Preparation method and application of severe alcoholic hepatitis animal model

Abstract

The invention discloses a preparation method and application of a severe alcoholic hepatitis animal model. The severe alcoholic hepatitis animal model prepared by the method provided by the invention can better simulate the clinical SAH pathogenesis process, especially better simulate the characteristics of higher clinical case fatality rate, remarkable liver inflammation, neutrophil infiltration and the like of SAH, and provides an important animal model tool for screening SAH prevention and treatment drugs.

Description

Technical Field

[0001] The present invention generally relates to the field of biomedical technology, and in particular to a method for preparing an animal model of severe alcoholic hepatitis and uses of the animal model prepared by the method. Background Art

[0002] Alcoholic liver disease (ALD) is a series of liver diseases caused by long-term excessive drinking. According to WHO statistics, there are more than 6 million patients with ALD in my country, accounting for 40% of the 150 million patients with ALD worldwide. The disease burden of ALD in my country cannot be ignored. ALD can be divided into five clinical types: mild ALD, alcoholic fatty liver, alcoholic hepatitis (AH), alcoholic liver fibrosis, and alcoholic cirrhosis. Among them, severe alcoholic hepatitis (SAH) is a progressive disease with liver failure on the basis of AH. The 30-day natural mortality rate can be as high as 35-50%, and the 90-day mortality rate can reach 70%. It is one of the main causes of death related to ALD.

[0003] The clinical pathological features of SAH are mainly excessive inflammatory response, a large number of neutrophil infiltration in the liver, inflammatory factor storm, systemic inflammation, etc. The inflammatory response and inducement of SAH are systemic immune inflammatory disorders. After alcohol intake, the liver is the main metabolic organ. The danger-associated molecular patterns (DAMPs) produced after alcohol metabolic toxins damage liver cells induce strong natural immune and inflammatory responses; and alcohol intake can also lead to intestinal barrier dysfunction, increase the translocation of intestinal bacteria and endotoxins as pathogen-associated molecular patterns (PAMPs), and then activate the immune response of the liver. The complex immune inflammatory response cascade amplification process and metabolic disorders based on this cause uncontrolled inflammation and cell energy crisis, and ultimately lead to massive cell death and organ failure.

[0004] For severe alcoholic hepatitis, there is currently a lack of an animal model that can better replicate clinical symptoms. Therefore, establishing a SAH animal model, especially a model that can simulate human drinking habits and the pathogenesis of SAH, has important theoretical value and practical significance. Summary of the invention

[0005] Based on this, the present application provides a method for preparing an animal model of severe alcoholic hepatitis, comprising:

[0006] Step 1): feeding the animals using the adaptive feeding procedure used to construct the NIAAA animal model;

[0007] Step 2): feeding the animals with the chronic alcohol feeding procedure used to construct the NIAAA animal model;

[0008] Step 3): After completing the feeding of step 1) and step 2), the animals are gavaged (ig) with 3-8 g / kg of alcohol, and 3-10 mg / kg of lipopolysaccharide is injected into the animals.

[0009] On the other hand, the present application also provides use of the severe alcoholic hepatitis animal model obtained by the preparation method described herein in screening or identifying drugs for preventing or treating severe alcoholic hepatitis.

[0010] The severe alcoholic hepatitis animal model prepared by the method of the present application can better simulate the pathogenesis of clinical SAH, especially better simulate the high clinical mortality rate, significant liver inflammation and neutrophil infiltration of SAH, and provides an important animal model tool for drug screening for the prevention and treatment of SAH.

[0011] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0013] Figure 1 The experimental design process of the SAH model in Example 1 of the present application is shown, wherein A: dose condition combination grouping; B: LPS action time grouping.

[0014] Figure 2 The statistics of the mortality rate of each group of mice in Example 1 of the present application are shown.

[0015] Figure 3 The H&E and IHC staining of liver tissues of each group of mice in Example 1 of the present application are shown. Among them, B: MPO histochemical quantitative analysis; C: Ly6G histochemical quantitative analysis.

[0016] Figure 4The effect of LPS stimulation time on mouse liver in Example 1 of the present application is shown. A: H&E staining and IHC staining of mouse liver tissue at different times; B: MPO histochemical quantitative analysis; C: Ly6G histochemical quantitative analysis; D: ALT levels in mouse serum at different times; E: AST levels in mouse serum at different times.

[0017] Figure 5 It shows that the liver function of SAH mice in Example 2 of the present application was significantly impaired. Among them, A: weight change during molding; B: liver index of SAH mice; C: serum ALT level of SAH mice; D: serum AST level of SAH mice; E: serum LDH level of SAH mice; F: serum DBIL level of SAH mice; G: serum TBA level of SAH mice; H: serum ALB level of SAH mice; I: serum CHE level of SAH mice.

[0018] Figure 6 It shows that the levels of serum inflammatory factors and chemokines in SAH mice in Example 2 of the present application were significantly increased. Among them, A: serum IL-1β level of SAH mice; B: serum IL-6 level of SAH mice; C: serum IL-18 level of SAH mice; D: serum TNF-α level of SAH mice; E: serum MIP-2 level of SAH mice; F: serum CCL2 level of SAH mice; G: serum CXCL1 level of SAH mice; H: serum CXCL7 level of SAH mice.

[0019] Figure 7 The present invention provides a process for establishing a SAH mouse model according to one embodiment of the present application. DETAILED DESCRIPTION

[0020] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0021] The terms "about" and "approximately" when used with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0022] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0023] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0024] At present, most studies on ALD use the chronic alcohol feeding plus acute alcohol gavage model (NIAAA model) established by the National Institute of Alcohol Abuse and Toxicology (NIAAA) of the National Institutes of Health, which can cause liver fatty degeneration and a small amount of inflammatory cell infiltration in the early stage of alcoholic hepatitis (the first attack). Lipopolysaccharide (LPS), as a cell wall component of Gram-negative bacteria, is the main toxic component of endotoxins. It can mediate inflammatory factors to destroy the integrity of the vascular endothelium, leading to apoptosis and necrosis of hepatocytes, activating inflammatory responses, and aggravating liver damage. On the basis of alcohol stimulation, LPS can further mediate inflammatory factors to destroy the vascular endothelium and enter the blood, and change the intestinal microbial structure, causing a "second attack" on the liver, aggravating liver inflammation and damage, which is more in line with the clinical characteristics of high mortality and large-scale inflammatory responses. At present, there are no reports of SAH animal models based on alcohol combined with LPS to simulate the "second attack" at home and abroad. The inventors of this application first established a SAH mouse model based on the NIAAA model by increasing the dose of acute large-dose intragastric alcohol on the last day and combining it with LPS stimulation. The optimal combination for simulating SAH was obtained from the perspective of different stimulation doses and times, combined with mortality, pathology, and liver function indicators (ALT, AST), and verified by comparing liver function indicators, inflammatory factors, and chemokine indicators with the control group. The establishment of this model can provide a good ideal animal model for the next step of research on the pathogenic mechanism of severe alcoholic hepatitis.

[0025] In one aspect, the present application provides a method for preparing an animal model of severe alcoholic hepatitis, comprising:

[0026] Step 1): feeding the animals using the adaptive feeding procedure used to construct the NIAAA animal model;

[0027] Step 2): feeding the animals with the chronic alcohol feeding procedure used to construct the NIAAA animal model;

[0028] Step 3): After completing the feeding of step 1) and step 2), the animals are gavaged (ig) with 3-8 g / kg of alcohol, and 3-10 mg / kg of lipopolysaccharide is injected into the animals.

[0029] In some embodiments, the adaptive feeding step is adaptive feeding the animal with liquid feed for 4-6 days;

[0030] The chronic alcohol feeding procedure continued to feed the animals with a liquid feed containing 3% to 7% alcohol (% v / v) for 8-12 days.

[0031] In some embodiments, in step 3), the animal is gavaged with 3 g / kg, 4 g / kg, 5 g / kg, 6 g / kg, 7 g / kg or 8 g / kg of alcohol, and the animal is injected with 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg of lipopolysaccharide.

[0032] In some embodiments, in step 3), the animal is gavaged with 7-8 g / kg of alcohol, and 4.5-5.5 mg / kg of lipopolysaccharide is injected into the animal.

[0033] In some embodiments, in step 3), the animal is gavaged with 7.5 g / kg of alcohol, and 5 mg / kg of lipopolysaccharide is injected into the animal.

[0034] In some embodiments, the animal is a mammal; preferably, the mammal is a rat, a mouse, a cat, a dog, a horse, a sheep, a cow, or a monkey; preferably, the mammal is a mouse.

[0035] In some embodiments, the liquid feed is a Lieber-DeCarli liquid feed. The ingredients of a Lieber-DeCarli liquid feed are known to those skilled in the art.

[0036] In some embodiments, other types of liquid feeds may also be used, such as Tsukamoto-French liquid feeds.

[0037] In some embodiments, the injection is intraperitoneal (ip).

[0038] In some embodiments, about 12 hours after step 3), the severe alcoholic hepatitis animal model is prepared.

[0039] In some embodiments, the method for preparing an animal model of severe alcoholic hepatitis described herein comprises:

[0040] 1) Adaptive feeding of mice with Lieber-DeCarli liquid diet for 5 days;

[0041] 2) continue to feed mice with Lieber-DeCarli liquid diet containing 5% alcohol (% v / v) for 10 days;

[0042] 3) After completing the feeding in step 2), the mice were gavaged with 7.5 g / kg of alcohol and injected intraperitoneally with 5 mg / kg of lipopolysaccharide. The model was established 12 hours later.

[0043] In some embodiments, various adaptive feeding steps 1) and chronic alcohol feeding steps 2) for constructing NIAAA animal models can be used. In some embodiments, the adaptive feeding step 1) is adaptive feeding with a liquid control feed for 5 days, or adaptive feeding (gradient transition) with a liquid alcohol feed with an alcohol concentration of 1% to 5% for 5 days, in order to adapt the animal to the liquid diet. After the 5-day adaptation period, the chronic alcohol feeding step 2) is feeding with a liquid feed containing 5% alcohol for 10 days.

[0044] In another aspect, the present application also provides an animal model of severe alcoholic hepatitis prepared using the method described herein.

[0045] In another aspect, the present application also provides use of the severe alcoholic hepatitis animal model prepared by the method described herein in the evaluation of therapeutic drugs for severe alcoholic hepatitis.

[0046] On the other hand, the present application also provides use of the severe alcoholic hepatitis animal model obtained by the preparation method described herein in screening or identifying drugs for preventing or treating severe alcoholic hepatitis.

[0047] Based on the NIAAA model, this application increases the dose of acute large-dose intragastric alcohol on the last day and combines LPS to stimulate mice. From the perspective of different stimulation doses and times, the best combination of simulated SAH is obtained by combining mortality, pathology and liver function indicators (ALT, AST). And the specific liver function indicators (ALT, AST, LDH, DBIL, TBA, CHE, ALB), inflammatory factors (IL-1β, IL-6, IL-18, TNF-) and chemokines (MIP-2, CCL2, CXCL1, CXCL7) were compared with the control group. It was found that the severe alcoholic hepatitis animal model prepared by the method of this application can better simulate the pathogenesis of clinical SAH, especially better simulate the high clinical mortality rate, significant liver inflammation and neutrophil infiltration of SAH, and provide an important animal model tool for drug screening for the prevention and treatment of SAH.

[0048] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with any other feature or element in any other embodiment, or may replace any other feature or element in any other embodiment.

[0049] The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements disclosed in the present application may also be combined with any conventional features or elements to form a unique invention scheme. Any features or elements of any embodiment may also be combined with features or elements from other invention schemes to form another unique invention scheme. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0050] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0051] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0052] Example

[0053] The relevant materials used in the examples are as follows:

[0054] 1. Reagents and Materials

[0055] Male C57BL / 6J mice, SPF grade, body weight 20-25g, 8-10 weeks old, were purchased from Weitonglihua (Beijing) Biotechnology Co., Ltd. The experimental animal production license number is SYXK (Beijing) 2022-0049. All mice were kept in an environment with a temperature of 22-25°C and a relative humidity of 40%-70%, with free access to food and water. Animal experiments were performed after 7 days of adaptive feeding. The experiment was approved by the University Ethics Committee of Capital Medical University (approval number AEEI-2023-102).

[0056] 99.5% vol pharmaceutical grade anhydrous alcohol was purchased from MacLean; Lipopolysaccharide (LPS) (Cat. No. L2880) was purchased from Sigma, USA; Lieber-DeCarli control feed (Cat. No. F1259SP) and Lieber-DeCarli alcohol feed (Cat. No. F1258SP) were purchased from Bio-Serv, USA; maltodextrin (Cat. No. M8450) was purchased from Beijing Solebow Technology Co., Ltd.; anti-myeloperoxidase (MPO) antibody (Cat. No. AB208670) was purchased from Abcam, UK; Ly6G antibody (Cat. No. 87048) was purchased from CST, USA; alanine aminotransferase assay kit (Cat. No. C009- 2-1), aspartate aminotransferase assay kit (catalog number: C010-2-1), lactate dehydrogenase assay kit (catalog number: A020-2-2), direct bilirubin assay kit (catalog number: C019-2-1), total bile acid assay kit (catalog number: E003-2-1), albumin assay kit (catalog number: A028-2-1), cholinesterase assay kit (catalog number: A023-2-1) were purchased from Nanjing Jiancheng Bioengineering Technology Co., Ltd.; mouse interleukin-1β ELISA quantitative kit (catalog number: KTE7005), mouse interleukin-6 ELISA quantitative kit (catalog number: KTE7009), mouse tumor necrosis factor-α ELISA quantitative kit (catalog number: HT7015), mouse chemokine CCL2 The ELISA quantitative kit (catalog number: KTE7001) was purchased from Wuhan Yacoin Biotechnology Co., Ltd.; the mouse interleukin-18 ELISA kit (catalog number: EK0433), mouse macrophage inflammatory protein-2 ELISA kit (catalog number: EK0452), mouse chemokine CXCL1 ELISA kit (catalog number: EK0732), and mouse chemokine CXCL7 ELISA kit (catalog number: EK0730) were purchased from Wuhan Boster Biotechnology Co., Ltd.

[0057] 2. Instruments

[0058] FlexStation 3 multifunctional microplate reader (MeiGu Molecular Instrument Co., Ltd.), Velocity 18R Pro high-speed refrigerated centrifuge (Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.), -20℃ refrigerator, 4℃ refrigerator, -80℃ ultra-low temperature refrigerator (Meiling Company).

[0059] Example 1. Establishment of conditions for establishing a severe alcoholic hepatitis mouse model

[0060] At present, most studies on ALD use the NIAAA model, that is, simply feeding with short-term chronic alcohol liquid feed and acute high-dose alcohol gavage on the last day cannot effectively simulate the pathological characteristics of SAH. However, alcohol intake can lead to intestinal barrier dysfunction, increase intestinal bacteria and endotoxins as pathogen-associated molecular patterns (PAMPs), and after alcohol stimulation, further administration of LPS, the main component of exogenous endotoxins, can aggravate liver damage and perform a "second strike", more effectively simulating the pathological characteristics of SAH. Based on the NIAAA model, this experiment gave different doses of alcohol and LPS on the last day for a combined study to explore the optimal combined dose for the construction of a SAH mouse model.

[0061] In this example, mice were randomly divided into 6 groups, each with 20 mice, namely, a control group (CON), a NIAAA group (Gao-Binge model), and four modified NIAAA model groups. Among them, the four modified NIAAA model groups respectively investigated different combinations of high-dose alcohol gavage and LPS intraperitoneal injection on the last day of the NIAAA model, which are:

[0062] Combination ① (Comb. 1): 5 g / kg alcohol i.g + 5 mg / kg LPS ip;

[0063] Combination ② (Comb. 2): 5 g / kg alcohol i.g + 10 mg / kg LPS ip;

[0064] Combination ③ (Comb. 3): 7.5 g / kg alcohol i.g + 5 mg / kg LPS ip;

[0065] Combination ④ (Comb.4): 7.5g / kg alcohol i.g + 10mg / kg LPS ip.

[0066] Specifically, the modeling is divided into four stages: the first stage (Preadaptation), the mice are fed adaptively for one week. The second stage (Liquid diet adaptation), the mice are fed with Lieber-DeCarli liquid diet (225g Lieber-DeCarli control diet, add 860mL tap water and mix thoroughly) for 5 days to adapt the mice to the liquid diet, ensuring that each mouse ingests 25mL of the control liquid diet. The third stage (Chronic alcohol liquid diet feeding), the control group mice are fed with the control liquid diet from the 6th day, and the other groups are given Lieber-DeCarli alcohol diet containing 5% alcohol every day (with the same total calories as the control group liquid diet) to ensure their free diet, giving each mouse 25mL, and the amount of liquid feed given every 24 hours is always kept the same as the model group, and feeding is continued for 10 days. In the fourth stage (Binge and LPS injection), gavage was performed at 7:00-9:00 am on the 16th day. The control group was gavaged with dextrin solution (each 10 mL of dextrin solution contained 4.5 g of dextrin); the other groups were gavaged with 31.5% alcohol solution. One hour later, the control group and the NIAAA group were intraperitoneally injected with normal saline (0.1 mL / 10 g), and the other groups were intraperitoneally injected with corresponding doses of LPS. The mice were killed 8 hours later. Figure 1 Serum and liver were collected for subsequent analysis.

[0067] After determining the optimal alcohol gavage dose and LPS intraperitoneal injection dose, we increased the duration of LPS action in mice and set the sampling time at 10 hours and 12 hours, respectively. Figure 1 B), serum and liver were collected for subsequent analysis until the optimal intervention dose and time for the SAH mouse model were determined.

[0068] Evaluate the effect of each condition group

[0069] Biological sample collection and processing: After the last administration, the mice were anesthetized with isoflurane and their eyeballs were removed to collect blood. The blood was placed in a 4°C refrigerator for 1 hour and the centrifuge conditions were set at 3500 r / min. -1 , centrifuge at 4℃ for 15min, take the upper serum, pack it, and freeze it at -80℃. After blood collection, cut the abdomen of the mouse, cut the complete liver tissue, and wash the blood on the surface of the liver with pre-cooled PBS. Then divide the liver into three parts, two of which are frozen in a -80℃ refrigerator, and one is fixed with 4% paraformaldehyde and stored at 4℃.

[0070] Index detection:

[0071] (1) Statistical analysis of mouse mortality in each group before sampling.

[0072] (2) Pathological observation of liver tissue: Mouse liver tissue was fixed with 4% paraformaldehyde, dehydrated with alcohol, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE) and immunohistochemistry (MPO, Ly6G). Pathological changes of liver tissue were observed under an optical microscope.

[0073] (3) Serum liver function index detection: Detect the levels of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), direct bilirubin (DBIL), total bile acid (TBA), albumin (ALB), and cholinesterase (CHE) in mouse serum.

[0074] Experimental Results

[0075] Clinically, SAH is characterized by rapid deterioration of liver inflammation, accompanied by high short-term mortality, and the 30-day natural mortality rate can be as high as 35%-50%. In this study, the last acute large-dose oral administration of alcohol was combined with LPS stimulation until the sampling (8 hours later). The results showed that the mortality rate of the control group (CON) was 0; when 5g / kg of alcohol was administered intragastricly (NIAAA, Comb.1, Comb.2), the mortality rate of each group was relatively low, all at 10% or below; the mortality rate of Comb.3 group was 35%, which was close to the clinical mortality rate of SAH; the mortality rate of Comb.4 group was 80%, which was too high and not conducive to animal experimental research. In summary, from the perspective of simulating the clinical mortality rate of SAH, 7.5g / kg alcohol i.g + 5mg / kg LPS ip is a better modeling condition. The mortality rate of each group is shown in Figure 2 .

[0076] H&E staining was used to observe the pathological changes of liver tissues in each group. The results showed that in the control group, the liver lobule structure was clear, the liver cells were arranged neatly, there was no deformation and necrosis, but there were very few fat vacuoles; in the NIAAA group, there was a large area of ​​loose liver cell cytoplasm, disordered liver lobule structure, and a large number of fat vacuoles; in the Comb.1 and Comb.2 groups, there was a large area of ​​loose liver cell cytoplasm, disordered liver lobule structure, a large number of fat vacuoles, and a small amount of neutrophil infiltration; in the Comb.3 and Comb.4 groups, there was a large area of ​​loose liver cell cytoplasm, large gaps, obvious fat vacuoles, and a large number of neutrophil aggregation ( Figure 3A in the figure). Specific immunohistochemical staining of neutrophils showed that the infiltration of neutrophils in the liver tissue of the control group and the NIAAA group was very rare; the infiltration of neutrophils in the liver tissue of the Comb.1 and Comb.2 groups was more obvious; the infiltration of neutrophils in the liver tissue of the Comb.3 and Comb.4 groups was extremely obvious ( Figure 3 A, B, and C in Figure 3). This is consistent with the excessive inflammatory response in SAH, with a large number of neutrophil infiltrations in the liver.

[0077] Based on the mortality statistics of the above groups of mice and the pathological changes of liver tissue, when the concentration of alcohol was 7.5g / kg and the concentration of LPS was 5mg / kg, the mice had a higher mortality rate, while when the concentration of alcohol was 7.5g / kg and the concentration of LPS was 10mg / kg, the mortality rate of mice was too high, which was not conducive to subsequent research. At the same time, the inflammatory response (neutrophil infiltration) in the liver was the most obvious. Therefore, a 7.5g / kg alcohol gavage dose combined with a 5mg / kg LPS dose was used for further model research.

[0078] The duration of action of LPS in vivo after intraperitoneal injection may have different effects on mice, including activation of acute inflammatory response, changes in pharmacodynamics, changes in liver function level, etc. In this study, after intraperitoneal injection of LPS 5mg / kg in mice, the time of sampling was increased from 8h to 10h and 12h respectively, and the liver tissue was subjected to H&E and immunohistochemical staining. The results showed that with the increase of time points, the looseness of hepatocyte cytoplasm, the larger the gap, the more fat vacuoles, and the more obvious the infiltration of neutrophils ( Figure 4 A, B and C in the figure). Serum was collected to detect the levels of ALT and AST in the liver function of mice. The results showed that the levels of ALT and AST increased at all time points compared with the control group. There were significant differences between 10h and 12h and the control group, and the increase at 12h was the most significant ( Figure 4 D and E in the figure). This indicates that the liver function of mice was most severely damaged when LPS was applied for 12 h, which can better simulate the state of massive neutrophil infiltration and inflammatory factor storm in the liver in the SAH model.

[0079] Example 2. Evaluation of severe alcoholic hepatitis mouse model

[0080] Index detection

[0081] (1) Serum liver function index detection: Detect the levels of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), direct bilirubin (DBIL), total bile acid (TBA), albumin (ALB), and cholinesterase (CHE) in mouse serum.

[0082] (2) Detection of inflammatory factor indicators in mouse serum: Detect the levels of IL-1β, IL-6, IL-18, and TNF-α in mouse serum.

[0083] (3) Detection of mouse serum chemokine indicators: Detect the levels of MIP-2, CCL2, CXCL1, and CXCL7 in mouse serum.

[0084] Experimental Results

[0085] The changes in the liver-to-body ratio and serum liver function indexes of mice can reflect the degree of liver damage, inflammatory response, fatty degeneration and other pathological conditions, providing important experimental data for the study of liver diseases. The liver-to-body ratio and serum liver function indexes of the model mice (SAH group) under the final conditions of the above study were compared with those of the control group. The results showed that there was no significant difference in weight changes between the control group and the SAH group during the entire feeding process, but the weight of the SAH group was slightly reduced ( Figure 5 A in the figure), while the liver index in the SAH group was significantly higher than that in the control group ( Figure 5 B). Among the liver function indicators, the levels of ALT, AST, LDH, and DBIL in the SAH group were significantly higher than those in the control group ( Figure 5 C, D, E and F), the TBA level increased most significantly ( Figure 5 G), the levels of ALB and CHE were significantly reduced ( Figure 5 H and I in the figure). The results showed that the liver function of mice in the SAH group was severely damaged. This result is consistent with the changes in liver function indicators of clinical SAH patients, indicating that the SAH mouse model is relatively successful.

[0086] Inflammatory factors and chemokines in serum are important components of the immune system. They play a key role in inflammatory responses, immune surveillance, tissue regeneration and other processes. Inflammatory factors play an important role in local and circulatory inflammatory responses. They can trigger the expression of vascular endothelial cells, enhance the expression of leukocyte adhesion molecules, and promote the infiltration of immune cells into infected or damaged sites; chemokines can induce directional cell migration and attract immune cells to migrate toward the source in the direction of increased chemokine concentration. The study detected the serum inflammatory factors and chemokines of mice in the control group and SAH group. The results showed that compared with the control group, the expression levels of IL-1β, IL-6, IL-18, CCL2, CXCL1, and CXCL7 in the serum of mice in the SAH group were significantly upregulated ( Figure 6 A, B, C, F, G and H in Figure ), the expression level of MIP-2 also increased significantly ( Figure 6 E), while the expression level of TNF-α increased, but there was no significant difference ( Figure 6 D). The results showed that the SAH model had an obvious excessive inflammatory response, with a large number of neutrophil infiltration in the liver and the appearance of an inflammatory factor storm, and the SAH mouse model was relatively successful.

[0087] In summary, based on the NIAAA model, the last intragastric alcohol concentration of 7.5 g / kg combined with intraperitoneal injection of LPS concentration of 5 mg / kg for 12 h was determined as the SAH mouse modeling condition, which can better simulate the pathogenesis of clinical SAH, especially the high clinical mortality rate, significant liver inflammation and neutrophil infiltration of SAH, and provide an important animal model tool for the screening of drugs for the prevention and treatment of SAH ( Figure 7 )

[0088] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for preparing an animal model of severe alcoholic hepatitis, comprising: Step 1): feeding the animal using an adaptive feeding procedure for constructing an NIAAA animal model; Step 2): feeding the animals with a chronic alcohol feeding procedure for constructing an NIAAA animal model; Step 3): After completing the feeding of step 1) and step 2), the animals are gavaged (ig) with 3-8 g / kg of alcohol, and 3-10 mg / kg of lipopolysaccharide is injected into the animals.

2. The preparation method according to claim 1, wherein The adaptive feeding step is to adaptively feed the animals with liquid feed for 4-6 days; The chronic alcohol feeding procedure continued to feed the animals with a liquid feed containing 3% to 7% alcohol (% v / v) for 8 to 12 days.

3. The preparation method according to claim 1 or 2, wherein In step 3), the animals are intragastrically administered with 7-8 g / kg of alcohol, and 4.5-5.5 mg / kg of lipopolysaccharide is injected into the animals.

4. The preparation method according to claim 3, wherein In step 3), 7.5 g / kg of alcohol was administered to the animal by gavage, and 5 mg / kg of lipopolysaccharide was injected into the animal.

5. The preparation method according to any one of claims 1 to 4, wherein The animal is a mammal.

6. The preparation method according to claim 5, wherein: The mammal is a rat, a mouse, a cat, a dog, a horse, a sheep, a cow or a monkey; preferably, the mammal is a mouse.

7. The preparation method according to any one of claims 1 to 6, wherein The liquid feed is Lieber-DeCarli liquid feed.

8. The preparation method according to any one of claims 1 to 7, wherein The injection was intraperitoneal.

9. The preparation method according to any one of claims 1 to 8, wherein About 12 hours after step 3), the severe alcoholic hepatitis animal model is prepared.

10. The preparation method according to any one of claims 1 to 9, wherein The method comprises: 1) Adaptive feeding of mice with Lieber-DeCarli liquid diet for 5 days; 2) continue to feed mice with Lieber-DeCarli liquid diet containing 5% alcohol (% v / v) for 10 days; 3) After completing the feeding in step 2), the mice were gavaged with 7.5 g / kg of alcohol and injected intraperitoneally with 5 mg / kg of lipopolysaccharide. The model was established 12 hours later.

11. Use of the severe alcoholic hepatitis animal model obtained by the preparation method according to any one of claims 1 to 10 in screening or identifying drugs for preventing or treating severe alcoholic hepatitis.

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