A method for establishing a rat model of damp-heat jaundice

Abstract

The present invention discloses a method for establishing a rat model of damp-heat jaundice syndrome, and the method includes three steps of pre-experimental treatment, control group treatment, and model group treatment, wherein the control group treatment is only gavaged with distilled water and olive oil, the model group is first gavaged with dried ginger solution and ethanol solution, and the olive oil solution containing α-naphthalene isothiocyanate is gavaged on the 15th and 16th days. By observing and evaluating the established animal model, based on the clinical characteristics of damp-heat jaundice syndrome in clinical traditional Chinese medicine, combined with the understanding of jaundice in modern medicine, the two groups of models established are preliminarily characterized by body weight, rectal temperature, clinical biochemical index determination and histopathological observation; by comparing the results, it is found that the damp-heat jaundice rat model established shows symptoms consistent with those of clinical damp-heat jaundice patients, indicating the effectiveness of the damp-heat jaundice rat model establishment method provided by the present invention, and the establishment of the model lays the foundation for metabolomics analysis.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, and in particular to a method for establishing a damp-heat jaundice syndrome rat model. Background Art

[0002] Jaundice, also known as yellow jaundice, is a disease characterized by yellow face, eyes, and skin, and yellow and red urine. Based on the experience of his predecessors, the medical saint Zhang Zhongjing created the five jaundice syndrome classification method in "Golden Chamber Synopsis·Jaundice Pulse Symptoms and Treatment Chapter 15", which classified jaundice into yellow jaundice, grain jaundice, alcohol jaundice, female labor jaundice, and black jaundice from the perspective of etiology; from the perspective of pathogenesis, it was classified into damp-heat jaundice, cold-damp jaundice, blood stasis jaundice, fire disaster jaundice, and female labor jaundice, creating a precedent for the diagnosis and treatment of jaundice; among them, the clinical symptoms of jaundice are jaundice, urination difficulties, abdominal distension, fever, and spontaneous sweating. There have been many discussions on the cause of jaundice, such as Han Dihe in the Song Dynasty and "Treatise on Febrile Diseases" for the first time, which classified jaundice into yin and yang, and the names of "yin jaundice" and "yang jaundice" were proposed. Since then, doctors have mostly classified jaundice into yin jaundice and yang jaundice, and they know that yang jaundice can be transformed into yin jaundice over time. In the book “Health Treasure Mirror: Jaundice” written by Luo Tianyi of Yuan Dynasty, it is pointed out that “when the middle yang is excessive, dampness turns into heat, and dampness and heat are the problem, it is Yang jaundice; when the middle yang is insufficient, dampness turns into cold, and cold and dampness are the problem, it is Yin jaundice”. The above data further clarified the pathogenesis of Yang jaundice and Yin jaundice, that is, the pathogenesis of damp-heat jaundice is damp-heat stagnation, and the pathogenesis of Yin jaundice is spleen yang deficiency and excessive cold and dampness. The modern textbook “Internal Medicine of Traditional Chinese Medicine” defines jaundice as “a disease with yellow eyes, yellow body and yellow urine as the main clinical manifestations due to liver failure to release, bile overflow, or blood failure and lack of color”.

[0003] Existing studies have shown that giving healthy rats a damp-heat environment, a high-fat, high-sugar diet, and dried ginger and aconite can cause animal models with damp-heat syndromes such as spleen and stomach damp-heat syndrome and deficiency-heat syndrome, but a high-fat, high-sugar diet may induce problems such as diabetes, fatty liver lesions, and renal vascular hypertension. For example, Li Lei et al. used a 1:0.5 mixture of 50% glucose and peanut oil to gavage rats to create a damp-heat background, and then gave them ANIT solution. Pathological examination of the liver of the model group rats found fatty degeneration of their hepatocytes; for example, Li Li et al. found that a high-fat, high-sugar diet can also induce diabetes in rats. Therefore, it is difficult to establish an animal model that meets the symptoms of damp-heat jaundice with a single method in the existing technology. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to propose a method for establishing a rat model of damp-heat jaundice. Based on the basic theory of Yang jaundice, mice are given different doses of dried ginger, ethanol, and ANIT solution to replicate an animal model of Yang jaundice combined with symptoms, which provides convenience for the clinical research of damp-heat jaundice.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for establishing a damp-heat jaundice rat model comprises the following steps:

[0007] (1) Pre-experimental treatment: 8-week-old rats were placed in metabolic cages for one week to adapt to the environment and then randomly divided into a control group and a model group, with 8 rats in each group;

[0008] (2) Treatment of the control group: Based on the rat body weight, the rats in the control group were given distilled water by gavage at 8-12 mL / kg for the first 14 days; on the 15th and 16th days, they were given olive oil by gavage at 8-12 mL / kg, and the experimental parameters were measured and recorded on time;

[0009] (3) Treatment of the model group: At the same time, based on the rat body weight, the model group rats were gavaged with dried ginger solution and ethanol solution at 0.8-1 g / kg and 0.65-0.75 g / kg, respectively, for the first 14 days. On the 15th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 10-11 mg / kg. On the 16th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 6-8 mg / kg. The experimental indicators were measured and recorded on time.

[0010] As a further preferred embodiment of the technical solution of the present invention, the rats in step (1) are male Wistar rats.

[0011] As a further preferred embodiment of the technical solution of the present invention, the amount of distilled water and olive oil used in step (2) is 10 mL / kg.

[0012] As a further preferred embodiment of the technical solution of the present invention, the dosage of the dried ginger solution in step (3) is 0.9 g / kg.

[0013] As a further preferred embodiment of the technical solution of the present invention, the preparation method of the dried ginger solution in step (3) is as follows: weigh 50.8 g of dried ginger medicinal material, add 10 times the amount of distilled water to soak for 1 hour, boil over high heat until boiling, boil over low heat for 1 hour, filter the filtrate through gauze into a beaker, add 10 times the amount of distilled water, repeat the above process twice, combine the three filtrates, and concentrate to 500 mL.

[0014] As a further preferred embodiment of the technical solution of the present invention, the volume concentration of the ethanol solution in step (3) is 11.8%, and the dosage is 0.69 g / kg.

[0015] As a further preferred embodiment of the technical solution of the present invention, the dosage of the olive oil solution containing α-naphthalene isothiocyanate on the 15th day in step (3) is 10.4 mg / kg.

[0016] As a further preferred embodiment of the technical solution of the present invention, the dosage of the olive oil solution containing α-naphthalene isothiocyanate on the 16th day in step (3) is 7 mg / kg.

[0017] As a further preferred embodiment of the technical solution of the present invention, the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 15th day in step (3) is 1.04 mg / mL, and the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 16th day is 0.7 mg / mL.

[0018] As a further preferred embodiment of the technical solution of the present invention, the preparation method of the olive oil solution containing α-naphthalene isothiocyanate is as follows: α-naphthalene isothiocyanate is accurately weighed according to a proportion, dissolved in olive oil, and then ultrasonically dispersed for 30 minutes to obtain the solution.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention establishes an animal model of damp-heat jaundice based on the TCM syndrome theory and a large number of previous studies. A small amount of dried ginger is given to rats to induce "heat" and a low dose of ethanol is given to rats to induce "dampness", thereby creating a "damp-heat" background. On this basis, ANIT oil solution that causes cholestatic liver damage is given to rats to establish an animal model of damp-heat jaundice.

[0021] At the same time, the present invention observes and evaluates the established animal model, based on the clinical characteristics of damp-heat jaundice syndrome in traditional Chinese medicine, combined with the understanding of jaundice in modern medicine, and preliminarily characterizes the two groups of models established by measuring body weight, rectal temperature, clinical biochemical indicators and histopathological observation. By comparing the results, it was found that the established damp-heat jaundice rat model showed symptoms consistent with those of clinical damp-heat jaundice patients, indicating the effectiveness of the damp-heat jaundice rat model establishment method provided by the present invention, and the establishment of the model laid the foundation for metabolomics analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a weight change trend diagram of the control group and the model group in Example 1; wherein □ represents the control group and ○ represents the model group;

[0023] Figure 2 A is the trend diagram of rectal temperature changes of rats in the control group and the model group; B is the thermal map of rectal temperature of rats in the control group and the model group; □ represents the control group, ○ represents the model group;

[0024] Figure 3 Energy metabolism curves of rats in the control group and model group within 48 hours on the 15th and 16th days of model replication;

[0025] Figure 4The box plots of various biochemical indicators in the serum of rats in the damp-heat jaundice model group and the control group after the model was successfully replicated; A: total bilirubin; B: direct bilirubin; C: total bile acid; D: γ-glutamyl transferase; E: alkaline phosphatase; F: alanine aminotransferase; G: aspartate aminotransferase; H: glutathione peroxidase; I: malondialdehyde;

[0026] Figure 5 HE staining results of liver tissues of rats in the control group and damp-heat jaundice model group; A: control group (×20), B: damp-heat jaundice model group (×20), C: control group (×200), D: damp-heat jaundice model group (×200);

[0027] Figure 6 These are the HE staining results of bile ducts of rats in the control group and damp-heat jaundice model group; A: control group (×20); B: damp-heat jaundice model group (×20); C: control group (×200); D: damp-heat jaundice model group (×200). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly explained, the present invention will be further explained in detail below in conjunction with the embodiments of the specification.

[0029] Among them, the information of the instruments, main chemical substances and rats in the present invention is as follows:

[0030] Metabolic analyzer, PRO-MRRM-8, Shanghai Yiyao Scientific Instrument Co., Ltd.;

[0031] Ultrasonic instrument, model KQ-250DB, Kunshan Ultrasonic Instrument Co., Ltd.;

[0032] α-Naphthalene isothiocyanate, Shanghai Jingchun Biochemical Technology Co., Ltd.;

[0033] Dried ginger, Beijing Tong Ren Tang Pharmacy Harbin Branch;

[0034] Rats, SPF grade, weighing 220±20g, were purchased from the Drug Safety Evaluation Center of Heilongjiang University of Chinese Medicine, certificate number: SCXK(Liao)2020-1003, and were kept in the animal room with 12h light and 12h dark, with free food and water intake.

[0035] Example 1

[0036] A method for establishing a damp-heat jaundice rat model comprises the following steps:

[0037] (1) Pre-experimental treatment: 8-week-old male Wistar rats were placed in metabolic cages to acclimate to the environment for one week and then randomly divided into a control group and a model group, with 8 rats in each group;

[0038] (2) Treatment of the control group: Based on the rat body weight, the rats in the control group were gavaged with distilled water at 10 mL / kg for the first 14 days; on the 15th and 16th days, they were gavaged with olive oil at 10 mL / kg, and the experimental parameters were measured and recorded on time;

[0039] (3) Treatment of the model group: At the same time, based on the rat body weight, the model group rats were gavaged with dried ginger solution and ethanol solution at 0.9 g / kg and 0.69 g / kg, respectively, for the first 14 days. On the 15th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 10.4 mg / kg. On the 16th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 7 mg / kg. The experimental indicators were measured and recorded on time.

[0040] As a further preferred embodiment of the technical solution of this embodiment, the preparation method of the dried ginger solution in step (3) is as follows: weigh 50.8 g of dried ginger medicinal material, add 10 times the amount of distilled water and soak for 1 hour, boil over high heat until boiling, boil over low heat for 1 hour, filter the filtrate through gauze into a beaker, add 10 times the amount of distilled water, repeat the above process twice, combine the three filtrates, and concentrate to 500 mL.

[0041] As a further preferred embodiment of the technical solution of this embodiment, the volume concentration of the ethanol solution in step (3) is 11.8%.

[0042] As a further preferred embodiment of the technical solution of this embodiment, the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 15th day in step (3) is 1.04 mg / mL, and the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 16th day is 0.7 mg / mL.

[0043] As a further preferred embodiment of the technical solution of this embodiment, the preparation method of the olive oil solution containing α-naphthalene isothiocyanate is as follows: α-naphthalene isothiocyanate is accurately weighed according to a proportion, dissolved in olive oil, and then ultrasonically dispersed for 30 minutes.

[0044] Example 2

[0045] A method for establishing a damp-heat jaundice rat model comprises the following steps:

[0046] (1) Pre-experimental treatment: 8-week-old male Wistar rats were placed in metabolic cages to acclimate to the environment for one week and then randomly divided into a control group and a model group, with 8 rats in each group;

[0047] (2) Treatment of the control group: Based on the rat body weight, the rats in the control group were gavaged with distilled water at 10 mL / kg for the first 14 days; on the 15th and 16th days, they were gavaged with olive oil at 10 mL / kg, and the experimental parameters were measured and recorded on time;

[0048] (3) Treatment of the model group: At the same time, based on the rat body weight, the model group rats were gavaged with dried ginger solution and ethanol solution at 0.85 g / kg and 0.72 g / kg, respectively, for the first 14 days. On the 15th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 10.2 mg / kg. On the 16th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 7.4 mg / kg. The experimental indicators were measured and recorded on time.

[0049] As a further preferred embodiment of the technical solution of this embodiment, the preparation method of the dried ginger solution in step (3) is as follows: weigh 50.8 g of dried ginger medicinal material, add 10 times the amount of distilled water and soak for 1 hour, boil over high heat until boiling, boil over low heat for 1 hour, filter the filtrate through gauze into a beaker, add 10 times the amount of distilled water, repeat the above process twice, combine the three filtrates, and concentrate to 500 mL.

[0050] As a further preferred embodiment of the technical solution of this embodiment, the volume concentration of the ethanol solution in step (3) is 11.8%.

[0051] As a further preferred embodiment of the technical solution of this embodiment, the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 15th day in step (3) is 1.04 mg / mL, and the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 16th day is 0.7 mg / mL.

[0052] As a further preferred embodiment of the technical solution of this embodiment, the preparation method of the olive oil solution containing α-naphthalene isothiocyanate is as follows: α-naphthalene isothiocyanate is accurately weighed according to a proportion, dissolved in olive oil, and then ultrasonically dispersed for 30 minutes.

[0053] Model evaluation method

[0054] The rats after the model was established in Example 1 were evaluated, and the specific evaluation indicators and methods were as follows:

[0055] (1) Evaluation based on physical sign monitoring

[0056] 1) Weight evaluation method

[0057] Before each intragastric administration on days 0, 3, 6, 9, 13, and 16 of model replication, the rats in each group were weighed using a small animal scale and the weight of each rat was recorded.

[0058] 2) Rectal temperature evaluation method

[0059] On days 0, 3, 6, 9, 13, and 16 of model replication, the rectal temperature of rats in each group was measured using a small animal thermometer before each gavage. Each measurement was repeated three times and recorded.

[0060] 3) Appearance behavior evaluation method

[0061] The appearance and behavior of rats in the control group and the model group were observed every two days. The observations included behavior, body shape, food intake, water intake, mental state and fur gloss.

[0062] 4) Energy metabolism monitoring methods

[0063] On the 15th day after the model replication, 4 rats were randomly selected from the control group and the model group and placed in a PRO-MRRM-8 small animal energy metabolism instrument to collect the rats' food intake, water intake, respiratory entropy changes, oxygen consumption, CO2 exhalation, H2O exhalation, and energy rate within 48 hours.

[0064] (2) Evaluation based on biochemical indicators

[0065] After the model was successfully replicated, blood samples were collected from rats in the control group and model group (each rat was fasted for 12 hours before sampling and had free access to water). 4% sodium pentobarbital solution (injection dose was 1.5 mL / kg) was injected into the rats' peritoneal cavity, and the rats were completely anesthetized by pinching the tail reaction. The abdomen was cut open along the abdominal suture, and about 8 mL of blood was collected through the abdominal aorta. After standing at room temperature (25°C) for 60 minutes, the blood was centrifuged at 4000 rpm / min for 15 minutes at 4°C, and the upper serum was divided into centrifuge tubes and stored in a -80°C refrigerator for later use. After thawing the serum samples at room temperature, the contents of total bilirubin (TBil), direct bilirubin (DBil), total bile acid (TBA), γ-glutamyl transferase (γ-GT), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate aminotransferase (GOT), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) in the serum of each group of rats were determined according to the instructions of the kit.

[0066] (3) Based on histopathological evaluation

[0067] After the model was successfully replicated, blood samples were collected from rats in the control group and the model group (each rat was fasted for 12 hours before sampling and had free access to water). 4% sodium pentobarbital solution (injection dose was 1.5 mL / kg) was injected into the rats' peritoneal cavity, and the rats were completely anesthetized after being judged by the tail pinching reaction. The abdomen was cut open along the abdominal suture, and after the blood of the rats was collected, the liver and bile duct of the rats were removed, rinsed in physiological saline, and the surface moisture was wiped clean with filter paper, and then put into 10% neutral formaldehyde for pathological detection.

[0068] Model evaluation results analysis

[0069] GraphPad Prism 9.0 (v9.0.1) was used to analyze the above data, and the results are presented as mean ± standard deviation. The other data were normalized with the values ​​of the control group as reference, and the statistical results were expressed as mean ± standard deviation. The Student's T test was used for comparison between groups. p<0.05 was considered statistically significant, and p<0.01 was considered extremely statistically significant. The specific analysis results are as follows:

[0070] (1) Weight evaluation results

[0071] The results of monitoring the rat weight showed that during the 16-day model replication period, the weight of the rats in the control group and the model group increased gradually; starting from the third day of model replication, the weight growth rate of the rats in the model group slowed down compared with the control group; after the ninth day of model replication, the weight of the rats in the model group was significantly lower than that of the rats in the control group, and the difference was significant (p<0.05); after the thirteenth day of model replication, the weight of the rats in the model group was significantly different from that in the control group (p<0.01). For specific results, see Figure 1 and Table 1. In the early stage of animal model replication, the experimental rats were gavaged, and the rats had not yet adapted. The weight gain rate of the two groups of rats was low. After the animals adapted, due to the mild effect of the damp-heat background modeling solution (dried ginger solution and ethanol solution), no significant difference was found in the first 9 days of model replication. The weight change trend of the model rats during the model replication period was similar to the clinical manifestation of weight loss in patients with damp-heat jaundice.

[0072] Table 1 Body weight results of rats in the control group and model group

[0073]

[0074] (2) Rectal temperature evaluation results

[0075] The results of monitoring the rectal temperature of rats showed that the rectal temperature of rats in the model group gradually increased during the 16-day model replication cycle. Starting from the 6th day of model replication, there was a significant difference in the rectal temperature of rats in the model group compared with the control group (p<0.05). The results showed that the rectal temperature of rats in the model group was gradually increasing. On the 9th day of model replication, there was an extremely significant difference in the model group compared with the control group (p<0.01). On the 16th day of model replication, after oral administration of ANIT modeling solution, the rectal temperature of rats in the damp-heat jaundice model group increased significantly compared with the control group. The specific results are shown in Figure 2 and Table 2. In the early stage of animal model replication, the overall trend of rectal temperature of the two groups of rats was similar. Since the damp-heat background modeling solution (dried ginger solution and ethanol solution) used had a relatively mild effect, no significant difference was found within the first 6 days of model replication. The trend of rectal temperature changes in the rat model during the model replication period was similar to the clinical manifestations of patients with damp-heat jaundice.

[0076] Table 2 Rectal temperature results of the model group and the control group

[0077]

[0078] (3) Results of appearance behavioral evaluation

[0079] In the early stage of rat model replication, the growth and mental state of rats in each group were good, they drank water normally, moved frequently, and had sharp eyes. During the period of intragastric administration of dried ginger ethanol solution, the rats in the model group gradually showed symptoms of weakened activity, curled up and moved less, dark red claws, nose and ears, reduced food intake, increased water intake, increased urine output, dull fur, etc., and the frequency of changing bedding gradually increased. After intragastric administration of ANIT oil solution, the above symptoms worsened.

[0080] (4) Energy metabolism monitoring results

[0081] The basal metabolic data of the model group and the control group rats on the 15th and 16th days of model replication were collected. The animal adaptation time data of the first 6 hours were removed from the original data, and the 48-hour data were selected and imported into GraphPad Prism to make a curve graph. The statistical results showed that the food intake of the model rats within 48 hours was lower than that of normal rats, while the water intake showed the opposite trend. Compared with the normal group rats, the food intake and water intake indicators of the damp-heat jaundice model rats showed significant differences (p<0.01). The respiratory entropy results show that after oral administration of the ANIT modeling solution, the respiratory entropy of the damp-heat jaundice model rats dropped sharply in a short period of time. In terms of the exhaled H2O rate, oxygen consumption rate and energy consumption rate, the rats in the damp-heat jaundice model group were higher than those in the control group. The results are as follows Figure 3 The experimental results are consistent with the clinical manifestations of patients with damp-heat jaundice, including loss of appetite, accelerated heat production and basal metabolism. In addition, the model rats drank more water and exhaled water at a high rate, which correspond to the physical signs of damp-heat jaundice caused by endogenous dampness and long-term heat.

[0082] (5) Biochemical index evaluation results

[0083] Compared with the control group, the levels of TBil, DBil, TBA, γ-GT, AKP, ALT, GOT, and MDA in the serum of the damp-heat jaundice model group were increased, with extremely significant differences (p<0.01); while the level of GSH-PX was significantly decreased, with extremely significant differences (p<0.01). Figure 4As shown in Table 3. Bilirubin is an important indicator for clinical determination of jaundice and an important indicator of liver function. Total bilirubin (TBil) is the sum of direct bilirubin and indirect bilirubin. The increase of TBil in the body reflects the damage of liver cells, liver dysfunction, and excretion disorder of intrahepatic bile duct. In clinical practice, damp-heat jaundice, acute icteric hepatitis, chronic active hepatitis, cirrhosis, liver cancer and other liver diseases are accompanied by the trend of increase of this biochemical index. Total bile acid (TBA) is one of the more sensitive and effective liver function tests. It is the product of cholesterol metabolism in the liver and the main component of bile. It is synthesized by liver cells and secreted into the duodenum with bile, and part of it is reabsorbed by the liver through the enterohepatic circulation. When liver cells are damaged or the bile duct is obstructed, it will cause bile acid metabolism disorder, and the total bile acid concentration in serum increases significantly with the degree of liver cell damage. γ-glutamyl transpeptidase (γ-GT) is widely distributed in the body, but γ-GT in serum mainly comes from the bile secretion side of the liver cell membrane and the epithelium of the small bile duct. When the extrahepatic bile duct and intrahepatic obstruction occur, the excretion of glutamyl transpeptidase will be blocked and reflux into the blood with bile, resulting in abnormal serum glutamyl transpeptidase, so it has strong specificity. Alkaline phosphatase (AKP) is an enzyme widely distributed in human liver, bones, intestines, kidneys, placenta and other tissues and excreted from the liver to the bile. It is used for the diagnosis and differential diagnosis of bone and hepatobiliary diseases, especially the identification of jaundice, and is mainly used for the examination of obstructive jaundice, liver cancer, cholestatic hepatitis, etc. During normal metabolism, it needs to be excreted from the liver by bile. When the liver tissue metabolism is abnormal and damaged, it will cause high AKP in serum. Obstructive jaundice, primary liver cancer, secondary liver cancer, and cholestatic hepatitis will all cause high AKP. Alanine aminotransferase (ALT) is recommended by the World Health Organization as the most sensitive indicator for liver function damage. It is mainly distributed in the cytoplasm of liver cells. If liver cells are damaged, ALT will be released into the blood, causing the blood content to increase. If 1% of liver cells are destroyed, the serum content can be doubled. The degree of increase is consistent with the degree of liver cell damage. Aspartate aminotransferase (GOT) is also one of the most commonly used liver function indicators and is a standard for reflecting liver cell necrosis. It is distributed in the mitochondria and cytoplasm of liver cells and is an intracellular functional enzyme. When liver cells are severely damaged, GOT in the cytoplasm and mitochondria will be released into the blood, causing the blood content to increase. Glutathione peroxidase (GSH-Px) is one of the indicators of the body's anti-peroxidation capacity. GSH-Px plays a key role in removing harmful peroxidation metabolites and protecting cells from free radical damage. When rats are given ANIT, a liver injury model is induced, which can cause membrane lipid peroxidation, causing liver cell degeneration and necrosis, leading to downregulation of GSH-Px activity. Malondialdehyde (MDA) is a product of lipid peroxidation degradation and is often used as a parameter of lipid peroxidation degree, indirectly reflecting the degree of free radical attack on body cells.

[0084] Table 3 Serum biochemical indexes in the model group and the control group after successful model replication

[0085]

[0086] (6) Histopathological test results of damp-heat jaundice rat model

[0087] The results of rat liver histopathology showed that the liver tissue capsule of the rats in the control group was composed of dense connective tissue rich in elastic fibers of uniform thickness, with clear hepatic lobule structure, central vein in the center, surrounded by hepatocyte cords and hepatic sinusoids arranged roughly radially, round and plump hepatocytes, regular and neat arrangement of hepatic plates, and no obvious expansion or compression of hepatic sinusoids; no obvious abnormalities in the portal tract area between adjacent hepatic lobules; no obvious inflammatory changes were observed ( Figure 5 A, Figure 5 C). Compared with the control group, the liver tissue pathological examination results of the model group rats showed more obvious inflammatory infiltration; more inflammatory cell infiltration foci (yellow arrows) and individual liver cell necrosis (black arrows) were seen locally ( Figure 5 B, Figure 5 D).

[0088] The results of bile duct pathology showed that the bile duct epithelial tissue of the rats in the control group was a single-layer columnar epithelium with intact epithelium and closely arranged epithelial cells without obvious degeneration, necrosis and shedding. The muscle layer was well-developed and thick, and the circular smooth muscle cells were arranged regularly. No obvious inflammatory cell infiltration was observed. Some shedding was visible in the cavity (black arrows) ( Figure 6 A, Figure 6 C). Compared with the control group, a small amount of inflammatory cell infiltration was observed in the bile duct tissue of the rats in the model group; the epithelium was mostly short columnar, with some epithelial cells falling off locally (black arrows), and a large amount of falling material was observed in the cavity; the local muscle layer was edematous (blue arrows), with loose structure, and a small amount of neutrophil infiltration was observed in the muscle layer (yellow arrows) ( Figure 6 B, Figure 6 D).

[0089] It can be seen from the above analysis and evaluation that the method for establishing a damp-heat jaundice rat model provided by the present invention is effective.

[0090] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a rat model of damp-heat jaundice syndrome, characterized in that: The steps include: (1) Pre-experimental treatment: 8-week-old rats were placed in metabolic cages to acclimate to the environment for one week and then randomly divided into a control group and a model group, with 8 rats in each group; (2) Treatment of the control group: Based on the rat body weight, the rats in the control group were gavaged with distilled water at a rate of 8-12 mL / kg for the first 14 days; on the 15th and 16th days, the rats were gavaged with olive oil at a rate of 8-12 mL / kg, and the experimental indicators were measured and recorded on time; (3) Treatment of the model group: At the same time, based on the rat body weight, the model group rats were gavaged with dried ginger solution and ethanol solution at 0.8-1 g / kg and 0.65-0.75 g / kg, respectively, for the first 14 days. On the 15th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 10-11 mg / kg. On the 16th day, they were gavaged with olive oil solution containing α-naphthalene isothiocyanate at 6-8 mg / kg. The experimental indicators were measured and recorded on time. The preparation method of the dried ginger solution in step (3) is as follows: weigh 50.8 g of dried ginger medicinal material, add 10 times the amount of distilled water to soak for 1 hour, boil over high heat until boiling, boil over low heat for 1 hour, filter the filtrate through gauze into a beaker, add 10 times the amount of distilled water, repeat the above process twice, combine the three filtrates, and concentrate to 500 mL.

2. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: The rats described in step (1) are male Wistar rats.

3. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: In step (2), the amount of distilled water and olive oil used is 10 mL / kg.

4. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: The amount of the dried ginger solution used in step (3) is 0.9 g / kg.

5. The method for establishing a rat model of damp-heat jaundice according to claim 1, characterized in that: The volume concentration of the ethanol solution in step (3) is 11.8%, and the dosage is 0.69 g / kg.

6. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: The dosage of the olive oil solution containing α-naphthalene isothiocyanate on the 15th day in step (3) is 10.4 mg / kg.

7. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: The dosage of the olive oil solution containing α-naphthalene isothiocyanate on the 16th day in step (3) is 7 mg / kg.

8. The method for establishing a damp-heat jaundice rat model according to claim 1, characterized in that: The concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 15th day in step (3) was 1.04 mg / mL, and the concentration of the olive oil solution containing α-naphthalene isothiocyanate used on the 16th day was 0.7 mg / mL.

9. The method for establishing a damp-heat jaundice rat model according to claim 7 or 8, characterized in that: The preparation method of the olive oil solution containing α-naphthalene isothiocyanate is as follows: α-naphthalene isothiocyanate is accurately weighed according to a proportion, dissolved in olive oil, and then ultrasonically dispersed for 30 minutes to obtain the solution.

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