Use of plpp2 as a target in the preparation of a drug for treating acute liver injury

Abstract

The application discloses application of PLPP2 as a target point in preparation of a drug for treating acute liver injury. The application first proposes that PLPP2 is a new potential target point for ALI treatment, and through in-vivo and in-vitro experimental systems, it is proved that up-regulation of PLPP2 can significantly reduce a liver injury area and reduce up-regulation of liver enzymes, thus providing a brand-new mechanism basis for ALI treatment, and having important theoretical innovativeness. The application clearly defines a key regulation role of PLPP2 in a repair role after ALI occurrence, thus providing a clear molecular target point for designing and optimizing small-molecule compounds, nucleic acid drugs and biological preparations around PLPP2, and being expected to give birth to a new generation of ALI targeted drugs with higher safety and more comprehensive effects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of PLPP2 as a novel target in drugs for the treatment of acute liver injury. Background Technology

[0002] Acute liver injury (ALI) is a clinically significant condition characterized by rapid hepatocellular damage that leads to impaired liver function within days or weeks. It is typically caused by drug toxicity (most notably acetaminophen (APAP) overdose, a leading cause of drug-induced liver injury (DILI)) as well as factors such as viral hepatitis, ischemia, or autoimmune reactions. Given its status as a liver emergency and a major cause of mortality, ALI is a serious concern.

[0003] ALI is associated with elevated transaminase levels, coagulation disorders, and severe acute liver failure (ALF). Despite the liver's strong intrinsic regenerative capacity, ALI impairs this capacity, making it a leading cause of liver emergencies and high mortality.

[0004] Liver regeneration involves a complex network of molecular regulations, in which phospholipid metabolism plays a crucial role. The dynamic regulation of phospholipid metabolism constitutes a fundamental, phylogenetically conserved mechanism that controls the efficiency of tissue repair and regeneration across a wide range of biological contexts. Although previous studies have identified the important roles of phosphatidylethanolamine (PE) and its associated autophagy in aging and cancer, the role of PE in ALI remains unclear.

[0005] This invention, through the establishment of an acute liver injury model and a PLPP2 gene knockdown mouse model, combined with molecular biology, proteomics, metabolomics, and histopathological analysis, has for the first time discovered that phospholipase 2 (PLPP2) is a key metabolic enzyme mediating PE biosynthesis and regulating liver repair and regeneration in the context of ALI, and elucidated its molecular mechanism of promoting hepatocyte autophagy and proliferation by regulating PE synthesis. Based on this, this invention provides the application of PLPP2 as a target in promoting liver regeneration, which has significant scientific value and clinical translational prospects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel therapeutic target for ALI. This invention offers PLPP2 as a new target in the treatment of acute liver injury. Through multi-model systematic studies, it is demonstrated for the first time that targeted upregulation of hepatocyte PLPP2 can significantly reduce the area of ​​liver damage in mouse models of drug-induced acute liver injury and liver ischemia-reperfusion injury. By mediating hepatocyte autophagy and proliferation, it promotes liver regeneration and repair, suggesting that PLPP2 is a key target for the treatment of ALI. This provides a theoretical basis and technical foundation for subsequent research and development of small molecule drugs, nucleic acid drugs, and biological agents targeting this target.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: the application of PLPP2 as a target in the preparation of drugs for treating acute liver injury.

[0008] Furthermore, the nucleotide sequence of PLPP2 is shown in SEQ ID NO. 1.

[0009] Application of reagents that upregulate PLPP2 expression or enhance PLPP2 protein function in the preparation of drugs for treating acute liver injury.

[0010] Furthermore, acute liver injury includes APAP-induced acute liver injury, CCL4-induced acute liver injury, and hepatic ischemia-reperfusion injury.

[0011] Furthermore, the nucleotide sequence of PLPP2 is shown in SEQ ID NO. 1, and the amino acid sequence of the PLPP2 protein is shown in SEQ ID NO. 2.

[0012] A drug for treating acute liver injury, comprising: the reagent that upregulates PLPP2 expression or enhances PLPP2 protein function.

[0013] Furthermore, drugs also include pharmaceutically acceptable carriers.

[0014] Furthermore, the dosage form of the drug is any one of tablets, granules, solutions, emulsions, suspensions, and capsules.

[0015] Application of PLPP2 as a novel target in the preparation of drugs for the treatment of acute liver injury.

[0016] The PLPP2 target, when specifically overexpressed in hepatocytes, can improve the pathological features of ALI, therefore drugs targeting PLPP2 can treat ALI.

[0017] In an in vitro model of acute hepatocyte injury, knocking down the PLPP2 gene significantly impairs autophagy during the hepatocyte repair phase.

[0018] Furthermore, PLPP2-specific knockout mice of hepatocytes were constructed using CRISPR / Cas9 technology to achieve the knockout of PLPP2 in mouse hepatocytes.

[0019] Furthermore, PLPP2 cDNA (NM_001302389.1) was delivered via an adeno-associated virus (AAV) vector to achieve overexpression of PLPP2 in mouse hepatocytes. The AAV vector was an AAV8 serotype with liver tissue tropism.

[0020] This target can be used to construct a biomarker model for patient prognosis. The model uses PLPP2 as the core biomarker. Upregulating PLPP2 can significantly reduce the area of ​​liver damage and promote liver tissue repair and regeneration.

[0021] The model analyzes the correlation between PLPP2 expression and patient clinicopathological features and prognosis by detecting the protein or transcriptional expression level of PLPP2 in patient liver tissue samples.

[0022] Compared with the prior art, the present invention has the following beneficial technical effects:

[0023] This invention is the first to propose PLPP2 as a novel potential target for ALI treatment, and through in vitro and in vivo experimental systems, it has been systematically demonstrated that upregulation of PLPP2 can significantly reduce the area of ​​liver damage and decrease the upregulation of liver enzymes, providing a completely new mechanistic basis for ALI treatment and possessing significant theoretical innovation. This invention clarifies the key regulatory role of PLPP2 in the repair process after ALI occurs, providing a clear molecular target for the design and optimization of small molecule compounds, nucleic acid drugs, and biological agents around PLPP2, and is expected to give rise to a new generation of ALI-targeting drugs with higher safety and more comprehensive effects.

[0024] In summary, this invention is the first to demonstrate that specific overexpression of PLPP2 in hepatocytes can improve ALI injury outcomes in in vitro acute hepatocyte injury models and ALI mouse models, suggesting that PLPP2 is a key target for the treatment of MASH, and providing a theoretical basis and technical foundation for subsequent research and development of small molecule drugs, nucleic acid drugs and biological agents targeting this target. Attached Figure Description

[0025] Figure 1This example demonstrates a combined proteomics and metabolomics screening approach (A. Heatmap showing the expression of genes related to apoptosis, necrosis, and inflammation pathways (n=3, proteomics); B. Heatmap showing the expression of genes related to cell proliferation pathways (n=3, proteomics); C. The intersection of enriched pathways obtained from three paired groups (48h vs 6h, 72h vs 6h, and 96h vs 6h) in a multi-omics combined analysis; D. Upregulated pathways co-enriched by proteomics and metabolomics in mouse livers at 48h and 6h after APAP administration; E. The three groups of samples at 48h vs 6h, 72h vs 6h, and 96h vs 6h). Volcano plot of differentially expressed phosphatidylethanolamine metabolism-related proteins at 6 h (n=3, proteomics); F, line plot of PLPP2 protein gradation change trend (n=3, proteomics); GH, representative confocal microscopy images and statistics of PLPP2 (green) stained THLE-2 cells after APAP (10mM) treatment (scale bar: 10μm); IJ, representative histological staining images and statistics of PLPP2 protein in mouse liver tissue obtained at different time points after APAP administration (scale bar: 50μm; n=8)).

[0026] Figure 2 The example illustrates how specific knockout of PLPP2 in hepatocytes exacerbates acute liver injury and delays liver tissue regeneration and repair (A, PLPP2 gene knockout strategy; B, Western blot detection of PLPP2 protein in PLPP2). FL / FL and Plpp2 ΔHep Expression in primary mouse hepatocytes (n=6); C, Plpp2 after 48 h of APAP (300 mg / kg) treatment FL / FL and Plpp2 ΔHep Serum ALT and AST levels in mice (n=8, ****p<0.0001); D. After treatment with a lethal dose of APAP (650 mg / kg), Plpp2 FL / FL and Plpp2 ΔHep Survival curves of mice (n=20, log-rank test); E, Plpp2 after 48 h of APAP (300 mg / kg) treatment. FL / FL and Plpp2 ΔHep Representative gross liver photographs and intrahepatic staining results from mice, including quantitative analysis results of H&E and Ki67 (scale bar: 50 μm; n=8; p<0.0001); F. After treatment with CCL4 (0.6 ml / kg, dissolved in olive oil), Plpp2 FL / FL and Plpp2 ΔHepRepresentative images and quantitative analysis of H&E and Ki67 staining in mouse liver (scale bar: 50 μm; n=6; p<0.0001); GH, after hepatic ischemia-reperfusion, Plpp2 FL / FL and Plpp2 ΔHep Representative images and quantitative analysis of H&E and Ki67 staining in mouse liver (scale bar: 50 μm; n=6; ***p<0.001; ****p<0.0001); I. Plpp2 after 48 h of APAP (300 mg / kg) treatment FL / FL and Plpp2 ΔHep Representative electron microscopy images and quantitative analysis of autophagosomes (including autophagosomes and lysosomes) in mouse liver tissue (n=6, ****p<0.0001)).

[0027] Figure 3 Examples of the study include: Overexpression of PLPP2 in hepatocytes promoting liver regeneration and repair after acute liver injury (A, Schematic diagram of adeno-associated virus intervention; B, Western blot analysis of PLPP2 protein expression in primary hepatocytes of AAV-GFP and AAV-Plpp2 mice (n=6); C, Fluorescence microscopy assessment of the effect of adeno-associated virus on mouse liver infection (scale bar, 50 μm); D, Serum ALT and AST levels in AAV-GFP and AAV-Plpp2 mice after 48 h of APAP (300 mg / kg) treatment (n=8, p<0.0001); E, Survival curves of AAV-GFP and AAV-Plpp2 mice after treatment with a lethal dose of APAP (650 mg / kg) (n=20, log-rank test); FG, Gross images and intrahepatic staining results of representative livers of AAV-GFP and AAV-Plpp2 mice after 48 h of APAP (300 mg / kg) treatment, including quantitative analysis results of H&E and Ki67 (scale bar: 50 μm; n=8; **p<0.01; ****p<0.0001); H, representative electron micrographs and quantitative analysis of autophagosomes (including autophagosomes and lysosomes) in liver tissue of AAV-GFP and AAV-Plpp2 mice after 48 h of APAP (300 mg / kg) treatment (n=6, ***p<0.001)).

[0028] Figure 4This example demonstrates the clinical sample validation of the association between PLPP2 and the clinical prognosis of patients with drug-induced liver injury (A, Schematic diagram of human liver specimen acquisition; B, Immunohistochemical staining results of PLPP2 protein in human liver samples from the HC and DILI groups (scale bar, 50 μm; HC and DILI represent individual samples from healthy controls and patients with drug-induced liver injury, respectively); C, Quantitative analysis results of PLPP2 in human liver samples from the HC and DILI groups (****p<0.0001); D, PLPP2 high Group and PLPP2 low E. Quantitative analysis of Ki67 in human liver samples (n=18, **p<0.01); F. Co-localization staining results of PLPP2 and HNF4a in human liver samples (scale bar, 50μm); G. Correlation analysis between PLPP2 expression level in human liver tissue and serum ALT and AST levels in patients. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0030] Example 1: Dynamic changes in liver injury and regeneration repair processes and PLPP2 expression after acute liver injury.

[0031] 1.1 Experimental Materials

[0032] Laboratory animals: Eight-week-old male C57BL / 6 mice, purchased from Jiangsu Jicui Biotechnology Co., Ltd. All mice were housed in the Fifth Animal Facility of Nanjing Medical University in a temperature- and light-controlled environment (20-22°C; 10:14 hour light-dark cycle) with free access to water. The experimental procedures strictly followed institutional animal care guidelines, and all procedures were approved by the Ethics Committee of the Experimental Animal Center of Nanjing Medical University.

[0033] Main reagents:

[0034] Acetaminophen (APAP, MCE, catalog number HY-66005);

[0035] PLPP2 antibody (Servicebio, catalog number GB113784-100);

[0036] 1.2 Experimental Methods

[0037] 1.2.1 Multi-omics sequencing and analysis after APAP-induced acute liver injury

[0038] To construct an ALI model, 8-week-old male mice were fasted overnight and then intraperitoneally injected with 300 mg / kg APAP. Except for non-experiment-related deaths or other diseases that may affect the experimental results, mice were sacrificed at specified time points (0 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 168 h after administration) and their livers were collected for multi-omics sequencing.

[0039] 1.2.2 Establishment of an in vitro APAP-induced acute hepatocyte injury model

[0040] THLE2 cells were seeded onto cell slides, air-dried, and fixed in paraformaldehyde at 20°C for 30 minutes. After washing with PBS, the cells were infiltrated with 0.3% Triton X-100 for 15 minutes, blocked with 10% bovine serum albumin at 20°C for 1 hour, stained with primary antibody overnight at 4°C, and then stained with fluorescently conjugated secondary antibody for 2 hours. Cell nuclei were stained with DAPI. Microscopic observation and image acquisition were performed, and quantitative analysis was conducted using ImageJ software.

[0041] 1.2.3 Immunohistochemical staining

[0042] After dewaxing with xylene and rehydration with graded ethanol, the sections were washed with PBS; subsequently, the sections were placed in 10 mM sodium citrate solution.

[0043] Antigen retrieval was performed by boiling in buffer (pH 6.0) for 10 minutes, followed by cooling to room temperature. After blocking with 10% goat serum, specific primary antibody was added, and the sections were incubated overnight at 4°C. Sections were then incubated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase activity, followed by the addition of the appropriate secondary antibody and incubation at room temperature for 60 minutes. Finally, hematoxylin was used for counterstaining. Microscopic observation and image acquisition were performed, and quantitative analysis was conducted using ImageJ software.

[0044] 1.3 Experimental Results and Analysis

[0045] Proteomic heatmaps showed that proteins involved in apoptosis, necrosis, and inflammation pathways were significantly upregulated within 48 hours after APAP administration. Figure 1 A), indicating that the damage phase mainly occurs within 48 hours. Increased expression of proteins associated with cell proliferation 48 hours after APAP administration predicts the onset of the repair phase. Figure 1 B).

[0046] Given that 6 hours represents the injury period, while 48, 72, and 96 hours correspond to the repair period, we analyzed the overlapping enrichment pathways between H48 vs. H6, H72 vs. H6, and H96 vs. H6. Figure 1 C). The results showed that the autophagy-related metabolite phosphatidylethanolamine (PE) was significantly enriched during the repair period. Figure 1 D).

[0047] To elucidate the regulatory mechanism of PE metabolic reprogramming in ALI, the dynamic changes in the expression of key PE metabolic enzymes throughout the continuous course of the disease were analyzed. Figure 1 E). Among them, PLPP2 showed the most significant differential expression, and it was significantly upregulated during the recovery period (E). Figure 1 F). In vitro hepatocyte immunofluorescence and in vivo mouse liver tissue immunohistochemical staining both confirmed that PLPP2 was downregulated during the injury phase and upregulated during the repair phase. Figure 1 (GJ). The above results suggest that PLPP2 may be involved in the regulation of liver regeneration and repair.

[0048] Example 2: PLPP2-specific knockout in hepatocytes inhibits liver regeneration and repair after acute liver injury and reduces survival rate.

[0049] 2.1 Experimental Materials

[0050] Laboratory animals: Plpp2 FL / FL and Plpp2 ΔHep The mice were constructed using CRISPR / Cas9 technology by Jiangsu Jicui Biotechnology Co., Ltd. and housed in the Fifth Animal Facility of Nanjing Medical University. The experimental procedures strictly followed institutional animal care guidelines, and all procedures were approved by the Ethics Committee of the Experimental Animal Center of Nanjing Medical University.

[0051] Main reagents:

[0052] Acetaminophen (APAP, MCE, catalog number HY-66005);

[0053] Carbon tetrachloride (CCL4, Sigma-Aldrich, catalog number 87030).

[0054] PLPP2 antibody (Servicebio, catalog number GB113784-100);

[0055] Ki67 antibody (Abcam, catalog number ab15580);

[0056] ACTIN antibody (CST, catalog number #3700);

[0057] Liver enzyme detection (automated detector, Olympus Corporation);

[0058] Electron microscopy fixative (Servicebio)

[0059] 2.2 Experimental Methods

[0060] 2.2.1 PLPP2 Knockout Efficiency Verification

[0061] Take Plpp2 FL / FL and Plpp2 ΔHep Mouse liver cells were sorted, and total protein was extracted for Western blotting. The primary antibody used in Western blotting was a PLPP2-specific antibody, incubated overnight at 4°C. The corresponding secondary antibody was incubated at room temperature for 1 hour. Immunoreactive bands were detected using Super Pico ECL chemiluminescence buffer, and the results were recorded using a Licor imaging system.

[0062] 2.2.2 Liver function tests

[0063] Mouse ocular blood was centrifuged at 8000 rpm for 15 minutes to obtain serum, which was then used to detect the concentrations of liver enzymes ALT and AST.

[0064] 2.2.3 Establishment of multiple acute liver injury models

[0065] To establish an APAP-induced acute liver injury model, 8-week-old male mice were fasted overnight and then injected intraperitoneally with 300 mg / kg (or a lethal dose of 650 mg / kg) of APAP.

[0066] To induce CCl4-induced liver injury, 8-week-old mice were given a single intraperitoneal injection of 0.6 ml / kg CCL4 (Sigma-Aldrich, 87030; dilution ratio 1:10, diluted in olive oil).

[0067] To establish a 70% hepatic ischemia-reperfusion injury (HIRI) model, mice were anesthetized and subjected to a midline abdominal incision. The portal vein and hepatic artery of the left and middle lobes of the liver were clamped using microvascular clips. The clips were removed after 60 minutes of ischemia, and the mice were sacrificed after 72 hours of reperfusion.

[0068] 2.2.4 Electron microscopy detection of autophagy function

[0069] Liver tissue specimens were obtained and fixed with electron microscopy fixative (3% glutaraldehyde). Images were obtained using a transmission electron microscope (HT7800, Hitachi, Japan). Two types of autophagic vesicles were observed, including autophagosomes (double-membrane, ribosome-free, with similar cytosol density) and lysosomes (single-membrane, with much lower optical cavity density, containing light or dense amorphous structures).

[0070] 2.2.5 Liver tissue H&E staining and immunohistochemical staining

[0071] Liver tissue was fixed overnight in 10% buffered formalin (Fisher-Scientific) and embedded in paraffin for sectioning. The tissue sections were stained with hematoxylin and eosin (H&E) for structural evaluation. For immunohistochemical staining, sections were dehydrated, and the antigen was incubated overnight at 4°C with the following primary antibodies. Sections were incubated with horseradish peroxidase-conjugated or fluorescence-conjugated secondary antibodies at 25°C for 1 hour. Microscopic observation and image acquisition were performed, and quantitative analysis was conducted using ImageJ software.

[0072] 2.3 Experimental Results and Analysis

[0073] With Plpp2 FL / FL Compared to mice, Plpp2 ΔHep The abundance of PLPP2 protein in mouse liver tissue was significantly reduced, verifying the successful construction of liver-specific PLPP2 knockout mice. Figure 2 B).

[0074] Compared to Plpp2 FL / FL Mouse, Plpp2 ΔHep Mice treated with APAP for 48 hours had higher serum ALT and AST levels, larger liver damage area, downregulated proliferative markers, and lower survival rates. Figure 2 CE). This demonstrates the crucial role of PLPP2 in promoting liver regeneration and repair in ALI. This conclusion was also validated in CCL4-induced acute liver injury and liver ischemia-reperfusion injury models. Figure 2 FH).

[0075] Transmission electron microscopy shows that compared to Plpp2 FL / FL Mouse, Plpp2 ΔHep Mice showed a significant reduction in autophagic vesicles after 48 hours of APAP treatment. Figure 2 (I) indicates that PLPP2 mediates liver repair and regeneration by influencing hepatocyte autophagy.

[0076] Example 3: Overexpression of hepatocyte PLPP2 promotes liver regeneration and repair and improves survival rate after acute liver injury.

[0077] 3.1 Experimental Materials

[0078] Adeno-associated virus: Both AAV2 / 8-TBG-Plpp2-EGFP and AAV2 / 8-TBG-EGFP were packaged by Jiman Biotechnology Co., Ltd.

[0079] Laboratory animals: Eight-week-old male C57BL / 6 mice were provided by Jiangsu Jicui Biotechnology Co., Ltd. and housed in the Wutai Animal Facility of Nanjing Medical University. The experimental procedures strictly followed the institutional animal care guidelines, and all experimental procedures were approved by the Ethics Committee of the Experimental Animal Center of Nanjing Medical University.

[0080] Main reagents:

[0081] Acetaminophen (APAP, MCE, catalog number HY-66005);

[0082] PLPP2 antibody (Servicebio, catalog number GB113784-100);

[0083] Ki67 antibody (Abcam, catalog number ab15580);

[0084] ACTIN antibody (CST, catalog number #3700)

[0085] Liver enzyme detection (automated detector, Olympus Corporation);

[0086] Electron microscopy fixative (Servicebio)

[0087] 3.2 Experimental Methods

[0088] 3.2.1 Validation of PLPP2 overexpression efficiency

[0089] To investigate liver-specific overexpression of Plpp2, a dose of 10*10 was administered via tail vein injection 28 days prior to APAP administration. 12 AAV2 / 8-TBG-Plpp2-EGFP and AAV2 / 8-TBG-EGFP were administered at vg / kg. Liver cells from AAV-Cre and AAV-Plpp2 mice were collected, primary liver cells were sorted, and total protein was extracted for Western blot analysis. The primary antibody used for Western blot analysis was a PLPP2-specific antibody, incubated overnight at 4°C. The corresponding secondary antibody was incubated at room temperature for 1 hour. Immunoreactive bands were detected using Super Pico ECL chemiluminescence buffer, and the results were recorded using a Licor imaging system.

[0090] 3.2.2 Liver function tests

[0091] Mouse ocular blood was centrifuged at 8000 rpm for 15 minutes to obtain serum, which was then used to detect the concentrations of liver enzymes ALT and AST.

[0092] 3.2.3 Establishment of an APAP-induced acute liver injury model

[0093] To establish an APAP-induced acute liver injury model, 8-week-old male mice were fasted overnight and then injected intraperitoneally with 300 mg / kg of APAP.

[0094] 3.2.4 Electron microscopy detection of autophagy function

[0095] Liver tissue specimens were obtained and fixed with electron microscopy fixative (3% glutaraldehyde). Images were obtained using a transmission electron microscope (HT7800, Hitachi, Japan). Two types of autophagic vesicles were observed, including autophagosomes (double-membrane, ribosome-free, with similar cytosol density) and lysosomes (single-membrane, with much lower optical cavity density, containing light or dense amorphous structures).

[0096] 3.2.5 Liver tissue H&E staining, immunohistochemistry, and immunofluorescence staining

[0097] Liver tissue was fixed overnight in 10% buffered formalin (Fisher-Scientific) and embedded in paraffin for sectioning. The tissue sections were stained with hematoxylin and eosin (H&E) for structural evaluation. For immunohistochemical and immunofluorescence staining, sections were dehydrated, and the antigens were incubated overnight at 4°C with the following primary antibodies. Sections were incubated with horseradish peroxidase-conjugated or fluorescence-conjugated secondary antibodies at 25°C for 1 hour. Microscopic observation and image acquisition were performed, and quantitative analysis was conducted using ImageJ software.

[0098] 3.3 Experimental Results and Analysis

[0099] Compared with AAV-Cre mice, the abundance of PLPP2 protein in the liver tissue of AAV-Plpp2 mice was significantly increased, verifying the successful construction of liver-specific PLPP2 overexpression mice. Figure 3 BC).

[0100] Compared to AAV-Cre mice, AAV-Plpp2 mice had lower serum ALT and AST levels after 48 hours of APAP treatment; smaller liver damage area; upregulated proliferation markers; and higher survival rate. Figure 3 This demonstrates the crucial role of PLPP2 in promoting liver regeneration and repair in ALI (DG).

[0101] Transmission electron microscopy showed that, compared with AAV-Cre mice, AAV-Plpp2 mice had a significant increase in autophagic vesicles after 48 h of APAP treatment. Figure 3 (H), indicating that PLPP2 mediates liver repair and regeneration by influencing hepatocyte autophagy.

[0102] Example 4: Clinical sample validation of the correlation between PLPP2 and clinical prognosis in patients with drug-induced liver injury

[0103] 4.1 Experimental Materials

[0104] Human liver tissue: Percutaneous liver biopsy was performed on 36 patients with drug-induced liver injury (DILI) at the First Affiliated Hospital of Nanjing Medical University. Thirty-one patients with hepatic hemangioma (HC) at the First Affiliated Hospital of Nanjing Medical University were selected as normal controls. Informed consent was obtained from each participant. This study followed the principles of the Declaration of Helsinki and was approved by the Research Ethics Committee of the First Affiliated Hospital of Nanjing Medical University.

[0105] Main reagents:

[0106] PLPP2 antibody (Invitrogen, catalog number PA5-98075);

[0107] Ki67 antibody (Abcam, catalog number ab15580);

[0108] HNF4a antibody (Servicebio, catalog number GB155549-100)

[0109] 4.2 Experimental Methods

[0110] 4.2.1 Immunohistochemical staining and immunofluorescence staining of liver tissue

[0111] Liver tissue was fixed overnight in 10% buffered formalin (Fisher-Scientific) and embedded in paraffin for sectioning. Before staining, sections were dehydrated, and the antigen was incubated overnight at 4°C with the following primary antibodies. Sections were then incubated with horseradish peroxidase-conjugated or fluorescently conjugated secondary antibodies at 25°C for 1 hour.

[0112] 4.3 Experimental Results and Analysis

[0113] Compared with HC, PLPP2 in hepatocytes of DlLl specimens was significantly upregulated ( Figure 4 BD). Importantly, patients with higher PLPP2 expression also had higher Ki67 levels (BD). Figure 4 E).

[0114] Peripheral blood was collected from patients with disseminated intravascular coagulation (DILI) to measure liver enzyme levels. Correlation analysis indicated that higher PLPP2 expression was associated with lower liver enzyme levels. Figure 4 F). These results suggest that PLPP2 plays an important protective role in ALI, and that PLPP2 expression is associated with a good patient prognosis.

Claims

1. Application of PLPP2 as a target in the preparation of drugs for treating acute liver injury.

2. The application according to claim 1, characterized in that: The nucleotide sequence of PLPP2 is shown in SEQ ID NO.

1.

3. Application of reagents that upregulate PLPP2 expression or enhance PLPP2 protein function in the preparation of drugs for treating acute liver injury.

4. The application according to claim 3, characterized in that: The acute liver injury includes APAP-induced acute liver injury, CCL4-induced acute liver injury, and hepatic ischemia-reperfusion injury.

5. The application according to claim 3, characterized in that: The nucleotide sequence of PLPP2 is shown in SEQ ID NO. 1, and the amino acid sequence of the PLPP2 protein is shown in SEQ ID NO.

2.

6. A drug for treating acute liver injury, characterized in that... include: The reagents that upregulate PLPP2 expression or enhance the function of PLPP2 protein.

7. The drug according to claim 6, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

8. The drug according to claim 6, characterized in that: The dosage form of the drug is any one of tablets, granules, solutions, emulsions, suspensions, and capsules.

Images