Construction method and application of liver heat rat model

Abstract

The invention provides a construction method and application of a liver-heat rat model, and relates to the technical field of medical animal experiment models.The construction method comprises the steps that adult rats are placed in a humid and hot environment with the temperature being 30 + / -2 DEG C and the humidity being 75 + / -5%, fed with high-fat feed, drunk with beer instead of water every day and subjected to gavage with baijiu twice every week, and the adult rats are totally fed for 12-16 weeks; and performing liver heat syndrome scoring and liver heat disease scoring on the rats obtained by feeding, and when the sum of the liver heat syndrome scores and the sum of the liver heat disease scores are both greater than or equal to a preset standard score, obtaining the liver heat rat model. According to the method, a disease modeling factor and syndrome modeling factor superposition method is adopted to establish a disease and syndrome combined'liver heat 'rat model, establishment of each syndrome macroscopic characterization index quantitative evaluation system is reasonably proposed, corresponding microscopic indexes are introduced to macroscopic characterization, and traditional Chinese medicine innovative research on liver disease prevention and treatment can be assisted.

Description

Technical Field

[0001] The invention relates to the technical field of medical animal model construction, and in particular to a construction method and application of a liver heat rat model. Background Art

[0002] Liver disease affects millions of people in my country and around the world. It is characterized by widespread incidence and high mortality. Currently, there is still a shortage of clinical drugs for the prevention and treatment of liver disease. The unique dialectical treatment system of traditional Chinese medicine is increasingly showing its unique clinical value.

[0003] The evaluation of drug efficacy based on animal models is the key to solving the problem of disease diagnosis and treatment. In recent years, the establishment and evaluation of animal models of liver disease have been continuously improved. However, the current modeling method uses disease modeling factors, which are separated from each other and cannot achieve the unity of "disease" and "syndrome", which limits its application in disease mechanism research and drug exploration.

[0004] "Liver heat" is one of the core pathogenesis of traditional Chinese medicine. It runs through different stages of various liver diseases. The causes are all related to factors such as improper diet and invasion of exogenous pathogens. At present, there is no establishment and evaluation of such animal model at home and abroad. Summary of the invention

[0005] In response to the problems mentioned in the above background technology, the present invention proposes a method for constructing and applying a liver heat rat model. The present invention adopts the method of superimposing disease modeling factors and syndrome modeling factors to establish a disease-syndrome combined "liver heat" rat model, and reasonably proposes to establish a quantitative evaluation system for the macroscopic characterization indicators of each syndrome and introduce corresponding microscopic indicators for the macroscopic characterization, intending to assist in innovative research on traditional Chinese medicine for the prevention and treatment of liver diseases.

[0006] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for constructing a liver heat rat model, comprising: Adult rats were placed in a hot and humid environment with a temperature of (30±2)℃ and a humidity of (75±5%), fed with a high-fat diet, given beer instead of water every day, and given liquor by gavage twice a week for a total of 12-16 weeks; The rats obtained by feeding were scored for liver heat syndrome and liver heat disease, wherein the liver heat syndrome score and liver heat disease score are both set with multiple observation items, and each observation item is set with multiple scoring levels for graded scoring; When the total liver heat syndrome score is greater than or equal to the preset syndrome standard score, and the total liver heat disease score is greater than or equal to the preset disease standard score, a liver heat rat model is obtained.

[0007] As a further illustration of the present invention, rats were fasted for 12 h before feeding with feed.

[0008] As a further illustration of the present invention, the feeding standard of the beer is: 500 mL / 5 / day.

[0009] As a further illustration of the present invention, the standard for intragastric administration of liquor is: intragastric administration of 5 mL / kg / time of liquor on Monday and Friday every week.

[0010] As a further illustration of the present invention, the liver heat syndrome scoring system includes 7 observation items, each of which includes 5 scoring levels and is graded; the liver heat disease scoring system includes 5 observation items, each of which includes 4 scoring levels and is graded.

[0011] As a further illustration of the present invention, the preset syndrome standard is divided into 10 points, and the preset disease standard is divided into 5 points.

[0012] As a further illustration of the present invention, the observation items in the liver-heat syndrome scoring are: nails, eyes, hair, tongue coating, urine, feces and emotional response, and the liver-heat syndrome scoring is performed by observing the status of each item of the rat.

[0013] As a further illustration of the present invention, when scoring liver heat syndrome, each observation item is scored according to the following criteria: Nail: 0 points for pale red; 1 point for pale white; 2 points for wrinkles and stripes; 3 points for dry and irregular nails; 4 points for bloodless, black, and easy to break; Eyes: 0 points for pale red; 1 point for bloodshot eyes; 2 points for dryness, redness, and swelling; 3 points for redness, swelling, and bulges; 4 points for redness, swelling, photophobia, tears, and secretions; Hair: 0 points for smooth and shiny hair; 1 point for rough and matte hair; 2 points for hair loss with dotted depressions; 3 points for irregular hair loss; 4 points for flaky hair loss and yellow hair color; Tongue coating: 0 points for ruddy and shiny; 1 point for light red and little tongue coating; 2 points for red tongue with yellow coating in the middle and back of the tongue; 3 points for stiff and straight tongue with greasy yellow coating in the middle and back of the tongue; 4 points for uneven, thick, greasy, yellow tongue with irregular edges; Urine: 0 points for light yellow and transparent; 1 point for yellowish and strong smell; 2 points for dark yellow, strong smell and turbidity; 3 points for yellowish brown, pungent and turbidity; 4 points for brown, pungent, small amount and thick urine. Feces: Dark brown, moderately soft and hard, with distinct particles: 0 points; yellowish brown, relatively soft, and smelly: 1 point; yellowish brown, shapeless, and smelly: 2 points; brownish yellow, relatively dry, with small particles: 3 points; brownish black, relatively hard, with food residues: 4 points; Emotional reaction: 0 points for quick reaction; 1 point for listlessness and poor appetite; 2 points for running and occasional limb twitching; 3 points for mania and biting; 4 points for listlessness, or running and biting.

[0014] As a further illustration of the present invention, the observation items in the liver heat disease score are: liver tissue morphology, serum biochemical indexes and liver pathological histological observation items: fatty lesions, inflammatory lesions and ballooning degree.

[0015] As a further illustration of the present invention, when scoring liver heat disease, each observation item is scored according to the following criteria: Liver tissue morphology: 0 points if the liver lobe is reddish brown and soft; 1 point if the liver lobe is brownish yellow and slightly larger; 2 points if the liver lobe is brownish yellow, enlarged, and hard; 3 points if the liver lobe is yellowish brown, dark, larger, and harder. Serum biochemical indexes: normal liver function and blood lipids are 0 points; abnormal liver function is 1 point; first-level abnormal liver function and blood lipids are 2 points; second-level abnormal liver function and blood lipids are 3 points; Fat lesions: <5% is 0 points; 5-35% is 1 point; 35% - 65% is 2 points; >65% is 3 points; Inflammatory lesions: no change, 0 points; 1-2 lesions, 1 point; 3-4 lesions, 2 points; > 4 lesions, 3 points; Ballooning: no change, 0 points; 1-2 lesions, 1 point; 3-4 lesions, 2 points; > 4 lesions, 3 points.

[0016] As a further illustration of the present invention, the serum biochemical indicators are specifically liver function indicators and blood lipid indicators; wherein the liver function indicators are AST, ALT, γ-GT, AKP; and the blood lipid indicators are TC, TG, HDL-C, LDL-C.

[0017] The second aspect of the present invention provides the use of any of the liver heat rat models described above in drugs for preventing or treating liver diseases caused by liver heat.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention adopts the method of superimposing disease modeling factors and syndrome modeling factors to establish a disease-syndrome combined "liver heat" rat model, and reasonably proposes to establish a quantitative evaluation system for the macroscopic characterization indicators of each syndrome and introduce corresponding microscopic indicators for the macroscopic characterization, which can contribute to the innovative research of traditional Chinese medicine for the prevention and treatment of liver diseases.

[0019] Other features and advantages of the technical solution will be described in the subsequent description, and partly become apparent from the description, or understood by implementing the technical solution. The purpose and other advantages of the technical solution can be achieved and obtained through the structures specifically pointed out in the written description and the drawings.

[0020] The technical solution of the present technical solution is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the technical solution and constitute a part of the specification. Together with the embodiments of the technical solution, they are used to explain the technical solution and do not constitute a limitation of the technical solution. In the accompanying drawings: Figure 1 The urine status of rats in the blank group and the model group; Figure 2 The fecal status of rats in the blank group and the model group; Figure 3 is the tongue coating status of rats in the model group; Figure 4 The eye and hair status of rats in the model group; Figure 5 The nail status of rats in the model group; Figure 6 is the emotional response of rats in the model group; Figure 7 Liver tissue morphology and liver index of rats in the blank group and model group; Figure 8 Liver function indexes and blood lipid indexes of rats in the blank group and model group; Fig. 9 The changes in liver tissue structure and fatty degeneration in the blank group and model group rats after Oil Red O staining; Fig.10 The changes in liver tissue structure and fatty degeneration in the rats in the blank group and model group after H&E staining. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present technical solution are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present technical solution, and are not used to limit the present technical solution.

[0023] The present invention provides a method for constructing a liver heat rat model, comprising the following steps: Step 1: Place adult rats in a hot and humid environment with a temperature of (30±2)℃ and a humidity of (75±5%), feed them with high-fat feed, give them beer instead of water every day, and give them liquor by gavage twice a week for a total of 12-16 weeks.

[0024] The present invention adopts a multi-factor (humid heat + high fat + beer + liquor) combination method to induce a "liver heat" model.

[0025] Among them, adult rats are preferably SD female rats. In order to better analyze the subsequent liver heat symptoms and disease scores, a normal group can be set for control when constructing the model. Specifically, the following grouping method can be used: 20 SD female rats are raised in a specific hot and humid environment (temperature: (30±2), humidity: (75±5%)). Randomly divided into a normal group (10 rats) and a model group (10 rats). In order to feed more scientifically, it is necessary to feed the model group and the normal group rats uniformly after fasting for 12 hours. The specific feeding standards are as follows: the model group is given a high-fat feed (such as MCD (methionine choline deficiency feed) without limit) every day, beer (Tsingtao beer, 500mL / 5 rats / day) instead of water, and Red Star Erguotou (56°) is gavaged (5 mL / kg / time) on Mondays and Fridays every week. The normal group is fed with ordinary feed and a normal diet for a total of 12 weeks.

[0026] It should be noted that both Tsingtao Beer and Red Star Erguotou can be replaced by corresponding types with the same alcohol content; the rat model prepared according to the present invention is stable within 12-16 weeks.

[0027] Step 2: The fed rats are scored for liver heat syndrome and liver heat disease, wherein the liver heat syndrome score and the liver heat disease score are each set with multiple observation items, and each observation item is set with multiple scoring levels for graded scoring; when the total liver heat syndrome score is greater than or equal to the preset syndrome standard score, and the total liver heat disease score is greater than or equal to the preset disease standard score, a liver heat rat model is obtained.

[0028] Specifically, there are 7 observation items in the liver heat syndrome scoring, namely nails, eyes, hair, tongue coating, urine, feces and emotional response. The liver heat syndrome score is performed by observing the status of each item of the rats. Each observation item is set with 5 scoring levels, namely 0 points, 1 point, 2 points, 3 points and 4 points, for graded scoring.

[0029] Specifically, there are five observation items in the liver heat disease scoring, namely, liver tissue morphology, serum biochemical indicators and liver pathological histology observation items: fatty lesions, inflammatory lesions and ballooning degree. Each observation item has four scoring levels, namely 0 points, 1 point, 2 points and 3 points, for graded scoring.

[0030] Specifically, the above-mentioned preset syndrome standard is divided into 10 points, and the above-mentioned preset disease standard is divided into 5 points.

[0031] The specific model evaluation process is as follows: NC is the blank group, and M or Model is the model group.

[0032] 2.1 Daily Observation The rats' mental state, activity, hair gloss, appetite, defecation and urination were observed and recorded daily. All rats were weighed once a week. Figure 1-Figure 6 As shown, the rats with "liver heat" showed a mixture of fatigue and mania; their hair was messy and yellow; their urine was yellow, their food was undigested, and their stools were dry; their tongue coating was thick, greasy, slightly yellow, and a little prickly, with skirt-like folds on the edges that were raised and drooping, and the middle part of the tongue was sunken; their eyes were dry, with bloodshot eyes and a foreign body sensation; their claws were wrinkled and easily broken, had irregular stripes, were dry and black, and their horns were arched.

[0033] 2.2 Liver tissue Observe the shape, size, color, texture, and cross-section of the liver, weigh it, and calculate the liver index. Figure 7 As shown, the livers of rats in the normal group were bright red with sharp edges and smooth surface when observed with the naked eye; the livers of rats in the model group were enlarged in size, with blunt edges, yellow-brown color, dark color, hard texture, and greasy surface when observed with the naked eye. 2.3 Serum index detection Blood was collected from the abdominal aorta of rats; centrifuged (800×g, 15 min) to separate serum; the microplate method was used to detect the content of liver injury indicators (AST, ALT, γ-GT, AKP) and blood lipid indicators (TC, TG, HDL-C, LDL-C) in the serum of rats and mice. Figure 8 As shown in the results, compared with the normal group, the levels of AST, ALT, TG, TC, LDL-C, GGT, and AKP in the serum of rats and mice in the model group were significantly increased (P<0.01), and the level of HDL-C was significantly decreased (P<0.01).

[0034] 2.4 Observation of liver pathological histology The liver lobes were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). H&E staining and Oil Red O staining were performed to observe the changes in liver tissue structure and fatty degeneration.

[0035] like Fig.10 As shown in the figure, H&E staining showed that the liver cells of the blank group rats were neatly arranged, the liver sinusoid structure was clear, and there was no cell degeneration or necrosis; compared with the blank group, the model group rats had significant liver tissue pathological changes such as unclear liver lobule structure, narrow liver sinusoids, loose cytoplasm, diffuse swelling of liver cells with flake balloon-like degeneration, pyknosis of a small number of liver cell nuclei, and lymphocyte infiltration foci in the liver lobules. Fig. 9 As shown, Oil Red O staining showed that a large number of lipid droplets appeared in the rats in the model group.

[0036] 2.5 Model scoring criteria formulation Table 1 Liver heat syndrome scoring table

[0037] Table 2 Hepatic heat disease scoring table

[0038] Model judgment criteria: According to the “Liver Heat Syndrome Scoring Table” and the “Liver Heat Disease Scoring Table”, the model judged that the patient’s liver heat syndrome score was ≥10 points and the disease score was greater than or equal to 5 points.

[0039] Mining of molecular biological indicators of "liver heat" model Transcriptomics was used to explore the potential molecular biological indicators of the model, providing supplement and reference for the exploration of the pharmacodynamic mechanism in model evaluation and application.

[0040] Liver tissues of the blank group and the model group were taken for transcriptome analysis, and P-value ≤ 0.05&|log2(fold change)|=3 was set as the condition for differential gene screening. KEGG enrichment analysis showed that PPAR signaling pathway was the dominant pathway, and further exploration revealed that genes Scd, Cyp7a1, Cyp4a2 and LOC120102953 in this pathway were significantly different. These four genes can be used as potential molecular biological evaluation indicators for the "liver heat" model.

[0041] Obviously, those skilled in the art can make various changes and modifications to the technical solution without departing from the spirit and scope of the technical solution. Thus, if these modifications and variations of the technical solution fall within the scope of the claims of the technical solution and their equivalents, the technical solution is also intended to include these modifications and variations.

Claims

1. A method for constructing a liver heat rat model, characterized in that: include: Adult rats were placed in a hot and humid environment with a temperature of (30±2)℃ and a humidity of (75±5%), fed with a high-fat diet, given beer instead of water every day, and given liquor by gavage twice a week for a total of 12-16 weeks; The rats obtained by feeding were scored for liver heat syndrome and liver heat disease, wherein the liver heat syndrome score and liver heat disease score are both set with multiple observation items, and each observation item is set with multiple scoring levels for graded scoring; When the total liver heat syndrome score is greater than or equal to the preset syndrome standard score, and the total liver heat disease score is greater than or equal to the preset disease standard score, a liver heat rat model is obtained.

2. The method for constructing a liver heat rat model according to claim 1, characterized in that: Before the rats were fed with feed, they were fasted for 12 h; the feeding standard of the beer was: 500 mL / 5 rats / day; the gavage standard of the liquor was: 5 mL / kg / time of liquor gavage on Mondays and Fridays every week.

3. The method for constructing a liver heat rat model according to claim 1, characterized in that: The liver-heat syndrome scoring system includes 7 observation items, each of which has 5 scoring levels and is scored in a graded manner; the liver-heat disease scoring system includes 5 observation items, each of which has 4 scoring levels and is scored in a graded manner.

4. The method for constructing a liver heat rat model according to claim 1, characterized in that: The preset syndrome standard is divided into 10 points, and the preset disease standard is divided into 5 points.

5. The method for constructing a liver heat rat model according to claim 1, characterized in that: The observation items in the liver-heat syndrome scoring are: nails, eyes, hair, tongue coating, urine, feces and emotional response, and the liver-heat syndrome scoring is performed by observing the status of each item of the rat.

6. The method for constructing a liver heat rat model according to claim 5, characterized in that: When scoring liver heat syndrome, score each observation item according to the following criteria: Nail: 0 points for pale red; 1 point for pale white; 2 points for wrinkles and stripes; 3 points for dry and irregular nails; 4 points for bloodless, black, and easy to break; Eyes: 0 points for pale red; 1 point for bloodshot eyes; 2 points for dryness, redness, and swelling; 3 points for redness, swelling, and bulges; 4 points for redness, swelling, photophobia, tears, and secretions; Hair: 0 points for smooth and shiny hair; 1 point for rough and matte hair; 2 points for hair loss with dotted depressions; 3 points for irregular hair loss; 4 points for flaky hair loss and yellow hair color; Tongue coating: 0 points for ruddy and shiny; 1 point for light red and little tongue coating; 2 points for red tongue with yellow coating in the middle and back of the tongue; 3 points for stiff and straight tongue with greasy yellow coating in the middle and back of the tongue; 4 points for uneven, thick, greasy, yellow tongue with irregular edges; Urine: 0 points for light yellow and transparent; 1 point for yellowish with strong smell; 2 points for dark yellow, strong smell and turbidity; 3 points for yellowish brown, pungent and turbidity; Brown, pungent, small amount and sticky, score 4; Feces: Dark brown, moderately soft and hard, with distinct particles: 0 points; yellowish brown, relatively soft, and smelly: 1 point; yellowish brown, shapeless, and smelly: 2 points; brownish yellow, relatively dry, with small particles: 3 points; brownish black, relatively hard, with food residues: 4 points; Emotional reaction: 0 points for quick reaction; 1 point for listlessness and poor appetite; 2 points for running and occasional limb twitching; 3 points for mania and biting; 4 points for listlessness, or running and biting.

7. The method for constructing a liver heat rat model according to claim 1, characterized in that: The observation items in the liver heat disease score are: liver tissue morphology, serum biochemical indexes and liver pathological histological observation items: fatty lesions, inflammatory lesions and ballooning degree.

8. The method for constructing a liver heat rat model according to claim 7, characterized in that: When scoring liver heat disease, score each observation item according to the following criteria: Liver tissue morphology: 0 points if the liver lobe is reddish brown and soft; 1 point if the liver lobe is brownish yellow and slightly larger; 2 points if the liver lobe is brownish yellow, enlarged, and hard; 3 points if the liver lobe is yellowish brown, dark, larger, and harder. Serum biochemical indexes: normal liver function and blood lipids are 0 points; abnormal liver function is 1 point; The first-grade abnormality of liver function and blood lipids is 2 points; the second-grade abnormality of liver function and blood lipids is 3 points; Fat lesions: <5% is 0 points; 5-35% is 1 point; 35% - 65% is 2 points; >65% is 3 points; Inflammatory lesions: no change, 0 points; 1-2 lesions, 1 point; 3-4 lesions, 2 points; > 4 lesions, 3 points; Ballooning: no change, 0 points; 1-2 lesions, 1 point; 3-4 lesions, 2 points; > 4 lesions, 3 points.

9. The method for constructing a liver heat rat model according to claim 7, characterized in that: The serum biochemical indicators specifically include liver function indicators and blood lipid indicators; the liver function indicators include AST, ALT, γ-GT, and AKP; the blood lipid indicators include TC, TG, HDL-C, and LDL-C.

10. Use of the liver heat rat model according to any one of claims 1 to 9 in a drug for preventing or treating liver diseases caused by liver heat.

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