Use of PXMP2 in the preparation of a medicament for treating and / or preventing acute drug-induced liver injury

Abstract

This invention discloses the application of PXMP2 in the preparation of drugs for the treatment and / or prevention of acute drug-induced liver injury. This invention provides the application of PXMP2 protein in the preparation of drugs for the treatment and / or prevention of drug-induced liver injury. This invention also provides the application of PXMP2-related biological materials in the preparation of drugs for the treatment and / or prevention of drug-induced liver injury; said PXMP2-related biological materials are: nucleic acid molecules encoding PXMP2 protein; expression cassettes containing said nucleic acid molecules; and recombinant vectors containing said nucleic acid molecules or said expression cassettes. This invention further provides the application of substances targeting PXMP2 protein or the PXMP2 gene in the preparation of products; said products are used to prepare animal models of drug-induced liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of PXMP2 in the preparation of treatments and / or preventions of acute drug-induced liver injury. Background Technology

[0002] Drug-induced liver injury (DILI) is a common and serious adverse drug reaction in clinical practice, which can lead to acute liver failure and even death. Acetaminophen (APAP) overdose is a significant cause of acute drug-induced liver injury and acute liver failure. APAP produces toxic intermediates during liver metabolism, which can trigger glutathione depletion, oxidative stress, lipid peroxidation, and mitochondrial dysfunction, ultimately leading to pathological processes such as hepatocyte necrosis and ferroptosis.

[0003] Current treatment options for drug-induced liver injury are limited. Clinically, treatment mainly relies on antidotes such as N-acetylcysteine, but their efficacy is closely related to the timing of administration, resulting in a limited therapeutic window and difficulty in meeting clinical needs. Therefore, identifying new therapeutic targets and intervention strategies is of great significance for the prevention and treatment of drug-induced liver injury. Summary of the Invention

[0004] The purpose of this invention is to provide the use of PXMP2 in the preparation of treatments and / or preventions of acute drug-induced liver injury.

[0005] This invention provides the use of PXMP2 protein in the preparation of medicaments for the treatment and / or prevention of drug-induced liver injury.

[0006] The present invention also provides the use of substances for increasing PXMP2 protein activity and / or increasing PXMP2 protein abundance in the preparation of medicaments for treating and / or preventing drug-induced liver injury.

[0007] This invention also provides the application of PXMP2-related biomaterials in the preparation of medicaments for the treatment and / or prevention of drug-induced liver injury; The PXMP2-related biomaterials are as follows (a1) or (a2) or (a3): (a1) A nucleic acid molecule, which is a nucleic acid molecule encoding the PXMP2 protein; (a2) An expression cassette having the nucleic acid molecule described in (a1); (a3) A recombinant vector having the nucleic acid molecule described in (a1) or the expression cassette described in (a2).

[0008] The present invention also provides products for treating and / or preventing drug-induced liver injury, which are as follows (b1) or (b2) or (b3): (b1) Products that include the PXMP2 protein; (b2) Products that include substances for increasing PXMP2 protein activity and / or for increasing PXMP2 protein abundance; (b3) Products that include the PXMP2-related biomaterials.

[0009] Specifically, the nucleic acid molecule is a DNA molecule.

[0010] When the nucleic acid molecule is a DNA molecule, the nucleic acid molecule encoding the PXMP2 protein is also called the PXMP2 gene.

[0011] As a specific example, the PXMP2 gene is shown in positions 902-1480 of SEQ ID NO: 3. As a specific example, the PXMP2 gene is shown in positions 902-1555 of SEQ ID NO: 3.

[0012] As a specific example, the recombinant vector is shown in SEQ ID NO: 3.

[0013] Specifically, the product in question is a medicine.

[0014] In some embodiments, the drug is used to treat and / or prevent drug-induced liver injury in animals with reduced PXMP2 gene expression.

[0015] Specifically, the drug-induced liver injury refers to acute drug-induced liver injury.

[0016] Specifically, the drug-induced liver injury refers to liver injury caused by acetaminophen.

[0017] Specifically, the drug-induced liver injury refers to hepatocellular damage caused by drugs (e.g., acetaminophen).

[0018] Specifically, the product is designed to alleviate liver cell damage.

[0019] Specifically, the product is designed to alleviate liver cell damage caused by acetaminophen.

[0020] The hepatocellular damage is manifested as (c1) and / or (c2) and / or (c3) and / or (c4) and / or (c5): (c1) Increased release of ALT and / or AST from hepatocytes; (c2) The number of peroxisomes in hepatocytes is reduced; (c3) Peroxisome damage in hepatocytes; (c4) Increased SOD activity and / or CAT activity and / or GPX activity and / or GSH level in hepatocytes; (c5) Disorder of antioxidant capacity of hepatocytes.

[0021] Specifically, the product's function is to inhibit the aforementioned liver cell damage.

[0022] Specifically, the product's effect is to improve the aforementioned liver cell damage.

[0023] Specifically, the treatment and / or prevention of drug-induced liver injury is achieved by maintaining peroxisome homeostasis and / or reducing oxidative stress levels.

[0024] Specifically, maintaining peroxisome homeostasis means "maintaining the structural integrity and / or quantity of peroxisomes" and / or "maintaining the level of peroxisome membrane protein PEX14".

[0025] Specifically, reducing oxidative stress levels means maintaining SOD activity and / or CAT activity and / or GSH levels and / or GPX activity.

[0026] This invention also provides the application of substances targeting PXMP2 protein or PXMP2 gene in the preparation of products; said products are used to prepare animal models of drug-induced liver injury.

[0027] This invention also provides the application of substances I and II in the preparation of a product; the product is used to prepare an animal model of drug-induced liver injury; substance I is a substance that targets the PXMP2 protein or the PXMP2 gene for inhibition; substance II is a substance that induces drug-induced liver injury. Specifically, substance II is acetaminophen.

[0028] The present invention also provides a composition for preparing an animal model of drug-induced liver injury, comprising substance I and substance II; wherein substance I is a substance that targets the PXMP2 protein or the PXMP2 gene for inhibition; and substance II is a substance that induces drug-induced liver injury. Specifically, substance II is acetaminophen.

[0029] This invention also provides the application of substances targeting the PXMP2 protein or PXMP2 gene in the preparation of products that aggravate the degree of liver injury in animal models of drug-induced liver injury. Specifically, the animal model of drug-induced liver injury is an animal model of drug-induced liver injury caused by acetaminophen. Specifically, the degree of liver injury in the animal model of aggravated drug-induced liver injury is manifested by a larger liver lesion area and / or higher serum ALT activity and / or higher serum AST activity.

[0030] Specifically, the animal is a mouse, such as a C57BL / 6N mouse.

[0031] In some embodiments, substance I is as follows (d1) or (d2): (d1) shRNA targeting the PXMP2 gene; (d2) Recombinant microorganisms expressing (d1) shRNA.

[0032] In one embodiment, the shRNA is as shown in SEQ ID NO: 1.

[0033] In one embodiment, the recombinant microorganism is a recombinant AAV virus.

[0034] In one embodiment, the recombinant microorganism is a recombinant AAV8 virus.

[0035] AAV refers to adeno-associated virus.

[0036] AAV8 refers to adeno-associated virus type 8.

[0037] Specifically, drug-induced liver injury refers to hepatocyte damage caused by drugs.

[0038] Specifically, the drug in question is acetaminophen.

[0039] PXMP2: Peroxisomal membrane protein 2.

[0040] Specifically, the PXMP2 protein is shown in NCBI Reference Sequence: NP_033019.2 (27-JUN-2025).

[0041] The PXMP2 protein is specifically either mouse-derived or human-derived.

[0042] The PXMP2 protein specifically comprises the following (e1) or (e2) or (e3): (e1) The protein represented by amino acid residues 1-193 in SEQ ID NO: 4; (e1) The protein shown in SEQ ID NO: 4; (e3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (e1) or (e2); (e4) is a protein that has more than 90% identity with (e1) or (e2) and has the same function.

[0043] The PXMP2 gene is a gene encoding the PXMP2 protein. As a specific example, the PXMP2 gene is shown in positions 902-1480 of SEQ ID NO: 3. As a specific example, the PXMP2 gene is shown in positions 902-1555 of SEQ ID NO: 3.

[0044] The inventors discovered in an APAP-induced liver injury model that knocking down PXMP2 exacerbates hepatocyte damage, disrupts peroxisome homeostasis, and enhances oxidative stress; while reintroducing PXMP2 significantly reverses these damaging phenotypes. This invention reveals for the first time that PXMP2 exerts a hepatoprotective effect by maintaining peroxisome function and inhibiting oxidative stress / ferroptosis, providing a novel target and strategy for the clinical prevention and treatment of drug-induced liver injury (DILI). Based on this, this invention claims the use of protecting the PXMP2 protein, its encoded nucleic acid, or reagents capable of upregulating PXMP2 expression or activity in the preparation of related drugs, diagnostic reagents, or screening systems. Attached Figure Description

[0045] Figure 1 The result diagram is from Example 1.

[0046] Figure 2 This is a result diagram of Example 2. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Results are expressed as mean ± standard error (mean ± SEM). A t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. PXMP2 specific antibody (PXMP2 Polyclonal antibody): Wuhan Sanying Biotechnology Co., Ltd., product catalog number 24801-1-AP. PEX14 primary antibody (PEX14 Polyclonal antibody): Wuhan Sanying Biotechnology Co., Ltd., product catalog number 10594-1-AP. Fluorescently labeled secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG): Wuhan Sanying Biotechnology Co., Ltd., product catalog number SA00001-2. DMEM medium: Gibco, catalog number C11995500BT. Opti-MEM medium: Gibco, catalog number 31985070. C57BL / 6N mice (SPF grade): SPAFE (Beijing) Biotechnology Co., Ltd., catalog number B203. Serum alanine aminotransferase (ALT) activity assay kit: Sigma-Aldrich, catalog number MAK052, follow the instructions. Serum glutamate-oxaloacetate (AST) activity assay kit: Sigma-Aldrich, catalog number MAK055, follow the instructions. Catalase (CAT) assay kit (visible light method): Nanjing Jiancheng Bioengineering Institute, catalog number A007-1-1, follow the instructions. Total SOD activity assay kit (WST-8 method): Beyotime Biotechnology, catalog number S0101M, follow the instructions. Glutathione peroxidase (GPX) assay kit: Beyotime Biotechnology, product catalog number S0056, operate according to the instructions. GSH and GSSG assay kit: Beyotime Biotechnology, product catalog number S0053, operate according to the instructions.

[0049] Acetaminophen (APAP): Sigma-Aldrich, catalog number PHR1005. Preparation of APAP solution: Dissolve acetaminophen in physiological saline in a water bath at 55-60℃ to a concentration of 22 mg / mL, then allow to cool naturally to 37℃ (this temperature is used for injection). Prepare and use immediately.

[0050] Example 1: Effect of downregulating PXMP2 expression on APAP-induced drug-induced acute liver injury Experimental animals: 6-8 week old male C57BL / 6N mice (SPF grade), weighing 18-22g.

[0051] Housing environment: SPF animal room, 12 hours of light / 12 hours of darkness, free access to food and water.

[0052] I. Obtaining a virus expressing shRNA Both the AAV8-shPxmp2 virus and the AAV8 control virus were packaged by Heyuan Biotechnology (Shanghai) Co., Ltd. During AAV virus packaging, the packaging plasmid encodes the target gene and two terminal inverted repeat sequences (ITRs, which play a decisive role in viral replication and packaging). The helper plasmid contains the cap (encoding viral capsid proteins) and rep (involved in viral replication) genes required for AAV packaging, as well as the adenovirus helper plasmid. After co-transfection of 293T cells with the three plasmids, AAV virus replication and packaging began. The AAV8-shPxmp2 virus expresses shRNA targeting the PXMP2 gene, as shown in SEQ ID NO: 1. The AAV8 control virus expresses shRNA targeting an irrelevant gene (i.e., a gene not present in the mouse genomic DNA), as shown in SEQ ID NO: 2.

[0053] II. Grouping The experimental animals were divided into four groups.

[0054] AAV8-PXMP2+APAP group: On day 1 of the experiment, mice were injected with AAV8-shPxmp2 virus via tail vein (the viral dose given to each mouse was 1×10⁻⁶). 12 vg, injection volume of 200 μL); on day 29 of the experiment, APAP solution was injected intraperitoneally (APAP dosage of 300 mg / kg body weight). APAP group: On day 1 of the experiment, mice were injected with AAV8 control virus via tail vein (the viral dose given to each mouse was 1×10⁻⁶). 12 vg, injection volume of 200 μL); on day 29 of the experiment, APAP solution was injected intraperitoneally (APAP dosage of 300 mg / kg body weight). AAV8-PXMP2 group: On day 1 of the experiment, mice were injected with AAV8-shPxmp2 virus via tail vein (the viral dose given to each mouse was 1×10⁻⁶). 12 vg, injection volume of 200 μL); on day 29 of the experiment, physiological saline was injected intraperitoneally (300 μL per mouse). Saline group: On day 1 of the experiment, mice were injected with AAV8 control virus via tail vein (the viral dose given to each mouse was 1×10⁻⁶). 12 vg, injection volume of 200 μL); on day 29 of the experiment, physiological saline was injected intraperitoneally (300 μL per mouse).

[0055] III. Testing Eight hours after the intraperitoneal injection in step two, the mice were anesthetized, blood was collected from the eyeballs and serum was obtained, and the liver was removed after dissection.

[0056] 1. Liver tissue pathological examination and assessment of lesion area Liver tissue was harvested, and paraffin sections (4 μm thick) were prepared. After H&E staining, the sections were observed under a light microscope. Special attention was paid to the necrotic area around the central vein of the hepatic lobules. Images of the necrotic area were recorded, and the area of ​​liver tissue damage was quantitatively assessed using ImageJ. Example images are shown below. Figure 1 A, the percentage of liver tissue damage area is shown in [the original text]. Figure 1 B (sample size N=3). In the Saline and AAV8-PXMP2 groups, the liver structure was intact with almost no obvious necrotic areas. In the APAP group, obvious necrotic areas appeared in the liver, indicating that APAP treatment induced liver tissue damage in mice. In the AAV8-PXMP2+APAP group, the necrotic area in the liver was further expanded compared to the APAP group, indicating that PXMP2 knockdown aggravated APAP-induced liver tissue damage.

[0057] 2. Liver functional enzyme activity detection Serum was collected, and ALT activity was detected using an ALT assay kit, while AST activity was detected using an AST assay kit. Results are shown below. Figure 1 C (sample size N=6) and Figure 1 D (sample size N=6). In the Saline and AAV8-PXMP2 groups, ALT and AST levels remained within normal ranges. Compared to the Saline group, the APAP group showed elevated ALT and AST levels, indicating that APAP stimulation led to hepatocyte damage accompanied by the release of related enzymes. Compared to the APAP group, the AAV8-PXMP2+APAP group showed further elevated ALT and AST levels, indicating that PXMP2 knockdown exacerbated APAP-induced liver function damage.

[0058] 3. PXMP2 knockdown verification Liver samples from mice in the Saline and AAV8-PXMP2 groups were collected, and total protein was extracted for Western blot analysis. The primary antibody used in the Western blot was a PXMP2-specific antibody. Example images are shown below. Figure 1E. Compared with the Saline group, the abundance of PXMP2 protein in liver tissue of the AAV8-PXMP2 group was significantly reduced, verifying the successful construction of liver-specific PXMP2 knockdown mice.

[0059] The results of Example 1 showed that PXMP2 knockdown mice had larger liver damage areas and higher serum ALT and AST activities under APAP stimulation, reflecting that APAP-induced drug-induced acute liver injury was more severe in PXMP2 knockdown mice, and verifying the key role of PXMP2 in maintaining liver homeostasis and protecting liver tissue from drug-induced damage.

[0060] Example 2: Protective effect of PXMP2 reinstatement against APAP-induced drug-induced acute hepatocellular injury The Flag-PXMP2 expression plasmid is a circular plasmid formed from double-stranded DNA molecules, and its full sequence is shown in SEQ ID NO: 3. Positions 902-1555 of SEQ ID NO: 3 form a coding frame, encoding the protein shown in SEQ ID NO: 4. Positions 1-193 of SEQ ID NO: 4 constitute the PXMP2 protein.

[0061] Experimental animals: 6-8 week old male C57BL / 6N mice (SPF grade), weighing 18-22g.

[0062] Housing environment: SPF animal room, 12 hours of light / 12 hours of darkness, free access to food and water.

[0063] I. Building a PXMP2 knockout mouse On day 1 of the experiment, the experimental animals were injected via tail vein with the AAV8-shPxmp2 virus from step one of Example 1 (the viral dose given to each mouse was 1 × 10⁻⁶). 12 vg, injection volume is 200μL).

[0064] II. Isolation of primary hepatocytes (collagenase perfusion method) On day 29 of the experiment, mice were anesthetized, their portal veins were exposed and cannulated, and the liver tissue was first perfused with pre-perfusion fluid to remove blood, followed by perfusion with a digestive solution containing collagenase to digest the liver tissue until it swelled and softened. The liver tissue was then harvested, minced, and gently pipetted to disperse the cells. After filtration through a sieve, the cells were centrifuged at low speed to enrich the hepatocytes. The cells were then resuspended in DMEM medium containing 10% FBS and seeded into culture dishes pre-coated with rat tail collagen. The cells were cultured at 37°C and 5% CO2 until adherence, and the supernatant (which also removed non-adherent cells and other contaminating cells) was discarded to obtain primary hepatocytes.

[0065] III. Experimental Group (shPxmp2+PXMP2+APAP group) 1. Take the primary hepatocytes obtained in step 2, resuspend them in Opti-MEM medium to obtain a cell suspension.

[0066] 2. Take 10 μL of Lipofectamin 2000 and add it to Opti-MEM medium to a final volume of 250 μL. Incubate at room temperature for 5 min to obtain liquid phase A. Take 4 μg of Flag-PXMP2 expression plasmid and add it to Opti-MEM medium to a final volume of 250 μL to obtain liquid phase B. Mix liquid phase A and liquid phase B thoroughly and incubate at room temperature for 20 min to obtain the transfection complex for one well.

[0067] 3. Take a 6-well plate, seed it with the cell suspension obtained in step 1 (1.5 mL / well), then add the transfection complex obtained in step 2, and incubate for 48 hours.

[0068] 4. After completing step 3, aspirate the culture supernatant, add DMEM medium containing 10 mM APAP, and incubate for 8 hours.

[0069] IV. Control group (shPxmp2+APAP group) 1. Take the primary hepatocytes obtained in step 2, resuspend them in Opti-MEM medium to obtain a cell suspension.

[0070] 2. Take a 6-well plate, seed it with the cell suspension obtained in step 1 (1.5 mL / well), then add 500 μL of Opti-MEM medium and incubate for 48 hours.

[0071] 3. After completing step 2, aspirate the culture supernatant, add DMEM medium containing 10 mM APAP, and incubate for 8 hours.

[0072] V. Detection and evaluation of changes in liver functional enzyme activity 1. Liver functional enzyme activity detection After completing step three or four, ALT activity was detected using an ALT assay kit, and AST activity was detected using an AST assay kit. Follow the kit instructions carefully, including collecting cells, lysing cells, collecting the supernatant, and detecting activity.

[0073] See results Figure 2 A (sample size N=6). Compared with the shPxmp2+APAP group, the shPxmp2+PXMP2+APAP group showed decreased ALT and AST activities, suggesting that PXMP2 supplementation can alleviate the degree of APAP-induced hepatocellular damage.

[0074] 2. Detection and assessment of changes in hepatocyte peroxisome homeostasis After completing steps three or four, cells were collected, fixed with 2.5% glutaraldehyde solution, rinsed with phosphate buffer, fixed with osmium tetroxide solution, dehydrated with graded ethanol, embedded in resin, and prepared into ultrathin sections. Finally, double-stained with uranium acetate and lead citrate. The number, morphology, and structural changes of peroxisomes were observed using transmission electron microscopy.

[0075] See example photos Figure 2 B. Compared with the shPxmp2+APAP group, the shPxmp2+PXMP2+APAP group showed an increase in the number of peroxisomes and a reduction in the degree of ultrastructural damage, suggesting that PXMP2 supplementation can improve APAP-induced peroxisome structural damage.

[0076] 3. Detection of PXMP2 abundance After completing step three or four, cells are collected, lysed, and total protein is extracted for Western blot. The primary antibody used for Western blot is a PXMP2-specific antibody.

[0077] See example photos Figure 2 C. Compared with the shPxmp2+APAP group, the abundance of PXMP2 protein in the cells of the shPxmp2+PXMP2+APAP group was significantly increased, indicating that Flag-PXMP2 replenishment was successful.

[0078] 4. Assessment of oxidative stress levels in liver tissue After completing steps three or four, cells were collected, fixed with 4% paraformaldehyde solution, washed with PBS buffer, and then blocked. Cells were then incubated sequentially with PEX14 primary antibody and fluorescently labeled secondary antibody, and finally counterstained with DAPI. The PEX14 fluorescence signal was observed and photographed using a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed using image analysis software.

[0079] See example photos Figure 2 D. The results showed that, compared with the shPxmp2+APAP group, the PEX14 fluorescence signal was enhanced in the shPxmp2+PXMP2+APAP group, suggesting that PXMP2 supplementation can improve APAP-induced peroxisome damage.

[0080] 5. Antioxidant index testing After completing step three or four, collect the cells. Detect SOD activity, CAT activity, GPX activity, and GSH levels using a kit.

[0081] See results Figure 2E (sample size N=6). The results showed that compared with the shPxmp2+APAP group, the shPxmp2+PXMP2+APAP group had decreased intracellular SOD, CAT and GPX enzyme activities, as well as decreased GSH levels, suggesting that PXMP2 supplementation can improve APAP-induced antioxidant capacity disorder.

[0082] The results of Example 2 showed that in PXMP2 knockdown primary hepatocytes, APAP treatment caused decreased cell viability, increased ALT / AST release, peroxisome ultrastructural damage, and downregulation of peroxisome-related protein expression, accompanied by abnormal changes in ferroptosis-related proteins and decreased antioxidant capacity. PXMP2 reinjection significantly improved the above-mentioned damage phenotypes, suggesting that PXMP2 plays an important role in resisting APAP-induced hepatocyte damage, maintaining peroxisome homeostasis, and regulating ferroptosis.

[0083] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of PXMP2 protein in the preparation of drugs for the treatment and / or prevention of drug-induced liver injury.

2. The use of substances for increasing PXMP2 protein activity and / or increasing PXMP2 protein abundance in the preparation of medicaments for treating and / or preventing drug-induced liver injury.

3. Application of PXMP2-related biomaterials in the preparation of drugs for the treatment and / or prevention of drug-induced liver injury; The PXMP2-related biomaterials are as follows (a1) or (a2) or (a3): (a1) A nucleic acid molecule, which is a nucleic acid molecule encoding the PXMP2 protein; (a2) An expression cassette having the nucleic acid molecule described in (a1); (a3) A recombinant vector having the nucleic acid molecule described in (a1) or the expression cassette described in (a2).

4. Products for the treatment and / or prevention of drug-induced liver injury, which are (b1) or (b2) or (b3) below: (b1) Products that include the PXMP2 protein; (b2) Products that include substances for increasing PXMP2 protein activity and / or for increasing PXMP2 protein abundance; (b3) A product comprising the PXMP2-related biomaterials as described in claim 3.

5. Use or product according to any one of claims 1 to 4, characterized in that: The drug-induced liver injury mentioned refers to liver injury caused by acetaminophen.

6. The application of substances that target PXMP2 protein or PXMP2 gene in the preparation of products; said products are used to prepare animal models of drug-induced liver injury.

7. Application of Substance I and Substance II in the preparation of the product; the product is used to prepare an animal model of drug-induced liver injury; Substance I is a substance that targets the PXMP2 protein or the PXMP2 gene for inhibition; Substance II is a substance that causes drug-induced liver injury.

8. A composition for preparing an animal model of drug-induced liver injury, comprising substance I and substance II; wherein substance I is a substance that inhibits PXMP2 protein or PXMP2 gene; and substance II is a substance that induces drug-induced liver injury.

9. Application of substances that target PXMP2 protein or PXMP2 gene in the preparation of products that aggravate the degree of liver injury in animal models of drug-induced liver injury.

10. Use or composition according to any one of claims 6 to 9, wherein: The drug-induced liver injury mentioned refers to liver injury caused by acetaminophen.

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