Application of GRPEL1 as a target and its activator in the preparation of drugs for the prevention or treatment of fatty liver disease related to metabolic dysfunction.

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of the target GRPEL1 and its activator in the preparation of drugs for the prevention or treatment of metabolic dysfunction-related fatty liver disease (MASLD). This invention demonstrates that under metabolic stress, GRPEL1 is pathologically downregulated in the liver, while upregulation of GRPEL1 significantly improves liver damage. It also demonstrates that the natural monomer imperatorin can act as a targeted activator of GRPEL1, enhancing the thermal stability of the target protein and inhibiting its degradation through direct physical binding. In vitro and in vivo experiments and gene knockdown confirmation show that imperatorin specifically depends on the GRPEL1 target to exert significant lipid-lowering, anti-inflammatory, and anti-fibrotic effects. This invention provides a clear therapeutic target and candidate drug for the prevention and treatment of MASLD, and has significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of GRPEL1 as a target and its activator in the preparation of drugs for the prevention or treatment of fatty liver disease related to metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is a chronic liver disease with an extremely high incidence worldwide. Its pathological progression typically begins with simple hepatic fat accumulation, gradually developing into metabolic dysfunction-associated steatohepatitis and liver fibrosis accompanied by inflammatory damage. In the pathological process of MASLD, the imbalance of hepatocyte homeostasis is the core hub driving lipid metabolism disorders and the inflammatory cascade. Currently, there are still no effective drugs in clinical practice that can directly target the repair of endogenous hepatocyte homeostasis.

[0003] GRPEL1 (GrpE protein homolog 1) is a chaperone protein located in the mitochondrial matrix, primarily involved in protein folding and homeostasis. However, under the pathological stress of MASLD, the abundance evolution of GRPEL1, its effectiveness as an intervention target, and specific small molecule activators targeting this target have not yet been publicly reported. Furthermore, although the natural small molecule imperatorin possesses certain biological activity, its exact efficacy and direct target in the treatment of MASLD have not yet been reported. Summary of the Invention

[0004] This invention aims to address the technical problems of the lack of endogenous homeostatic repair targets for MASLD and the unclear pharmacological mechanism of the natural monomer imperatorin. This invention confirms that GRPEL1 is an effective target for intervening in the MASLD process and discovers that imperatorin can act as a targeted activator of GRPEL1, enhancing the stability of the target protein through direct physical binding and exerting significant therapeutic effects in the prevention and treatment of MASLD.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the invention provides the use of the GRPEL1 protein or its encoding gene in the preparation of a medicament for the prevention or treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

[0007] Preferably, the amino acid sequence of the GRPEL1 protein is shown in SEQ ID NO: 1; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0008] A second aspect of the invention provides the use of imperatorin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

[0009] Preferably, the drug for preventing or treating fatty liver disease related to metabolic dysfunction is a drug used to increase the abundance of GRPEL1 protein in hepatocytes and improve liver damage.

[0010] Preferably, the imperatorin or its pharmaceutically acceptable salt enhances the thermal stability of the GRPEL1 protein and inhibits its protein degradation under pathological conditions by non-covalently binding to the GRPEL1 protein, thereby exerting a therapeutic effect specifically dependent on the GRPEL1 target.

[0011] In a third aspect of the invention, in any of the applications described above, the MASLD includes simple hepatic steatosis, metabolic dysfunction-related steatohepatitis, and liver fibrosis.

[0012] In a fourth aspect of the invention, in the applications described in any of the foregoing aspects, the prevention or treatment exhibits one or more of the following pharmacological effects:

[0013] (1) Reduce the activity of alanine aminotransferase and / or aspartate aminotransferase in serum;

[0014] (2) Improve liver steatosis and reduce or eliminate the pathological accumulation of neutral lipid droplets in hepatocytes;

[0015] (3) Reduce liver inflammation, inhibit the expression of monocyte chemoattractant protein-1 in liver tissue, and / or inhibit the infiltration of Ly6C positive inflammatory cells;

[0016] (4) Improve liver fibrosis, reduce the levels of type IV collagen, laminin and / or hyaluronic acid in serum, or reduce the content of hydroxyproline in liver tissue.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention is the first to confirm that the downregulation of GRPEL1 protein under pathological stress is a key factor driving the progression of MASLD. Through bidirectional verification of gene knockdown and overexpression in vivo, the core role of GRPEL1 in resisting liver lipid overload and tissue damage was confirmed, providing a definite endogenous intervention target for the treatment of MASLD.

[0019] (2) The white peucedanum alcohol provided by this invention can comprehensively improve multiple pathological phenotypes such as lipid accumulation, inflammation and tissue fibrosis. In vivo gene knockdown animal experiments have confirmed that its macroscopic efficacy specifically depends on the expression of GRPEL1 protein. This targeting dependence reduces the potential risks of non-specific intervention and improves the reliability of the drug.

[0020] (3) This invention elucidates the microscopic mechanism of imperatorin as a GRPEL1 targeting activator from a biophysical perspective, and confirms that it inhibits the protein-level degradation of the target protein through direct physical binding, thus overcoming the technical defects of the vague target of traditional natural products. Attached Figure Description

[0021] Figure 1 This is a validation diagram illustrating how metabolic stress triggers GRPEL1 loss in hepatocytes and that hepatocyte-specific knockdown of Grpel1 exacerbates liver damage in mice. Specifically: A shows the GRPEL1 protein level in mouse liver tissue after different weeks of MD diet induction; B shows the GRPEL1 protein level in primary hepatocytes after MD diet treatment; C and D are statistical graphs of serum ALT and AST activities in mice after 4 weeks of MD diet induction; E is a pathological image of mouse liver tissue stained with H&E after 4 weeks of MD diet induction (scale bar = 50 μm); F is a statistical graph of NAS scores for each group; and G is a pathological image of liver tissue stained with Oil Red O (scale bar = 10 μm).

[0022] Figure 2 This figure shows the results of hepatocyte-specific overexpression of Grpel1 improving diet-induced liver injury. Specifically: A and B are statistical graphs of serum ALT and AST activities in mice after 16 weeks of MD diet induction; C is a pathological image of liver tissue stained with H&E (scale bar = 50 μm) from each group of mice; D is a statistical graph of NAS scores; and E is a pathological image of liver tissue stained with Oil Red O (scale bar = 10 μm) from each group of mice.

[0023] Figure 3 This image is an assessment of how overexpression of Grpel1 reduces the progression of inflammatory infiltration and fibrosis in liver tissue. In the image: A and B are immunohistochemical images of MCP-1 and Ly6C in mouse liver tissue after 16 weeks of MD diet induction (scale bar = 50 μm); C–E are statistical graphs of serum CIV, LN, and HA levels in mice, respectively; F is a statistical graph of hydroxyproline content in mouse liver tissue.

[0024] Figure 4 This diagram illustrates the physical binding of imperatorin to GRPEL1 protein and its effect on increasing the abundance of the target protein in vivo. Specifically: A is a simulated molecular docking diagram of imperatorin and GRPEL1; B is a CETSA experiment validating the targeted binding in live cells; and C is a diagram showing the effect of imperatorin on the abundance of GRPEL1 protein in the liver tissue of 16-week-old MD diet mice.

[0025] Figure 5 This is a confirmatory graph showing the GRPEL1 target specificity dependence of the efficacy of purslane in improving liver damage. Specifically: A is the in vivo knockdown efficiency verification graph mediated by AAV; B and C are statistical graphs of serum ALT and AST in mice of each group after 16 weeks of MD diet induction; D is a comparison of H&E staining in liver tissue of mice of each group (scale bar = 50 μm); E is a statistical graph of NAS scores; F is a comparison of Oil Red O staining in liver tissue (scale bar = 10 μm); G and H are immunohistochemical images of MCP-1 and Ly6C in liver tissue, respectively (scale bar = 50 μm); I and J are statistical graphs of serum CIV and HA levels, respectively; K is a statistical graph of hydroxyproline content in liver tissue. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional molecular biology, cell biology, and animal experimental methods in the art. All animal experimental protocols were approved by the ethics committee and strictly followed. The GRPEL1 protein involved in this invention has the amino acid sequence shown in SEQ ID NO: 1; the nucleotide sequence (CDS region) of its encoding gene is shown in SEQ ID NO: 2.

[0027] The amino acid sequence of the GRPEL1 protein (SEQ ID NO: 1)

[0028] MAARCVRLARRSLPALALSFRPSPRLLCTATKQKNNGQNLDEDLGHCEPKTDPPSADKTLLEEKAKLEEQLRETMEKYKRALADTENLRQRSQKLVEEAKLYGIQGFC KDLLEVADILEKATQSVPKEEISNNNPHLKSLYEGLVMTEVQIQKVFTKHGLLRLDPIGAKFDPYEHEALFHTPVEGKEPGTVALVSKVGYKLHGRTLRPALVGVVKDA

[0029] The nucleotide sequence of the gene encoding the GRPEL1 protein (SEQ ID NO: 2)

[0030] ATGGCGGCTCAGTGCGTGAGGTTGGCGGCGCAGTCTTCCTGCTTTGGCGTTGTCTCTCAGGCCATCTCCCCGGTTGTTGTGCACAGCCACGAAACAAAAGAACAGTGGCCAGAACCTGGAAGAGGACATGGGTCAGAGTGAACAGAAGGCAGATCCTCCTG CTACAGAGAAGACCCTCCTGGAAGAGAAGGTCAAGTTGGAGGAACAGCTGAAGGAGACTGTGGAAAAATATAAACGAGCTTTGGCAGACACTGAGAACTTACGGCAGAGGAGCCAGAAATTGGTGGAGGAGGCAAAATTATACGGCATTCAAGCCTTCTGCAAG GACTTGTTGGAGGTGGCAGACGTTCTGGAGAAGGCAACACAGTGTGTTCCAAAAGAAGAAATTAAAGACGATAACCCTCACCTGAAGAACCTCTATGAGGGGCTGGTCATGACTGAAGTCCAGATCCAGAAGGTGTTCACAAAAGCATGGCTTGCTCAAGTTGA ACCCTGTCGGAGCCAAGTTCGACCCTTATGAACATGAGGCCTTGTTCCACACACCGGTTGAGGGGAAGGAGCCAGGCACAGTGGCCCTAGTTAGCAAAGTGGGGTACAAGCTGCATGGGCGCACTCTGAGACCCGCCCTGGTGGGGGTGGTGAAGGAAGCTTAG

[0031] Statistical analysis:

[0032] All experimental data in this invention are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPadPrism software. Student's t-test was used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups, combined with Tukey's or Dunnett's multiple comparison test. Two-way ANOVA was used for CETSA data. A p-value < 0.05 was considered statistically significant.

[0033] Example 1: Evolution of GRPEL1 expression in the progression of MASLD and the promoting effect of knockdown on disease course

[0034] 1 Experimental Methods

[0035] 1.1 Animal Model Construction and Processing

[0036] Eight-week-old male ICR mice (purchased from the Experimental Animal Center of Nantong University) were housed in an SPF-grade environment with a temperature of 21–25°C, relative humidity of 50–60%, and a 12-hour light / dark cycle. Two weeks before dietary induction, mice were injected via tail vein with recombinant adeno-associated virus (rAAV-TBG) carrying a hepatocyte-specific thyroxine-binding globulin promoter. The core target sequence of the virus mediating the knockdown of the mouse Grpel1 gene (rAAV8-TBG-shGrpel1) was 5'-GCCAAGTTCGACCCTTACGAA-3'; the sequence of the negative control virus (rAAV8-TBG-shNC) was 5'-TTCTCCGAACGTGTCACGT-3'. The injection dose was 2 × 10⁻⁶. 11 vg / mouse. The model group mice were fed a high-fat, high-sugar diet containing 1.25% cholesterol (catalog number: TD.160785.PWD, purchased from Envigo) for different weeks to simulate the disease progression of MASLD.

[0037] 1.2 Isolation of primary hepatocytes

[0038] After dietary intervention, primary mouse hepatocytes were isolated using a two-step collagenase perfusion method. Mice were anesthetized, and blood was removed by injecting calcium-free buffer via the inferior vena cava. Subsequently, in situ perfusion was performed at 37°C using 0.5 mg / mL type IV collagenase (catalog number: C4-28, purchased from Sigma-Aldrich). The digested liver was excised, dispersed, filtered, and hepatocytes were collected by centrifugation, ensuring cell purity greater than 95%.

[0039] 1.3 Biochemical indicators and immunoblotting detection

[0040] Mouse serum was collected, and the activities of alanine aminotransferase (ALT, catalog number: C009-2-1) and aspartate aminotransferase (AST, catalog number: C010-2-1) were measured strictly according to the kit instructions (Nanjing Jiancheng). Total protein was extracted from liver tissue and primary hepatocytes, and immunoblotting was performed using 1:1000 dilution of anti-GRPEL1 primary antibody (catalog number: 12720-1-AP, purchased from Proteintech) and 1:10000 dilution of anti-Actin primary antibody (catalog number: sc-47778, purchased from Santa Cruz Biotechnology).

[0041] 1.4 Organizational Morphological Assessment

[0042] Liver tissue was fixed in 4% paraformaldehyde for 24 h. Some tissue samples were dehydrated, embedded in paraffin, and prepared into 4 μm sections for H&E staining. NAFLD activity scores (NAS scores) were calculated by double-blind researchers based on criteria of fatty degeneration (0–3), intralobular inflammation (0–3), and ballooning degeneration (0–2). Another portion of the fixed tissue was dehydrated in 30% sucrose, embedded in OCT, and cut into 8 μm frozen sections for Oil Red O staining to visualize neutral lipid droplets.

[0043] 2. Experimental Results

[0044] The experiment used Western blotting to dynamically detect changes in GRPEL1 protein abundance during pathological progression. Data showed that with increasing weeks of MASLD-induced dietary treatment, the level of GRPEL1 protein in mouse liver tissue exhibited a progressively significant decrease. Figure 1 A). In primary hepatocytes isolated after MD diet treatment, the expression level of GRPEL1 protein was also significantly lost ( Figure 1 As shown in B). Based on the confirmed protein downregulation, in vivo gene knockdown experiments showed that after 4 weeks of MD diet induction, the serum ALT and AST activities of mice with hepatocyte-specific Grpel1 knockdown were significantly increased compared with the control virus group. Figure 1 C, 1D). Histological staining further showed that the liver tissue of knockdown mice exhibited more severe hepatocyte ballooning degeneration and structural damage in H&E staining (C, 1D). Figure 1 E), the NAS score subsequently deteriorated significantly ( Figure 1 F). Oil Red O staining visually confirmed that the reduction of GRPEL1 led to a significant increase in the pathological accumulation area of ​​neutral lipid droplets in hepatocytes. Figure 1 G).

[0045] 3. Experimental Conclusions

[0046] Metabolic stress triggers the pathological loss of GRPEL1 in liver tissue and primary hepatocytes, and the reduced abundance of GRPEL1 in hepatocytes is a key rate-limiting factor driving the aggravation of liver functional damage and the deterioration of lipid degeneration.

[0047] Example 2: Hepatocyte-specific overexpression of Grpel1 improves diet-induced liver injury

[0048] 1 Experimental Methods

[0049] 1.1 Animal Model Construction

[0050] A recombinant adeno-associated virus containing the complete GRPEL1 coding sequence was constructed using the rAAV8-TBG system. The full-length GRPEL1 coding sequence for overexpression in the insert vector is shown in SEQ ID NO: 2. A mouse model of hepatocyte-specific overexpression of the Grpel1 gene was established by intravenous injection of rAAV8-TBG-Grpel1 into 8-week-old male ICR mice via tail vein, with rAAV8-TBG-NC serving as a control. The injection dose was 2 × 10⁻⁶. 11 vg / mouse. Two weeks after viral infection, mice were fed a high-fat, high-sugar MD diet containing 1.25% cholesterol for 16 weeks to establish a MASLD model.

[0051] 1.2 Efficacy testing and pathological evaluation

[0052] After modeling, blood was collected from mice to separate serum, and ALT and AST activities were detected using a biochemical kit to assess liver function. Liver lobe tissue from the same location was fixed with 4% paraformaldehyde, and 4 μm paraffin sections were prepared for H&E staining and NAS score calculation. 8 μm frozen sections were prepared for Oil Red O staining to assess lipid droplet clearance.

[0053] 2. Experimental Results

[0054] In a 16-week MD diet-induced chronic liver injury model, overexpression of Grpel1 demonstrated a significant hepatoprotective effect. Biochemical assays showed that, compared with the model control group, serum ALT and AST activities in Grpel1-overexpressing mice were significantly reduced. Figure 2 (A, 2B) This suggests that the damage to hepatocytes has been effectively repaired. Regarding liver tissue morphology, H&E staining results showed a significant reduction in steatosis and ballooning degeneration in the liver tissue of overexpressing mice, with cells becoming more orderly arranged. Figure 2 C), the quantified NAS score showed a significant decrease compared to the model group ( Figure 2 D). Further tissue lipid-specific staining confirmed that upregulation of GRPEL1 could extremely effectively clear the large-scale accumulation of neutral lipid droplets in hepatocytes caused by a high-fat, high-sugar diet, significantly reducing the Oil Red O positive area. Figure 2 E).

[0055] 3. Experimental Conclusions

[0056] By specifically increasing the protein abundance of GRPEL1 in hepatocytes through genetic means, liver function damage caused by long-term metabolic stress can be effectively reversed and pathological lipid overload in the liver can be significantly alleviated.

[0057] Example 3: Overexpression of Grpel1 reduces inflammatory infiltration and fibrosis in liver tissue

[0058] 1 Experimental Methods

[0059] 1.1 Immunohistochemical staining

[0060] The 4 μm paraffin sections fixed in Example 2 were dewaxed and hydrated, and then subjected to antigen retrieval via microwave in citrate buffer (pH 6.0). After blocking non-specific binding sites with 3% BSA, the sections were incubated overnight at 4°C with anti-MCP-1 primary antibody (1:200 dilution; catalog number: GB11199, purchased from Servicebio) and anti-Ly6C primary antibody (1:800 dilution; catalog number: GB115601, purchased from Servicebio). Subsequently, they were incubated with HRP-conjugated secondary antibody (catalog number: G1213, purchased from Servicebio), developed using a DAB substrate kit, and counterstained with hematoxylin.

[0061] 1.2 Quantitative analysis of biochemical indicators of fibrosis

[0062] Following the operating instructions of the commercially available kit (Nanjing Jiancheng), the concentrations of type IV collagen (CIV; catalog number: H145-1-2), laminin (LN; catalog number: H148-1-2), and hyaluronic acid (HA; catalog number: H141-1-2) in the serum of mice in each group were measured. Simultaneously, an appropriate amount of liver tissue homogenate was weighed, and the absolute content of hydroxyproline in the liver tissue was quantitatively determined using a kit (A030-2-1).

[0063] 2. Experimental Results

[0064] In the assessment of core pathological markers in the progression stage of MASLD, upregulation of GRPEL1 also played a significant interventional role. Immunohistochemical staining results showed that overexpression of Grpel1 significantly reduced the positive expression area of ​​chemokine MCP-1 in the liver tissue of model mice. Figure 3 A), and significantly inhibited the infiltration of Ly6C-positive inflammatory monocytes / macrophages in the liver parenchyma ( Figure 3 B). In systemic and local antifibrotic assessments, the serum levels of CIV, LN, and HA, markers reflecting fibrotic activity, were significantly lower in overexpressing mice compared to the model group. Figure 3 C~3E); at the same time, the content of hydroxyproline, a key biochemical indicator reflecting the total amount of collagen deposition in the liver, was also significantly inhibited (C~3E); Figure 3 F).

[0065] 3. Experimental Conclusions

[0066] Targeted upregulation of GRPEL1 can effectively block the inflammatory cascade and local immune cell infiltration in the process of MASLD, and significantly reduce collagen deposition and fibrotic damage in liver tissue.

[0067] Example 4: Physical binding and stability identification of imperatorin as a GRPEL1 targeting activator

[0068] 1 Experimental Methods

[0069] 1.1 Molecular docking simulation

[0070] Binding modes were analyzed using Molecular Operating Environment (MOE) 2019 software. The crystal structure of human GRPEL1 (ID: 9BLS) was downloaded from the PDB database, and the three-dimensional structure of imperatorin (CID: 15296614) was downloaded from PubChem. After removing water molecules, the ligands were subjected to energy minimization using the Amber10:EHT force field, and docking was performed using the Protonate3D module to output the lowest binding energy conformation.

[0071] 1.2 Cell thermal migration analysis (CETSA)

[0072] AML12 mouse hepatocytes were cultured in DMEM / F-12 medium containing 10% FBS. Cells were incubated with 20 μM imperatorin or DMSO-containing solvent for 1 h, then resuspended in PBS containing protease inhibitors and aliquoted. Each tube was heated at a temperature gradient of 50–70 °C for 3 min, followed by cooling at 4 °C for 3 min. Cells were lysed by three liquid nitrogen / 37 °C freeze-thaw cycles. Soluble proteins were extracted by centrifugation at 20,000 × g for 20 min at 4 °C and analyzed by Western blotting.

[0073] 1.3 In vivo targeted intervention

[0074] Working solutions were prepared by dissolving ≥98% pure imperatorin (catalog number: TN7760, purchased from TargetMol) in 5% DMSO, 40% PEG300, 5% Tween 80, and 50% physiological saline. MD-fed mice were intraperitoneally injected daily with imperatorin (20 mg / kg) or an equivalent volume of solvent for 16 consecutive weeks. Liver tissue proteins were extracted to verify the expression abundance of GRPEL1.

[0075] 2. Experimental Results

[0076] Structural biology simulations based on molecular docking showed that imperatorin could precisely fit into the binding pocket of the GRPEL1 protein, exhibiting a highly matched physical binding conformation, with a docking energy as low as -4.7105 kcal / mol. Figure 4A). CETSA experiments in a live cell system further confirmed that, under a set temperature gradient, treatment with imperatorin significantly increased the thermosoluble proportion of GRPEL1 protein in AML12 cells, confirming that the two are physically bound intracellularly and enhance the thermal stability of the target protein. Figure 4 B). Based on this mechanism, long-term in vivo drug intervention experiments showed that the intervention of imperatorin successfully and significantly reversed the loss of target proteins induced by the MD diet, and the protein expression abundance of GRPEL1 in mouse liver tissue was significantly increased. Figure 4 C).

[0077] 3. Experimental Conclusions

[0078] Imperatorin is a specific target activator of GRPEL1. It significantly enhances the expression stability of GRPEL1 in vitro and in vivo by directly physical binding to inhibit the degradation of the target protein under pathological conditions.

[0079] Example 5: Verification of the specific dependence of the efficacy of angelica dahurica in improving liver damage on the GRPEL1 target.

[0080] 1 Experimental Methods

[0081] 1.1 Targeted Blockade Model and Drug Administration

[0082] Male ICR mice were divided into a control group (injected with rAAV8-TBG-shNC) and a specific knockdown group (injected with rAAV8-TBG-shGrpel1). After the model was established, all mice were fed an MD diet and simultaneously received daily intraperitoneal injections of 20 mg / kg of Impatiens balsamina or an equivalent amount of solvent for 16 weeks.

[0083] 1.2 Comprehensive Pharmacodynamic Validation

[0084] After the intervention, liver tissue was harvested for immunoblotting analysis to verify the in vivo knockdown efficiency. Serum was extracted, and ALT and AST activities, as well as the levels of CIV and HA fibrosis markers, were measured according to the kit standards. Liver pathological sections were prepared, and H&E staining was used to assess NAS scores, while Oil Red O staining was used to assess lipid droplet area. Immunohistochemical staining was used to assess the expression of MCP-1 and Ly6C in liver tissue, and the absolute content of hydroxyproline in the liver was measured.

[0085] 2. Experimental Results

[0086] In vivo knockdown efficiency verification showed that injection of shGrpel1 virus successfully and stably inhibited the expression of GRPEL1 in mouse liver ( Figure 5A). In shNC mice that retained GRPEL1 expression, imperatorin exhibited significant efficacy, not only significantly reducing serum ALT and AST activities in the model mice ( Figure 5 B, 5C), and significantly improved fatty degeneration and reduced NAS score in terms of histological morphology. Figure 5 D, 5E), cleared the large-scale accumulation of neutral lipid droplets ( Figure 5 F). Simultaneously, the drug successfully inhibited MCP-1 and Ly6C-mediated inflammatory cell infiltration in shNC group mice. Figure 5 G, 5H), and significantly reduced serum CIV, HA levels and liver tissue hydroxyproline content (G ...). Figure 5 (I~5K). However, in mice with specifically knocked-down Grpel1, the above-mentioned effects of imperatorin were significantly blocked, and their serum transaminase levels, histopathological damage, inflammatory factor infiltration range, and various fibrosis markers were consistent with the untreated knockdown model group, showing no signs of improvement.

[0087] 3. Experimental Conclusions

[0088] The comprehensive protective effects of imperatorin in improving liver tissue damage, resisting lipid accumulation, anti-inflammation and anti-fibrosis are highly and specifically dependent on the presence of the GRPEL1 target.

[0089] In summary, this invention, through in vitro and in vivo experiments, demonstrates that the loss of GRPEL1 expression is closely related to the progression of liver damage in metabolic dysfunction-associated fatty liver disease (MASLD). Upregulating its protein abundance can significantly reduce lipid accumulation, inflammatory infiltration, and fibrosis in liver tissue, indicating that GRPEL1 is an effective pharmacodynamic target for intervention in MASLD. Furthermore, this invention reveals that the natural monomer imperatorin can directly physically bind to GRPEL1 and enhance its thermal stability. Animal experiments confirm that the ameliorative effect of imperatorin on liver function damage, lipid metabolism disorders, and tissue fibrosis in MASLD model mice specifically depends on the presence of the GRPEL1 target. The implementation of this invention provides experimental evidence with a clear mechanism of action and candidate drug molecules for targeted therapy of MASLD.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention; however, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of GRPEL1 protein or its encoding gene in the preparation of drugs for the prevention or treatment of fatty liver disease related to metabolic dysfunction.

2. The application according to claim 1, characterized in that, The amino acid sequence of the GRPEL1 protein is shown in SEQ ID NO: 1; the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. The use of imperatorin or its pharmaceutically acceptable salt in the preparation of drugs for the prevention or treatment of fatty liver disease related to metabolic dysfunction.

4. The application according to claim 1 or 3, characterized in that, The metabolic dysfunction-related fatty liver disease includes simple hepatic fat accumulation, metabolic dysfunction-related steatohepatitis, and liver fibrosis.

5. The application according to claim 3, characterized in that, The drug mentioned for the prevention or treatment of fatty liver disease related to metabolic dysfunction is used to increase the abundance of GRPEL1 protein in hepatocytes and improve liver damage.

6. The application according to claim 5, characterized in that, The purpureusol or its pharmaceutically acceptable salt enhances the thermal stability of the GRPEL1 protein and inhibits its protein-level degradation under pathological conditions by non-covalently binding to the GRPEL1 protein, thereby exerting a therapeutic effect specifically dependent on the GRPEL1 target.

7. The application according to claim 3, characterized in that, The prevention or treatment manifests as one or more of the following effects: (1) reducing the activity of alanine aminotransferase and / or aspartate aminotransferase in serum; (2) improving hepatic steatosis and reducing or clearing the pathological accumulation of neutral lipid droplets in hepatocytes; (3) alleviating liver inflammation, inhibiting the expression of monocyte chemotactic protein-1 in liver tissue, and / or inhibiting the infiltration of Ly6C positive inflammatory cells; (4) improving liver fibrosis, reducing the levels of type IV collagen, laminin and / or hyaluronic acid in serum, or reducing the content of hydroxyproline in liver tissue.

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