0.031 parts of vitamin B12, 35 to 35.5 parts of niacin, 25 to 25.5 parts of calcium pantothenate, 0.5 to 0.51 parts of folic acid, 0.1 to 0.11 parts of biotin, 50 to 50.5 parts of methionine, and 50 to 50.5 parts of lysine; The experimental animal premix consists of 1 to 1.05 parts of the vitamin additive, 1 to 1.05 parts of the trace element additive, 10 to 10.5 parts of salt, and 88 to 89 parts of corn flour in parts by mass.

5. The Bama pig model according to claim 1, characterized in that: The healthy Bama pig is a common-grade Bama pig of 4 to 6 months old and weighs 20 to 30 kg.

6. The Bama pig model according to claim 1, characterized in that: The Bama pigs in the model group were first fed with ordinary feed for 1.0 to 1.1 weeks, and then fed with high-fat feed until the obesity level of the Bama pigs in the model group was greater than 20%; The feeding comprises giving common feed or high-fat feed every day, and the weight of the common feed or high-fat feed used for feeding is 3.0-3.01% of the body weight of the Bama pig in that week.

7. Use of the Bama pig model according to any one of claims 1 to 6 in preclinical research on the weight loss drug COST.

8. The use according to claim 7, characterized in that: The Bama pig model described in any one of claims 1 to 6 is used as a model group, and the Bama pigs fed with ordinary feed are used as a control group; The model group was randomly divided into: model control group, positive drug orlistat group and COST group; 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 is observed daily, and the weight and body size indicators are measured weekly; blood biochemical tests are performed monthly, and the indicators of the blood biochemical tests include fasting blood sugar, aspartate aminotransferase, alanine aminotransferase, triglycerides, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol; after the weight loss period, the weight loss effect is judged based on the physiological state, weight, body size, and serum biochemical indicators; After the weight loss period, the Bama pigs were killed, the back fat thickness and subcutaneous fat layer thickness were measured, and then the samples were collected; The sampling includes taking out organs, adipose tissue and cecal contents; The organs are weighed, embedded and cryopreserved; The organs include: heart, liver, kidney, duodenum, ileum, colon; The adipose tissue includes: perirenal fat, abdominal subcutaneous fat, and greater omentum fat; The adipose tissue adopts a fatty acid analysis method based on a GC-MS / MS platform to detect and analyze free fatty acids in the adipose tissue, and study the molecular level mechanism of COST in lipid metabolism and synthesis.

9. The use according to claim 8, characterized in that: The free fatty acid detection and analysis specifically comprises the following steps: S1, sample pretreatment; S2. The sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with a specification of 30 m × 0.25 mm × 0.25 μm. The carrier gas was high-purity helium. The heating procedure was as follows: Start at 40°C for 2 minutes, increase to 200°C at 30°C / min, continue for 1 minute, increase to 240°C at 10°C / min, continue for 1 minute, and finally increase to 285°C at 5°C / min, continue for 3 minutes; flow rate: 1.0mL / min; injection port temperature: 230°C; injection volume: 1.0μL; The GC-EI-MS system was an Agilent 8890-5977B GC-MS system. After the GC detection was completed, the EI-MS parameters were set as follows: temperature: 230°C; ionization voltage: 70 eV; transfer line temperature: 240°C; quadrupole temperature: 150°C; solvent delay: 4 minutes; scan mode: SIM; S3. Perform statistical analysis.

10. The use according to claim 9, characterized in that: The sample pre-processing in step S1 specifically includes the following steps: After taking out the ultra-low temperature stored sample, thaw it on ice, grind it into powder, add methanol, methyl tert-butyl ether, and 36% phosphoric acid for extraction; centrifuge to remove the supernatant, blow dry, and add 15% boron trifluoride methanol solution; keep it in an oven at 60-65°C for 30-40 minutes; after cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and remove the n-hexane layer solution for analysis on the machine.

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