Manufacturing method of qi deficiency and blood stasis syndrome large animal model

Abstract

The invention relates to the technical field of medical evaluation and detection, in particular to a Qi deficiency and blood stasis syndrome large animal model making method which comprises the following steps: step 1, feed proportioning and feeding: miniature pigs are continuously fed with high-calorie feed for 6 months, and the high-calorie feed comprises the following components in percentage by weight: 10% of beef tallow, 6% of peanut oil, 0.5% of cholate, 3% of cholesterol and the balance of water; the acesulfame potassium accounts for 2.5%, and the basal feed accounts for 78%. According to the preparation method of the qi deficiency and blood stasis syndrome model, animals are subjected to long-term chronic high-fat diet, stomach impairment and spleen impairment according to the traditional Chinese medicine theories of'fat, sweet and thick taste impairment on spleen and qi 'and'qi consumption due to fatigue', so that the source of vital energy can be reduced, and the blood stasis syndrome caused by phlegm-dampness can be helped to occur; exhaustive swimming is used for increasing the consumption of healthy qi, qi is the commander of blood, qi deficiency causes inability to promote blood circulation, and blood in meridians is stagnated and deposited to form blood stasis.

Description

Technical Field

[0001] The invention relates to the field of medical evaluation and detection technology, and in particular to a method for preparing a large animal model of qi deficiency and blood stasis syndrome. Background Art

[0002] Qi deficiency and blood stasis syndrome refers to the presence of both qi deficiency and blood stasis. Qi deficiency and blood stasis syndrome is a common symptom in many diseases in TCM clinics, and is more common in cardiovascular and cerebrovascular diseases in the elderly, such as coronary heart disease and angina pectoris. This syndrome is characterized by both deficiency and excess, and is characterized by the simultaneous presence of qi deficiency and blood stasis syndrome.

[0003] Qi is the leader of blood, and blood is the mother of Qi. Qi deficiency can cause blood stasis. If blood stasis lasts for a long time, the collaterals will be blocked, blood deficiency and Qi depletion can also cause Qi damage. It clarifies the logical dialectical relationship between Qi deficiency and blood stasis, and the inevitable connection between the function of Qi and blood stasis.

[0004] At present, there are many methods for making animal models of "Qi deficiency and blood stasis syndrome" that have been reported, which can be divided into single-factor modeling methods and multi-factor combined modeling methods. Common single-factor modeling methods are mainly simple exhaustive exercise, such as exhaustive swimming and running. Zhang Hongyu et al. selected Wistar Bama miniature pigs to swim for 14 days to simulate the occurrence of Qi deficiency syndrome caused by extreme fatigue, and obtained Bama miniature pigs with Qi deficiency and blood stasis syndrome. (Zhang Hongyu, Zhang Xiaohua. Effect of Buyang Huanwu Decoction on Hemorheology of Bama Miniature Pigs with Qi Deficiency and Blood Stasis Syndrome [J]. Journal of Yunnan University of Traditional Chinese Medicine, 2000, 23(3):10-11.) The model obtained by this method can simulate the process of "Qi deficiency" leading to "blood stasis", but the model animals cannot well reflect the clinical characteristics of Qi deficiency and blood stasis syndrome. For example, the model animals did not show clinical manifestations of blood stasis syndrome such as purple tongue, and the recovery time of the model animals was short, which was not very close to clinical reality.

[0005] The modeling method combining multiple factors can be divided into etiological modeling, modeling combined with modern pathological factors, and modeling combined with symptoms. Modeling based on etiology mainly includes comprehensive exhaustive exercise, improper diet, panic, cold and dampness, etc. Pang Shuling et al. used hunger (eating 1 / 3 of the normal amount), fatigue, and cold (swimming in cold water at 14-16℃) to act on Wistar Bama miniature pigs to create a model of qi deficiency and blood stasis. (Pang Shuling, Gao Jinliang. Simulation of qi deficiency and blood stasis in middle-aged Bama miniature pigs and its mechanism [J]. Journal of Tianjin University of Traditional Chinese Medicine, 1997 (3): 28-30.) The model Bama miniature pigs obtained by this method showed a low viscosity state of blood, which is not directly consistent with the characteristics of blood stasis and cannot well reflect the clinical characteristics of qi deficiency and blood stasis. In addition, this type of modeling method has many artificial interference factors and poor repeatability of model results. Modeling combined with modern pathological factors mainly combines the pathological factors of blood stasis syndrome with etiological modeling. Cai Guangxian et al. prepared a model of Qi deficiency and blood stasis by exhaustive swimming plus subcutaneous injection of adrenaline. The results showed that the blood rheology parameters of the animals were increased after modeling. This type of method can simulate the required symptoms of Qi deficiency and blood stasis, but it is only a short-term state, easy to recover naturally, and there are individual differences in the response of animals to drugs, low model stability, and difficult to repeat experimental results. The symptom-combined modeling method refers to the modeling method of modern medical diseases combined with the etiology and pathogenesis of traditional Chinese medicine. Wang Jian et al. selected middle-aged and elderly Bama miniature pigs and used starvation (controlling the amount of basic feed), fatigue (swimming), high-fat diet (instilled with fat emulsion) and ligation of the left common carotid artery to create an animal model of Qi deficiency and blood stasis in ischemic stroke. The model Bama miniature pigs showed symptoms of hemiplegia, obvious cerebral edema, and the blood was highly concentrated, sticky, coagulated, and aggregated, which was considered to be consistent with the clinical characteristics of Qi deficiency and blood stasis in ischemic stroke. (Wang Jian, Hu Jianpeng. Preliminary study on animal model of qi deficiency and blood stasis in ischemic stroke [J]. Journal of Anhui University of Traditional Chinese Medicine, 1999(2):46-49.) However, this type of modeling method has obvious shortcomings: recreating the disease model on the basis of multi-factor modeling to create a disease-combined animal model often causes excessive damage to the experimental animals, resulting in high animal mortality and poor experimental feasibility.

[0006] Therefore, it is of great significance to explore a method for preparing an animal model of Qi deficiency and blood stasis syndrome that is simple to operate, has a low model mortality rate, good repeatability and stability, and has long-lasting symptom characteristics and is close to clinical practice. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a model of qi deficiency and blood stasis syndrome. According to the theories of traditional Chinese medicine such as "fat, sweet and greasy food injure the spleen and damage qi" and "exhaustion leads to qi depletion", animals are fed a long-term chronic high-fat diet to hinder the stomach and injure the spleen, which can reduce the source of their vitality and promote the generation of phlegm and dampness leading to the occurrence of blood stasis syndrome; exhaustion swimming is performed to increase the consumption of their vital energy, "qi is the commander of blood", and qi deficiency is unable to promote blood circulation, so the blood in the meridians stagnates and accumulates to form blood stasis, so as to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing a large animal model of Qi deficiency and blood stasis syndrome, comprising the following steps:

[0009] Step 1, feed proportion and feeding: feeding the miniature pigs with high-calorie feed for 6 months, wherein the components of the high-calorie feed are 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed by weight;

[0010] Step 2: A method for establishing a hyperlipidemia model of Bama miniature pigs by feeding a high-calorie feed, comprising the following steps:

[0011] (1) Select healthy Bama miniature pigs, aged 6-8 months, weighing 29.88 kg ± 2.27 kg, male, with normal intestinal flora;

[0012] (2) The Bama miniature pigs were fed with the aforementioned high-calorie diet for 48 weeks, and their body weight was measured daily. The daily food intake was 3% of the measured body weight, and they were allowed to drink water freely;

[0013] (3) Regularly test the blood disease indicators of Bama miniature pigs.

[0014] Preferably, the specific formula of the basic feed is as follows: 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt, and 0.42% premix.

[0015] Preferably, the percentages of the components in the premix are: lysine 0.20%, multivitamins 0.05%, trace elements 0.10%, choline 0.05%, and phytase 0.02%.

[0016] Preferably, the multiple vitamins in the premix are: vitamin A, vitamin D-3, vitamin E, vitamin K-3, pantothenic acid, niacinamide, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, and biotin.

[0017] Preferably, the trace elements are: copper sulfate pentahydrate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, 1% calcium iodate, and 1% sodium selenite.

[0018] Preferably, the method for establishing a Bama miniature pig hyperlipidemia model by feeding a high-calorie feed in step 2 comprises the following steps:

[0019] Experimental material selection: 55 healthy Bama miniature pigs, aged 6-8 months, weighing 29.88kg±2.27kg, male, with normal intestinal flora;

[0020] Experimental method: High-calorie feed feeding: The weight percentage of each component in the high-calorie feed formula is: 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed. The model group Bama miniature pigs were fed for 48 weeks. The body weight of the Bama miniature pigs was measured daily, and the food intake was 3% of the measured body weight. Water was available for free drinking.

[0021] Preferably, the raw materials of the basic feed are 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt and 0.42% premix.

[0022] Preferably, the weight percentage of each component in the premix to the high-fat feed is: lysine 0.20%, multivitamins 0.05%, trace elements 0.10%, choline 0.05%, phytase 0.02%, a total of 0.42%.

[0023] Preferably, the multivitamins in the premix include: 12500IU vitamin A, 5500IU vitamin D-3, 60g vitamin E, 10g vitamin K-3, 10g pantothenic acid, 60g niacinamide, 10g vitamin B-1, 30g vitamin B-2, 10g vitamin B-6, 0.1g vitamin B-12, and 0.6g biotin; trace elements include: 200g copper sulfate pentahydrate, 260g ferrous sulfate monohydrate, 260g zinc sulfate monohydrate, 80g manganese sulfate monohydrate, 5g 1% calcium iodate, 3g 1% sodium selenite, and feed carriers: bentonite and bran.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present application discloses a method for preparing a model of qi deficiency and blood stasis syndrome. According to the theories of traditional Chinese medicine such as "fat, sweet and greasy food injure the spleen and damage qi" and "exhaustion leads to qi depletion", animals are fed a long-term chronic high-fat diet, which hinders the stomach and injures the spleen, which can reduce the source of their vitality and promote the generation of phlegm and dampness leading to the occurrence of blood stasis syndrome; exhaustion swimming is used to increase the consumption of their vital energy, and "qi is the commander of blood". If qi is deficient, it will be unable to promote blood circulation, and the blood in the meridians will stagnate and accumulate to form blood stasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The present invention is a flow chart of the method for preparing a large animal model of Qi deficiency and blood stasis syndrome. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] See also Figure 1 , the present invention provides a technical solution:

[0030] A method for preparing a large animal model of Qi deficiency and blood stasis syndrome comprises the following steps:

[0031] Step 1, feed proportion and feeding: feeding the miniature pigs with high-calorie feed for 6 months, wherein the components of the high-calorie feed are 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed by weight;

[0032] Step 2: A method for establishing a hyperlipidemia model of Bama miniature pigs by feeding a high-calorie feed, comprising the following steps:

[0033] (1) Select healthy Bama miniature pigs, aged 6-8 months, weighing 29.88 kg ± 2.27 kg, male, with normal intestinal flora;

[0034] (2) The Bama miniature pigs were fed with the aforementioned high-calorie diet for 48 weeks, and their body weight was measured daily. The daily food intake was 3% of the measured body weight, and they were allowed to drink water freely;

[0035] (3) Regularly test the blood disease indicators of Bama miniature pigs.

[0036] The specific formula of the basic feed is as follows: 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt, and 0.42% premix.

[0037] The percentages of the components in the premix are: 0.20% lysine, 0.05% multivitamins, 0.10% trace elements, 0.05% choline and 0.02% phytase.

[0038] The multiple vitamins in the premix are: vitamin A, vitamin D-3, vitamin E, vitamin K-3, pantothenic acid, niacinamide, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, and biotin.

[0039] Preferably, the trace elements are: copper sulfate pentahydrate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, 1% calcium iodate, and 1% sodium selenite.

[0040] The second step is a method for establishing a hyperlipidemia model of Bama miniature pigs by feeding high-calorie feed, comprising the following steps:

[0041] Experimental material selection: 55 healthy Bama miniature pigs, aged 6-8 months, weighing 29.88kg±2.27kg, male, with normal intestinal flora;

[0042] Experimental method: High-calorie feed feeding: The weight percentage of each component in the high-calorie feed formula is: 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed. The model group Bama miniature pigs were fed for 48 weeks. The body weight of the Bama miniature pigs was measured daily, and the food intake was 3% of the measured body weight. Water was available for free drinking.

[0043] The raw materials of the basic feed are 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt and 0.42% premix.

[0044] The weight percentage of each component in the premix to the high-fat feed is: 0.20% lysine, 0.05% multivitamins, 0.10% trace elements, 0.05% choline, 0.02% phytase, which is 0.42% in total.

[0045] The multivitamins in the premix include: 12500 IU vitamin A, 5500 IU vitamin D-3, 60 g vitamin E, 10 g vitamin K-3, 10 g pantothenic acid, 60 g nicotinamide, 10 g vitamin B-1, 30 g vitamin B-2, 10 g vitamin B-6, 0.1 g vitamin B-12, and 0.6 g biotin; the trace elements include: 200 g copper sulfate pentahydrate, 260 g ferrous sulfate monohydrate, 260 g zinc sulfate monohydrate, 80 g manganese sulfate monohydrate, 5 g 1% calcium iodate, and 3 g 1% sodium selenite; and the feed carrier includes bentonite and bran.

[0046] Indicator detection method.

[0047] Evaluation of "Qi Deficiency"

[0048] Compared with the normal group, the model group showed the following symptoms of qi deficiency: thin body, rough and dull fur, weakness,

[0049] Reluctance to walk, sometimes accompanied by diarrhea, loose stools and other symptoms, in severe cases even manifested as rectal prolapse and hernia.

[0050] Physical and chemical testing.

[0051] All miniature pigs were blooded from the ear vein at 24, 28, 36, and 48 weeks (fasted for 16 hours before blood collection), and serum was routinely prepared. Cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected by automatic biochemical analyzer; oxidized low-density lipoprotein (ox-LDL), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and P-selectin (sP-selectin) were determined by ELISA, and the specific operations were strictly carried out according to the instructions of the kit.

[0052] HE staining of coronary arteries.

[0053] At 24 weeks, one miniature pig in the normal group and two miniature pigs in the model group were killed by bleeding from the portal vein under anesthesia. At 48 weeks, all the miniature pigs were killed, and the anterior descending branch of the coronary artery was quickly isolated. After fixation with 4% paraformaldehyde, the samples were routinely paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) for pathological histological analysis, mainly to observe the degree of luminal stenosis and endothelial damage.

[0054] In order to better understand the essence of the present invention, the present invention is described in the form of embodiments below. It must be noted that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0055] Example 1: Qi deficiency and blood stasis

[0056] Method 1:

[0057] (1) Prepare a high-fat feed with a total energy of 4.4 kcal / g and a calorie ratio of: protein 17%, fat 37%, and carbohydrate 46%.

[0058] (2) Bama miniature pigs were selected and continuously fed with a high-fat diet.

[0059] (3) After that, the pigs were subjected to exhaustive swimming every day for 2 weeks. Specifically, the pigs were placed in a pool with a diameter of 160 cm, a height of 50 cm, a depth of 40 cm, and a water temperature of 19°C in the morning for swimming. The pigs were allowed to swim until they sank naturally (the nose tip was submerged in the water) for 10 seconds before stopping. After the experiment, the animal model of Qi deficiency and blood stasis was obtained.

[0060] Method 2:

[0061] (1) Prepare a high-fat feed with a total energy of 4.2 kcal / g and a calorie ratio of: protein 15%, fat 35%, and carbohydrate 50%.

[0062] (2) Select Bama miniature pigs and continuously feed them with a high-fat diet.

[0063] (3) After that, the pigs were subjected to exhaustive swimming every day for 1 week. Specifically, the pigs were placed in a pool with a diameter of 160 cm, a height of 50 cm, a depth of 40 cm, and a water temperature of 17°C in the morning for swimming. The pigs were allowed to swim until they sank naturally (the nose tip was submerged in the water) for 10 seconds before stopping. After the experiment, the animal model of Qi deficiency and blood stasis was obtained.

[0064] Method 3:

[0065] (1) Prepare a high-fat feed with a total energy of 4.6 kcal / g and a calorie ratio of: protein 19%, fat 39%, and carbohydrate 42%.

[0066] (2) 8-week-old SPF SD Bama miniature pigs were selected and continuously fed with a high-fat diet.

[0067] (3) After that, the pigs were subjected to exhaustive swimming every day for 4 weeks. Specifically, the pigs were placed in a pool with a diameter of 160 cm, a height of 50 cm, a depth of 40 cm, and a water temperature of 21°C in the morning for swimming. The pigs were allowed to swim until they sank naturally (the nose tip was submerged in the water) for 10 seconds before stopping. After the experiment, the animal model of Qi deficiency and blood stasis was obtained.

[0068] Characteristic indicators include: Qi deficiency symptoms: a. Fatigue and lethargy, mental depression, curled limbs, poor activity, reduced or disappeared aggressive and confrontational behaviors, no obvious escape attempt when placed outside the cage alone; b. Loss of appetite, feces appear different degrees of looseness; c. Sparse hair, easy to fall off, or curled, dry and yellow, lose moisture; d. Easily startled and trembling when hearing special sounds, and often show restlessness and running around or curling up and trembling when hearing other Bama miniature pigs swimming. Blood stasis symptoms: a. The tongue of the experimental Bama miniature pigs is obviously dark purple compared with the control group; b. Subcutaneous bruises or blue spots appear from the tip to the root of the tail to different degrees; c. The eyeballs turn dark red or light red compared with the bright red of the control group; d. The hind limbs are often difficult and slow to move.

[0069] The oxidative stress indicators were superoxide dismutase (SOD) and malondialdehyde (MDA); the myocardial enzyme spectrum indicators were lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB); the liver function indicators were alanine aminotransferase (ALT) and aspartate aminotransferase (AST); the renal function indicators were creatinine (Cr), urea nitrogen (BUN), and uric acid (UA); the immune inflammation indicators were interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α ) complement 3 (C3), immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM); endothelial function indicators include endothelin-1 (ET-1), thromboxane A2 (TXA2), prostacyclin (PGI2); blood lipid indicators include total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG); blood rheology indicators include 3 coagulation items (APTT, PT, Fg), whole blood viscosity and plasma viscosity.

[0070] The data were presented as mean ± standard deviation. The inter-group differences were statistically analyzed using independent sample t-test with SPSS19.0 software. P values ​​less than 0.05 or 0.01 were considered statistically significant.

[0071] Experimental Results

[0072] Evaluation of "Qi Deficiency"

[0073] Compared with normal Bama miniature pigs, the model Bama miniature pigs showed the following symptoms of qi deficiency: a. fatigue and lethargy, mental depression, curled limbs, poor activity, reduced or disappeared aggressive and confrontational behaviors, and no obvious escape attempt when placed alone outside the cage; b. loss of appetite, and feces appeared to be loose to varying degrees; c. sparse hair that was easy to fall off, or curled up, dry and yellow and lost its luster; d. The pigs were easily startled and trembled when hearing special sounds, and would become agitated and run around or curl up and tremble when hearing the sound of other Bama miniature pigs swimming.

[0074] Oxidative stress test results

[0075] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0076] The experimental results showed that compared with the blank group, the SOD activity of the model Bama miniature pigs was significantly decreased (P<0.01), and the MDA content was significantly increased (P<0.05), indicating that the body was in a state of unbalanced free radical metabolism, the body's free radical scavenging ability was reduced, the increase in free radicals aggravated the body's peroxidation damage, and the degree of body weakness was severe.

[0077] Myocardial enzyme spectrum test results

[0078] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0079] The experimental results showed that compared with the blank group, the levels of LDH and CK-MB in the model Bama miniature pigs were significantly increased (P<0.01, P<0.05), indicating that the myocardium was damaged by hypoxia, the myocardial energy metabolism was disordered, and the heart qi was depleted.

[0080] Liver and kidney function test results

[0081] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0082] The experimental results showed that compared with the blank group, the ALT, AST, Cr and BUN of the model Bama miniature pigs were significantly increased (P<0.01, P<0.01, P<0.05, P<0.01), indicating that their liver and kidney functions were impaired, which is the modern medical manifestation of "liver qi deficiency" and "kidney qi deficiency" in traditional Chinese medicine.

[0083] Inflammatory factor test results

[0084] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0085] The experimental results showed that compared with the blank group, the levels of IL-1β, TNF-α, IL-6 and IL-8 in the model group Bama miniature pigs were significantly increased (P<0.01, P<0.01, P<0.01, P<0.05), indicating that the model Bama miniature pigs had obvious inflammatory response.

[0086] Immune response test results

[0087] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0088] The experimental results showed that compared with the blank group, the levels of IgA, IgM, IgG and C3 in the model group Bama miniature pigs were significantly increased (P<0.01, P<0.01, P<0.05, P<0.05), indicating that the immune response of the model Bama miniature pigs was upregulated. The model Bama miniature pigs had chronic inflammation and immune disorder, which was the modern medical manifestation of "deficiency of Wei Qi" in "deficiency of Qi" in traditional Chinese medicine.

[0089] Evaluation of "blood stasis"

[0090] Compared with normal Bama miniature pigs, the model Bama miniature pigs showed the following blood stasis symptoms: a. The tongue of the experimental Bama miniature pigs was obviously dark purple compared with that of the control group; b. Subcutaneous bruises or blue spots appeared from the tip to the root of the tail to varying degrees; c. The eyeballs turned dark red or light red compared with the bright red of the control group; d. The hind limbs often had difficulty and slow movements.

[0091] Endothelial function of Bama miniature pigs in the model group

[0092] The experimental results showed that compared with the blank group, the ET-1 content of Bama miniature pigs in the model group was significantly increased (P<0.01), the PGI2 content was significantly decreased (P<0.05), and the TXA2 content was significantly increased (P<0.01), indicating that the vascular endothelium of the model Bama miniature pigs was damaged, the vascular contraction function was impaired, thrombosis was prone to occur, leading to blood stasis.

[0093] Blood lipid test results

[0094] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0095] The experimental results showed that compared with the blank group, the TG, TC, and LDL-C levels of Bama miniature pigs in the model group were significantly increased (P<0.05, P<0.01, P<0.01), and the HDL-C content was significantly decreased (P<0.01). The results showed that the blood lipid metabolism of the model Bama miniature pigs was abnormal, and sputum was accumulated in the inner wall of blood vessels, which could easily cause atherosclerosis.

[0096] Coagulation test results

[0097] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0098] The experimental results showed that compared with the blank group, the PT of Bama miniature pigs in the model group was significantly reduced (P<0.05), indicating that they were in a hypercoagulable state, indicating that their blood was in a hypercoagulable state and showed symptoms of "blood stasis".

[0099] Whole blood viscosity and plasma viscosity test results

[0100] Note: P<0.05 and P<0.01 vs blank group (n=10)

[0101] The experimental results showed that compared with the blank group, the whole blood viscosity of the Bama miniature pigs in the model group at low, medium and high shear rates increased significantly (P<0.01), indicating that their blood was viscous and had symptoms of "blood stasis".

[0102] Results and Discussion

[0103] From the results of superficial observations, it can be seen that the model animals obtained by this method showed sensitive manifestations of the symptoms of "Qi deficiency" and "blood stasis", mainly manifested as fatigue and drowsiness, mental depression, curled limbs, sparse hair that is easy to fall off, or curled and dry, withered and yellow hair that loses its moisture, and a tongue that is obviously dark purple and dull, etc.

[0104] From the results of blood biochemical index detection, it can be seen that compared with the blank group, the SOD of the model animals obtained by this method was significantly decreased, and the MDA was significantly increased, indicating that the model animals were in a state of oxidative stress disorder and the body was severely damaged, which is a typical manifestation of "Qi deficiency" in traditional Chinese medicine; the LDH and CK-MB of the model animals obtained by this method were significantly increased, indicating that their myocardium was hypoxic and damaged, and the heart qi was severely depleted; the ALT, AST, Crea, and UA of the model animals obtained by this method were significantly increased, indicating that their liver and kidney functions were impaired, which is the modern medical manifestation of "liver qi deficiency" and "kidney qi deficiency" in the "Qi deficiency" of traditional Chinese medicine; the four inflammatory factors in the model animals obtained by this method were significantly increased, and the four immune protein indicators were significantly different from those of normal Bama miniature pigs, indicating that the model Bama miniature pigs had chronic inflammation and disordered immune status, which is the modern medical manifestation of "Wei Qi deficiency" in the "Qi deficiency" of traditional Chinese medicine.

[0105] Compared with the blank group, the ET-1 and TXA2 of the model animals obtained by this method were significantly increased, and PGI2 was significantly decreased, indicating that the vascular endothelium of the model Bama miniature pigs was damaged, the vasomotor function was impaired, and thrombosis was prone to occur, leading to blood stasis. Compared with the blank group, the TG, TC, and LDL-C of the model animals obtained by this method were significantly increased, and HDL-C was significantly decreased. The blood lipid metabolism was abnormal, and phlegm was accumulated on the inner wall of the blood vessels, which was easy to cause atherosclerosis. Compared with the blank group, the WBV of the model animals obtained by this method at different shear rates was significantly increased, indicating that their blood was viscous, and PT was significantly decreased, indicating that they were in a state of hypercoagulability, which are typical manifestations of "blood stasis" in traditional Chinese medicine.

[0106] In summary, the model produced by this method showed obvious symptoms of qi deficiency and blood stasis, indicating that the animal model of qi deficiency and blood stasis was successfully produced.

[0107] Similarly, the Qi deficiency and blood stasis syndrome animal model prepared by other methods in Example 1 was used for the above evaluation. The experimental results were similar to those in Example 2, and the symptom manifestations and biochemical indicators were consistent. This proves that the Qi deficiency and blood stasis syndrome model and its preparation method of the present invention are stable, feasible, and highly reproducible.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a large animal model of Qi deficiency and blood stasis syndrome, characterized in that: The steps include: Step 1, feed proportion and feeding: feeding the miniature pigs with high-calorie feed for 6 months, wherein the components of the high-calorie feed are 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed by weight; Step 2: A method for establishing a hyperlipidemia model of Bama miniature pigs by feeding a high-calorie feed, comprising the following steps: (1) Select healthy Bama miniature pigs, aged 6-8 months, weighing 29.88 kg ± 2.27 kg, male, with normal intestinal flora; (2) The Bama miniature pigs were fed with the aforementioned high-calorie diet for 48 weeks, and their body weight was measured daily. The daily food intake was 3% of the measured body weight, and they were allowed to drink water freely; (3) Regularly test the blood disease indicators of Bama miniature pigs.

2. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 1, characterized in that: The specific formula of the basic feed is as follows: 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt, and 0.42% premix.

3. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 2, characterized in that: The percentages of the components in the premix are as follows: lysine 0.20%, multivitamins 0.05%, trace elements 0.10%, choline 0.05%, and phytase 0.02%.

4. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 3, characterized in that: The multiple vitamins in the premix are: vitamin A, vitamin D-3, vitamin E, vitamin K-3, pantothenic acid, niacinamide, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, and biotin.

5. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 4, characterized in that: The trace elements are: copper sulfate pentahydrate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, 1% calcium iodate, and 1% sodium selenite.

6. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 1, characterized in that: The second step is a method for establishing a hyperlipidemia model of Bama miniature pigs by feeding high-calorie feed, comprising the following steps: Experimental material selection: 55 healthy Bama miniature pigs, aged 6-8 months, weighing 29.88kg±2.27kg, male, with normal intestinal flora; Experimental method: High-calorie feed feeding: The weight percentage of each component in the high-calorie feed formula is: 10% butter, 6% peanut oil, 0.5% bile salt, 3% cholesterol, 2.5% acesulfame potassium, and 78% basic feed. The model group Bama miniature pigs were fed for 48 weeks. The body weight of the Bama miniature pigs was measured daily, and the food intake was 3% of the measured body weight. Water was available for free drinking.

7. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 6, characterized in that: The raw materials of the basic feed are 80.20% corn, 18.00% soybean cake, 0.30% fish meal, 0.40% calcium hydrogen, 0.40% stone powder, 0.28% salt and 0.42% premix.

8. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 7, characterized in that: The weight percentage of each component in the premix to the high-fat feed is: 0.20% lysine, 0.05% multivitamins, 0.10% trace elements, 0.05% choline, 0.02% phytase, which is 0.42% in total.

9. The method for preparing a large animal model of Qi deficiency and blood stasis syndrome according to claim 8, characterized in that: The multivitamins in the premix include: 12500 IU vitamin A, 5500 IU vitamin D-3, 60 g vitamin E, 10 g vitamin K-3, 10 g pantothenic acid, 60 g nicotinamide, 10 g vitamin B-1, 30 g vitamin B-2, 10 g vitamin B-6, 0.1 g vitamin B-12, and 0.6 g biotin; the trace elements include: 200 g copper sulfate pentahydrate, 260 g ferrous sulfate monohydrate, 260 g zinc sulfate monohydrate, 80 g manganese sulfate monohydrate, 5 g 1% calcium iodate, and 3 g 1% sodium selenite; and the feed carrier includes bentonite and bran.

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