Method for constructing a mouse model simulating human non-obese nafld under normal diet conditions

Abstract

This invention discloses a method for constructing a mouse model of non-obese NAFLD in humans under normal dietary conditions. The method involves injecting adeno-associated virus (adenovirus) shIDO2-AAV8 into mice via the tail vein to target and silence IDO2 gene expression in the liver, while simultaneously administering a normal diet. The adenovirus shIDO2-AAV8 is obtained by recombining an IDO2-shRNA sequence inserted into an AAV8 vector. This invention, through tail vein injection of adenovirus shIDO2-AAV8 combined with a normal diet, can construct a mouse model simulating non-obese NAFLD in humans. Compared with traditional dietary induction methods and combined induction methods, the dietary conditions are consistent with daily human intake, more closely reflecting the actual pathological situation in humans, and more closely reproducing the pathogenesis environment and physical state of non-obese NAFLD in humans, making the model more clinically valuable.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a mouse model that simulates non-obese NAFLD in humans under normal dietary conditions. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a type of liver injury associated with metabolic disorders and genetic susceptibility. In most cases, NAFLD is related to overnutrition and obesity, but a portion of NAFLD patients have a normal BMI, termed non-obese NAFLD. Current research shows that non-obese NAFLD accounts for a significant portion of NAFLD cases in my country and has a high mortality rate. Non-obese NAFLD is gradually attracting attention, but its exact pathogenesis remains unclear and requires further investigation. Reliable animal models are a crucial foundation for conducting this research.

[0003] Currently, the main methods for constructing NAFLD mouse models fall into four categories: dietary induction, chemical induction, gene editing, and combined models. In dietary induction models, a high-fat diet (HFD) simulates the background of human metabolic syndrome by feeding animals a high-fat diet (e.g., 60% fat for energy), inducing simple fatty liver in 12-16 weeks, but with milder fibrosis and inflammation, and a longer cycle. A methionine-choline deficiency (MCD) diet rapidly induces steatosis, inflammation, and fibrosis (4-8 weeks) by blocking lipoprotein synthesis, but animals experience severe weight loss and lack insulin resistance, inconsistent with human metabolic characteristics. A high-fructose diet, while rapidly inducing steatosis, has a weak inflammatory response and is only suitable for drug screening. In chemical induction models, carbon tetrachloride (CCl4) activates oxidative stress and inflammation through hepatotoxicity, leading to fibrosis in 8 weeks, but it is highly toxic, has a high mortality rate, and its pathological mechanism differs significantly from that in humans. While tetracycline and streptozotocin (STZ) have lower toxicity, STZ damages pancreatic β-cell function and cannot simulate systemic insulin resistance. In gene-edited models, ob / ob (leptin deficiency) and db / db (leptin receptor deficiency) mice naturally develop obesity and insulin resistance, but they are unlikely to develop significant NASH when used alone, requiring a "second hit" in combination with an MCD diet or a high-fat diet; ApoE - / - and Ldlr - / - Mice on a high-fat, high-cholesterol diet can develop NASH with atherosclerosis, making it suitable for studying the association with metabolic syndrome. However, the genetic background is unique, and the cost is high. Composite models optimize pathological features by combining dietary, chemical, or genetic methods. For example, a high-fat diet combined with low-dose CCl4 can rapidly induce fibrosis, while the combination of the MCD diet and ob / ob mice can more closely resemble the metabolic and pathological manifestations of human NASH.

[0004] In scientific research, the most commonly used methods for constructing NAFLD mouse models are dietary induction and combined induction. However, the dietary components in these two methods are very different from the daily human diet and can cause significant weight gain or loss. Neither of these methods can simulate the pathological process of non-obese NAFLD caused by the normal diet of humans.

[0005] In summary, how to provide a method for constructing a mouse model of non-obese NAFLD in humans under normal dietary conditions, which can better simulate the pathological process of non-obese NAFLD induced by human daily diet, is an urgent problem to be solved.

[0006] Indoleamine 2,3-deoxygenase 2 (IDO2) is a recently discovered rate-limiting enzyme in tryptophan metabolism. Existing research mainly focuses on its role in immune regulation, such as inflammation, tumors, and autoimmune diseases. There are currently no reports on the role of IDO2 in hepatic lipid metabolism. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a method for constructing a mouse model of non-obese NAFLD in humans under normal dietary conditions. By injecting adeno-associated virus shIDO2-AAV8 into the tail vein, shIDO2-AAV8 can target the liver in mice and specifically silence the gene expression of IDO2 in the liver. Combined with a normal diet, a mouse model of non-obese NAFLD in humans can be constructed. Compared with traditional diet induction methods and combined induction methods, the dietary conditions are consistent with the daily intake of humans, which is more in line with the actual pathology of humans and more closely restores the pathogenesis environment and physical state of non-obese NAFLD in humans, making the model more clinically valuable.

[0008] During their research, the inventors first discovered that IDO2 plays a crucial role in hepatic lipid metabolism. This invention aims to simulate a mouse model of non-obese NAFLD in humans under normal dietary conditions by targeting and silencing the expression of the hepatic IDO2 gene.

[0009] The technical solution of the present invention is as follows:

[0010] A method for constructing a mouse model of non-obese NAFLD in humans under normal diet conditions involves targeting the liver in mice to specifically silence the gene expression of IDO2 in the liver while administering a normal diet.

[0011] Preferred methods for specifically silencing IDO2 gene expression in the liver include: injecting adeno-associated virus shIDO2-AAV8 into the tail vein of mice.

[0012] The adeno-associated virus shIDO2-AAV8 is an AAV8 vector cloned with IDO2-shRNA (a nucleotide sequence of shRNA that specifically silences IDO2).

[0013] Preferably, the AAV8 vector includes a U6 promoter.

[0014] Preferably, the sense strand of the IDO2-shRNA comprises the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0015] Preferably, the sequence of the adeno-associated virus shIDO2-AAV8 is shown in SEQ ID NO: 3.

[0016] Preferably, the adeno-associated virus shIDO2-AAV8 is injected once, with a titer of not less than 1E+12GC / mL, and the injection dose is 200μL / mouse.

[0017] Preferably, when the shIDO2-AAV8-specific silencing of IDO2 gene expression in the liver is achieved for 2 to 3 weeks, a mouse model simulating non-obese NAFLD in humans under normal diet conditions can be successfully constructed.

[0018] Preferably, the general diet includes feeding with conventional rodent feed (without added cholesterol, sucrose, or high-fat ingredients).

[0019] Preferably, the adeno-associated virus shIDO2-AAV8 construction method includes the following steps: designing the sequence of IDO2-shRNA and synthesizing the target gene for later use; linearizing the vector using AAV8 as a vector at a downstream site of the U6 promoter; and then performing a recombination reaction between the linearized vector and the target gene.

[0020] The present invention also provides a mouse model that simulates human non-obese NAFLD under normal diet conditions obtained by the construction method described above.

[0021] The present invention also provides a mouse model of non-obese NAFLD in humans under normal diet conditions obtained by the construction method described above, and its application in screening or preparing drugs for the prevention or treatment of non-obese NAFLD.

[0022] Technical effects of the present invention:

[0023] 1. The mouse model construction method of this invention is more closely aligned with actual human pathology, solving the core problems of traditional dietary induction and combined induction methods. Existing methods rely on special diets, whose composition differs greatly from daily human diets, and easily lead to significant weight gain or loss in mice, failing to simulate the pathological process of non-obese NAFLD under normal human dietary conditions. This invention, through tail vein injection of adeno-associated virus shIDO2-AAV8, targets and silences IDO2 gene expression in the liver, and combines this with a normal diet to construct a mouse model simulating non-obese human NAFLD. The dietary conditions are consistent with daily human intake, more closely reproducing the pathogenesis environment and physical state of non-obese human NAFLD, making the model more clinically valuable.

[0024] 2. This invention constructs adeno-associated virus shIDO2-AAV8 through a recombination reaction. It can target the liver in mice, specifically silencing the gene expression of IDO2 in the liver, and stably induces significant fat accumulation in the mouse liver within a short period, rapidly achieving the pathological characteristics of NAFLD. Compared to traditional methods, which may suffer from long induction periods and unstable pathological manifestations, this invention significantly improves model construction efficiency, ensuring the timeliness and accuracy of subsequent studies.

[0025] 3. This invention constructs a non-obese NAFLD mouse model and detects hepatic IDO2 gene expression, monitoring body weight, liver function, blood glucose, and pathological changes in liver tissue. It is suitable for research on non-obese NAFLD. Non-obese NAFLD accounts for a significant proportion of the human population, but related research is limited due to a lack of suitable animal models. This invention fills this gap, providing an ideal experimental model for exploring the pathogenesis of non-obese NAFLD, screening targeted therapeutic drugs, and verifying the effectiveness of intervention programs. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the adeno-associated virus shIDO2-AAV8 recombinantly constructed according to the present invention;

[0027] Figure 2 The gene sequencing results are for the adeno-associated virus shIDO2-AAV8 recombinantly constructed according to this invention;

[0028] Figure 3 This is a diagram showing the protein expression of IDO2 in the liver after the adeno-associated virus shIDO2-AAV8 targets and silences IDO2 according to the present invention.

[0029] Figure 4 This invention describes the liver fat accumulation after the liver is silenced by targeting adeno-associated virus shIDO2-AAV8. Detailed Implementation

[0030] The above-mentioned scheme is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. All technical means of silencing or knocking out the IDO2 gene can achieve the purpose of the invention. For example, shRNAs with different nucleic acid sequences can be designed for the IDO2 mRNA nucleic acid sequence; shRNA nucleic acid sequences that can specifically silence IDO2 gene expression can be cloned into other adeno-associated viruses, such as AVV9, AAV-ZK2, etc.; liver-specific IDO2 knockout (IDO2-LKO) mice can be constructed using Cre-loxP conditional knockout technology, which can also successfully establish a non-obese NAFLD animal model; the implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in routine experiments.

[0031] Unless otherwise specified in the following examples, all raw materials are commercially available or prepared by conventional methods in the art.

[0032] Example 1: Construction of adeno-associated virus shIDO2-AAV8

[0033] 1. Experimental materials:

[0034] The nucleotide sequence of the positive strand of the target gene IDO2-shRNA is as follows:

[0035] 5'-GUCAUGUCCUGCACCCUAA[dT][dT]-3' (SEQ ID NO: 1).

[0036] The nucleotide sequence of the antisense strand of the target gene IDO2-shRNA is as follows:

[0037] 5'-UUAGGGUGCAGGACAUGAC[dT][dT]-3' (SEQ ID NO: 2).

[0038] The adeno-associated virus shIDO2-AAV8 of the present invention was constructed by Guangzhou Paizhen Biotechnology Co., Ltd., and the construction method is a conventional gene recombination method in the art. The nucleotide sequence of shIDO2-AAV8 is shown in SEQ ID NO: 3.

[0039] The selected vector was AAV8[scAAV.CAG.EGFP.WPRE.SV40pA], purchased from Guangzhou Paizhen Biotechnology Co., Ltd., which contains the U6 promoter.

[0040] 2. Construction steps of adeno-associated virus shIDO2-AAV8

[0041] (1) Obtaining the target gene

[0042] Design an effective IDO2-shRNA sequence, with the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 2, to obtain the target gene fragment; after the target gene fragment is verified to be correct, it is directly used in subsequent recombination reactions.

[0043] (2) Preparation of linearized carriers

[0044] AAV8 was selected as the vector backbone. Restriction endonucleases were used to select the restriction sites downstream of the U6 promoter for linearization. The linearized vector fragments were purified and recovered for subsequent recombination reactions.

[0045] (3) Homologous recombination and transformation

[0046] The target gene IDO2-shRNA sequence fragment synthesized in step (1) was recombined in vitro with the linearized vector obtained in step (2) to construct adeno-associated virus shIDO2-AAV8. A schematic diagram of shIDO2-AAV8 is shown below. Figure 1 [C-12412-1scAAV.U6.shRNA(midol2).CAG.EGFP.WPRE.SV40pA], target gene IDO2-shRNA (see...) Figure 1 The insertion site of the shRNA (mIdol2) is downstream of the Human U6 Promoter. The reaction product was transformed into competent cells and cultured on selective plates containing ampicillin.

[0047] (4) Screening and validation of positive clones

[0048] Colony PCR and enzyme digestion were used for preliminary screening of positive clones. Sanger sequencing confirmed that the target gene sequence was correctly inserted into the AAV8 vector without mutation. The sequencing results of shIDO2-AAV8 are shown below. Figure 2 The target gene sequence IDO2-shRNA is visible (see...) Figure 2 The shRNA (mIdol2) has been successfully inserted downstream of the Human U6 Promoter.

[0049] Example 2: Construction of a mouse model simulating non-obese NAFLD in humans under normal diet conditions (targeting liver-specific silencing of IDO2 gene expression in the liver).

[0050] Male rodent mice, 6-8 weeks old and weighing 18-20 grams, were selected as experimental animals. Mice were randomly divided into two groups: a non-obese NAFD mouse model group (shIDO2-AAV8) and a negative control group (NS). The mice were injected with shIDO2-AAV8 and blank-AAV8, respectively, and fed a standard maintenance diet (conventional rodent diet without added cholesterol, sucrose, or high-fat components), with free access to food and water. The injection site was the tail vein, using an insulin injection needle (1 mL), with an injection volume of 200 μL of virus.

[0051] The adeno-associated virus shIDO2-AAV8 is a double-stranded RNA that specifically degrades IDO2 mRNA, while blank-AAV8 is an empty vector. Both viruses are stored at a concentration / titer of 1E+13 GC / mL, aliquoted into 50 μL tubes, and stored at -80°C. Before use, remove the virus from the ultra-low temperature freezer and thaw it on ice. Add 450 μL of sterile PBS (pre-chilled on ice) to each tube of virus, bringing the total virus volume to 500 μL, and adjust the virus concentration / titer to 1E+12 GC / mL.

[0052] Example 3: Detection of liver silencing after shIDO2-AAV8 targeting.

[0053] 1. Detection of IDO2 mRNA expression in mouse liver using q-PCR

[0054] A non-obese NAFLD mouse model was constructed according to the method in Example 2. Three weeks after each group of mice were injected with the virus, the livers of the mice were collected, and the mRNA expression of IDO2 in the mouse liver was detected by real-time quantitative PCR.

[0055] The specific steps of real-time quantitative PCR are as follows: extract total RNA from liver tissue; determine RNA concentration; reverse transcribe into cDNA; amplify using real-time quantitative PCR. Primer sequences are shown in Table 1, reaction system in Table 2, and reaction procedure in Table 3. Finally, 2... -△△Ct The relative expression level of IDO2 mRNA was calculated using the method, and the results are shown in Table 4. Compared with the negative control group (NS), the relative expression level of IDO2 mRNA in the liver tissue of the model group (shIDO2-AAV8) decreased by about 60%.

[0056] Table 1 q-PCR primer sequences

[0057]

[0058] Table 2 q-PCR reaction system (20 μL)

[0059]

[0060] Table 3 q-PCR reaction procedure

[0061]

[0062] Table 4. Relative mRNA expression of IDO2 in mouse liver

[0063]

[0064] 2. Western blotting was used to detect the expression level of IDO2 protein in mouse liver tissue.

[0065] A non-obese NAFLD mouse model was constructed according to the method in Example 2. Two weeks after viral injection in each group, the livers of the mice were collected, and liver tissue proteins were extracted under ice bath conditions. The protein concentration in the liver tissue was determined by BSA. Vertical electrophoresis was performed; the membrane was transferred using an electroporation apparatus; the membrane was blocked at room temperature for 1 hour; internal control or IDO2 primary antibody was added, and then the membrane was placed in a 4°C refrigerator overnight; the primary antibody was collected, and the membrane was washed three times with PBST, 10 min each time; secondary antibody incubation was performed at room temperature for 1 hour; the membrane was washed; ECL staining was performed; and the membrane was photographed. The results of the Western blot assay are shown in Table 5 and... Figure 3 As shown, compared with the negative control group, the relative expression level of IDO2 protein in the liver of the model group was significantly reduced.

[0066] Table 5. Protein expression of IDO2 in mouse liver

[0067]

[0068] 3. Monitoring of fasting body weight and blood glucose in mice

[0069] Mice were fasted for 8-12 hours prior to the experiment (provided only with water). A calibrated portable blood glucose meter, matching test strips, 28-30G lancets, and alcohol swabs were prepared. During the experiment, the mice were restrained (using a restraint device or manually, exposing the tail). The tail tip was disinfected 1-2 cm with 75% alcohol; if the veins were not clearly visible, a warm compress could be applied for 10-20 seconds. Then, the lancet was inserted 1-2 mm into the side of the tail, and 2 μL of blood was gently squeezed from the tail base and dripped onto the test strip. The blood glucose meter displayed the value after 5-10 seconds. After blood collection, pressure was applied with a cotton swab for 30 seconds to 1 minute to stop the bleeding. The mice were then returned to their cages and given water. Mouse number, weight, fasting time, and blood glucose levels were recorded weekly. Three weeks after modeling, the body weight of the model group (after targeting and silencing the liver IDO2 gene) was slightly lower than that of the negative control group (see Table 6); the fasting blood glucose of the model group mice was significantly higher (see Table 7).

[0070] Table 6. Body weight of experimental mice

[0071]

[0072] Table 7 Fasting blood glucose in experimental mice

[0073]

[0074] 4. Detection of liver weight, liver index, liver function and blood lipid levels in mice.

[0075] A non-obese NAFLD mouse model was constructed according to the method in Example 2. Three weeks after viral injection in each group, the mice were sacrificed. First, mouse serum was obtained, following these steps: The mice were fasted (for the duration as needed). Capillary tubes (0.5-1 mm inner diameter), centrifuge tubes, alcohol swabs, and cervical dislocation tools were prepared. After sterilizing the worktable, the mouse's head was held in place with gloves, and the left thumb and forefinger were used to gently press the eye sockets to make the eyeballs protrude. The capillary tube was inserted along the inner wall of the eye socket (approximately 3-5 mm) using the right hand, and blood was collected into centrifuge tubes using negative pressure (0.2-0.5 mL per mouse). The blood was allowed to stand at room temperature for 30-60 minutes to coagulate naturally, then centrifuged at 3000-4000 rpm for 10-15 minutes. After centrifugation, the upper pale yellow serum was aspirated into new centrifuge tubes using a pipette (avoiding aspiration of red blood cells). The tubes were labeled and stored refrigerated or frozen as needed.

[0076] After blood collection, mice were euthanized by cervical dislocation, and their liver tissue was completely removed, weighed, and the liver index (liver weight / body weight) was calculated. The results are shown in Table 8. Compared with the negative control group, the model group mice showed a significant increase in liver weight and liver index. Liver function (aspartate aminotransferase AST, alanine aminotransferase ALT) and four lipid parameters (triglycerides (TG), total cholesterol (TC), high-density cholesterol, and low-density cholesterol) were measured in each mouse using an automated biochemical analyzer. The results are shown in Table 8. Compared with the negative control group, the serum ALT and AST levels in the liver-targeted IDO2 silencing group were significantly increased, while triglyceride levels showed no significant difference, and cholesterol levels were significantly increased.

[0077] Table 8. Liver weight, liver index, liver function, and blood lipids in experimental mice.

[0078]

[0079] 5. Histopathological examination of mouse liver tissue

[0080] A non-obese mouse model of NAFLD was constructed according to the method in Example 2. Liver tissue pathological examination was performed one, two, and three weeks after viral injection in each group. The effect of targeted liver IDO2 silencing on the liver was analyzed using hematoxylin-eosin staining (HE staining). The specific steps are as follows: liver tissue was harvested; fixed; embedded; sectioned; dewaxed; stained; dehydrated, cleared, and mounted. HE results are shown in […]. Figure 4 Compared with the negative control group, small lipid droplets were observed in hepatocytes of mice in the model group one week after IDO2 gene silencing, the central venous structure of the liver lobules was clear, and there was no inflammatory cell infiltration. At two weeks, the volume of fat vacuoles in hepatocytes increased and their distribution became more widespread. The liver plates were slightly loose but the structure was basically intact, and there was still no obvious inflammation, indicating simple non-alcoholic fatty liver (NAFL). At three weeks, the fat vacuoles in hepatocytes further increased, with dense vacuoles, mild compression of the hepatic sinusoids, and scattered inflammatory cell infiltration in local areas (not limited to the portal area), consistent with the manifestation of intralobular inflammation. According to the NAS scoring system, the steatosis (3 points) + lobular inflammation (1 point) in the pathological section at three weeks reached the diagnostic threshold of early non-alcoholic steatohepatitis (NASH) (total score ≥4 points), consistent with the pathological characteristics of early NASH.

[0081] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model simulating non-obese NAFLD in humans under normal dietary conditions, characterized by: In mice, the liver was targeted to specifically silence the gene expression of IDO2 in the liver while the mice were given a normal diet. Methods for specifically silencing IDO2 gene expression in the liver include: injecting adeno-associated virus shIDO2-AAV8 into the tail vein of mice; The adeno-associated virus shIDO2-AAV8 is an AAV8 vector cloned with IDO2-shRNA, which can specifically silence IDO2. When the adeno-associated virus shIDO2-AAV8 specifically silences the IDO2 gene expression in the liver for 2-3 weeks, a mouse model simulating human non-obese NAFLD under normal diet conditions can be successfully constructed.

2. The construction method according to claim 1, characterized in that: The general diet includes feeding rodents with conventional rodent feed.

3. The construction method according to claim 1, characterized in that: The AAV8 vector includes the U6 promoter.

4. The construction method according to claim 1, characterized in that: The sense strand of the IDO2-shRNA includes the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 2; the sequence of the adeno-associated virus shIDO2-AAV8 is shown in SEQ ID NO:

3.

5. The construction method according to claim 1, characterized in that: The adeno-associated virus shIDO2-AAV8 was injected once, with a titer of not less than 1E+12GC / mL, and the injection dose was 200μL per mouse.

6. The construction method according to claim 3, characterized in that: The method for constructing adeno-associated virus shIDO2-AAV8 includes the following steps: designing the sequence of IDO2-shRNA and synthesizing the target gene for later use; linearizing the vector using AAV8 as a vector at a downstream site of the U6 promoter; and then performing a recombination reaction between the linearized vector and the target gene.

7. The application of a mouse model simulating non-obese NAFLD in humans under normal diet conditions obtained by the construction method according to claim 1 in screening or preparing drugs for the prevention or treatment of non-obese NAFLD.

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