Establishment method of mouse acute hepatic failure model based on short-term fasting and small-dose PloyI: C / D-GalN induction

Abstract

The invention discloses a method for establishing a mouse acute hepatic failure model based on short-term fasting and small-dose PloyI: C / D-GalN induction, and the mouse acute hepatic failure model can be successfully established only by providing 24-hour fasting for a 5-6w c57BL / 6J mouse, then injecting 0.014 [mu] g / g of PloyI: C drug into the tail vein of the mouse and injecting 0.56 mg / g of D-GalN drug into the intraperitoneal cavity of the mouse. Through short-term fasting pretreatment, physiological adjustment of an organism in a stress state is simulated, so that subsequent small-dose PloyI: C / D-GalN induced liver injury is closer to the physiological process of actual disease occurrence and development. Compared with traditional single large-dose drug induction, the method has the advantages that non-characteristic damage caused by excessive drugs is reduced, and the stability and repeatability of the model are improved. The method is simple to operate, special equipment is not needed, and the experiment cost is reduced due to the fact that the dosage of the medicine is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a method for establishing a mouse acute liver failure model based on short-term fasting and low-dose PloyI:C / D-GalN induction. Background Art

[0002] Acute liver failure (ALF) refers to a reversible syndrome in which patients without chronic liver disease suddenly develop a large number of hepatocyte apoptosis and necrosis, with significant abnormal liver function, and rapidly progress to hepatic encephalopathy in a short period of time (8 weeks). Acute liver failure may also lead to hepatic encephalopathy, coagulopathy, multiple organ failure and ultimately death within a few days. Common causes of ALF in adults include drug toxicity, hepatotropic and non-hepatotropic viruses, herbal medicines and dietary supplements, anti-tuberculosis drugs and autoimmune hepatitis.

[0003] Animal liver injury is the result of various liver diseases, and the prevention and treatment of liver injury is still a serious issue. It is of great practical significance to establish an experimental animal model of liver injury, study the pathogenesis of liver disease, screen liver-protective drugs, and explore the principle of liver protection. In order to deeply study its pathogenesis and explore effective treatment strategies, it is crucial to establish a reliable animal model. Traditionally, the use of a large dose of PloyI:C / D-GalN to induce acute liver failure in mice can trigger liver injury, but this method may deviate from the physiological changes of the natural course of the disease due to excessive drug dosage, limiting the application value of the model.

[0004] Based on this, the present invention proposes a method for establishing a mouse model that is more physiologically relevant, simple to operate, and can effectively simulate the pathological process of human acute liver failure, providing a more ideal animal model for the study of the pathogenesis of acute liver failure, drug screening, and evaluation of therapeutic effects. Summary of the invention

[0005] In view of the above analysis, the present invention proposes an innovative model establishment method, which successfully simulates an acute liver failure mouse model that is closer to the physiological state by combining short-term fasting pretreatment with low-dose PloyI:C / D-GalN injection. The present invention is achieved by the following technical means:

[0006] The present invention first discloses a method for establishing an acute liver failure model in mice induced by short-term fasting and low-dose PloyI:C / D-GalN, wherein:

[0007] The short-term fasting time is 24h;

[0008] The small dose is: the dosage of PloyI:C (polyinosinic-polycytidylic acid) is calculated based on 0.014ug / g of mouse body weight (i.e., 0.014ug of drug is injected per gram of body weight), and the dosage of D-GalN (D-galactosamine) is calculated based on 0.56mg / g of mouse body weight (i.e., 0.56mg of drug is injected per gram of body weight).

[0009] Furthermore, the acute liver failure model animal is a 5-6w c57BL / 6J mouse.

[0010] Furthermore, the administration method of PloyI:C is tail vein injection, and the administration method of D-GalN is intraperitoneal injection.

[0011] Furthermore, the administration sequence is to first administer PloyI:C and then administer D-GalN.

[0012] Furthermore, 18 hours after modeling, biochemical indexes and liver pathology examinations were performed on the experimental mice.

[0013] Furthermore, the biochemical index detection includes: serum ALT, serum AST, serum tumor necrosis factor α (TNF-α), mouse serum interleukin 6 (IL-6), and mouse serum interleukin 10 (IL-10).

[0014] The present invention also discloses a product for AE, wherein:

[0015] A is to study the mechanism of acute liver failure;

[0016] B is to screen drugs for the treatment of acute liver failure;

[0017] C is to screen drugs for preventing acute liver failure;

[0018] D is a drug for preparing a drug for treating acute liver failure;

[0019] E is a drug for the preparation of a drug for preventing acute liver failure;

[0020] The product includes any mouse acute liver failure model obtained by the above-mentioned construction method.

[0021] The beneficial effects of the present invention are:

[0022] 1. Enhanced physiological relevance: The present invention simulates the physiological adjustment of the body under stress through short-term fasting pretreatment, so that the subsequent liver damage induced by a small dose of PloyI:C / D-GalN is closer to the physiological process of actual disease occurrence and development.

[0023] 2. Improved model stability: Compared with single large-dose drug induction, this method reduces non-specific damage caused by drug overdose and improves the stability and repeatability of the model.

[0024] 3. Easy operation and reduced cost: This method is easy to operate, does not require special equipment, and reduces experimental costs by reducing the amount of drugs used. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the specific implementation methods of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0026] Figure 1 To study the effects of short-term fasting and low-dose PloyI:C / D-GalN on serum ALT in mice with acute liver failure.

[0027] Figure 2 To investigate the effects of short-term fasting and low-dose PloyI:C / D-GalN on serum AST in mice with acute liver failure.

[0028] Figure 3 To study the effects of short-term fasting and low-dose PloyI:C / D-GalN-induced acute liver failure model on serum tumor necrosis factor α (TNF-α) in mice;

[0029] Figure 4 To study the effects of short-term fasting and low-dose PloyI:C / D-GalN on serum interleukin-6 (IL-6) in mice with acute liver failure.

[0030] Figure 5 To study the effects of short-term fasting and low-dose PloyI:C / D-GalN on serum interleukin-10 (IL-10) in mice with acute liver failure.

[0031] Figure 6 Liver tissue pathology of mice with acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for establishing an acute liver failure model in mice induced by short-term fasting and low-dose PloyI:C / D-GalN

[0035] 1. Short-term fasting treatment: The mice in the experimental group were subjected to a short-term fasting treatment of 24 hours to slightly reduce the basal metabolic rate and liver glycogen reserve of the mice, creating a more sensitive physiological environment for subsequent liver damage induction.

[0036] 2. Low-dose PloyI:C / D-GalN injection: After the fasting period, the experimental group was given a small dose of PloyI:C (0.014ug / g) via tail vein injection and D-GalN (0.56mg / g) via intraperitoneal injection, which is lower than the traditional dose. By precisely controlling the dose, it is ensured that the apoptosis and necrosis of hepatocytes can be effectively triggered while maintaining the physiological relevance of the model and reducing nonspecific damage.

[0037] 3. Model evaluation: The serum ALT and AST levels of the mice in the experimental group were increased, the serum inflammatory cytokine levels were increased, and the pathological damage of liver tissue was aggravated, indicating that the acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice was successfully established.

[0038] 4. The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0039] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods.

[0040] The specific plan is as follows:

[0041] 1. Materials and reagents:

[0042] 1.1 Animals:

[0043] Male C57BL / 6J mice, SPF level, 5-6 weeks old, weighing 17-18g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and fed adaptively in an SPF laboratory for one week. Laboratory Animal Production License No.: SCXK (Xiang) 2019-0004.

[0044] 1.2 Reagents:

[0045] PloyI:C, D-GalN, Sigma; ALT, AST test kits, Nanjing Jiancheng Bioengineering Institute; TNF-α ELISA kit, Shenzhen Dakowe Biotechnology Co., Ltd.; IL-6 ELISA kit, Shenzhen Dakowe Biotechnology Co., Ltd.; IL-10 ELISA kit, Shenzhen Dakowe Biotechnology Co., Ltd.; 4% paraformaldehyde, Biosharp; hematoxylin and eosin staining solution, phosphate buffer (powder), neutral gum, Shanghai Bioengineering.

[0046] 1.3 Instrument consumables:

[0047] Electronic analytical balance, Shanghai Jingtian Electronic Instrument Co., Ltd.; low-temperature high-speed centrifuge, Eppendorf, Germany; microplate reader, Molecular Devices, USA; vortex oscillator, Scientific Industries, USA; 0.22um bacteria filter, Millipore; disposable sterile syringe (1 ml), Shandong Weigao Group Co., Ltd.

[0048] Preparation of related solutions: Preparation of PloyI:C and D-GalN solutions: Weigh PloyI:C and D-GalN powders on a clean bench, dissolve them in DD water, and prepare working solutions with concentrations of 1ug / 200ul and 50mg / ml respectively. Mix them on a vortex machine, filter them with a 0.22um filter and use them directly. Prepare and use them immediately.

[0049] 2. Methods

[0050] (1) Method for establishing acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice: C57BL / 6J mice weighing 17-18 g at 5-6 weeks were randomly divided into a free-feeding group (AL) and a short-term fasting group (STF). After the mice in the experimental group completed 24 hours of fasting, PloyI:C / D-GalN was injected intraperitoneally and intravenously to construct a free-feeding liver injury group (AL+PloyI:C / D-GalN) and a short-term fasting liver injury group (STF+PloyI:C / D-GalN). The mice in the AL and AL+PloyI:C / D-GalN groups were fed freely, while the mice in the STF and STF+PloyI:C / D-GalN groups were fasted for 24 hours but not water deprivation.

[0051] (2) Detection of serum ALT and AST levels: 18 hours after modeling, serum ALT and AST levels were detected. After anesthetizing the mice, the eyeballs were bled, and the mixture was allowed to stand at room temperature for 30 minutes. The mixture was centrifuged at 4°C, 3000 rpm, and the supernatant was taken and diluted to a reasonable multiple before detection using ALT and AST kits.

[0052] (3) Detection of serum inflammatory factors TNF-α, IL-6, and IL-10 levels: 18 hours after modeling, serum TNF-α, IL-6, and IL-10 levels were detected. After anesthesia, the mice were bled from the eyeballs, left to stand at room temperature for 30 minutes, centrifuged at 4°C, 4000 rpm for 10 minutes, and the supernatant was taken and diluted to a reasonable multiple before detection using an ELISA kit.

[0053] (4) Hematoxylin-eosin (H&E) staining of liver tissue: 18 hours after the modeling of mice, the right middle lobe of the liver tissue was removed from the anesthetized mice and fixed in 4% paraformaldehyde for 48 hours. The tissue was dehydrated in a glass jar with an alcohol gradient, embedded in paraffin and sliced. The tissue was stained with hematoxylin-eosin and sealed with neutral resin, and the pathological changes of liver tissue were observed under an optical microscope.

[0054] 3. Results

[0055] 3.1 Results of serum ALT and AST in acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice

[0056] Mice were randomly divided into AL group, STF group, AL+PloyI:C / D-GalN group, and STF+PloyI:C / D-GalN group, N = 5. The two AL groups of mice were fed freely, and the two STF groups of mice were fasted for 24 hours. The AL+PloyI:C / D-GalN group and the STF+PloyI:C / D-GalN group were injected with PloyI:C (0.014ug / g) by tail vein and D-GalN (0.56mg / g) by intraperitoneal injection.

[0057] 18 hours after modeling, serum ALT and AST levels were detected: after anesthetizing the mice, the eyeballs were bled and the mixture was allowed to stand at room temperature for 30 minutes. Then, the mixture was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was taken and diluted to an appropriate multiple before detection using ALT and AST kits.

[0058] The results are as follows Figure 1 , Figure 2 As shown: ALT and AST levels of mice in the STF+PloyI:C / D-GalN model group were significantly increased (P<0.05). The experimental results showed that the acute liver failure model of mice induced by short-term fasting and low-dose PloyI:C / D-GalN was successfully established.

[0059] 3.2 Results of serum TNF-α, IL-6, and IL-10 in mice with acute liver failure induced by short-term fasting and low-dose PloyI:C / D-GalN

[0060] 18 h after modeling, blood was collected from mouse eyes, left to stand at room temperature for 30 min, centrifuged at 4°C, 4000 rpm for 10 min, and the supernatant was taken and diluted to an appropriate multiple before detection using TNF-α, IL-6, and IL-10 kits.

[0061] The results are as follows Figure 3 , Figure 4 , Figure 5 As shown: TNF-α, IL-6, and IL-10 in the STF+PloyI:C / D-GalN model group were significantly higher than those in the AL+PloyI:C / D-GalN group (P<0.05). The experimental results showed that the acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice was successfully established.

[0062] 3.3 Pathological results of liver tissues in mice with acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN

[0063] The mouse livers were taken for H&E staining 18 hours after modeling, and the pathological changes of liver tissue were observed under an optical microscope.

[0064] The results are as follows Figure 5 As shown: The hepatocytes in the AL group and the STF drug group had clear outlines and normal morphological structures. The hepatic blood vessels and hepatic sinusoids of the mice in the STF+PloyI:C / D-GalN model group were obviously congested and hemorrhagic, and the necrotic area was infiltrated with inflammatory cells, while the hepatic hemorrhage and inflammatory infiltration of the mice in the AL+PloyI:C / D-GalN group were milder. The results suggest that the acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice was successfully established.

[0065] In this study, we detected the serum ALT and AST levels of mice with liver injury induced by short-term fasting and low-dose PloyI:C / D-GalN, detected the serum TNF-α, IL-6, and IL-10 levels of mice, and observed the pathological manifestations of liver tissue. We found that short-term fasting and low-dose PloyI:C / D-GalN can increase the serum ALT and AST of mice with acute liver failure model, increase the serum TNF-α, IL-6, and IL-10 levels, and aggravate the pathological manifestations of liver tissue. This confirmed that the acute liver failure model induced by short-term fasting and low-dose PloyI:C / D-GalN in mice was successfully established.

[0066] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of equivalent transformations, and these equivalent transformations all belong to the protection scope of the present invention. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations. In addition, the various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for establishing an acute liver failure model in mice induced by short-term fasting and low-dose PloyI:C / D-GalN, wherein: The short-term fasting time is 24h; The small dose is: the dosage of PloyI:C is calculated based on the weight of the mouse at 0.014 ug / g, and the dosage of D-GalN is calculated based on the weight of the mouse at 0.56 mg / g.

2. The method according to claim 1, wherein: The acute liver failure model animal is a 5-6w c57BL / 6J mouse.

3. The method according to claim 1, wherein: The administration method of PloyI:C is tail vein injection, and the administration method of D-GalN is intraperitoneal injection.

4. The method according to claim 1, wherein: The administration order is to first administer PloyI:C and then administer D-GalN.

5. The method according to claim 1, wherein: The establishment method also includes: 18 hours after modeling, biochemical indexes and liver pathology examinations were performed on the experimental mice.

6. The method according to claim 5, wherein: The biochemical index detection includes: serum ALT, serum AST, serum tumor necrosis factor α, serum interleukin 6, and serum interleukin 10.

7. A product for AE, wherein: A is to study the mechanism of acute liver failure; B is to screen drugs for the treatment of acute liver failure; C is to screen drugs for preventing acute liver failure; D is a drug for the preparation of a drug for the treatment of acute liver failure; E is a drug for the preparation of a drug for preventing acute liver failure; The product includes a mouse acute liver failure model obtained by the construction method described in any one of claims 1 to 6.

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