Bama miniature pig model for pre-clinical research of weight loss drug cost and use

Abstract

A Bama miniature pig model for pre-clinical research of a weight loss drug COST and a use. Healthy Bama miniature pigs are first subjected to adaptive feeding with an ordinary feed, and then fed with a high-fat feed for modeling, and obesity-related indicators of the Bama miniature pigs are measured at fixed weekly intervals. The high-fat feed comprises: an ordinary feed, beef tallow, margarine, sucrose, casein, experimental animal premix, microcrystalline cellulose, calcium bicarbonate, gypenoside, mint leaves, maslinic acid, ursolic acid, Atractylodes macrocephala, and nutmeg.

Description

A Bama pig model for preclinical research on the cost of weight-loss drugs and its application Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a Bama pig model and its application in preclinical research on the cost of weight loss drugs. Background Technology

[0002] According to the World Health Organization (WHO) definition, obesity refers to an excessive accumulation of body fat, an abnormal condition that can impair health, and was officially listed as an endocrine disorder in 1997. Obesity is associated with an increased risk of premature death and increases the incidence of various complications, such as type 2 diabetes, sleep apnea syndrome, atherosclerosis, and non-alcoholic fatty liver disease. Furthermore, obesity increases the risk of various cancers and negatively impacts patients' mental health, emotions, and cognitive abilities. Currently, 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 treatments.

[0003] Mouse models are commonly used mammalian models in the screening and preclinical research stages of new drugs. However, the physiological differences between mice and humans limit their use in disease simulation. For example, in the study of metabolic diseases such as diabetes, due to the differences in glucose and lipid metabolism between mice and humans, even if mice are induced to exhibit the corresponding phenotypes through drugs or genetic means, the disease development process and pathophysiological mechanisms in mice are not entirely consistent with those in humans, meaning that the research results cannot be directly applied to humans.

[0004] In preclinical drug research, differences in drug-metabolizing enzyme systems and drug transporters between mice and humans lead to variations in drug metabolism and pharmacokinetic characteristics. Consequently, some drugs effective in mouse models may not perform well or produce different adverse reactions in human clinical trials, impacting the success rate of drug development and the accuracy of safety assessments. Therefore, an animal model with closer 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 those in humans on a timescale. Their organs are more similar to humans in size, structure, and physiological function, and their nutritional needs and intake patterns are similar to those of humans. Their absorption and metabolic responses to different nutrients are also more closely approximated, allowing for a more accurate simulation of human physiological conditions. By controlling the intake and composition of Bama pig feed, the mechanisms of obesity that closely resemble human physiological conditions can be better simulated, providing more reliable data support for research.

[0006] CN110862988A discloses an sgRNA and its CREBRF point mutant Bama miniature pig and its application. However, gene editing for modeling is costly, and using gene-edited animal models to screen drugs may pose long-term safety issues. The edited gene may produce some unknown and potential effects, such as inducing tumors or immune system abnormalities. These problems may be overlooked during drug screening but will be exposed in subsequent clinical trials or clinical applications.

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

[0008] In summary, there is currently a lack of standardized methods for constructing Bama pig models that can successfully build stable obesity phenotypes in a short period of time. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a Bama pig model for preclinical research on the cost of weight loss drugs. This model has a short construction time and a stable obesity phenotype, providing a reliable animal model for preclinical research on new drugs for treating obesity. It also provides 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 treating obesity.

[0010] To achieve the above objectives, the first aspect of this invention provides a method for constructing a Bama pig model, as detailed below:

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

[0012] The control group of Bama pigs was fed ordinary feed;

[0013] The model group of Bama pigs was first fed ordinary feed, and then switched to high-fat feed. Obesity-related indicators were detected in both the control group and the model group of Bama pigs at fixed time periods each week. When the obesity rate of the model group of Bama pigs was greater than 20%, the Bama pig model was obtained.

[0014] The formula for calculating obesity 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%;

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

[0016] Preferably, the high-fat feed further includes the following components in parts by weight: 0.1-0.3 parts of Gynostemma pentaphyllum saponins, 0.5-1.0 parts of peppermint leaves, 0.2-0.3 parts of hawthorn 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 combination of Gynostemma pentaphyllum saponins, Atractylodes macrocephala, and nutmeg in this invention can promote the absorption of nutrients from the high-fat feed by Bama pigs. Because high-fat feed contains a large amount of oil, long-term feeding of high-fat feed to Bama pigs can cause indigestion and reduced absorption efficiency. The preferred combination of Gynostemma pentaphyllum saponins, Atractylodes macrocephala, and nutmeg in this invention is more suitable for the constitution of Bama pigs and has a better effect on promoting their digestion and absorption. At the same time, the addition of peppermint leaves, hawthorn acid, and ursolic acid improves the palatability of the feed, and hawthorn acid and ursolic acid can also promote the secretion of digestive juices in Bama pigs, further promoting their digestion and absorption.

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

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

[0019] The vitamin additive is composed of the following components in parts by weight: Vitamin A 19-19.5 parts; Vitamin D3 0.4-0.45 parts; Vitamin E 16.75-16.8 parts; Vitamin K 35-5.1 parts; Vitamin B12 2.1 parts; Vitamin B2 16-16.5 parts; Vitamin C 10-10.1 parts; Vitamin B6 6-6.05 parts; Vitamin B12 0.03-0.031 parts; Niacin 35-35.5 parts; Calcium pantothenate 25-25.5 parts; Folic acid 0.5-0.51 parts; Biotin 0.1-0.11 parts; Methionine 50-50.5 parts; Lysine 50-50.5 parts.

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

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

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

[0023] The feeding involves giving the pigs regular feed or high-fat feed daily, with the weight of the regular feed or high-fat feed being 3.0 to 3.01% of the Bama pigs' body weight for the week.

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

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

[0026] Preferably, the application uses the Bama pig model according to any one of claims 1 to 6 as the model group and uses Bama pigs fed with ordinary feed as the control group;

[0027] The model group was 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 were given drug administration intervention, which was carried out during the weight loss period;

[0029] During the weight loss period, the physiological status of Bama pigs was observed daily, with weekly weight weighing and body size measurements; monthly blood biochemistry tests were conducted, including fasting blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C); at the end of the weight loss period, the weight loss effect was assessed based on the physiological status, weight, body size, and serum biochemistry indicators.

[0030] After the weight loss period, the Bama pigs were euthanized, and the thickness of their back fat and subcutaneous fat layer was measured before samples were taken.

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

[0032] The organs were weighed, embedded, and cryopreserved.

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

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

[0035] The adipose tissue was analyzed using a fatty acid analysis method based on the GC-MS / MS platform to detect and analyze free fatty acids, and to study the molecular mechanism of COST in lipid metabolism and synthesis.

[0036] Preferably, the detection and analysis of free fatty acids specifically includes the following steps:

[0037] S1, Sample pretreatment;

[0038] S2. Sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with dimensions of 30m × 0.25mm × 0.25μm; the carrier gas was high-purity helium; the heating program was as follows:

[0039] Starting at 40℃ and holding for 2 minutes, the temperature was increased at 30℃ / min to 200℃ and held for 1 minute, then increased at 10℃ / min to 240℃ and held for 1 minute, and finally increased at 5℃ / min to 285℃ and held for 3 minutes; flow rate: 1.0 mL / min; injection port temperature: 230℃; injection volume: 1.0 μL;

[0040] The GC-EI-MS system is an Agilent 8890-5977B GC-MS system. After GC detection, the EI-MS parameters are set as follows: Temperature: 230℃; Ionization voltage: 70eV; Transmission line temperature: 240℃; Quadrupole temperature: 150℃; Solvent delay: 4 minutes; Scan mode: SIM.

[0041] S3. Perform statistical analysis.

[0042] Preferably, the sample preprocessing in step S1 specifically includes the following steps:

[0043] After removing the ultra-low temperature preserved sample, thaw it on ice, grind it into powder, and extract it with methanol, methyl tert-butyl ether, and 36% phosphoric acid. Centrifuge, transfer the supernatant, dry it, and add 15% boron trifluoride methanol solution. Keep it in an oven at 60-65℃ for 30-40 minutes. After cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and transfer the n-hexane layer solution for instrumental analysis.

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

[0045] (1) This invention provides a method for constructing a Bama pig model. By feeding a self-made high-fat feed, the ratio of lipids and sugars is reasonably matched, and natural ingredients such as peppermint leaves and hawthorn acid are added to promote the appetite and digestive and absorptive capacity of Bama pigs. After the model is completed, each Bama pig individual maintains a high weight state. The weight gain of individuals in the group is relatively stable between the current week and the previous week. A stable obesity phenotype Bama pig model can be successfully constructed within 12 weeks, which shortens the modeling time of the Bama pig obesity model and improves the modeling efficiency.

[0046] (2) This invention provides a gas chromatography-mass spectrometry method for detecting free fatty acids in Bama pigs, which can analyze the composition and content of free fatty acid metabolites in the adipose tissue of Bama pigs in each group, thereby helping to further explore the effects of drugs on specific differential metabolites of Bama pig fat, and contributing to preclinical drug research. Attached Figure Description

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

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

[0049] Figure 3 shows the body length (A), neck circumference (B), chest circumference (C), abdominal circumference (D), and hip circumference (E) 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 shows the obesity (A), backfat (B), and abdominal subcutaneous fat thickness (C) 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 shows the PCA scores of the mass spectrometry data of Bama pig samples in each group.

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

[0053] Figure 7 shows 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 8 shows the levels 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 shows the liver of Bama pigs after drug administration, HE staining 100X (A) and Oil Red O staining 200X (B) (n=3);

[0056] Figure 10 shows HE staining of the heart (200X) and kidney (100X) of Bama pigs (n=3).

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

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

[0059] Figure 13 shows the 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 shows the serum AST(A) and ALT(B) levels 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] Twelve healthy, 5-month-old, ordinary Bama pigs weighing 20-30kg were selected. Three were randomly selected as the control group and the remaining nine were the model group.

[0066] The control group of Bama pigs were fed regular feed, with a daily feed weight of 3% of the piglets' body weight for the week.

[0067] The model group of Bama pigs were fed ordinary feed for one week, and then switched to high-fat feed, with the daily feed weight being 3% of the pig's body weight for that week.

[0068] At fixed times each week, the weight and body size of Bama pigs in the control group and the model group were measured, and 2 mL of blood was collected from the anterior vena cava of the Bama pigs. The supernatant was then used for blood lipid testing.

[0069] When the obesity rate of Bama pigs in the model group exceeds 20%, the Bama pig model is obtained.

[0070] The formula for calculating obesity 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] The specific steps for preparing ordinary feed are as follows:

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

[0073] The specific steps for preparing a high-fat feed are as follows:

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

[0075] The trace element additives consist of 7.7–12 parts copper, 41–61 parts iron, 22–33 parts zinc, 14.1–21 parts manganese, 0.17–0.33 parts iodine, 0.12–0.23 parts selenium, 0.07–0.13 parts cobalt, and 9.46–14.19 parts water by mass.

[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; 35 parts of vitamin K; 12 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] Experimental Example 1: Obesity Detection in Bama Pigs

[0088] 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.

[0089] Table 2. Results of Obesity Detection in Bama Pigs

[0090] As shown in the table above, Example 1 of the present invention achieved a obesity rate of 20% after 10.5 weeks of feeding with a high-fat diet, while Comparative Examples 1-4 all took 13-14 weeks. Comparative Example 1 lacked nutmeg, Comparative Example 2 lacked gypenosides, and Comparative Example 3 lacked atractylodes macrocephala. The missing components in the high-fat diets of Comparative Examples 1-3 affected the absorption of nutrients in the feed by Bama pigs, thereby reducing their weight gain rate. Atractylodes macrocephala, nutmeg, and gypenosides work synergistically, and all three are indispensable. They can regulate the gastrointestinal tract and promote digestion in Bama pigs, thus promoting the digestion and absorption of nutrients in the feed. The high-fat diet used in Comparative Example 4 lacked peppermint leaves, hawthorn acid, and ursolic acid, which affected the palatability of the feed. Replacing peppermint leaves with patchouli leaves, hawthorn acid with malic acid, and ursolic acid with citric acid did not promote the secretion of digestive juices in Bama pigs, resulting in loss of appetite and reducing the weight gain rate of Bama pigs in Comparative Example 4. In summary, the high-fat feed of this invention, while being highly palatable, utilizes three natural medicinal ingredients—Atractylodes macrocephala, nutmeg, and Gynostemma pentaphyllum saponins—to synergistically enhance the digestion and absorption of Bama pigs, thereby achieving the effect of rapid weight gain in Bama pigs.

[0091] Example 2: Application of the Bama pig model in preclinical studies of the weight-loss drug COST

[0092] Experimental methods: The control group Bama pigs and the model group Bama pigs from Example 1 were used to conduct a preclinical study on the weight loss drug COST.

[0093] The Bama pigs in the model group were randomly divided into: the model control group (3 pigs), the positive drug orlistat group (3 pigs), and the COST group (3 pigs);

[0094] The model control group, the positive control group (orlistat), and the COST group underwent drug intervention during the weight loss period. During this period, the physiological status of the Bama pigs was monitored daily, with weekly weight and body size measurements. Monthly blood biochemical tests were performed, including fasting blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). The weight loss effect was assessed at the end of the weight loss period based on the observed physiological status, weight, body size, and serum biochemical indicators.

[0095] After the weight loss period, Bama pigs were euthanized, and the thickness of backfat and subcutaneous fat layer was measured. Then, samples were collected, including organs, adipose tissue, and cecal contents. Organs included the heart, liver, kidneys, duodenum, ileum, and colon; adipose tissue included perirenal fat, abdominal subcutaneous fat, and greater omentum fat. Organs were weighed, embedded, and cryopreserved. Fatty acid analysis of the adipose tissue was performed using a GC-MS / MS platform to detect and analyze free fatty acids.

[0096] The detection and analysis of free fatty acids includes the following steps:

[0097] S1. After removing the ultra-low temperature preserved sample, thaw it on ice, grind it into powder, add methanol, methyl tert-butyl ether, and 36% phosphoric acid for extraction; centrifuge, transfer the supernatant, blow dry, and add 15% boron trifluoride methanol solution; keep in an oven at 60-65℃ for 30-40 min; after cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and transfer the n-hexane layer solution for instrumental analysis;

[0098] S2. Sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with dimensions of 30m × 0.25mm × 0.25μm; the carrier gas was high-purity helium; the heating program was as follows:

[0099] Starting at 40℃ and holding for 2 minutes, the temperature was increased at 30℃ / min to 200℃ and held for 1 minute, then increased at 10℃ / min to 240℃ and held for 1 minute, and finally increased at 5℃ / min to 285℃ and held for 3 minutes; flow rate: 1.0 mL / min; injection port temperature: 230℃; injection volume: 1.0 μL;

[0100] The GC-EI-MS system used was an Agilent 8890-5977B GC-MS system. After GC detection, the EI-MS parameters were set as follows: Temperature: 230℃; Ionization voltage: 70eV; Transfer line temperature: 240℃; Quadrupole temperature: 150℃; Solvent delay: 4 minutes; Scan mode: SIM.

[0101] S3. Perform statistical analysis.

[0102] The specific experimental procedure is as follows:

[0103] 1. Grouping and administration of experimental animals

[0104] The Bama pig model successfully established in Example 1 was randomly divided into three experimental groups: a model control group (Model group), a positive control group (Orlistat group), and a chitosan oligosaccharide group (COST group). Each group consisted of three obese Bama pigs (2 females and 1 male). After grouping, the Orlistat group and the COST group were administered orlistat and chitosan oligosaccharide, respectively. The dosage was determined according to the conversion factor Rab = 1.370 from adult to miniature pig of standard weight. The Control group and the Model group were fed the corresponding starch feed. This stage was called the weight loss period. Since oral administration to animal models during the weight loss period could easily lead to drug waste, the drug was first mixed with 1 / 10 of the daily feed and completely consumed before feeding the remaining 9 / 10 of the feed to ensure that the drug was fully consumed. The administration period lasted for 12 weeks.

[0105] 2. Observation indicators and material collection

[0106] Throughout the experiment, the physiological state of the Bama pigs needed to be observed daily, including whether they exhibited lethargy, reduced appetite, dry and dull coat, loose stools, or pale mouth. The pigs were weighed and their body size measured weekly after fasting. Blood was collected monthly from the anterior vena cava of the pigs, and the supernatant was used for blood biochemical analysis, including fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), total 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 were assessed based on the general condition, weight, body size, and serum biochemical indicators of the Bama pigs.

[0107] After the 12-week drug administration period, Bama pigs were fasted for 16 hours but allowed free access to water to maintain a relatively fasting state. Before sampling, a final weighing, body measurement, and venous blood sampling were performed. The pigs were then anesthetized with isoflurane and euthanized by bleeding. The backfat thickness and subcutaneous fat layer thickness of each pig were measured. After measurement, all organs were completely removed, weighed, embedded, and cryopreserved. Organs requiring sampling included: heart, liver, kidney, duodenum, ileum, and colon. Adipose tissue samples included: perirenal fat, abdominal subcutaneous fat, and greater omentum fat. Additionally, Bama pig feces (cecal contents) were cryopreserved for subsequent experimental research.

[0108] (1) Observation results of daily behavior and condition of Bama pigs

[0109] Throughout the experiment, the daily behavior and condition of the Bama pigs in each group were closely observed. Piglets in the Control group, fed a standard diet, exhibited good mental state, good appetite, wagged their tails, showed self-cleaning behavior, had pale nasal discs, glossy fur, and formed feces. Piglets in the Model group showed generally poor mental state, preferred to lie down and were less active, later exhibiting a lack of appetite. When fed, their eyes only followed the feeder, they moved slowly, had dry noses, reddish nasal discs with ecchymosis, loose skin, dull fur, numerous fat spots on their backs, and dark-colored feces. Piglets in the Orlistat group were good mental state, active, wagged their tails, were often irritable, had moist noses, pale nasal discs, relatively dry fur, and fewer fat spots on their backs than in the Model group. Their feces were pale and oily. Piglets in the Cost group were good mental state, wagged their tails, showed waiting behavior for feeding, had moist noses, pale nasal discs, relatively dry fur, and fewer fat spots on their backs than in the Model group. Their feces were formed and slightly oily. In terms of physical characteristics, the piglets in the Model group were bulky, with their abdominal skin close to the ground when standing upright, and they had difficulty standing on their hind legs; the piglets in the Control group were relatively small and had a symmetrical body shape; the piglets in the Orlistat and COST groups were similar in size, had no difficulty standing, and could walk upright, as shown in Figure 2.

[0110] (2) Table of weight and weight gain of Bama pigs

[0111] 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 data on weight and weight gain. The results are shown in Figure 1 and Table 3.

[0112] Table 3. Body weight and weight gain of Bama pigs in each group after drug administration.

[0113] As shown in the table above, compared with the Control group, the Model group fed a high-fat diet showed a significant increase in body weight, with a large increase in weight; while the Orlistat and COST groups, which were fed the same high-fat diet, showed a significant decrease in body weight compared with the Model group, with a slow increase in weight. This indicates that orlistat and chitosan oligosaccharide can reduce the body weight of Bama pigs to a certain extent, thereby improving obesity symptoms.

[0114] 3. Measurement of body size index

[0115] Methods for assessing body fat distribution applicable to humans are also applicable to animal models. In clinical practice, height and weight are commonly used indicators for analyzing overall body fat composition, sometimes including waist circumference and skinfold thickness. This experiment measured multiple circumferences in Bama pigs, including body length, neck circumference, chest circumference, abdominal circumference, and waist circumference, as follows.

[0116] Body length: Measure the straight-line distance along the back of the Bama pig from the midpoint of the line connecting the base of the pig's two ears to the first marking on the base of the tail. This is the body length.

[0117] Neck circumference: Use a measuring tape to measure the circumference around the neck of the Bama pig, starting from the back edge of the ear. The tape should be loose enough to fit snugly against the fur.

[0118] Chest circumference: The chest circumference is the length measured around the body with the back edge of the shoulder blade of the Bama pig as the boundary.

[0119] Abdominal circumference: The abdominal circumference is the distance around the lowest point of the abdomen of a Bama pig measured with a tape measure.

[0120] Waist circumference: The circumference measured around the body of a Bama pig, close to the base of its hind legs, is the waist circumference.

[0121] Body size data is an important indicator for measuring the body shape of miniature pigs. In this experiment, the body length, neck circumference, chest circumference, abdominal circumference and waist circumference of Bama pigs were measured. Among them, body length is the longitudinal height and the other four body size indicators are the transverse circumference.

[0122] The measurement results are shown in Figure 3 and Table 4.

[0123] Table 4. Results of body size information of Bama pigs in each group after drug administration (n=3, mean±SEM)

[0124] As shown in the table above, the body length of Bama pigs in the Control group was smaller than that of the other groups fed a high-fat diet. However, there was no significant difference among the Model, Orlistat, and COST groups, which were also fed a high-fat diet. This indicates that drug intervention did not inhibit the growth of Bama pig body length, and body length is strongly correlated with dietary factors. Regarding neck circumference, the neck circumference of the Control group was significantly smaller than that of the Model group, showing a significant difference. The neck circumferences of the Orlistat and COST groups were also significantly smaller than those of the Model group, showing a significant difference from the Model group. There was no significant difference between the Orlistat and COST groups. Compared to the Control group, the chest circumference of Bama pigs in the Model group was significantly increased, while the chest circumference of the Orlistat and COST groups was significantly decreased relative to the Model group. Regarding abdominal circumference, compared to the Control group, the abdominal circumference of Bama pigs in the Model group was significantly increased, while the abdominal circumference of the Orlistat and COST groups was also significantly decreased relative to the Model group. Data analysis of waist circumference showed that the waist circumference of Bama pigs in the Control group was significantly smaller than that in the Model group, while the waist circumference of piglets in the Orlistat and COST groups was significantly smaller than that in the Model group. These data all indicate that feeding high-fat diets accelerates obesity in Bama pigs, while chitosan oligosaccharides can significantly improve the degree of obesity induced by high-fat diets.

[0125] 4. Measurement of subcutaneous fat thickness

[0126] Back fat thickness: After euthanizing the Bama pig, the thickness of subcutaneous fat on the back was measured using calipers at three locations: the posterior edge of the scapula, the last rib, and approximately 4 cm from the midline of the back at the lumbosacral junction. The average of these three measurements was then calculated; this average is called the "BFT" index. BFT is of great significance in measuring the subcutaneous fat content of the back.

[0127] Subcutaneous fat thickness in the abdomen: After the pigs were euthanized, the thickness of subcutaneous fat at the lowest point of the abdomen was measured using calipers. In obese animal models, the degree of obesity is often used as an indicator of whether the model has been successfully established and whether weight loss has been achieved; a degree of obesity greater than 20% indicates obesity.

[0128] Table 5. Results of obesity and fat thickness in Bama pigs after drug administration (n=2-3, mean±SEM)

[0129] As shown in the table above, after 12 weeks of drug intervention, the obesity rate in the Orlistat and COST groups decreased to below 20%, while the obesity rate in the Model group continued to increase, exceeding 30%. As shown in Figure 4B, compared to the Control group, the backfat thickness in the Model group was significantly increased. Further comparison between the Model group and the Orlistat and COST groups showed that the backfat thickness in the latter two groups was significantly decreased. This indicates that subcutaneous fat deposition in Bama pigs was relatively lower after chitosan oligosaccharide feeding. As shown in Figure 4C, compared to the Control group, the abdominal subcutaneous fat thickness in the Model group was significantly increased. Further comparison between the Model group and the Orlistat and COST groups showed that the abdominal fat thickness in the latter two groups was significantly decreased. All these results indicate that the obesity status of Bama pigs was improved to some extent after orlistat and chitosan oligosaccharide intervention.

[0130] 5. Biochemical index determination

[0131] Blood collection method: Lay the Bama pig down in a supine position, exposing the chest cavity. Disinfect the puncture site with 75% ethanol. Use a sterile 10mL syringe to insert the needle vertically into the anterior vena cava sinus and slowly draw 2mL of blood.

[0132] The drawn venous blood is slowly injected into the blood collection tube to avoid blood cell rupture and hemolysis. The blood in the tube is then immediately centrifuged at 3500 rpm for 15 minutes to obtain the supernatant serum for blood index testing.

[0133] Blood parameters: According to the corresponding instructions for use, use a standard kit to measure the levels of TG, TC, HDL-C, LDL-C, FFA, ALT, AST, and GLU in serum samples, and use an ELISA kit to measure the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α), fasting insulin (Insulin), and endotoxin (LPS) in serum samples.

[0134] Liver / Adipose Tissue Indicators: Using a 0.100g analytical balance, weigh 0.100g of liver or adipose tissue into 1.5mL EP tubes. Add 0.9mL of physiological saline and 2 grinding beads to the tubes. Homogenize the prepared tubes in an automated homogenizer. After homogenization, centrifuge the tubes containing the tissue at 4℃ and 2500r / min for 10min. Collect the supernatant after centrifugation. Measure Bama pig liver indicators (GLU, AST, ALT, TG, TC, LDL-C) using standard kits and adipose tissue levels (IL-1β, IL-6, IL-10, TNF-α) using ELISA kits.

[0135] The experimental results are shown in Figures 7, 8, 13, 14 and Tables 6-9.

[0136] Table 6. Results of blood glucose-related indicators in Bama pigs after drug administration (n=3, mean±SEM)

[0137] Table 7. Results of serum AST and ALT levels in Bama pigs after drug administration (n=3, mean±SEM)

[0138] Table 8. Results of changes in serum lipid levels in Bama pigs after drug administration (n=3, mean±SEM)

[0139] Table 9. Results of liver biochemical indicators in Bama pigs after drug administration (n=3, mean±SEM)

[0140] 6. Pathological sections and staining

[0141] HE staining: The embedding cassette containing the tissue was fixed in 4% paraformaldehyde solution for 24 hours, and then placed in an automated dehydrator for stepwise dehydration. After dehydration, the tissue was embedded in paraffin. After pre-cooling, the embedded paraffin tissue was sectioned using a microtome. The slides containing the tissue were stained with hematoxylin and eosin, following the same staining procedure and time as previously experienced in the laboratory. Finally, the stained slides were stored in a slide cassette for subsequent microscopic observation.

[0142] Oil Red O staining: The embedding cassette containing the tissue was fixed in 4% paraformaldehyde solution for 24 hours. Then, the cassette was sequentially immersed in 15% sucrose solution for 14 hours, 20% sucrose solution for 14 hours, and 30% sucrose solution for 14 hours, for stepwise dehydration. After dehydration, the tissue was embedded using OCT embedding medium. The embedded tissue was then stored at -20°C. Sections with a thickness of 8 μm were then prepared using a cryostat. The sections were stained with Oil Red O staining solution. Finally, the stained sections were stored in a slide cassette for subsequent microscopic observation.

[0143] To further understand the deposition of lipid droplets in the liver tissue of obese Bama pigs, paraffin sections of livers from each group of Bama pigs were stained with hematoxylin and eosin (HE), and frozen sections were stained with Oil Red O. HE staining of Bama pig livers is shown in Figure 9(A). In the Control group, Bama pig liver cells were regularly arranged with uniform cytoplasm, while in the Model group, mouse liver cells contained lipid droplet vacuoles of varying sizes. After COST intervention, the number of lipid vacuoles in the COST group was significantly reduced compared to the Model group, and the arrangement of liver cells was significantly improved. Oil Red O staining of Bama pig livers is shown in Figure 9(B). Compared to the Control group, the Model group showed a large number of red lipid droplets in its liver, while the number of lipid droplets in the COST group was significantly reduced. Combining the results of HE and Oil Red O staining, chitosan oligosaccharide can alleviate steatosis in the liver of obese Bama pigs and reduce lipid droplet accumulation in the liver.

[0144] In Figure 10(A), the cardiomyocytes of all four groups of Bama pigs showed normal morphology, and no hypertrophy was observed. In Figure 10(B), the kidney cells of all four groups of Bama pigs showed normal morphology, with glomeruli and corpuscles clearly visible and nuclear membranes clearly defined. Based on HE staining and the appearance of the kidneys in each group of Bama pigs, the kidney tissue was determined to be in a normal state. Therefore, based on the pathological sections, we preliminarily conclude that chitosan oligosaccharide has no significant toxic side effects on the heart and kidney tissues of Bama pigs.

[0145] Adipose tissue is an important indicator of obesity. In this study, subcutaneous fat and perirenal fat (fat around the kidneys) of Bama pigs in each group were stained with hematoxylin and eosin (HE). As shown in Figure 11(A), the subcutaneous fat cells in the Control group were uniform in size and numerous, while the subcutaneous fat cells in the Model group were larger and had indistinct nuclear membranes. COST administration significantly increased the number of cells and reduced the size of fat cells. In perirenal fat (Figure 11(B), it can be seen that the perirenal fat cells in the Model group were large, few in number, and had indistinct nuclear membranes, while both the Control and COST groups significantly improved the condition of perirenal fat cells in the Model group.

[0146] To observe the morphology and structure of the Bama pig intestine, HE staining was performed on the ileum and colon tissues. Figure 12(A) shows the staining of the Bama pig ileum tissue. In the Model group, the intestinal villi and intestinal muscle layer of the Bama pigs were damaged, the barrier function was weakened, and the intestinal crypt depth was shallower. Compared with the Model group, the intestinal muscle layer of the Control and Cost groups was relatively intact, and the crypt depth was deeper. In Figure 12(B) of the HE staining of the Bama pig colon, adipocytes in the colon tissue of the Bama pigs in the Model group had infiltrated into the intestinal muscle layer, and the goblet cells secreting mucus had abnormal morphology and reduced number. Compared with the Model group, the number of goblet cells in the Control and Cost groups was significantly increased, and they could secrete more mucus, which helps reduce the damage of fecal pellets to the intestinal mucosa. This indicates that chitosan oligosaccharide can alleviate the damage of a high-fat diet to the intestines of Bama pigs and is beneficial to intestinal metabolism.

[0147] In conclusion, feeding chitosan oligosaccharides can reduce the weight and weight gain of obese Bama pigs, lower blood liver lipids, reduce blood glucose, improve inflammation in serum and adipose tissue, improve liver function, and remodel the intestinal structure of piglets, thus exhibiting certain weight-loss activity, laying the foundation for further exploration of the mechanism.

[0148] 7. Detection of free fatty acid composition in Bama pig fat

[0149] Fatty acids (FA) exist widely in various forms within organisms and participate in lipid metabolism as key compounds. Fatty acid detection employs mass spectrometry in selected ion monitoring (SIM) mode. After sample molecules are ionized, only pre-selected target ions can reach the detector via a quadrupole, while other ions cannot. Selected ion monitoring effectively removes matrix interference, improves sensitivity, and facilitates better quantitative and qualitative analysis of free fatty acid components in Bama pigs. This experiment analyzed the free fatty acid components of adipose tissue from various groups of Bama miniature pigs using GC-MS / MS platform methods, further exploring the molecular mechanism of COST in lipid metabolism and synthesis.

[0150] This experiment detected 48 free fatty acids in the adipose tissue of Bama pigs. The composition information is shown in the table below.

[0151] Table 10 Information on 48 Fatty Acids

[0152] A total of 12 samples from 4 groups were selected for testing. Sample information is shown in the table below.

[0153] Table 11 Sample Information Table

[0154] The specific steps for detecting free fatty acids are as follows:

[0155] 7.1 Sample Pretreatment

[0156] (1) Remove the sample from the -80℃ freezer and thaw it on ice (all subsequent operations must be performed on ice).

[0157] (2) Take out the cryopreserved biological sample and grind it with a grinder (30Hz, 1min) until it becomes powder.

[0158] (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.

[0159] (4) Vortex for 3 minutes, then centrifuge at 4℃ and 12000r / min for 5 minutes.

[0160] (5) Take 200 μL of supernatant and blow it dry with a nitrogen blower, then add 300 μL of 15% boron trifluoride methanol solution.

[0161] (6) Vortex for 3 minutes, then keep in an oven at 60℃ for 30 minutes.

[0162] (7) Cool to room temperature and accurately add 500 μL of n-hexane solution and 200 μL of saturated sodium chloride solution.

[0163] (8) Vortex for 3 min, centrifuge at 4℃ and 12000 r / min for 5 min, and then transfer 100 μL of the n-hexane layer solution for analysis.

[0164] 7.2 Chromatographic and Mass Spectrometric Acquisition and Analysis

[0165] 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 analytical 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: starting at 40℃ (2 min), increasing at 30℃ / min to 200℃ (1 min), then at 10℃ / min to 240℃ (1 min), and finally at 5℃ / min to 285℃ (3 min); Flow rate: 1.0 mL / min; Injector temperature: 230℃; Injection volume: 1.0 μL

[0166] 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 minutes; scan mode: SIM.

[0167] 7.3 Statistical Analysis

[0168] (1) Principal Component Analysis (PCA)

[0169] Unsupervised principal component analysis (PCA) is performed using the statistical function prcomp at www.r-project.org. The data needs to be scaled to unit variance before performing unsupervised PCA.

[0170] (2) Hierarchical cluster analysis and Pearson correlation coefficient

[0171] A dendritic heatmap was generated based on hierarchical cluster analysis (HCA) of samples and metabolites, while the Pearson correlation coefficients (PCCs) between samples were calculated using the `cor` function in R and presented only as heatmaps. Both hierarchical cluster analysis and Pearson correlation coefficients were performed using the R package `pheatmap`. For hierarchical cluster analysis, the normalized signal intensity of metabolites (scaled to unit variance) was visualized as a color gradient.

[0172] 7.4 Results of the detection of different fatty acid contents in the adipose tissue of Bama pigs

[0173] This experiment detected the main components of free fatty acids in the adipose tissue of Bama pigs in different groups, and the results were grouped and statistically analyzed. The results are shown in the table below. By comparing the results, it was found that the contents of 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 were significantly increased in the Model group compared with the Control group. It can be inferred that the high-fat diet induces obesity in Bama pigs by interfering with the normal lipid metabolism process of Bama pigs by increasing the synthesis of these fatty acid components. Significant differences were found between the COST group and the Model group in the levels of certain fatty acids. 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, and C22-1n9, the COST group showed a significantly lower content in Bama pig tissues compared to the Model group. Furthermore, the levels of most of these components were significantly higher in the Model group than in the Control group. This indicates that COST administration can reduce the levels of free fatty acids that contribute to obesity, thereby alleviating the fat accumulation induced by a high-fat diet.

[0174] Table 12 Content of free fatty acids in adipose tissue of Bama pigs (n=3, mean±SD)

[0175] 7.5 Principal Component Analysis of Fat Samples from Bama Pigs

[0176] Multivariate statistical analysis can reduce the dimensionality of high-dimensional and complex data while preserving as much of the original data as possible. This method helps to accurately build mathematical models, thereby summarizing and analyzing the metabolic profile characteristics of samples. Principal Component Analysis (PCA) is an unsupervised pattern recognition method for multidimensional data statistical analysis. It transforms a set of potentially correlated variables into a set of linearly uncorrelated variables through orthogonal transformation; this transformed set of variables is called the principal components. The results are shown in Figure 5. In 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 explanatory power of the principal component on the dataset. Each point in the figure represents a sample; samples in the same group are represented by the same color. "Group" indicates grouping.

[0177] Principal component analysis (PCA) was performed on adipose tissue samples from Bama pigs in each group to understand the overall metabolic differences among groups and within groups. PCA results showed metabolomic trends between groups, indicating the metabolomic status within each group of adipose tissue. QC samples served as quality control samples. As shown in Figure 5, the PCA score plot revealed a significant shift in principal components between the Control and Model groups, indicating a significant metabolic difference between the two groups. This suggests that lipid metabolism in the Model group's adipose tissue was significantly disrupted. However, in the Bama pig samples treated with COST, the principal components of metabolites were closer to those in the Control group. In conclusion, COST treatment can partially restore the metabolic characteristics of Bama pig adipose tissue and alleviate the metabolic differences between the Control and Model groups, suggesting that COST may have a certain influence on regulating lipid metabolism in Bama pig adipose tissue.

[0178] 7.6 Cluster analysis of metabolites in fat samples from Bama pigs

[0179] Cluster analysis is a multivariate statistical analysis method for classification. This method categorizes individuals or samples based on their characteristics, aiming to maximize homogeneity within the same category and heterogeneity between categories. Cluster analysis provides a more intuitive way to observe the accumulation of lipid metabolites among different Bama pig samples.

[0180] In this experiment, the fatty acid metabolite data of Bama pigs were processed using unit variance scaling (UV), and a clustering heatmap was plotted using an R program script, as shown in Figure 6.

[0181] As shown in Figure 6, 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 showed higher levels in the Bama pig samples of the Model group, significantly higher than those of the Control group. However, in the COST-treated group, the clustered levels of these fatty acids were significantly lower than those in the Model group. This is consistent with the results of metabolite detection in individual samples mentioned earlier, further supporting the conclusion 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 lead to obesity, thereby alleviating the fat accumulation state induced by a high-fat diet.

[0182] Free fatty acid metabolites play a crucial role in obesity. In obese individuals, free fatty acid levels are typically elevated. These free fatty acids are products of triglyceride hydrolysis within adipocytes and can be transported to other tissues via the bloodstream, serving as an energy source. However, excessively high levels of free fatty acids can lead to problems such as inflammatory responses in adipocytes, insulin resistance, and fat accumulation. Studies have shown that excessive free fatty acids can promote the enlargement and increase the number of adipocytes, leading to hypertrophy and excessive accumulation of adipose tissue. In other words, free fatty acid metabolites in obesity may also affect the physiological functions of adipose tissue by influencing adipocyte proliferation and differentiation, potentially disrupting normal metabolic functions. Furthermore, excessive free fatty acids can interfere with lipid synthesis and degradation pathways, leading to lipid metabolism disorders and inflammatory responses in adipose tissue, thereby exacerbating the development of obesity.

[0183] In conclusion, COST treatment played a role in restoring the metabolic characteristics of adipose tissue in Bama pigs, helping to reduce the metabolic differences between the Control and Model groups. These findings provide new clues for researching treatment strategies for obesity-related metabolic disorders and warrant further investigation and exploration.

Claims

1. A Bama pig model for preclinical studies of the cost of weight-loss drugs, characterized in that, The method for constructing the Bama pig model includes the following steps: Healthy Bama pigs were selected and divided into a control group and a model group. The control group of Bama pigs was fed ordinary feed; The model group of Bama pigs was first fed ordinary feed, and then switched to high-fat feed. Obesity-related indicators were detected in both the control group and the model group of Bama pigs at fixed time periods each week. When the obesity rate of the model group of Bama pigs was greater than 20%, the Bama pig model was obtained. The formula for calculating obesity 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%; By weight, the high-fat feed comprises the following components: 49-50 parts of the ordinary feed, 9-10 parts of butter, 10-11 parts of margarine, 14-15 parts of sucrose, 9-10 parts of casein, 2-2.5 parts of laboratory animal premix, 1-1.5 parts of microcrystalline cellulose, and 2-2.5 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 Gynostemma pentaphyllum saponins, 0.5-1.0 parts of peppermint leaves, 0.2-0.3 parts of hawthorn 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 corn, 3-4 parts fish meal, 8-9 parts soybean meal, 9-10 parts rice bran, 18-20 parts wheat bran, 30-32 parts alfalfa meal, 2-3 parts calcium bicarbonate, 0.3-0.4 parts iodized salt, 0.03-0.04 parts trace element additives, and 0.03-0.032 parts vitamin additives.

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

5. The Bama pig model according to claim 1, characterized in that, The healthy Bama pigs mentioned are ordinary Bama pigs aged 4 to 6 months, weighing 20 to 30 kg.

6. The Bama pig model according to claim 1, characterized in that, The model group of Bama pigs was first fed ordinary feed for 1.0 to 1.1 weeks, and then fed high-fat feed until the obesity rate of the model group of Bama pigs was greater than 20%. The feeding involves giving the pigs regular feed or high-fat feed daily, with the weight of the regular feed or high-fat feed being 3.0 to 3.01% of the Bama pigs' body weight for the week.

7. The application of the Bama pig model as described in any one of claims 1 to 6 in the preclinical study of the weight-loss drug COST.

8. The application according to claim 7, characterized in that, The Bama pig model according to any one of claims 1 to 6 was used as the model group, and Bama pigs fed with ordinary feed were used as the control group. The model group was 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 were given drug administration intervention, which was carried out during the weight loss period; During the weight loss period, the physiological status of Bama pigs was observed daily, with weekly weight weighing and body size measurements; monthly blood biochemistry tests were conducted, including fasting blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C); at the end of the weight loss period, the weight loss effect was assessed based on the physiological status, weight, body size, and serum biochemistry indicators. After the weight loss period, the Bama pigs were euthanized, and the thickness of their back fat and subcutaneous fat layer was measured before samples were taken. The sampling includes removing organs, adipose tissue, and cecal contents; The organs were weighed, embedded, and cryopreserved. The organs include: heart, liver, kidney, duodenum, ileum, and colon; The adipose tissue includes: perirenal fat, abdominal subcutaneous fat, and greater omentum fat; The adipose tissue was analyzed using a fatty acid analysis method based on the GC-MS / MS platform to detect and analyze free fatty acids, and to study the molecular mechanism of COST in lipid metabolism and synthesis.

9. The application according to claim 8, characterized in that, The detection and analysis of free fatty acids specifically includes the following steps: S1, Sample pretreatment; S2. Sample derivatives were analyzed using a GC-EI-MS system. The GC column used was a DB-5MS capillary column with dimensions of 30m × 0.25mm × 0.25μm; the carrier gas was high-purity helium; the heating program was as follows: Starting at 40℃ and holding for 2 minutes, the temperature was increased at 30℃ / min to 200℃ and held for 1 minute, then increased at 10℃ / min to 240℃ and held for 1 minute, and finally increased at 5℃ / min to 285℃ and held 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. After GC detection, the EI-MS parameters are set as follows: Temperature: 230℃; Ionization voltage: 70eV; Transmission line temperature: 240℃; Quadrupole temperature: 150℃; Solvent delay: 4 minutes; Scan mode: SIM. S3. Perform statistical analysis.

10. The application according to claim 9, characterized in that, Step S1, the sample preprocessing, specifically includes the following steps: After removing the ultra-low temperature preserved sample, thaw it on ice, grind it into powder, and extract it with methanol, methyl tert-butyl ether, and 36% phosphoric acid. Centrifuge, transfer the supernatant, dry it, and add 15% boron trifluoride methanol solution. Keep it in an oven at 60-65℃ for 30-40 minutes. After cooling to room temperature, add n-hexane solution and saturated sodium chloride solution, mix well, centrifuge, and transfer the n-hexane layer solution for instrumental analysis.

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