Use of arginine or polyamine metabolites for the preparation of a medicament for preventing, alleviating or treating copper death-related diseases

Abstract

The application discloses application of arginine or polyamine metabolites in preparation of medicines for preventing, alleviating or treating copper death related diseases, and specifically discloses that arginine or polyamine metabolites can activate polyamine synthesis metabolic pathways, up-regulate expression of key enzymes in polyamine synthesis, restore Fe-S protein homeostasis caused by copper overload, thereby antagonizing copper death, promoting copper discharge from the liver, and reducing tissue damage and inflammation. ‑ / ‑ In a Wilson disease model mouse, arginine intervention significantly improves liver tissue pathological morphology, reduces transaminase and inflammatory factor levels, and reduces liver copper accumulation. The application provides a safe and effective treatment strategy based on metabolic regulation for Wilson disease and other copper metabolism disorder diseases.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to the application of arginine or polyamine metabolites in the preparation of drugs for the prevention, relief or treatment of copper death-related diseases. Background Technology

[0002] Copper is an essential trace element for organisms, playing a crucial role in various physiological processes. However, an imbalance in intracellular copper ion homeostasis can lead to severe cytotoxicity. In recent years, research has discovered a unique regulatory cell death mechanism induced by intracellular copper ion overload—cuproptosis. Its key characteristics include oligomerization of lipoyl-modified proteins (such as DLAT) in mitochondria, and downregulation and loss of function of iron-sulfur cluster (Fe-S) proteins (such as ACO2 and FDX1).

[0003] Wilson's disease (WD) is an autosomal recessive genetic disorder caused by a mutation in the ATP7B gene. It is characterized by the pathological accumulation of copper in vital organs such as the liver and brain, ultimately leading to progressive liver damage, cirrhosis, and neurodegenerative changes. Currently, clinical treatment for Wilson's disease primarily relies on a low-copper diet and copper chelators (such as penicillamine and tricentylin). However, long-term use of chelators has serious toxic side effects (such as kidney damage and worsening of neurological symptoms), and its efficacy is limited for some advanced-stage patients.

[0004] Although existing studies have suggested that certain polyamine compounds (such as trientine, which has polyamine structural features) have the potential to antagonize copper death, direct supplementation of polyamines (such as spermidine and spermine) faces multiple challenges in clinical application. First, natural polyamines are easily oxidized in vitro, making formulation development difficult and costly. Second, high concentrations of exogenous polyamines have significant cytotoxicity, and direct supplementation often struggles to balance safety and efficacy. Finally, simply supplementing downstream products may lead to imbalances in intracellular feedback regulation, making it difficult to maintain long-term metabolic homeostasis.

[0005] Therefore, developing a novel drug regimen that can effectively antagonize copper death, has high safety and low cost, and can improve copper metabolism disorders through endogenous regulation is a technical challenge that urgently needs to be solved in the current biomedical field. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of arginine or polyamine metabolites in the preparation of medicaments for the prevention, relief or treatment of copper death-related diseases.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of arginine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, relief or treatment of copper death-related diseases, wherein the arginine antagonizes copper death by activating a polyamine anabolic pathway.

[0009] Preferably, the copper-related death disease is Wilson's disease, copper metabolism disorder, or neurodegenerative disease associated with copper accumulation.

[0010] Preferably, arginine activates endogenous polyamine biosynthesis by upregulating the expression of key enzymes in polyamine synthesis, including arginase 1, ornithine decarboxylase 1, S-adenosylmethionine decarboxylase 1, spermine synthase, and spermine synthase.

[0011] Preferably, arginine restores the downregulation of Fe-S protein expression caused by copper overload by activating the polyamine synthesis pathway, wherein the Fe-S protein includes POLD1, ACO2 and FDX1.

[0012] Preferably, the drug is used to alleviate liver tissue damage and inflammation, including improving hepatocyte pathological morphology, reducing serum alanine aminotransferase and aspartate aminotransferase levels, and downregulating the expression of local liver pro-inflammatory cytokines IL-6, IL-1β and TNF-α.

[0013] Preferably, the drug is used to promote the excretion of copper from the liver and reduce the copper content in liver tissue.

[0014] In a second aspect, the present invention provides the use of polyamine metabolites of arginine or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention, relief or treatment of copper death-related diseases, said polyamine metabolites including ornithine, putrescine, spermidine and spermine.

[0015] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier, diluent, or excipient, wherein the active ingredient is selected from one or more of arginine, ornithine, putrescine, spermidine, spermine, and their pharmaceutically acceptable salts.

[0016] Preferably, the pharmaceutical composition is a tablet, capsule, granule, oral liquid, or injection.

[0017] Preferably, the pharmaceutical composition further comprises a copper chelating agent, an anti-inflammatory drug, or a hepatoprotective drug, wherein the copper chelating agent is penicillamine or trientine.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention reveals for the first time the complete mechanism by which arginine exerts its protective effect through the arginine-polyamine anabolic metabolic axis. Unlike traditional chelators that rely solely on chemical copper excretion, this invention specifically maintains the homeostasis of Fe-S proteins (such as ACO2 and FDX1) through endogenous metabolic reprogramming and precisely promotes copper excretion from liver tissue, providing a novel metabolic regulation and treatment strategy for copper metabolism disorders. Both cell and animal experiments have confirmed that arginine and its polyamine metabolites can significantly antagonize copper death induced by copper overload. In Wilson's disease (WD) mouse models, the proposed approach not only effectively reduces serum transaminase (ALT, AST) and pro-inflammatory cytokine levels, alleviating hepatocyte edema and inflammatory infiltration, but also significantly reduces collagen fiber deposition and reverses the progression of liver fibrosis.

[0020] Compared to existing chelating agents in clinical practice such as penicillamine and tricentine, the arginine used in this invention, as an endogenous amino acid in the human body, has core advantages such as good biocompatibility, low toxicity and side effects, and a wide therapeutic window. Furthermore, arginine and its metabolites are widely available, have mature synthesis processes, and are inexpensive, greatly reducing the long-term medication burden for patients. Attached Figure Description

[0021] Figure 1 The figure shows the effect of arginine and its polyamine metabolites on the viability of copper-dead cells. Specifically, A and E represent the effect of arginine (arg) on ​​Es / Cu-induced Huh-7 cell viability, while polyamine synthetase inhibitors (nor-NOHA, DFMO, MCHA, APCHA, SAM486A) can weaken or even eliminate the copper-dead rescuing function of arginine; F represents ornithine rescuing copper death, but the ornithine decarboxylase inhibitor DFMO weakens this function; G represents putrescine rescuing copper death, but the spermidine synthase inhibitor MCHA weakens this function; H represents SAM rescuing copper death, but the SAM decarboxylase inhibitor SAM486A weakens this function; and I represents spermine rescuing copper death.

[0022] Figure 2 The graph shows the effect of copper overload on intracellular polyamine metabolism levels and synthase expression; AD are bar charts showing the changes in the relative contents of arginine, putrescine, spermidine, and spermine in cells after copper treatment; EF are Western blotting images showing the effect of arginine supplementation on the protein expression of polyamine synthetases (ARG1, ODC1, AMD1, SRM, SMS).

[0023] Figure 3The image shows the Western Blot bands representing the effects of arginine combined with a polyamine synthase inhibitor on Fe-S protein levels. In this image, A represents the results of combined application of the arginase inhibitor nor-NOHA; B represents the results of combined application of the ODC1 inhibitor DFMO; C represents the results of combined application of the AMD1 inhibitor SAM486A; D represents the results of combined application of the SRM inhibitor MCHA; and E represents the results of combined application of the SMS inhibitor APCHA.

[0024] Figure 4 The images show the effects of arginine intervention on the pathological morphology of liver tissue in WD model mice; where A is the HE staining image (100× and 400×) of liver tissue from each group of mice; and B is the Masson staining image (100× and 400×) of liver tissue from each group of mice.

[0025] Figure 5 The bar chart shows the effects of arginine on serum biochemistry and inflammatory factors in WD model mice; where A represents serum alanine aminotransferase (ALT) level; B represents serum aspartate aminotransferase (AST) level; C represents liver tissue IL-6 level; D represents liver tissue TNF-α level; and E represents liver tissue IL-1β level.

[0026] Figure 6 The graph shows the effect of arginine on copper distribution in WD model mice; where A is a bar chart of copper content in liver tissue; B is a bar chart of copper content in serum; and C is a morphological observation of copper staining in liver tissue, with the orange-yellow color indicated by the arrow representing positive copper staining (400×).

[0027] Figure 7 The figure shows the effect of arginine on polyamine metabolic homeostasis in liver tissue of WD model mice; where AD are bar charts showing the relative contents of arginine, putrescine, spermidine and spermine in liver tissue, respectively; EF is a Western blot chart of polyamine synthesis metabolic enzyme protein expression in liver tissue.

[0028] Figure 8 The image shows the effect of arginine on the Fe-S protein level in the liver tissue of WD model mice, illustrating the expression of POLD1, ACO2, and FDX1 proteins. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments.

[0030] To verify the mechanism by which arginine antagonizes copper death by activating the polyamine biosynthesis pathway, this invention sequentially conducted cellular efficacy verification (Example 1), cellular mechanism studies (Examples 2-3), and animal-level therapeutic efficacy verification (Example 4). The cellular experiments used an Es / Cu-induced Huh-7 copper death cell model; the animal experiments used ATP7B... - / - In Wilson's disease model mice, arginine was administered by gavage at a dose of 300 mg / kg / day for 8 consecutive weeks.

[0031] The following is a detailed explanation.

[0032] Example 1: Arginine rescues copper death in a polyamine anabolic dependence.

[0033] 1.1 Cell Culture Conditions

[0034] 1.2 Preparation of Es / Cu treatment solution: Ellisclomol (Es; GLPBIO, GC13885) was dissolved in dimethyl sulfoxide (DMSO), and copper chloride (Sigma-Aldrich, 222011) (CuCl2, Cu) was dissolved in pure water to prepare a high-concentration stock solution. Immediately before use, the above solutions were used to prepare an equimolar concentration 250 nM mixed solution (Es / Cu) to induce copper death in Huh-7 cells.

[0035] 1.3 Arginine and Polyamine Treatment: Arginine (MCE, HY-N0455), ornithine (MCE, HY-B1352), putrescine (MCE, HY-N2407), spermidine (MCE, HY-B1776), and spermine (MCE, HY-B1777) were all purchased from MCE Company. Arginase 1 (ARG1) inhibitor Nω-hydroxy or L-arginine hydrochloride (nor-NOHA) was purchased from GlpBio, catalog number GC10628. Ornithine decarboxylase (AMD1) inhibitor efornithine (DFMO; MCE, HY-B0744), S-adenosylmethionine decarboxylase 1 (AMD1) inhibitor (SAM486A; MCE, HY-13746), spermine synthase (SRM) inhibitor trans-4-methylcyclohexylamine (MCHA; MCE, HY-W010538), and spermine synthase (SMS) inhibitor N-(3-aminopropyl)cyclohexylamine (APCHA; MCE, HY-W015514) were all purchased from MCE.

[0036] Huh-7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (Sangon Biotech, E600001). Cells were treated with equimolar amounts of 0.25 μM irimol and copper chloride (Es / Cu) alone or with different concentrations of arginine, ornithine, putrescine, and spermine in combination. Cell viability was assessed using the CCK-8 assay.

[0037] 1.4 CCK-8 testing steps:

[0038] Huh-7 cells of passages 3-7 with good cell morphology and growth rate were seeded into 96-well plates at a density of 30% per well, with at least three replicates per group. PBS was added to the outermost well to prevent culture medium evaporation. The 96-well plates were then incubated at 37°C with 5% CO2. Cell culture continued until the cell density reached approximately 40%–50%.

[0039] The intervention was performed as described above. After 24 hours, 100 μL of complete culture medium containing 10% CCK8 reagent was added to each well by changing the medium. The 96-well plate containing the CCK8 reaction solution was then placed in a 37°C incubator for about 20 minutes in the dark with aluminum foil. The OD value was then detected using a multi-functional microplate reader set to a wavelength of 450 nm.

[0040] 1.5 Results Analysis:

[0041] like Figure 1 As shown, compared with the Es / Cu treatment group, the cell viability increased by 33.24% in the 80 μM arginine treatment group, 35.02% in the 100 μM ornithine treatment group, 35.66% in the 80 μM putrescine treatment group, and 27.11% in the 20 μM spermine treatment group, all of which were statistically significant (P < 0.05). Inhibitors of polyamine biosynthesis enzymes, including the ARG1 inhibitor nor-NOHA, the AMD1 inhibitor DFMO, the AMD1 inhibitor SAM486A, the SRM inhibitor MCHA, and the SMS inhibitor APCHA, all inhibited the copper-rescuing function of arginine. DFMO and MCHA also inhibited the copper-rescuing function of ornithine and putrescine, respectively. These results indicate that arginine and its polyamine metabolic products, ornithine, putrescine, and spermine, can effectively rescue copper death. Arginine rescues copper death in a polyamine biosynthesis-dependent manner.

[0042] Example 2: Detection of polyamine metabolites and synthases

[0043] 2.1 Sakaguchi Reaction for Arginine Detection: After drug treatment, cells in 6-well plates were washed twice with 1× phosphate-buffered saline (PBS), digested with trypsin, and collected by centrifugation at 12,000 rpm for 5 minutes at 4°C. The precipitate was resuspended in 50 μL of 2% perchloric acid, sonicated on ice (30% amplitude, 1 minute total time, 2 seconds working / 2 seconds rest), and then incubated on a 360° rotating shaker at 4°C for 2 hours. The lysis buffer was centrifuged at 12,000 rpm for 45 minutes at 4°C, and the supernatant was collected.

[0044] Take 40 μL of the sample to be tested or arginine standard, add 40 μL of sodium hydroxide-urea solution, heat in a boiling water bath for 6 minutes, then cool on ice; add 80 μL of freshly prepared Sakaguchi reagent, and incubate at room temperature in the dark for 20 minutes. Add 80 μL of fresh sodium hypochlorite solution, vortex vigorously for 30 seconds to start color development; then add 80 μL of 10% hydrochloric acid to stop the reaction, let stand at room temperature for 2 minutes, and then briefly centrifuge. Measure the absorbance at 515 nm using a microplate reader, and calculate the arginine concentration based on the standard curve.

[0045] The results are as follows Figure 2 As shown in A, treatment with 250 nM Es / Cu for 24 h significantly reduced intracellular arginine content.

[0046] 2.2 Thin-layer chromatography for the detection of putrescine, spermidine, and spermine: The content of polyamines was determined by thin-layer chromatography (TLC). A simplified procedure is as follows: The prepared sample lysis buffer (or polyamine standard) was mixed with 18.6 mM dansyl chloride and supersaturated sodium carbonate solution at a volume ratio of 1:2:1. After thorough vortexing, the mixture was incubated at 60°C for 25 min. Then, 0.5 times the volume of 1.3 M L-proline solution was added, vortexed, and allowed to stand at room temperature for 30 min. Next, 2.5 times the volume of benzene solution was added, vortexed, and allowed to stand for 20 min. The mixture was then centrifuged at 12000 rpm for 10 min at room temperature to separate the upper organic phase.

[0047] 4 μL of organic phase was spotted onto a thin-layer chromatography plate: 2 μL was spotted in multiple spots, 1.5 cm from the bottom of the plate, with each spot allowed to dry in between. The spot diameter was controlled to be ≤2 mm and the spot spacing ≥1.5 cm. Cyclohexane:ethyl acetate = 3:2 (v / v) was used as the developing solvent, and the plate was developed in a pre-saturated, closed chromatography vessel for approximately 30 min (the solvent level was below the spotting line). Immediately after development, the solvent front was marked. After drying the thin-layer plate, it was placed under UV light for development and photographed. The spot gray values ​​were analyzed, normalized to the cell count, and the relative polyamine content was calculated.

[0048] The results are as follows Figure 2 As shown in BD, treatment with 250 nM Es / Cu for 24 h significantly reduced the levels of putrescine, spermine, and spermine in cells.

[0049] 2.3 Western Blot detection of ARG1, ODC1, AMD1, SRM, and SMS

[0050] Proteins were extracted from Huh-7 cells using pre-chilled IP cell lysis buffer. Protein samples were mixed with 5× loading buffer (Coolaber, SL1170) and boiled, followed by separation on a 4–20% polyacrylamide gel. The proteins were then transferred to a PVDF membrane (Merck Millipore, ISEQ00010). After blocking with 2.5% BSA, the membranes were incubated overnight at 4°C with the following primary antibodies: ARG1 (Proteintech, 15646-1-AP), AMD1 (Proteintech, 11052-1-AP), ODC1 (Proteintech, 67336-Ig), SRM (Proteintech, 19858-1-AP), SMS (Proteintech, 15979-1-AP), GAPDH (Proteintech, 60004-1-Ig), β-actin (ZSGB-BIO, TA-09), and α-tubulin (Abcam, ab18251). After washing, the membranes were incubated with the corresponding secondary antibodies: goat anti-rabbit IgG (ZSGB-BIO, ZB-5301) or goat anti-mouse IgG (ZSGB-BIO, ZB-5305). Finally, the protein signal was developed and acquired using a chemiluminescence detection kit (Bio-Rad, 1705061).

[0051] like Figure 2 As shown in the CD, compared with the solvent control group, the relative contents of arginine, putrescine, spermidine, and spermine in the Es / Cu treatment group decreased by 96.51%, 53.77%, 34.48%, and 27.83%, respectively, with statistically significant differences (P < 0.05). Western blot results showed that Es / Cu treatment significantly downregulated the protein expression of ARG1, ODC1, AMD1, SRM, and SMS in Huh-7 cells, and exogenous arginine supplementation could reverse the downregulation of these enzymes caused by Es / Cu, restoring them to near-normal levels. The results indicate that copper overload inhibits polyamine metabolism, and arginine can restore the expression of polyamine synthases.

[0052] Example 3: Detection of Fe-S protein levels

[0053] 3.1 Protein extraction: Same as in Example 2.

[0054] 3.2 Western Blot Detection of POLD1, ACO2, and FDX1

[0055] The primary antibodies used were: Aconitase 2 (ACO2, Proteintech, 11134-1-AP), FDX1 (Abmart, T510671F), and POLD1 (Proteintech, 15646-1-AP).

[0056] 3.3 Polyamine synthase inhibitor combined treatment experiment: Same as Example 1.

[0057] like Figure 3 As shown, Es / Cu treatment significantly downregulated the expression levels of Fe-S proteins POLD1, ACO2, and FDX1 in Huh-7 cells; exogenous arginine supplementation significantly reversed this downregulation. Combined application of polyamine synthase inhibitors nor-NOHA, DFMO, SAM486A, MCHA, and APCHA on top of arginine could partially or completely eliminate the function of arginine in restoring Fe-S protein levels. These results indicate that arginine maintains Fe-S protein homeostasis through the polyamine biosynthesis pathway.

[0058] Example 4: Arginine Intervention Experiment in Wilson's Disease Model Mice

[0059] 4.1 Animal grouping and administration

[0060] With homozygous ATP7B - / - Mice serve as a model animal for Wilson's disease. ATP7B - / - The mice were donated by Professor Shi Junping's research group at the Affiliated Hospital of Hangzhou Normal University. The mice were bred and raised at the Experimental Animal Center of Hangzhou Normal University. Experimental animal use license number: SYXK (Zhejiang) 2025-0037. All animal experiments in this invention complied with the animal welfare and ethics requirements of Hangzhou Normal University. 12-week-old ATP7B mice - / - Mice were randomly divided into an arginine intervention group (WD-Arg, 300 mg / kg / day by gavage) and a solvent control group (WD-NS, equal volume of physiological saline), with wild-type mice (WT) of the same strain serving as blank controls. Each group consisted of 7-10 mice. Treatment lasted 8 weeks, with body weight recorded weekly. Mice were euthanized 24 hours after the last administration, and serum, liver, and brain tissue were collected for analysis.

[0061] 4.2 Liver histopathological examination (HE, Masson)

[0062] like Figure 4As shown, H&E staining of liver tissue revealed typical liver injury characteristics in WD model mice. Compared with the WT group, the WD-NS group showed diffuse hepatocyte edema and ballooning degeneration, with abundant inflammatory cell infiltration in the portal areas. In contrast, the WD-Arg group showed significantly reduced hepatocyte degeneration and inflammatory infiltration. Masson staining confirmed significant collagen fiber hyperplasia in the liver of the WD-NS group, indicating liver fibrosis; in contrast, the WD-Arg group showed a significantly reduced area of ​​collagen fiber deposition, effectively alleviating the degree of fibrosis. These results indicate that arginine intervention improved the pathological morphology of liver tissue in WD model mice, reduced inflammatory cell infiltration, and decreased the level of liver fibrosis.

[0063] 4.3 Serum biochemical marker detection (ALT, AST)

[0064] A portion of liver tissue was taken and added to physiological saline to prepare a 10% tissue homogenate. After centrifugation, the supernatant was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a fully automated biochemical analyzer (HITACHI 3100).

[0065] like Figure 5 As shown in AB, compared with the WD-NS group mice, the activities of ALT and AST in the serum of the WD-Arg group were significantly reduced (P < 0.05).

[0066] 4.4 Detection of inflammatory factors (IL-6, IL-1β, TNF-α)

[0067] The levels of IL-6, IL-1β and TNF-α inflammatory factors were detected by ELISA. All test kits were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., with catalog numbers Cat.#mI063159, Cat.#mI106733 and Cat.#mI1002095, respectively.

[0068] like Figure 5 As shown in the CE diagram, the levels of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in the liver tissue homogenate supernatant of the WD-Arg group were significantly lower than those of the WD-NS group (P < 0.05).

[0069] The above results indicate that arginine intervention effectively alleviated hepatocyte damage and reduced local inflammatory response in the liver in WD model mice.

[0070] 4.5 Detection of copper content in liver tissue (ICP-MS, tissue copper staining)

[0071] 4.5.1 Determination of Copper Content by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[0072] Accurately transfer 50 mg of tissue sample and 100 μL of serum into a clean, dry polytetrachloroethylene (PTFE) microwave tube. Then add 10 mL of analytical grade concentrated nitric acid, mix thoroughly, and shake for 1 min. After acid removal, allow the microwave tube to cool completely before capping. Afterward, place the PTFE digestion tube in a 120°C reactor for pre-digestion for 20 min. Stop digestion when a small amount of yellow fumes is observed, then remove and cool to room temperature.

[0073] Add 5 mL of high-purity concentrated nitric acid to the microwave tube that has been digested, tighten the cap, and then add it to the microwave digester. Digest the tube according to the preset digestion temperature program.

[0074] After digestion, allow the temperature to drop to room temperature, then slowly open the lid. Next, place the digestion vessel at 180°C for 30 minutes to remove the acid. Continue heating until only about 1 mL of liquid remains and the digest is clear and transparent. Remove the vessel and allow it to cool to room temperature.

[0075] Finally, the digested sample was diluted three times with ultrapure water to a final volume of 50 mL in centrifuge tubes. The mixture was shaken for 1 min, and then filtered through a 0.22 μm pore size membrane to remove impurities. The sample was diluted according to its concentration before analysis. Copper ion detection was performed using inductively coupled plasma mass spectrometry (ICP-MS). A standard curve was also plotted for quantification.

[0076] 4.5.2 Rhodanine copper staining to observe copper distribution in liver tissue

[0077] Liver tissue sections were dewaxed to water and briefly washed with 95% ethanol. The sections were then immersed in rhodanine staining solution, sealed, and stained at 37°C for 24 hours or in a 60°C water bath for 3 hours. The sections were then removed, briefly washed with 95% ethanol, and rinsed with running water for 10 seconds. Hematoxylin (Mayer) staining solution was added for counterstaining for 2 minutes, followed by rinsing with running water for 3 minutes. The sections were then routinely dehydrated and cleared, mounted with mounting adhesive, and examined under a microscope.

[0078] like Figure 6 As shown, compared with the WD-NS group, the copper content in the liver tissue of mice in the WD-Arg group was significantly decreased, while the copper content in the serum did not change significantly. Tissue copper staining results showed a significant reduction in copper granules in the WD-Arg group, consistent with the decrease in tissue copper content. These results indicate that arginine intervention can effectively reduce copper accumulation in the liver and promote copper excretion from the liver.

[0079] 4.6 Detection of polyamine metabolites in liver tissue

[0080] The contents of arginine and polyamines in liver tissue were detected by Sakaguchi reaction and wavelet chromatography, respectively.

[0081] like Figure 7 As shown, compared with the wild-type mouse (WT) group, the WD model mice showed an increase in arginine, an upstream metabolite of polyamine synthesis in the liver, while the content of putrescine remained unchanged, and the content of downstream products spermidine and spermine was significantly decreased. The arginine content in the liver of the arginine intervention group was not different from that of the WT group. Compared with the WD-NS group, the WD-Arg group showed a decrease in putrescine, an upstream metabolite of polyamine synthesis, while the content of downstream products spermidine and spermine increased. Western blot results showed that arginine upregulated the levels of polyamine metabolic enzymes. These results indicate that arginine reduces the content of arginine and putrescine, upstream metabolites of polyamine metabolism, and increases the content of downstream products spermidine and spermine in the WD model mice, thus maintaining polyamine metabolic homeostasis.

[0082] 4.7 Detection of Fe-S protein in liver tissue

[0083] like Figure 8 As shown, compared with the WD-NS group, the WD-Arg group showed increased expression levels of ACO2 and FDX1 in the liver, with statistically significant differences (P < 0.05). These results indicate that arginine intervention helps improve impaired Fe-S protein homeostasis in the Wilson's disease mouse model.

Claims

1. The use of arginine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, relief or treatment of copper-related death diseases, characterized in that, Arginine antagonizes copper death by activating the polyamine anabolic pathway.

2. The application according to claim 1, characterized in that, The copper-related death diseases mentioned are Wilson's disease, copper metabolism disorders, or neurodegenerative diseases associated with copper accumulation.

3. The application according to claim 1, characterized in that, Arginine activates endogenous polyamine biosynthesis by upregulating the expression of key enzymes in polyamine synthesis, including arginase 1, ornithine decarboxylase 1, S-adenosylmethionine decarboxylase 1, spermine synthase, and spermine synthase.

4. The application according to claim 3, characterized in that, Arginine restores the downregulation of Fe-S protein expression caused by copper overload by activating the polyamine synthesis pathway. The Fe-S proteins include POLD1, ACO2, and FDX1.

5. The application according to claim 1, characterized in that, The drug is used to reduce liver tissue damage and inflammation, including improving hepatocyte pathological morphology, reducing serum alanine aminotransferase and aspartate aminotransferase levels, and downregulating the expression of local liver pro-inflammatory cytokines IL-6, IL-1β and TNF-α.

6. The application according to claim 5, characterized in that, The drug is used to promote the excretion of copper from the liver and reduce the copper content in liver tissue.

7. The use of polyamine metabolites of arginine or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention, relief or treatment of copper death-related diseases, characterized in that, The polyamine metabolites include ornithine, putrescine, spermidine, and spermine.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier, diluent, or excipient, wherein the active ingredient is selected from one or more of arginine, ornithine, putrescine, spermidine, spermine, and their pharmaceutically acceptable salts.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, granules, oral liquids, or injections.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further comprises a copper chelating agent, an anti-inflammatory drug, or a hepatoprotective drug, wherein the copper chelating agent is penicillamine or trientine.

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