CN113424795B

CN113424795B - Construction method and application of acute pancreatitis animal model

Info

Publication number: CN113424795B
Application number: CN202110567952.1A
Authority: CN
Inventors: 杨鑫敏
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2022-09-09
Anticipated expiration: 2041-05-24
Also published as: CN113424795A

Abstract

The invention provides a construction method of an animal model of acute pancreatitis, which induces animals to form acute pancreatitis by using high-fat feed and alcohol together. Obesity and alcohol caused by high-fat feed can synergistically cause pathological damage to the pancreas of a mouse, local inflammation reaction of the pancreas is caused, and lung and general inflammation reaction can be caused. The animal model is closer to human morbidity due to large amount of drinking in the basic obesity state, has obvious pancreatic injury and is accompanied with the expression of multi-organ dysfunction syndrome, and accords with the clinical characteristic that the obese acute pancreatitis patient is easy to become severe. The animal model has the advantages of simple modeling method, good repeatability and strong clinical relevance, can be used for researching pathogenesis of obesity and alcohol-related acute pancreatitis, and provides an important basis for screening of treatment targets and research of medical transformation.

Description

Construction method and application of acute pancreatitis animal model

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of an acute pancreatitis animal model.

Background

Acute Pancreatitis (AP) is an inflammatory injury of the pancreas, such as edema, hemorrhage and necrosis, caused by the self-digestion of pancreatic tissues due to various causes. With the change of life style of people, the incidence of AP is increasing year by year. AP is a common disease in digestive internal medicine, which is mainly manifested by acute and continuous middle and upper abdominal pain, nausea, vomiting and the like, the rise of blood amylase or lipase is more than 3 times of the upper limit of a normal value, and the inflammation of a severe patient is affected by typical imaging change, often combined with systemic or local complications, and the prognosis is poor.

The research on the pathogenesis and the drug treatment of the AP is extremely important, ethical disputes exist in clinical research, and the animal model can well simulate the pathogenesis of the AP and provide a good research tool for researching the pathogenesis, the drug treatment, the prevention and the nursing and the like of the AP.

The existing AP animal model mainly adopts a method that the animal is acted by physical, chemical, biological and compound pathogenic factors to cause the local damage of the pancreas of the animal with or without the damage of other organ functions of the whole body, and has the characteristics similar to the generation of human AP. For example, the most common of them is the induction of the intraperitoneal injection of ranophanin (chemical medicine), which is simple and easy to repeat, but has no clinical relevance; secondly, bile acid salt (physical combined chemical medicine) is also commonly injected in a pancreatic bile duct retrograde manner, and the model has certain clinical relevance with biliary AP, but is complex to operate and not easy to repeat; the rest AP models have no clinical relevance basically, so that the current research uses less, and better reference is difficult to be provided for the treatment means of AP in actual clinical.

For many years, a great deal of research on AP shows that the pathogenic causes of the AP comprise biliary tract diseases, alcohol, hyperlipidemia and the like, and obesity and fatty liver are also classified as risk factors for AP prognosis. However, the research on the specific association mechanism of the above-mentioned factors with AP is still unclear, and thus it is difficult to effectively guide the establishment of animal models with high clinical relevance and the development of new drugs for treating pancreatitis.

Therefore, there is a need to develop an AP animal model based on risk factors of AP occurrence, with strong clinical relevance and significant AP characteristics.

Disclosure of Invention

The invention aims to provide an acute pancreatitis animal model and a construction method and application thereof.

The invention provides a construction method of an animal model of acute pancreatitis, which induces animals to form acute pancreatitis by using high-fat feed and alcohol.

Further, the method comprises the following steps:

(1) high fat diet: continuously feeding animals with high-fat feed for 10-15 weeks; the high-fat feed is a feed with the energy supply ratio of fat higher than 45%.

(2) Alcohol induction: the animals were given an ethanol solution.

Further, the high fat diet of step (1) is a diet having a fat functional ratio of 60%.

Further, the period of feeding in step (1) is 12 weeks.

Further, the ethanol solution in the step (2) is a 35-45% ethanol solution prepared by water or normal saline, preferably a 37.5% ethanol solution prepared by normal saline.

Further, the method for administering an ethanol solution according to step (2) is: performing intragastric administration or injection; the injection is intraperitoneal injection or intravenous injection.

Further, the method for administering the ethanol solution in the step (2) is intraperitoneal injection, wherein the number of intraperitoneal injections is 1-3, and the intervals of the intraperitoneal injections are 1 hour; the injection dosage of each time is 5-15 mu L/g;

preferably, the number of intraperitoneal injections is 2, with 1 hour interval; the injection dose is 10 μ L/g.

Further, 1 hour after the second injection in step (2), injecting normal saline into the abdominal cavity; the injection dosage of the physiological saline is 5-15 mu L/g, and preferably 10 mu L/g.

Further, the animal is any one of a mouse, a rat, a dog, and a pig, and is preferably a mouse.

The invention also provides application of the acute pancreatitis animal model in acute pancreatitis disease research, which aims at diagnosis or treatment of non-diseases.

Furthermore, the research on the acute pancreatitis disease is a research on an acute pancreatitis treatment mechanism or a research on screening drugs for treating the acute pancreatitis disease.

The experimental result shows that the animal model has the following beneficial effects: based on the high prevalence rate of obesity, a plurality of complications and the epidemiological burden of alcoholism, aiming at the current clinical situations that the pathogenesis of AP is not completely clear and the AP has no specific treatment drugs, obesity and alcohol are common risk factors of AP, and an obesity alcoholic AP model is creatively established. The model simulates the morbidity of a large amount of drinking under the basic obesity state, and is closer to the human morbidity. The pancreatic injury is obvious and accompanied with the manifestation of multiple organ dysfunction syndrome, and the clinical characteristics of easy intensification of obese AP patients are met. The animal model of the invention has simple modeling method, high success rate, good repeatability and strong clinical relevance, can be used for researching pathogenesis of obesity and alcohol-related acute pancreatitis, and provides an important basis for screening of treatment targets and research of medical transformation.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

Fig. 1 is a schematic diagram of animal model induction of obese alcoholic acute pancreatitis. HFD, high fat diet; EtOH, alcohol.

FIG. 2 shows the change in body weight and fat in mice. After 12 weeks of high fat diet feeding, mice were (a) changed in body weight and (B) changed in abdominal fat. P < 0.001 compared to control group. CD, control diet; HFD, high fat diet.

FIG. 3 shows the change in serum amylase and lipase levels in mice. After induction of the obese alcoholic AP model, serum was tested for (a) pancreatic amylase and (B) lipase levels. Data are presented as mean ± sem. P < 0.01, P < 0.001, compared to the blank control group; compared with the alcohol control group, # P < 0.05. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1.

FIG. 4 shows pathological changes of mouse pancreas. (A) Pancreatic histopathological section HE was stained and scored double-blindly for (B) edema, (C) inflammatory infiltration, (D) necrosis, and (E) total score, respectively. Data are presented as mean ± sem. P < 0.05, P < 0.01, P < 0.001, compared to the blank control group; compared with the alcohol control group, the # P is less than 0.001. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1.

FIG. 5 shows the change in systemic inflammatory response in mice. (A) Pancreatic tissue MPO levels, (B) lung tissue MPO levels and (C) serum IL-6 levels. Data are presented as mean ± sem. P < 0.05, P < 0.01, P < 0.001, as compared to the blank control group; compared with the alcohol control group, # P < 0.05, # P < 0.01, # P < 0.001. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1; MPO, myeloperoxidase; IL-6, interleukin-6.

FIG. 6 shows the changes in liver and kidney function of mice. (A) Serum CREA levels, (B) serum UREA levels, (C) serum ALT levels and (D) serum AST levels. Data are presented as mean ± sem. P < 0.01, P < 0.001, compared to the blank control group; compared with the alcohol control group, # P < 0.05, # P < 0.01. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1; CREA, creatinine; UREA, UREA; ALT, glutamic-pyruvic transaminase; AST, glutamic-oxaloacetic transaminase.

FIG. 7 shows the changes in serum lactate dehydrogenase and calcium ion levels in mice. (A) Serum lactate dehydrogenase levels and (B) serum calcium ion levels. Data are presented as mean ± sem. Compared with the blank control group, the composition of the composition, ^＊＊ P＜0.01， ^＊＊＊ p is less than 0.001; compared with the alcohol control group, ^# P＜0.05， ^### p is less than 0.001. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1; LDH, lactate dehydrogenase; ca ²⁺ And calcium ions.

FIG. 8 shows changes in circulating mouse serum histones. (A) Detecting histone expression in serum by Western blot, and (B) analyzing and counting grey scale values of the result of the Western blot. Data are presented as mean ± sem. Compared with the blank control group, the composition of the composition, ^＊＊＊ p is less than 0.001; compared with the alcohol control group, ^### p is less than 0.001. CD, blank control; HFD, high fat control; EtOH, alcohol control; HFD + EtOH, mouse model of obese alcoholic acute pancreatitis modeled in example 1.

Detailed Description

1. Laboratory animal

SPF male C57BL/6J mice (4-5 weeks) were purchased from Beijing Huafukang Biotech GmbH, Inc., license: SCXK (Kyoto) 2020-. The animal rooms are raised in cages in the experimental animal center of the Huaxi hospital, Sichuan university, 5 cages are used for each, the temperature is kept at 25 +/-2 ℃, the illumination is controlled (12h day/night circulation), food and water are taken freely, and the experiment is started formally after 1 week of adaptive feeding. The experiment is approved by the ethical committee of the experimental animal center of western hospital, Sichuan university (ethical number 2021016A), and all animal experiments and related operations are performed according to school and national standards.

Example 1 construction of mouse model for acute pancreatitis

The mice are continuously fed with high-fat feed for 12 weeks, and the feed is added every week to ensure the quality of the mice. The high-fat feed adopted in this example is 60% high-fat feed (HFD, fat supply ratio 60%, H10060, huafukang, beijing) in obesity model series feeds (DIO series feeds); the specific composition and energy supply ratio of the feed are shown in table 1. Alcohol induction was performed after 12 weeks, and all mice were fasted for 12h before molding without water deprivation. A37.5% ethanol solution is prepared by normal saline, and is used as it is, the 37.5% ethanol solution (10 mu L/g) is injected into abdominal cavity of mouse for 2 times at intervals of 1 h. After the last alcohol injection, water is supplemented for 1h, namely, normal saline (10 mu L/g) is injected into the abdominal cavity. The induction scheme is shown in FIG. 1.

TABLE 1 feed composition and energy supply ratio

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 synergistic induction of pathological changes in mouse pancreas and injury of multiple organ functions by obesity and alcohol

1. Animal grouping and treatment (8 animals per group)

(1) Obese alcoholic acute pancreatitis group (i.e., example 1): the high fat diet was fed for 12 weeks, and 37.5% ethanol solution was administered for 2 consecutive injections at 1h intervals. The specific procedure was as in example 1.

(2) Blank control group: the normal diet (using control feed (CD, fat energy supply ratio of 10%, H10010, Huafukang, Beijing), the specific composition and energy supply ratio of the feed are shown in Table 1) was fed for 12 weeks, and the same amount of physiological saline as the ethanol solution used in example 1 was continuously injected 2 times at intervals of 1 hour, and the rest of the procedure was the same as that of example 1.

(3) High fat control group: after feeding the diet with high fat for 12 weeks, the same amount of physiological saline as the ethanol solution used in example 1 was continuously injected 2 times at 1-hour intervals, and the rest of the procedure was the same as in example 1.

(4) Alcohol control group: the normal diet was fed for 12 weeks, and 37.5% ethanol solution equivalent to that used in example 1 was continuously injected 2 times at 1-hour intervals, and the rest was the same as in example 1.

2. Method for taking materials

After the modeling was completed, the material was taken 12h after the first alcohol injection. Blood sampling is carried out after isoflurane gas is adopted to induce anesthesia, and the specific operation is as follows: the mice are placed in an anesthesia induction box for anesthesia, the mice are maintained by a mask after anesthesia, the thoracic cavity is quickly opened, the heart is exposed and cut open, blood is quickly sucked by a 1mL syringe, after standing at room temperature for 30min, the mice are centrifuged at 1500g at room temperature for 15min, the upper serum is sucked and frozen in a refrigerator at the temperature of minus 80 ℃, and the levels of serum biochemistry, interleukin 6 (IL-6) and circulating histone are measured. Taking out the right lower lung after blood collection, rinsing the right lower lung in normal saline, sucking the lung by using filter paper, putting the lung into liquid nitrogen for quick freezing, and storing the lung in a refrigerator at the temperature of-80 ℃ for measuring Myeloperoxidase (MPO). Then, rapidly opening the abdominal cavity, dividing pancreatic tissues into three parts, putting the complete pancreatic part into an embedding box, soaking the pancreatic part into 10% neutral formalin solution for fixing for more than 48h, dehydrating, embedding paraffin, and staining by hematoxylin-eosin (HE); and taking out the other two pancreatic tissues, quickly freezing by using liquid nitrogen, and freezing and storing in a refrigerator at the temperature of-80 ℃ for experiments such as MPO (maximum oxygen production) determination and the like.

3. Results of the experiment

(1) High fat diet induced obesity

Obesity is manifested by excessive weight gain and excessive accumulation of body fat. As shown in FIG. 2A, there was no significant difference in body weight between the high fat diet and the control diet group mice before the start of feeding (20.9vs 21.2g, P > 0.05). After feeding for 12 weeks on two different diets, the body weight increased compared to before feeding. The weight of the high fat diet group increased to 32.9g, which is significantly higher than that of the control diet group (26.5g, P < 0.001). It was seen by dissection (fig. 2B) that mice on the high fat diet showed significant fat accumulation, particularly a significant increase in visceral fat.

The results show that the high-fat diet can cause the obesity of the mice, and provides an obesity basic state for a model for further inducing the obesity alcoholic acute pancreatitis.

(2) Serum amylase, lipase and pathological changes of pancreas

The increase of the levels of circulating amylase and lipase is an important mark of AP, and clinical guidelines take one of the diagnostic criteria of increasing the levels of amylase and lipase to more than 3 times of the upper limit of normal. The levels of pancreatic amylase and pancreatic lipase in serum are detected by using a full-automatic biochemical analyzer, and the results are shown in fig. 3A-B, and the levels of amylase and lipase in a high-fat control group and a blank control group have no obvious difference, which indicates that the successful construction of an AP model cannot be realized only by adopting high-fat induction.

After alcohol injection, the levels of amylase and lipase in each group were increased, and compared with the blank control group, the levels were statistically significant (P < 0.01). Wherein, amylase in the obese alcoholic acute pancreatitis group is increased to 21768U/L, lipase level is increased to 1595U/L, and the levels are respectively obviously higher than that of an alcohol control group (P is less than 0.05), which indicates that high-fat diet and alcohol induction generate obvious synergistic effect, and an animal model with obvious AP characteristic can be successfully constructed.

Pathological diagnosis is often considered as a golden standard of disease, pathological changes of mouse pancreatic tissues are further observed, and the pancreatic tissues are observed under a microscope after being stained by paraffin-embedded sections HE. The result is shown in fig. 4A, and it can be seen that acinar cells of pancreatic tissues of the blank control group and the high-fat control group are complete and regularly arranged, and there are no interstitial edema, inflammatory cell infiltration and cell necrosis, which indicates that successful construction of the AP model cannot be achieved only by high-fat induction.

After the alcohol injection, the gap of pancreatic tissues is slightly increased in an alcohol control group, and scattered acinar cells are subjected to edema separation; and the pancreatic tissue clearance of the obese alcoholic AP group induced by high fat and alcohol is obviously increased, acinar cell edema is separated, inflammatory cell infiltration can be seen between the duct and the lobule, and acinar cell necrosis can be seen locally. Two pathologists used a double-blind method to stain pathological sections of mouse pancreatic tissue HE, and scored from three aspects of edema, inflammatory infiltration and necrosis. The results are shown in fig. 4B-E, the edema, inflammatory infiltration, necrosis and total components of the high-fat control group and the blank control group are not obviously different, the edema, necrosis and total components of the alcohol control group are slightly higher than those of the blank control group (P is less than 0.05), and the inflammatory infiltration is not obviously different compared with the blank control group, which indicates that the AP model with obvious pathological characteristics cannot be constructed by using only alcohol induction.

Edema, inflammatory infiltration, necrosis and total components of the high-fat diet and alcohol co-induced obesity alcoholic acute pancreatitis group are respectively 1.98, 1.10, 1.27 and 4.35 which are all obviously higher than those of a blank control group (P is less than 0.001), and furthermore, all pancreas pathological scoring indexes of the obesity alcoholic AP group are all obviously higher than those of an alcohol control group (P is less than 0.001). The results show that the cooperation of obesity and alcohol can cause the increase of the levels of serum amylase and lipase of mice and pathological damage of pancreas, which marks the occurrence of acute pancreatitis.

(3) Changes in systemic inflammatory response

When AP occurs, the pancreas is inflamed locally and the inflammatory response can progress systemically, causing distal organs to become involved, the lung being the most vulnerable. Changes in MPO levels and activity represent the functional and active state of neutrophils, and thus MPO may serve as an inflammatory marker. Numerous studies have shown that when AP occurs, MPO activity is significantly elevated in pancreatic and lung tissues, suggesting a massive neutrophil infiltration in pancreatic and lung tissues. IL-6, a member of cytokines, is involved in a number of inflammatory diseases. When AP occurs, circulating IL-6 levels are closely correlated with the severity of AP. Therefore, we examined the MPO activity of pancreatic tissue to reflect local inflammatory responses of the pancreas and examined MPO activity and circulating IL-6 levels in lung tissue to reflect systemic inflammatory responses.

The results are shown in fig. 5A-B, where there was no significant difference in MPO levels in the pancreas and lung tissues between the high-fat control group and the blank control group, indicating that the inflammatory response was not significant using only high-fat induction. After the alcohol injection is given, the MPO level of pancreas and lung tissues of each group is increased, and the MPO level has statistical significance (P is less than 0.05) compared with that of a blank control group, which indicates that the single use of alcohol for induction can promote the generation of inflammatory reaction to a certain extent, but the effect is poor. MPO level of the obese alcoholic acute pancreatitis group induced by both high fat and alcohol is 100 percent and is obviously higher than that of the alcohol control group (P is less than 0.05).

The IL-6 level in the serum of the mice was measured by a commercial kit (FIG. 5C), and there was no significant difference between the high-fat control group and the blank control group, and also between the alcohol control group and the blank group. It shows that the induction of only alcohol or only high fat can not lead to obvious inflammatory reaction. After the mice are given alcohol injection based on the induction of high-fat diet, the serum IL-6 level of the obese alcoholic AP group of the mice can be increased to 139.71pg/mL (P < 0.01) from 16.97pg/mL of a blank control group, and is obviously higher than that of an alcohol control group (P < 0.01).

The above results indicate that obesity and alcohol in combination can cause elevation of MPO level in pancreas, lung and IL-6 level in serum, which is the manifestation of local and systemic inflammatory reaction in acute pancreatitis. By the method for cooperatively inducing high-fat diet and alcohol, an AP model with obvious characteristics can be effectively constructed.

(4) Changes in multiple organ dysfunction

AP can further develop into severe AP, with organ failure occurring in one or more organs, and the incidence of renal function impairment is second only to lung dysfunction. We measured Creatinine (CREA) and UREA (UREA) in mouse serum to reflect impairment of renal function.

The results are shown in fig. 6A, B, in which there was no significant difference between the high-fat control group, the alcohol control group and the blank control group, indicating that the effect of either high-fat induction or alcohol induction on renal function injury was not significant. After the mice are given alcohol injection based on the induction of high-fat diet, the serum CREA and UREA levels of the obese alcoholic AP group of the mice are obviously increased (P < 0.01) compared with that of a blank control group and are obviously higher than that of an alcohol control group (P < 0.05), which indicates that the co-induction of high fat and alcohol obviously promotes the renal function injury.

Alcohol can cause liver damage, the latter also commonly seen in severe AP patients. We tested serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) to reflect impaired liver function.

The results are shown in fig. 6C, D, where the high-fat control group and the blank control group have no significant difference, and serum ALT and AST levels after alcohol injection are significantly increased (P < 0.001), while the obese alcoholic AP group is significantly increased (P < 0.01) compared with the alcohol control group, indicating that co-induction of high fat and alcohol significantly promotes liver function damage.

Severe AP is often accompanied by a series of biomarker changes. Lactate Dehydrogenase (LDH) is an indicator of cell death.

The results of measuring the LDH level in the serum are shown in FIG. 7A, the high-fat control group and the blank control group have no obvious difference, and the LDH level in the serum is increased (P < 0.001) after the alcohol injection, while the obese alcoholic AP group is more significantly increased (P < 0.001) compared with the alcohol control group. A decrease in blood calcium is a severe manifestation of AP disease, suggesting a poor prognosis. As shown in fig. 7B, there was no significant difference between the high-fat control group and the blank control group, and after alcohol injection, the blood calcium level of the alcohol control group was not significantly changed, whereas the blood calcium level of the obese alcoholic AP group was significantly reduced (P < 0.05) compared to both the blank control group and the alcohol control group. It is shown that the co-induction of high fat and alcohol significantly promotes the development of severe AP.

Histones are basic structural proteins highly conserved in eukaryotic cell nuclei, and are released outside cells into circulation, i.e., circulating histones, during tissue damage and cell death. Circulating histones were significantly elevated in severe AP.

The circulating histone levels are detected by an immunoblotting method, and a standard substance is used for quantitative analysis, the result is shown in fig. 8A, B, the serum of a blank control group and the serum of a high-fat control group have almost no histone, the serum histone level of an alcohol control group is slightly higher than that of the blank control group after alcohol injection, but the difference is not statistically significant, and the serum histone level of an obese alcoholic AP group induced by both high fat and alcohol is obviously higher than that of the blank control group and the alcohol control group (P is less than 0.001), so that the generation of the AP can be effectively caused by the co-induction of the high-fat diet and the alcohol.

The results show that the synergy of obesity and alcohol can cause the liver and kidney function abnormality of mice, and the multiple organ functional injury and the severe manifestation of acute pancreatitis, such as the rise of serum lactate dehydrogenase, the reduction of blood calcium, the increase of circulating histone and the like, are in line with the clinical characteristics of easy severe manifestation of obese AP patients, and have strong clinical relevance.

In general, 8 mice of the obese alcoholic acute pancreatitis group of the invention, which were modeled according to the method of example 1, were successfully modeled to prepare an animal model with significant AP characteristics, indicating that the method of the invention has high modeling success rate and good repeatability.

In conclusion, the invention provides a construction method of an animal model of the obese alcoholic acute pancreatitis, obesity and alcohol can cooperatively cause pathological injury of the pancreas of a mouse to cause local inflammatory reaction of the pancreas and can cause inflammatory reaction of the lung and the whole body.

Claims

1. A method for constructing an animal model of acute pancreatitis is characterized in that high-fat feed and alcohol are used for inducing animals to form the acute pancreatitis, and the method comprises the following steps:

(1) high fat diet: continuously feeding animals with high-fat feed for 10-15 weeks; the high-fat feed is a feed with the energy supply ratio of fat higher than 45%;

(2) alcohol induction: administering an ethanol solution to the animal; the ethanol solution is 35-45% ethanol solution prepared by water or normal saline; the method for administering the ethanol solution is intraperitoneal injection, wherein the number of intraperitoneal injection is 1-3, and each time interval is 1 hour; the injection dosage of each time is 5-15 mu L/g; injecting normal saline into the abdominal cavity 1 hour after the last injection of the ethanol solution into the abdominal cavity; the injection dosage of the normal saline is 5-15 mu L/g;

the animal is a mouse.

2. The method of claim 1, wherein the high fat diet of step (1) is a diet having a fat energy ratio of 60%.

3. The method of claim 1, wherein the period of feeding in step (1) is 12 weeks.

4. The method of claim 1, wherein the ethanol solution of step (2) is a 37.5% ethanol solution in normal saline.

5. The method of claim 1, wherein the number of intraperitoneal injections in step (2) is 2, each time at 1 hour intervals; the injection dose is 10 μ L/g.

6. The method of claim 1, wherein the normal saline is injected in a dose of 10 μ L/g.

7. Use of an animal model of acute pancreatitis prepared by the method of any one of claims 1-6 in the study of acute pancreatitis, for the purpose of diagnosis or treatment of non-diseases.

8. The use of claim 7, wherein the acute pancreatitis disease study is a study screening for drugs to treat acute pancreatitis disease.