Use of vglL4 in metabolic-related fatty liver disease

Abstract

This invention discloses the application of VGLL4 in metabolic-associated fatty liver disease (MAFLD), relating to the field of biomedical technology. This invention is the first to discover that the molecular VGLL4 can be used to construct a novel MAFLD animal model with a complete pathological phenotype and clearly defined targets. This model exhibits typical phenotypes, including hepatic steatosis, insulin resistance, inflammatory response, and progression to steatohepatitis (MASH), without requiring a high-fat diet or chemical stimulation. By targeting the VGLL4-TEAD4 interaction or Pdk4, the aforementioned pathological phenotypes can be reversed. This invention provides a novel MAFLD model and intervention strategy, laying the foundation for further clinical trials and drug development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of VGLL4 in metabolic-associated fatty liver disease. Background Technology

[0002] Metabolic fatty liver disease (MAFLD) is a chronic liver disease characterized by abnormal fat deposition in the liver and closely associated with metabolic abnormalities such as obesity, insulin resistance, type 2 diabetes, and dyslipidemia. Current interventions mainly include lifestyle management and drug therapy targeting metabolic abnormalities. However, effective intervention targets for key molecular processes in the development and progression of fatty liver disease remain limited. Therefore, elucidating the core regulatory mechanisms of fatty liver disease development and progression and discovering new therapeutic targets are of great significance.

[0003] The development of metabolic-related fatty liver disease involves multiple biological processes, including lipid synthesis, fatty acid oxidation, inflammatory responses, cellular stress, and fibrosis. In recent years, the Hippo signaling pathway has been identified as being involved in liver development, regeneration, metabolic homeostasis, and the progression of liver diseases. The core kinase of the Hippo pathway and its downstream effector molecule YAP / TAZ can influence hepatocyte proliferation, inflammatory responses, fibrosis, and lipid metabolism by regulating TEAD-mediated transcriptional programs.

[0004] VGLL4 is a transcriptional cofactor that interacts with TEAD transcription factors and, under certain conditions, competitively regulates TEAD-dependent gene transcription with YAP / TAZ. Current research primarily focuses on the role of VGLL4 in tumorigenesis, cell proliferation, and tissue homeostasis regulation, while its function in hepatic lipid metabolism, hepatic steatosis, and the development of metabolic-related fatty liver disease remains insufficiently investigated. In particular, current technologies have not clarified whether alterations in VGLL4 expression levels can regulate the fatty liver phenotype, nor have they disclosed technical solutions based on VGLL4 as a target for fatty liver prevention and treatment or for constructing fatty liver models.

[0005] Currently, animal models of fatty liver mainly include diet-induced models and genetically modified models. Diet-induced models, such as high-fat diet models, can simulate some metabolic abnormalities, but the modeling cycle is long, and phenotypic stability is greatly affected by animal strain, feeding conditions, and the modeling period. Methionine-choline deficiency diet models can induce hepatic steatosis and inflammatory damage relatively quickly, but are often accompanied by weight loss, making it difficult to fully simulate the metabolic background of human metabolic-related fatty liver disease. Although genetically modified models can be used to study the function of specific genes, some models have problems such as systemic genetic alterations, excessive phenotypic weight, or complex mechanisms, making it difficult to meet the needs of rapid, stable, and controllable research on the pathogenesis of fatty liver and screening for intervention targets. Therefore, those skilled in the art are dedicated to developing a new method for constructing metabolic-related fatty liver disease models with clearly defined targets and corresponding drugs. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide the application of VGLL4 in metabolic-related fatty liver disease.

[0007] To achieve the above objectives, this invention provides the role of VGLL4 in metabolic-related fatty liver disease.

[0008] Applications.

[0009] Furthermore, the model is a transgenic mouse that specifically overexpresses the VGLL4 gene in hepatocytes.

[0010] Furthermore, the model was constructed by specifically overexpressing the VGLL4 gene in mouse hepatocytes.

[0011] Furthermore, the application of the VGLL4 gene in the preparation of drugs for the treatment of metabolic-related fatty liver disease.

[0012] Furthermore, the drug used to treat metabolic-related fatty liver disease is a drug that can inhibit the overexpression of the VGLL4 gene.

[0013] Furthermore, the drug for treating metabolic-related fatty liver disease is a drug that can inhibit the interaction between VGLL4 and TEAD4.

[0014] Furthermore, the drug that can block the interaction between VGLL4 and TEAD4 is Super-TDU.

[0015] Furthermore, the drug used to treat metabolic-related fatty liver disease is a drug that can inhibit PDK4 expression.

[0016] Furthermore, the drug that inhibits PDK4 expression is a shRNA that targets PDK4.

[0017] Furthermore, a drug for treating metabolic-associated fatty liver disease includes an active ingredient that inhibits VGLL4 gene overexpression, blocks the interaction between VGLL4 and TEAD4, or inhibits PDK4 expression.

[0018] Experiments have confirmed that the VGLL4 protein can bind to the TEAD4 transcription factor through its TDU domain, and further interact with CEBPA to form the VGLL4 / TEAD4 / CEBPA transcriptional regulatory complex. This complex binds to downstream metabolism-related genes (especially...). Pdk4 The promoter subregion of ) promotes Pdk4Transcriptional activation of PDK4 is a key factor. Upregulation of PDK4 can disrupt hepatocyte lipid and glucose homeostasis, specifically by promoting fatty acid synthesis (upregulating FASN, ACC1, SCD1, etc.), increasing fatty acid uptake (upregulating Fapp1, Cd36, etc.), and exacerbating hepatic lipid deposition. Simultaneously, it inhibits the insulin signaling pathway (reducing p-AKT and p-IRS1), leading to insulin resistance, and upregulates key gluconeogenesis enzymes (PEPCK, G6Pase), further aggravating glucose metabolism abnormalities. Furthermore, excessive lipid accumulation can induce inflammatory responses and hepatocyte damage, ultimately leading to a MAFLD-like pathological phenotype.

[0019] Based on the above-mentioned mechanism of action, knocking out the VGLL4 gene to block the interaction between VGLL4 and TEAD4, or knocking down downstream PDK4, can disrupt the formation or function of the VGLL4 / TEAD4 / CEBPA transcriptional complex, thereby alleviating hepatic steatosis, insulin resistance, and inflammatory damage. Therefore, VGLL4, TEAD4, CEBPA, PDK4, and the VGLL4 / TEAD4 / CEBPA complex can all serve as molecular targets for MAFLD model construction, drug screening, and therapeutic intervention.

[0020] Technical effects: 1. This invention is the first to apply VGLL4 to the construction of a metabolic-associated fatty liver disease (MAFLD) model. Hepatocyte-specific overexpression of VGLL4 can induce a complete MAFLD pathological phenotype. This model, with VGLL4 as the driving target, differs from diet-induced models, exhibiting comprehensive pathological characteristics. It can be used for MAFLD mechanism research, as well as for efficacy evaluation of anti-fatty liver drugs and VGLL4 / TEAD4 / PDK4-related targeted agents.

[0021] 2. Based on the VGLL4-TEAD4-CEBPA-PDK4 action axis, this invention confirms that preparations such as VGLL4 expression inhibitors, Super-TDU blocking peptides, and PDK4 inhibitors can reduce the content of lipids in liver tissue and serum, improve insulin resistance, and reduce liver inflammation and damage, and can be used for the prevention, relief and treatment of MAFLD.

[0022] 3. The model construction method of the present invention can be used to develop the MAFLD in vitro screening platform and efficacy evaluation system. It can be applied to the fields of fatty liver model preparation, anti-fatty liver drug screening, targeted drug development and efficacy evaluation. It has high transformation and application value and good prospects for promotion. Attached Figure Description

[0023] Figure 1The results of VGLL4 expression and functional validation in human MAFLD samples and in vitro models are as follows: A is a volcano plot of MAFLD patient sample database; B is the VGLL4 protein expression level detected by Western blot in clinical liver tissue; C is the results of immunofluorescence co-localization staining; D is Oil Red and BODIPY staining images of VGLL4 overexpression and knockdown in L02 cell line; E and F are the WB and RT-qPCR results of VGLL4 overexpression and knockdown, respectively.

[0024] Figure 2 The liver phenotype results of mice overexpressing VGLL4 in hepatocytes are as follows: A) Comparison of liver appearance between the control group (CTRL) and the overexpression group (OE); B) Weight change trend of mice from week 4 to week 16; C) Percentage of liver weight to body weight; D) Quantitative detection results of triglyceride (TG) content in mouse liver tissue; E) Quantitative detection results of total cholesterol (TC) content in mouse liver tissue; F) Detection results of total cholesterol level in mouse peripheral serum; G) Detection results of alanine aminotransferase (ALT) level in mouse serum; H I represents the results of serum aspartate aminotransferase (AST) levels in mice; I represents the results of Oil Red O and H&E staining of frozen liver sections; J represents the relative mRNA expression levels of lipid metabolism-related genes in the liver; K represents the expression of key lipid metabolism proteins in liver tissue detected by Western blotting; L represents the blood glucose change curves at different time points after intraperitoneal injection of glucose in mice and the statistical bar chart of the area under the GTT blood glucose curve; M represents the blood glucose change curves at different time points after intraperitoneal injection of insulin in mice and the statistical bar chart of the area under the ITT blood glucose curve; N represents the activation status of the hepatic insulin signaling pathway detected by Western blot; O represents the expression patterns of inflammation and fibrosis-related genes in liver tissue as shown by a heatmap.

[0025] Figure 3 Results of the investigation into the mechanism of fatty liver in mice with VGLL4 gene-induced hepatocyte overexpression: A represents the control group (CTRL) and VGLL4 overexpression (VGLL4 OE) cells, respectively, with TEADs (sh TEADs ) or switch to control (sh Luc Oil Red O and BODIPY staining results; B represents the detection of lipid synthesis genes in cells of each group. FASN, ACACA, SREBF1 )and PDK4C shows the mRNA level of VGLL4; D shows the design diagram of wild-type VGLL4 (containing TDU1 and TDU2 domains) and two mutants; E shows the effect of VGLL4 mutants on lipid accumulation and gene expression; F shows the bioinformatics enrichment analysis results of potential VGLL4 binding proteins; G shows the enrichment of FLAG-CEBPA using FLAG magnetic beads, and the detection of protein expression in the input and precipitate (IP) by Western blot; H shows the transcriptomic differential gene volcano plot of VGLL4 OE vs CTRL obtained by RNA-seq analysis; I shows the validation of VGLL4 overexpression by RT-qPCR. Pdk4 Changes in mRNA expression levels; J represents chromatin immunoprecipitation assays, using HA-tagged antibodies to enrich VGLL4-bound DNA fragments; KL shows the phenotypic characteristics and molecular-level changes of PDK4 overexpression alone; MN represents PDK4 knockdown (sh) on the basis of VGLL4 OE. PDK4 Phenotypic characteristics and molecular-level changes of ) ; O is a dual-luciferase reporter gene assay, used to detect PDK4 Transcriptional activity of the promoter.

[0026] Figure 4 Results of the protective effect of Vgll4 knockout in hepatocytes against high-fat diet-induced fatty liver: A shows the timeline of gene knockout and high-fat diet (HFD) induction in mice; B shows the body weight of mice during 16 weeks of HFD feeding; C shows the gross morphology of the livers in both groups of mice; DE shows the absolute liver weight and liver weight / body weight ratio; F shows the content of liver triglycerides; GH shows the serum transaminase levels in both groups of mice; I shows Oil Red and H&E staining of the livers in both groups of mice; JK shows the expression and knockout efficiency verification of key lipid metabolism proteins; L shows the RT-qPCR results of key genes for fatty acid synthesis and fatty acid uptake in the livers of both groups of mice; MN shows the insulin tolerance test and glucose tolerance test and their corresponding areas under the curve; WB and statistical results of the activation status of the OP insulin signaling pathway; QR shows the expression of PEPCK2, a key rate-limiting enzyme in gluconeogenesis; S shows the PAS staining results of glycogen reserves in the livers of both groups of mice.

[0027] Figure 5To investigate the mitigating effect of targeting the interaction between VGLL4 and TEAD4 on fatty liver induced by VGLL4 overexpression: A shows the experimental grouping and treatment process. Mice were divided into three groups: control group (CTRL NC), overexpression group (OE NC), and overexpression treatment group (OE Super-TDU). Mice were injected with adenovirus (ADV) at week 8 to induce overexpression, and were simultaneously given Super-TDU or the negative control (NC). Finally, mice were sacrificed (Sac) at week 16 and samples were collected. B shows the appearance of the livers of the three groups of mice. C shows the percentage of liver weight / body weight in each group of mice. D shows the quantitative detection of triglyceride (TG) content in liver tissue. E shows the detection of total cholesterol (TC) content in liver tissue. F shows the total cholesterol content in peripheral serum of the two groups of mice. G shows serum ALT activity. H shows serum AST activity. I shows the Oil Red staining and H&E staining of liver tissue histopathological sections of the three groups of mice. J shows the transcriptional level of key genes detected by RT-qPCR.

[0028] Figure 6 Results of the effect of Pdk4 knockdown on fatty liver induced by VGLL4 overexpression: A shows the experimental design and drug administration timeline for Pdk4 knockdown in mice. Mice were divided into three groups: control group (CTRL NC), VGLL4 overexpression group (OE NC), and VGLL4 overexpression and PDK4 knockdown group (OE sh). Pdk4 (); Pdk4 knockdown was induced by injection of adeno-associated virus (AAV) at week 8, and mice were sacrificed (Sac) at week 16; B shows the gross morphology of the mouse liver; C shows the liver weight to body weight ratio bar chart; DF shows the quantitative detection of lipid components in the liver and serum; GH shows the detection results of serum liver function transaminase levels; I shows the histopathological staining observation results of the liver tissue of the three groups of mice; JK shows the expression of key lipid metabolism proteins and their statistical results; L shows the detection results of transcription levels of lipid metabolism-related genes in the liver of the three groups of mice; MN shows the glucose and insulin tolerance test and the corresponding area under the curve; OP shows the protein level changes of key proteins in the insulin signaling pathway and their corresponding statistical results; QR shows the protein level changes of the key gluconeogenesis enzyme PEPCK2 and their corresponding statistical results; S shows the expression of PDK4 in liver samples from patients with fatty liver. Detailed Implementation

[0029] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0030] Example 1: Expression characteristics of VGLL4 in human MAFLD liver tissue and its effect on lipid accumulation in human hepatocytes

[0031] The purpose of this embodiment is to verify the expression characteristics of VGLL4 in the liver tissue of human MAFLD patients and to explore the effects of VGLL4 gain and loss on lipid accumulation in an in vitro human hepatocyte model.

[0032] I. Experimental Materials and Methods

[0033] 1. Bioinformatics Analysis: Gene expression matrices of liver tissues from patients with metabolic-associated fatty liver disease (MAFLD) and normal controls were obtained using the public dataset NCBI GEO (GSE135251). The limma package in R (v4.2.1) was used to analyze the difference in VGLL4 mRNA expression levels between the two groups, with P < 0.05 considered statistically significant.

[0034] 2. Patient Sample Collection and Ethics: All experiments involving human clinical samples were approved by the Human Research Ethics Committee of Zhongshan Hospital, Fudan University (Approval No.: B2025-687). Before tissue collection, each patient or their legal representative signed an informed consent form covering tissue acquisition and clinical information collection. Liver samples (including tissue from MAFLD patients and normal control tissues) were obtained from distal adjacent or non-lesion areas of patients who underwent liver cancer or liver cyst resection surgery at the Department of Hepatobiliary Surgery, Zhongshan Hospital. Exclusion criteria: Fatty liver samples caused by excessive alcohol consumption, drug / toxin exposure, autoimmune liver disease, or hepatitis C / B.

[0035] 3. Immunofluorescence staining (IF): Freshly isolated human liver tissue samples were fixed overnight (16 hours) at 4°C in 4% paraformaldehyde (PFA, purchased from Sigma-Aldrich). After fixation, the tissues were washed thoroughly three times with 1×PBS for 10 minutes each time. The tissues were then transferred to 30% sucrose solution (dissolved in 1×PBS) and dehydrated and equilibrated at 4°C for 48–72 hours until the tissues had completely settled to the bottom of the tube. The treated tissues were embedded using OCT embedding medium (Catalog No.: H-LD-803, purchased from Thermo) and prepared into 10 mm thick sections using a cryostat. Frozen sections of m were prepared. After the sections were brought to room temperature, they were washed three times with 1×PBS for 5 minutes each time. They were then blocked and permeabilized for 1 hour at room temperature using blocking and permeabilization buffer (formulation: 1×PBS solution containing 3% bovine serum albumin (BSA) and 1% Triton X-100). After blocking, primary antibody was added: rabbit anti-human VGLL4 primary antibody (purchased from Abclonal, A18248, dilution 1:500), and incubated overnight (18 hours) at 4°C. The next day, the sections were washed three times with PBS (PBST) containing 0.1% Triton X-100 for 10 minutes each time. Donkey anti-rabbit secondary antibody (purchased from Jackson, dilution 1:1000) was then added, and incubated for 1 hour at room temperature in the dark. After secondary antibody incubation, the sections were washed three times with PBST and mounted using anti-fluorescence quenching mounting medium containing DAPI (purchased from Beyotime). Fluorescence images were acquired under a Leica STELLARIS 5 confocal microscope.

[0036] 4. Cell model construction and induction of steatosis: The human normal liver cell line L02 (purchased from the Cell Bank of the Chinese Academy of Sciences) was used as the in vitro experimental subject in this experiment.

[0037] Construction of a stable VGLL4 overexpression cell line (VGLL4-OE): The full-length human VGLL4 coding region (CDS) was cloned into a lentiviral vector, and overexpressing lentivirus was prepared by co-transfecting HEK293T cells with packaging plasmids (psPAX2, pMD2.G). L02 cells were infected with the lentivirus, and puromycin was added for drug selection for 7 days after infection, resulting in a stable VGLL4 overexpressing VGLL4 VGLL4-OE cell line. An empty vector lentiviral selection strain was used as a negative control (CON-OE).

[0038] Construction of a stable VGLL4 knockdown transgenic cell line (VGLL4-KD): A shRNA sequence targeting human VGLL4 (5'-GTTCTGTGCTATGAAGGTGAA-3') was designed and cloned into the pLKO.1-puro vector to prepare a knockdown lentivirus. L02 cells were infected and selected with puromycin to obtain a stable VGLL4 knockdown VGLL4-KD cell line. A lentivirus strain targeting a non-specific luciferase-specific shRNA sequence was used as a negative control (CON-KD).

[0039] In vitro induction of fatty degeneration (PA / OA stimulation): Palmitic acid was prepared as a 100 mM stock solution (dissolved in 0.1 M NaOH, heated at 95 °C for 30 min); oleic acid was prepared as a 100 mM stock solution (dissolved in 0.1 M NaOH, heated at 95 °C for 10 min). Subsequently, the fatty acid solutions were separately mixed with 10% fat-free bovine serum albumin (BSA) solution to form fatty acid-BSA complexes (final concentration 10 mM stock solution). This was then subjected to 0.22... Sterilization was achieved by filtration through a membrane filter. L02 cells were first starved for 12 hours in RPMI 1640 medium containing 1% fetal bovine serum (FBS). Subsequently, a mixed fatty acid working solution was prepared at a molar ratio of OA:PA = 2:1, with a final concentration of 0.6 mM (containing 0.4 mM OA and 0.2 mM PA), and the cells were continuously stimulated for 24 hours to induce fatty degeneration.

[0040] 5. Oil Red O Staining: After treatment, cell samples or liver tissue sections are first washed with 1×PBS, then pretreated with 60% isopropanol solution at room temperature for 1 minute. Preparation of Oil Red O working solution: Weigh 0.3g of Oil Red O powder and dissolve it in 50mL of isopropanol as a saturated stock solution. Before the experiment, mix the stock solution with distilled water at a ratio of 3:2, let it stand at room temperature for 10 minutes, and then filter it through ordinary filter paper to obtain the Oil Red O working solution. Immerse the tissue or cell samples in the freshly prepared Oil Red O working solution and stain at room temperature in the dark for 15 minutes. After staining, immediately rinse quickly with 60% isopropanol solution for 5 seconds to remove non-specific dye background, then wash three times with 1×PBS until the background is completely clear. Lightly counterstain the cell nuclei with hematoxylin staining solution for 1 minute, then rinse with running water to regain blue color. Finally, acquire bright-field images under an Olympus BX53 microscope.

[0041] 6. Assay for cellular fatty acid uptake capacity (BODIPY staining): After 20 hours of PA / OA stimulation, L02 cells were directly added to the culture medium with green fluorescently labeled long-chain fatty acid analog BODIPY FL C12 (catalog number: D3835, purchased from Thermo Fisher Scientific, final concentration 0.6%). The cells (M) were placed in a 37°C cell culture incubator and incubated for another 4 hours. After incubation, the cells were washed three times with 1×PBS. They were then fixed with 4% PFA at room temperature for 15 minutes, washed with PBS, and green fluorescence images were acquired under an inverted fluorescence microscope. The intensity of green fluorescence was directly proportional to the cells' fatty acid uptake and endocytic accumulation capacity.

[0042] 7. RT-qPCR: Total RNA was first extracted from liver tissue or cells using TRIzol reagent (Catalog No.: R401-01, purchased from Vazyme). RNA purity and integrity were assessed using a spectrophotometer or bioanalyzer (OD260 / 280 = 1.8–2.2, OD260 / 230 ≥ 2.0). Subsequently, the total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Catalog No.: r323-01, purchased from Vazyme). In the real-time quantitative PCR reaction, SYBR green MasterMix (Catalog No.: Q511-03, purchased from Vazyme) was used, with β-actin or ACTB as internal control genes for standardization.

[0043] 8. Protein Immunoblot: First, collect tissue or cell samples and lyse them with lysis buffer containing protease inhibitors (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA). Centrifuge at 12,000 × g for 10 minutes at 4°C and collect the supernatant as the protein sample. Protein concentration is quantified using a BCA kit (catalog number: P0009, purchased from Beyotime). Then, add loading buffer containing SDS, glycerol, bromophenol blue, and β-mercaptoethanol, and denature at 95°C for 15 minutes. Separate proteins by SDS-PAGE electrophoresis according to molecular weight, and then transfer them to a PVDF membrane. Block the membrane in TBST buffer containing 5% skim milk powder for 1 hour, then add primary antibody and incubate overnight at 4°C. The next day, wash the membrane three times with TBST for 5 minutes each time, then incubate with HRP-labeled secondary antibody matched to the primary antibody source for 1 hour. After washing, the target protein signal was detected using a chemiluminescent (ECL) substrate and acquired using an imaging system.

[0044] 9. All data in this embodiment were tested using an independent samples t-test.

[0045] II. Results

[0046] from Figure 1 It can be seen that, compared with the normal control group, the relative expression level of VGLL4 mRNA in the liver tissue of MAFLD patients was significantly upregulated. Figure 1 A), the VGLL4 expression level in some MAFLD patients was significantly higher than that in the liver tissue of healthy controls. Figure 1 B).

[0047] Figure 1The results showed that the fluorescence signal of VGLL4 was strongly increased in MAFLD tissue, and the increased cells had round, multinucleated nuclei, indicating that VGLL4 was highly expressed in human MAFLD liver tissue and was mainly located in hepatocytes.

[0048] from Figure 1 As shown in Figure D, under PA / OA mixed fatty acid stimulation, the number and volume of lipid droplets stained red with Oil Red O increased in VGLL4-OE cells stably overexpressing VGLL4, indicating abnormal accumulation of neutral lipids; while VGLL4-KD cells stably knocked down VGLL4 significantly reduced lipid droplet deposition caused by fatty acid overload. Dynamic results from the BODIPY FL C12 fluorescent long-chain fatty acid uptake assay also showed that VGLL4-OE led to increased intensity of intracellular green fluorescent particles, suggesting significantly enhanced fatty acid uptake activity; while knockdown of VGLL4 significantly reduced intracellular green fatty acid endocytosis fluorescence.

[0049] Figure 1 EH data showed that overexpression of VGLL4 significantly upregulated the mRNA and protein expression levels of the core enzymes in fatty acid synthesis; conversely, knockdown of VGLL4 inhibited the expression of these indicators, and gene expression and VGLL4 levels showed a strong positive correlation.

[0050] The above results indicate that VGLL4 is upregulated in the liver tissue of human MAFLD patients and promotes lipid absorption, lipid droplet accumulation and release of inflammatory factors in vitro, suggesting that VGLL4 has a very clear pathological role in promoting fatty liver formation.

[0051] Example 2: Construction of a MAFLD mouse model by specific overexpression of VGLL4 in hepatocytes

[0052] The purpose of this experiment is to verify that hepatocyte-specific VGLL4 overexpression can induce MAFLD-related phenotypes in mice.

[0053] I. Experimental Materials and Methods

[0054] 1. Mouse Model and Grouping: The LAP-tTA transgenic mice and TetO-Vgll4 transgenic mice used in this invention were both C57BL / 6J background mice. LAP-tTA mice were mated with TetO-Vgll4 mice to construct the Tet-off system: under normal drinking water conditions and without doxycycline (Dox) supplementation, tTA binds to the tetO element, initiating specific overexpression of the Vgll4 gene in the mouse liver. This genotype group was defined as the overexpression group (OE). Lignate mice not carrying the LAP-tTA allele served as the control group (CTRL). Mice were housed in a specific pathogen-free (SPF) environment with a 12-hour light / dark cycle at a room temperature of 22±2°C. Both groups of mice were basal fed with standard rodent diet after weaning until 16 weeks of age.

[0055] 2. Determination of Triglyceride and Cholesterol Content in Mouse Liver: Liver tissue was rapidly separated after sacrifice of mice at 16 weeks of age and gently rinsed with pre-cooled PBS to remove surface blood. Fresh liver tissue samples of similar weight from the same location were collected and their weights recorded. Triglyceride (TG) and total cholesterol (TC) content assay kits manufactured by Solarbio were used for detection.

[0056] 3. Detection of serum biochemical indicators in mice: After sacrifice, whole blood was collected from the heart and placed in anticoagulant-free tubes, allowing it to coagulate naturally at room temperature for 30 minutes. The blood was then centrifuged at 3,000×g at room temperature for 10 minutes, and the supernatant serum was collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were used as indicators of liver function, and serum total cholesterol (CHOL) levels were used as indicators of blood lipids, all accurately measured using a fully automated biochemical analyzer.

[0057] 4. Oil Red Staining and Hematoxylin-Eosin (H&E) Staining: Oil Red Staining: The specific steps are the same as in Example 1. H&E Staining: First, mouse liver tissue was removed and fixed in 4% paraformaldehyde for about 24 hours to ensure sufficient preservation of tissue morphology. After fixation, the tissue was sequentially dehydrated with different concentrations of ethanol, then cleared with xylene, and subsequently embedded in paraffin. The embedded liver tissue was cut into tissue sections of about 3 μm thickness using a paraffin microtome and attached to glass slides. After drying, the sections were ready for use. Before staining, the sections were sequentially dewaxed with xylene and rehydrated to distilled water through a gradient of ethanol. Then, hematoxylin staining was used to stain the cell nuclei. After staining, the sections were rinsed with running water and subjected to differentiation and blueing treatment to make the cell nuclei appear clearly blue-purple. Afterward, eosin staining was used to counterstain the cytoplasm, extracellular matrix, and other tissue structures to make them appear in different shades of pink or red. After staining, the sections were dehydrated again with graded ethanol, cleared with xylene, and mounted with neutral resin or other mounting medium. Finally, images were observed and acquired under an optical microscope to assess pathological changes such as the overall structure of the liver tissue, hepatocyte arrangement, fat vacuolation, inflammatory cell infiltration, and the degree of tissue damage. This method can visually reflect the fatty degeneration and pathological damage of the liver tissue in different experimental groups of mice.

[0058] 5. Gene / protein expression: The specific steps are the same as in Example 1.

[0059] 6. Metabolic Assays: The Insulin Tolerance Test (ITT) and Glucose Tolerance Test (GTT) are used to evaluate insulin sensitivity and glucose metabolism in mice. For the GTT, mice are typically fasted overnight before the experiment but allowed free access to water. After fasting, the basal blood glucose level is measured as the 0-minute blood glucose level, followed by an intraperitoneal injection of glucose solution at a dose of 1.5 g / kg body weight. Blood glucose levels are measured at 15, 30, 60, 90, and 120 minutes after injection using a Roche blood glucose meter and dedicated test strips, and the changes in blood glucose at each time point are recorded. For the ITT, mice are typically fasted for 4 hours before the experiment begins. Similarly, the basal blood glucose level is measured first, followed by an intraperitoneal injection of insulin at the experimentally designed dose, typically 0.5 IU / kg or 1.5 IU / kg body weight. After insulin injection, blood is collected from the tail tip at 15, 30, 60, 90, and 120 minutes, and blood glucose concentration is measured using a blood glucose meter and test strips. During the experiment, it is important to maintain the stability of the mice and avoid excessive stress that could affect the blood glucose results. Finally, blood glucose curves were plotted based on blood glucose values ​​at each time point, and the area under the curve (AUC) was calculated to compare differences in glucose tolerance and insulin sensitivity among different experimental groups. An elevated GTT curve or an increased AUC usually indicates decreased glucose clearance, while a weakened decrease in blood glucose or an increased AUC after insulin injection in the ITT indicates increased insulin resistance.

[0060] 7. In this embodiment, the detection results of 2C-H, 2J, 2L and 2M were all tested using independent samples t-tests; the data of 2B and 2L-M were tested using two-way ANOVA.

[0061] II. Results

[0062] from Figure 2 AC analysis revealed that under standard dietary conditions at 16 weeks of age, the livers of mice in the OE group appeared paler than those in the CTRL group, and the OE mice exhibited significantly higher absolute liver weight and liver weight / body weight ratio. Figure 2 DH staining revealed significantly elevated levels of triglycerides and total cholesterol in the liver tissue of OE mice; simultaneously, serum total cholesterol, peripheral blood transaminases ALT and AST were also significantly elevated in OE mice, indicating severe liver damage. Oil Red O and H&E staining confirmed this. Figure 2 I) Fat vacuoles and bright red lipid droplets were found in the liver tissue of OE mice. Figure 2 JK data indicate that Vgll4 was successfully overexpressed and upregulated the mRNA and protein abundance of the core rate-limiting enzyme in lipogenesis. From Figure 2 According to LM, in the in vivo metabolic experiment, the OE group mice showed severe glucose intolerance and insulin resistance, and their GTT and ITT dynamic blood glucose curves were all shifted upwards, with their areas under the curves being significantly higher than those of the control group. Figure 2 N indicates that the core kinase activity of the insulin pathway in the liver of OE group mice was inhibited, manifested by increased phosphorylation of the p-IRS1 (Tyr307) inhibitory site, while the phosphorylation activation level of the downstream key molecule p-AKT (Ser473) was significantly reduced. Figure 2 The O indicates that the expression levels of multiple inflammatory factors in the liver of mice in the OE group were significantly increased, accompanied by obvious immune infiltration and activation.

[0063] The above results demonstrate that, under standard dietary conditions, specific overexpression of VGLL4 in hepatocytes alone can directly induce stable fatty liver degeneration, systemic insulin resistance, and intrahepatic inflammatory pathological phenotypes in mice.

[0064] Example 3: Validation of the mechanism by which VGLL4 overexpression in hepatocytes induces fatty liver

[0065] I. Experimental Materials and Methods

[0066] 1. Construction of stable cell mutants: The specific steps are basically the same as in Example 1. To accurately define the domains, this example also constructed a mutant form of mouse Vgll4 and packaged it as a lentivirus: a mutant strain lacking the Tondu domain (VGLL4-ΔTDU). For the endogenous TEAD family, hybrid targeting shRNA lentiviruses (shTEADs) targeting TEAD1 / 2 / 3 / 4 were designed.

[0067] 2. Co-immunoprecipitation (Co-IP): HEK293T cells were transfected with target expression plasmids (HA-VGLL4, MYC-TEAD4, FLAG-CEBPA). After 48 hours of culture, cells were collected and 1 mL of ice-cold IP lysis buffer (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA, and containing 1× protease inhibitor mixture) was added. The cells were centrifuged at 12,000×g for 10 minutes at 4°C, and the supernatant was collected as the total protein sample. The protein sample was divided into an input group and an immunoprecipitation group. 20 μL of magnetic beads conjugated with primary antibodies (anti-FLAG magnetic beads, catalog number: B26101; anti-MYC magnetic beads, catalog number: B26301) were added to the immunoprecipitation group. The cells were incubated at room temperature for 1.5 hours to allow the target protein to bind to the antibody. Subsequently, the magnetic beads were washed 3–5 times with pre-chilled IP lysis buffer to remove non-specifically bound proteins. After washing, the magnetic beads were added to the SDS-PAGE loading buffer and heated to denature them. The supernatant was then collected for SDS-PAGE electrophoresis separation.

[0068] 3. ChIP-qPCR: First, AML12 cells were seeded in 10cm culture dishes. Once the cell density reached a suitable level, the cells were transfected with the appropriate expression plasmid or treated accordingly according to the experimental design. After approximately 48 hours of treatment, the cells were washed twice with PBS, followed by the addition of 1% formaldehyde and cross-linking at 37°C for approximately 15 minutes to form a stable cross-linked complex between intracellular proteins and DNA. After cross-linking, glycine was added to a final concentration of 0.125M, and the cells were incubated at 37°C for approximately 5 minutes to terminate the cross-linking reaction. Cells were then collected and lysed using pre-chilled cell lysis buffer containing protease inhibitors to prevent protein degradation. After cell lysis, the cells were sonicated on ice to break down chromatin DNA into fragments approximately 200–500 bp in size. The sonication conditions were set to 25 cycles, with each cycle lasting 10 seconds and a 20-second interval, at approximately 25% power. The entire process was kept at a low temperature to avoid sample overheating and subsequent protein or DNA degradation. After sonication, the samples were centrifuged at 12,000×g for 10 minutes at 4°C, and the supernatant was collected as soluble chromatin samples. A small amount of chromatin was used as an input control, and the remaining samples were incubated overnight at 4°C with either a specific antibody against the target protein or a normal IgG negative control antibody to allow the antibody to fully bind to the target protein-DNA complex. The next day, Dynabeads Protein A magnetic beads were added, and incubation continued at 4°C for approximately 2 hours to enrich the antibody-bound chromatin complex. Subsequently, the magnetic beads were washed sequentially with low-salt and high-salt washing buffers to remove non-specifically bound components. After washing, the protein-DNA complex was eluted from the magnetic beads with elution buffer, and NaCl was added for overnight incubation at 65°C to reverse cross-linking. Then, Proteinase K (20 mg / mL) and EDTA were added, and incubation continued at 55°C for approximately 2 hours to remove protein components. The obtained DNA fragments were purified using a PCR purification kit and used as qPCR templates for detection. For qPCR detection, specific primers targeting the promoter region of the target gene were used for amplification. The reaction system employed SYBR Green quantitative PCR and amplification was performed on a real-time quantitative PCR instrument. Finally, the relative enrichment of the target protein in the corresponding DNA region was calculated based on the IgG negative control. By comparing the ChIP-qPCR enrichment levels of the target protein in the Pdk4 promoter region among different experimental groups, changes in the binding affinity of proteins such as VGLL4, TEAD4, or CEBPA to the target gene promoter could be evaluated, thereby analyzing their roles in Pdk4 transcriptional regulation.

[0069] 4. Dual-luciferase reporter: Pdk4The gene promoter fragment was cloned into the pGL3-basic vector as a luciferase reporter plasmid, while the Renilla luciferase plasmid was used as an internal control to correct for transfection efficiency and cell state differences. L02 cells were seeded in 24-well plates. After cell adhesion, the Firefly reporter plasmid, Renilla internal control plasmid, and expression plasmids required for the experimental group (such as VGLL4, TEAD4, and CEBPA) were co-transfected into the cells using ExFect (Catalog No.: T101, Vazyme). The total amount of plasmid was kept consistent by adding an equal amount of empty vector. Twelve hours after transfection, the cells were cultured in standard medium for another 36 hours to ensure sufficient expression of the target protein and its effect on promoter activity. Subsequently, the cells were collected and lysed thoroughly with lysis buffer to obtain cell lysates. The activities of Firefly and Renilla luciferase were measured sequentially using a dual-luciferase assay system, and the luminescence signals were read using a Promega GloMax 20 / 20 Luminometer according to the manufacturer's instructions. In the experimental results, Firefly luciferase activity was normalized using Renilla luciferase activity to obtain relative promoter activity. By comparing the relative luciferase activities under different treatment groups or different protein expression conditions, the regulatory effects of proteins such as VGLL4, TEAD4, and CEBPA on the transcriptional activity of the Pdk4 promoter can be quantitatively assessed, and the reproducibility of the results was verified through multiple independent experiments.

[0070] 5. In this embodiment, the 3I-J detection results were tested using an independent samples t-test; the 3B, 3E, 3L and 3N-O data were all analyzed using one-way ANOVA.

[0071] II. Results

[0072] from Figure 3 As shown in AB, knocking out the entire TEAD family of genes (shTEADs) using shRNA in cell lines stably overexpressing VGLL4 significantly alleviated the excessive accumulation of red neutral lipid droplets caused by VGLL4 overexpression, and the expression of downstream lipid synthesis-related enzymes also decreased significantly. The mutant phenotypic reversion results showed ( Figure 3 CE), the stable cell line lacking the TEAD4 interaction domain (ΔTDU) could not further promote the accumulation of red lipid droplets and fatty acid uptake in hepatocytes like VGLL4, and the downstream PDK4 transcriptional activation was weakened. Upstream transcription factor enrichment analysis of differentially expressed genes (DEGs) in overexpressing mice and controls revealed ( Figure 3 F), the key lipid regulator CEBPA showed a high enrichment count and a significant correlation score. From Figure 3G indicates that EAD4 and CEBPA interact, and the physical binding interaction between CEBPA and TEAD4 is significantly enhanced with increasing HA-VGLL4 dose gradient, while the protein abundance of CEBPA itself remains unchanged. This suggests that TEAD4 acts as a scaffold linker protein mediating the assembly of the "VGLL4 / TEAD4 / CEBPA" ternary transcriptional complex.

[0073] from Figure 3 HI revealed that Pdk4 mRNA was significantly upregulated in livers with VGLL4 overexpression. From... Figure 3 J indicates that ChIP-qPCR chromatin immunoprecipitation results confirmed that the binding enrichment of anti-HA-VGLL4 specific antibody in the specific response region of the mouse Pdk4 promoter was significantly higher than that in the negative control IgG group, confirming that VGLL4 directly and physically binds to the promoter region of Pdk4. From Figure 3 KN studies show that PDK4 overexpression effectively mimics the phenotype of massive red lipid droplet accumulation caused by VGLL4 overexpression; conversely, targeted knockout of Pdk4 (shPdk4) using shRNA in VGLL4-overexpressing cell lines significantly alleviates the lipid accumulation and synthase activation phenotype caused by VGLL4 overexpression. This indicates that VGLL4 function depends on its downstream target, Pdk4. Dual-luciferase reporter gene assays show that ( Figure 3 The addition of wild-type VGLL4 can dose-dependently promote the luciferase luminescence ratio of the Pdk4 promoter.

[0074] The above results indicate that the VGLL4 / TEAD4 / CEBPA-PDK4 axis is a novel core pathway for promoting MAFLD metabolic remodeling that is not limited by the classical Hippo pathway and is discovered for the first time in this invention.

[0075] Example 4: Effect of VGLL4 knockout on high-fat meal-induced fatty liver

[0076] The purpose of this embodiment is to determine the necessity of VGLL4 in the development of fatty liver.

[0077] I. Experimental Materials and Methods

[0078] 1. Experimental animals: Mx1-Cre mice and Vgll4 f / f Mice were the key experimental material used in this study to construct a conditional Vgll4 gene knockout model. In this study, Mx1-Cre and... Vgll4 f / f The combined use of mice is primarily for research purposes. Vgll4 The effects of deficiency on high-fat diet-induced hepatic steatosis and abnormal glucose and lipid metabolism. Specifically, in 8-week-old males, Mx1-Cre; Vgll4 f / f (HKO) mice and Vgll4 f / f Flox / Flox control mice received intraperitoneal injections of poly I:C (2 mg / ml; catalog number: GE27-4732-01, Sigma-Aldrich) at a dose of 13 mg / kg body weight, administered every 24 hours for a total of two injections. After induction, the mice continued to be fed a high-fat diet or undergo other experimental treatments, and liver and serum samples were collected at set time points to detect changes in liver lipid deposition, serum biochemical indicators, glucose metabolism function, and related gene and protein expression.

[0079] 2. Induction and Phenotypic Detection of High-Fat Diet (HFD) Model: After poly I:C induction, mice in both the flox / flox control group and the HKO group were switched to a high-fat diet. Both groups of mice were fed a high-fat diet for 16 consecutive weeks (until 25 weeks of age). Absolute body weight was measured every two weeks during the feeding period.

[0080] 3. Other experimental methods: Mice were sacrificed at the end of 16 weeks of feeding. All biochemical and pathological measurements (including the determination of absolute TG / TC levels in whole liver using the Solarbiok kit, serum AST / ALT transaminase activity, serum total cholesterol, whole liver H&E paraffin staining pathology, Oil Red O staining of frozen sections, GTT / ITT metabolic clearance assay, whole liver protein extraction, and Western blot separation) were performed with identical parameters, procedures, and formulations, strictly following the descriptions in Examples 1 and 2. In addition, an endogenous periodate-Schiff (PAS) glycogen-specific staining kit (catalog number: C0142, purchased from Beyotime) was used to assess liver glycogen homeostasis.

[0081] 4. In this embodiment, the detection data of 4D-H, 4K-L, 4M-N, 4P and 4R were tested using independent samples t-tests; the results of 4B and 4M-N were tested using two-way ANOVA.

[0082] II. Results

[0083] from Figure 4 AC data shows that, throughout the entire timeline, during the long-term HFD feeding period, the absolute weight gain of the HKO knockout group mice was significantly inhibited from week 8 onwards; after 16 weeks of feeding, the livers of the flux / flox control group mice were enlarged and turbidly yellowish-white, while the livers of the HKO group mice were dark red and without swelling. Figure 4DH studies showed that Vgll4 knockout significantly reduced the absolute liver weight and liver weight / body weight percentage decrease caused by HFD feeding; simultaneously, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels decreased due to Vgll4 knockout. Figure 4 IL analysis revealed that the hepatic lobules in the flux / flox group were filled with large fat vacuoles and extensively stained with Oil Red O, while the hepatocytes in the HKO group showed less fat accumulation compared to the control group. Furthermore, Western blot and RT-qPCR results of whole liver lysis products were consistent: Vgll4 was conditionally knocked out in vivo in the HKO group, significantly alleviating and weakening the abnormally elevated mRNA and protein expression levels of this key rate-limiting enzyme in fatty acid metabolism in the livers of high-fat-fed mice.

[0084] from Figure 4 MS analysis revealed that in the metabolic clearance assay, the ITT and GTT blood glucose curves showed that Vgll4 knockout significantly suppressed the overall dynamic blood glucose trajectory in mice, indicating that HKO alleviated systemic insulin resistance and glucose metabolism disorders induced by HFD feeding. Simultaneously, Western blotting analysis of the insulin signaling pathway showed increased phosphorylation activation of the downstream activating kinase p-AKT (Ser473) compared to the control group; biochemical analysis of key gluconeogenesis enzymes showed decreased PEPCK2 protein levels; and PAS glycogen-specific staining confirmed the presence of extremely abundant deep purple-red glycogen granules in hepatocytes of the HKO group, demonstrating that Vgll4 knockout restored normal carbohydrate homeostasis in the liver.

[0085] The above results demonstrate that targeted knockout of endogenous Vgll4 is an extremely effective intervention strategy for treating obesity, fatty liver degeneration, liver damage, and systemic insulin resistance caused by a high-fat diet.

[0086] Example 5: Detection of the effect of targeting the interaction between VGLL4 and TEAD4 on alleviating fatty liver caused by VGLL4 overexpression.

[0087] I. Experimental Materials and Methods

[0088] 1. Definition of Recombinant Adenovirus (ADV): ​​This example aims to demonstrate the pharmacological intervention effect of blocking transcription complex assembly. Super-TDU is a reported small-molecule blocking peptide whose core sequence mimics the tandem TDU domain of mouse VGLL4. This peptide can competitively bind to endogenous TEAD4 with extremely high affinity, thereby specifically blocking the physical interaction between VGLL4 and TEAD4 in spatial conformation. The fragment encoding the Super-TDU sequence was homologously recombinantly cloned into an adenovirus vector to construct a recombinant adenovirus tool, which was custom-named ADV-Super-TDU. An empty vector adenovirus without the peptide coding sequence was used as a negative control and named ADV-NC.

[0089] 2. The tail vein injection administration procedure used the OE overexpression mouse system described in Example 2. Mice were randomly divided into three groups: Control group (CTRL NC group): control mice, which received a single tail vein injection of 200 μL of sterile saline-diluted empty vector virus ADV-NC. Overexpression pathology group (OE NC group): OE mice, which received a single tail vein injection of an equal volume and dose of empty vector virus ADV-NC. Overexpression blocking treatment group (OE Super-TDU group): OE mice, which received a slow tail vein injection of ADV-Super-TDU. The single injection dose per mouse was strictly limited to 5 × 10^8 PFU. After the injection administration, the mice in all three groups were fed under standard conditions for 8 weeks. The mice were sacrificed at 16 weeks of age, and liver and serum samples were collected for comprehensive analysis.

[0090] 3. Other experimental procedures: The quantitative and histopathological morphological procedures after euthanizing the mice were exactly the same in terms of reaction solution formulation, extraction purity control and circulation parameters, and strictly followed the specific limitations of Examples 1, 2 and 4.

[0091] 4. In this embodiment, the detection results of 5C-H and 5J were both analyzed using one-way ANOVA.

[0092] II. Results

[0093] from Figure 5 According to AC, the experimental procedure and timeline show that after 8 weeks of drug administration, the livers of mice in the OE Super-TDU group injected with ADV-SuperTDU did not show the same white liver phenotype as those in the OE NC group mice. Nevertheless, the percentage of absolute liver weight to body weight in the overexpression blocking treatment group was not significantly lower than that in the overexpression pathology group.

[0094] from Figure 5DH indicates that dissolving the interaction between TEAD4 and VGLL4 (OE Super-TDU group) significantly reduced liver triglyceride content, liver tissue total cholesterol content, and serum total cholesterol levels in overexpressing mice; at the same time, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in peripheral blood circulation were significantly lower than those in the OE NC group.

[0095] from Figure 5 According to IJ, H&E and Oil Red O sections showed that the balloon-like vacuoles and bright red lipid droplets in the hepatocytes of the OE Super-TDU group had significantly decreased. At the same time, the relative quantitative statistics of RNA RT-qPCR showed that Super-TDU could alleviate the increased expression of fatty acid metabolism enzymes and uptake-related genes in the liver of overexpressing mice.

[0096] The above experimental results demonstrate that targeting and relieving the ternary complex interaction between endogenous VGLL4 and TEAD4 is a completely effective intervention and treatment strategy for fatty liver in animals.

[0097] Example 6: Detection of the effect of Pdk4 knockdown on fatty liver induced by VGLL4 overexpression

[0098] I. Experimental Materials and Methods

[0099] 1. Construction of an in vivo targeted knockdown tool for adeno-associated virus (AAV): A core small interfering RNA sequence (5'-GAAATTGGATCCTATCACA-3') targeting the mouse Pdk4 gene transcript was designed and cloned into the AAV2 / 8-shRNA expression vector. Recombinant packaging and iodixanol gradient ultracentrifugation purification of the AAV2 / 8 serotype were completed at the gene editing core facility of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. A viral tool with Pdk4 knockdown targeting was obtained and named AAV2 / 8-shPdk4. A nonspecific empty vector virus without a polypeptide sequence was used as a negative control and named AAV2 / 8-NC.

[0100] 2. The tail vein injection administration procedure used the OE overexpression mouse system described in Example 2. Mice were uniformly grouped and received injections via the tail vein. Control group (CTRL NC group): CTRL mice received a single bolus injection of AAV2 / 8-NC virus via the tail vein. Overexpression group (OE NC group): OE mice received a single bolus injection of AAV2 / 8-NC virus via the tail vein. Gene knockdown group (OE shPdk4 group): OE mice received a precise injection of the AAV2 / 8-shPdk4 vector via the tail vein. The injection dose for all three groups was 5 × 10^11 viral genomes (vg) per mouse. After administration, all three groups of mice were routinely fed under standard conditions for 8 weeks. Mice were sacrificed at 16 weeks of age, and liver and serum samples were collected.

[0101] 3. The parameters, conditions, and lysis components of all other biochemical, pathological, and metabolic tolerance tests performed on mice were exactly the same as those described in Examples 1, 2, and 4.

[0102] 4. In this embodiment, the detection results of 6C-H, 6K-N, 6P and 6R were all analyzed using one-way ANOVA; the data of 6M and 6N were all analyzed using two-way ANOVA.

[0103] II. Results

[0104] Intervention process and drug administration timeline ( Figure 6 A) indicates that after liver-specific knockdown of PDK4, OE sh Pdk4 The whitening pathological phenotype caused by overexpression in the livers of the mice in this group was weakened, and the overall color returned to a healthy, rosy hue. Figure 6 B); at the same time, the bar chart statistics confirm that OE sh Pdk4 The liver weight / body weight percentage ratio of the mice in the control group was significantly lower than that in the OE NC group. Figure 6 C). Figure 6 DH results indicate that specific knockout of Pdk4 significantly reduced the absolute levels of liver triglycerides, total liver cholesterol, and serum total cholesterol levels in peripheral blood circulation observed in overexpressing mice; simultaneously, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were also significantly reduced. Liver microscopic histological staining results ( Figure 6 In I), H&E and Oil Red O sections confirmed that the ballooning fat vacuoles in the overexpression group (OE NC) significantly regressed after PDK4 knockdown, with only a small number of bright red lipid droplets remaining in the liver lobules.

[0105] from Figure 6 According to JL, Western blot electrophoresis of total protein cleavage products from liver tissue and the relative quantitative grayscale statistics of each band density show that PDK4, when introduced into AAV-sh... Pdk4In the subsequent groups, the expression levels of key enzymes involved in fatty acid metabolism and synthesis (FASN, ACC1, and SCD1) activated by VGLL4 overexpression were effectively knocked down, and the protein expression levels were also reduced. RT-qPCR simultaneously confirmed that the transcripts of these lipid synthesis and peripheral uptake-related genes were significantly downregulated.

[0106] from Figure 6 MR analysis revealed that, in the glucose tolerance test, a comparison of the dynamic blood glucose curves and AUC areas between the GTT and ITT showed that OE sh Pdk4 Compared with the OE NC pathological group, the group showed significant relief of glucose metabolism abnormalities and insulin resistance phenotypes. Simultaneously, Western blotting confirmed a further upregulation of phosphorylation levels of the core insulin downstream conduction kinase compared to the OE NC group; gluconeogenesis rate-limiting enzyme expression analysis showed a significant improvement in abnormal gluconeogenesis activation.

[0107] from Figure 6 As can be seen from S, the endogenous protein expression level of PDK4 protein in liver tissue samples from human patients with fatty liver disease (MAFLD) was significantly higher than that in normal healthy human livers.

[0108] This embodiment demonstrates through downstream knockout experiments that the downstream target PDK4 is a necessary target for mediating upstream VGLL4-induced fatty liver, liver damage, and abnormal total glucose metabolism.

[0109] The above results indicate that hepatocyte-specific VGLL4 overexpression can rapidly induce animal and cell models that highly mimic human metabolic-related fatty liver disease. These models exhibit typical phenotypes, including hepatic steatosis, insulin resistance, inflammatory response, and progression to steatohepatitis, without requiring a high-fat diet or chemical stimulation. The mechanism of action involves VGLL4 acting as a transcriptional coactivator to enhance the interaction between TEAD4 and CEBPA, directly upregulating downstream Pdk4 expression, leading to a shift in carbon flow towards lipid synthesis and enhanced gluconeogenesis. Effects include significantly increased hepatic TG / TC ratio, lipid droplet accumulation, insulin signaling inhibition, and upregulation of inflammatory factors. Targeting the VGLL4-TEAD4 interaction or Pdk4 can reverse the aforementioned pathological phenotypes. The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Application of VGLL4 gene in constructing a metabolic-related fatty liver disease model.

2. The application as described in claim 1, wherein the model is a transgenic mouse that specifically overexpresses the VGLL4 gene in liver parenchymal cells.

3. A method for constructing a metabolic-associated fatty liver disease model, characterized in that, The model was constructed by specifically overexpressing the VGLL4 gene in mouse hepatocytes.

4. Application of VGLL4 gene in the preparation of drugs for the treatment of metabolic-related fatty liver disease.

5. The application as described in claim 4, wherein the drug for treating metabolic-related fatty liver disease is a drug capable of inhibiting VGLL4 gene overexpression.

6. The application as described in claim 4, wherein the drug for treating metabolic-related fatty liver disease is a drug capable of inhibiting the interaction between VGLL4 and TEAD4.

7. The application as described in claim 6, wherein the drug capable of blocking the interaction between VGLL4 and TEAD4 is a Super-TDU.

8. The application as described in claim 4, wherein the drug for treating metabolic-related fatty liver disease is a drug capable of inhibiting PDK4 expression.

9. The application as described in claim 8, wherein the drug inhibiting PDK4 expression is a shRNA targeting PDK4.

10. A drug for treating metabolic-associated fatty liver disease, characterized in that, The drug comprises an active ingredient that inhibits VGLL4 gene overexpression, blocks the interaction between VGLL4 and TEAD4, or inhibits PDK4 expression.

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