Application of guanidinoacetic acid in preparation of medicine for preventing / treating metabolism-related liver diseases

Abstract

The invention provides an application of guanidinoacetic acid in preparation of a medicine for preventing / treating metabolism-related liver diseases, and experiments clearly prove that GAA has a remarkable treatment effect on MASH for the first time and can relieve fatty degeneration, inflammation and fibrosis of the liver; gAA has a multi-target action mechanism, can regulate lipid metabolism, inflammation and oxidative stress, and is comprehensive in curative effect; gAA is a natural metabolite, the metabolic pathway of GAA is clear, and the long-term safety of GAA is verified in the field of feed additives. Safety is high, and potential toxic and side effects are small; gAA is relocated from a nutritional supplement to a MASLD / MASH treatment drug, and a brand new treatment strategy and a drug development direction are provided for MASH.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of guanidino acetic acid in the preparation of a drug for preventing / treating metabolic-related liver disease. BACKGROUND

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex metabolic disease defined as hepatic steatosis due to metabolic dysfunction without excess alcohol consumption or other secondary causes, and is closely related to metabolic risk factors such as obesity and type 2 diabetes.

[0003] MASLD includes metabolic dysfunction-associated fatty liver (MASL), metabolic dysfunction-associated steatohepatitis (MASH) and related cirrhosis. Its main histological features are steatosis, inflammation and fibrosis. MASL is defined as steatosis in more than 5% of hepatocytes, and hepatocytes are not ballooned. In about 20% of MASL patients, steatosis can progress to MASH. MASH is one of the progressive forms of MASLD, characterized by hepatocyte injury, and histologically showing hepatic steatosis with inflammation and hepatocyte ballooning. About 25% of MASH patients can develop into advanced liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC).

[0004] The pathogenesis of MASLD is very complex. Studies have shown that metabolic disorder is the cause of MASLD, and inflammation and fibrosis triggered by metabolic disorder are the core of the pathogenesis of MASH. This metabolic disorder is caused by the overload of the liver's ability to handle major energy metabolism substrates (such as carbohydrates and fatty acids), which manifests as systemic metabolic disorders such as lipid metabolism, amino acid metabolism, and glucose metabolism. This further leads to endoplasmic reticulum stress, inflammation and hepatocyte death, activates pathways such as nuclear factor kappa-B (NF-κB), and promotes the transformation of hepatic stellate cells into myofibroblast-like cells, leading to fibrosis. Studies have shown that MASLD is a manifestation of metabolic syndrome in the liver and is a systemic disease that affects various organs, including the pancreas, kidneys, adipose tissue and intestinal tract. However, at present, there are very limited effective therapeutic drugs for MASH. Most clinical trial results show that the in-development drugs based on existing targets have the problem of low patient response rate, and there is an urgent need to find new intervention targets and measures.

[0005] Guanidoacetic acid (GAA) is a direct precursor of creatine synthesis in animals. The metabolic process of GAA in vivo is first catalyzed by glycine and arginine under the action of arginine-glycine amidinotransferase (L-arginine glycine amidinotransferase, AGAT) to form GAA and ornithine, which is mainly carried out in the kidney and liver. The generated GAA is transported to the liver through the blood circulation, and creatine is generated under the action of guanidinoacetate methltransferase (guanidinoacetate methltransferase, GAMT), which is mainly carried out in the liver. Creatine is commonly used as a sports supplement and is often used for muscle gain. After the generation of creatine, it is transported to target organs such as muscles by SLC6A8 and other transporters to play a role (such as Figure 1 In addition to being a precursor of creatine, GAA has many physiological functions, including promoting insulin secretion, affecting neural regulation, changing arginine metabolism, and affecting the body's oxidation and antioxidant system.

[0006] Studies have found that supplementing GAA in the diet can inhibit liver steatosis and lipid deposition in high-fat diet (HFD) fed mice, and may promote the expression of browning markers and mitochondrial-related genes in iWAT. In addition, supplementing GAA also significantly reduces the levels of inflammatory factors, including IL-4, TNF-α, IL-1β and IL-6. This suggests that GAA may play an important role in improving HFD-induced obesity and related metabolic diseases, but whether GAA can improve MASH and its mechanism of action have not been determined. SUMMARY

[0007] The present application provides a use of guanidoacetic acid in the preparation of a drug for preventing / treating metabolic-related liver disease.

[0008] The first aspect of the present application provides a use of guanidoacetic acid in the preparation of a drug for preventing / treating metabolic-related liver disease.

[0009] The use described above is set as metabolic dysfunction-related fatty liver disease, including metabolic dysfunction-related fatty liver, metabolic dysfunction-related fatty hepatitis and its related cirrhosis.

[0010] The use described above is that guanidoacetic acid reduces the content of triglyceride and total cholesterol in liver tissue, and reduces the expression of lipid synthesis-related genes of MASLD.

[0011] The dose of guanidoacetic acid in the use described above is set to 5-50 mg / kg.

[0012] The use as described above, wherein the guanidino acetic acid is the only active ingredient in the preparation of a drug for preventing / treating metabolic associated liver disease.

[0013] The second aspect of the present application provides the use of guanidino acetic acid in the preparation of a drug for lipid accumulation, inflammation and fibrosis.

[0014] The third aspect of the present application provides a drug for preventing / treating metabolic associated liver disease, comprising guanidino acetic acid or a salt thereof, and at least one pharmaceutically acceptable carrier.

[0015] The pharmaceutical composition as described above, wherein the guanidino acetic acid is C3H7N3O2, and its structural formula is as shown in Figure 2

[0016] The pharmaceutical composition as described above, wherein the MASH is treated by regulating lipid metabolism, inhibiting inflammation and oxidative stress.

[0017] The pharmaceutical composition as described above, wherein the activity or expression of the key enzyme for GAA synthesis is regulated to enhance the effect of guanidino acetic acid.

[0018] The pharmaceutical composition as described above, wherein the administration route of the drug is set as one of oral administration, drinking water dilution, injection or sustained release preparation.

[0019] ​The application discloses application of guanidinoacetic acid (GAA) in preparation of drugs for preventing and treating metabolic-related liver diseases (especially MASLD / MASH), breaks through the traditional cognition of GAA as a nutritional supplement in the prior art, and proves that GAA can significantly improve the core pathological characteristics in various MASH animal models such as genetic (db / db) and diet-induced (GAN diet) through a series of in-vivo experiments, and the specific performance is that GAA can effectively reduce liver cell steatosis, inhibit inflammatory infiltration in the lobule, and delay and even reverse the progress of liver fibrosis. More importantly, the application first systematically clarifies the multi-target mechanism of GAA in playing a therapeutic role, that is, GAA can simultaneously regulate liver lipid metabolism (such as reducing triglyceride and total cholesterol, and lowering the expression of key genes in lipid synthesis), inhibit the activation of a key inflammatory pathway, and improve the liver oxidative stress level. The multi-pathway synergistic effect makes GAA have an incomparable comprehensive curative effect advantage over single-target drugs in dealing with the complex disease MASLD / MASH. In addition, the treatment strategy proposed in the application has high clinical transformation potential and safety. GAA itself is a natural metabolite existing in the human body, the synthesis and metabolic pathway of which is clear, and especially, the safety of long-term use has been fully verified in the practice of large-scale animal husbandry as a feed additive, which indicates that the potential toxic side effect risk of GAA as a new type of human drug will be far lower than that of a synthetic drug with a new chemical structure.

[0020] The concept, specific structure and generated technical effects of the application will be further described below with reference to the drawings, so as to fully understand the purpose, features and effects of the application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a schematic diagram of biosynthesis and metabolism of GAA in the background art of the application;

[0022] Figure 2 is a schematic diagram of the structural formula of guanidinoacetic acid in the embodiment of the application;

[0023] Figure 3 A is a schematic diagram of GAA intervention in a short-term mouse model of MASLD; Figure 3 B is a comparison of liver indexes of GAA intervention in a short-term mouse model of MASLD; data is represented by mean value ± SEM nP < 0.05, * P < 0.05, ** P < 0.01 and *** P < 0.001;

[0024] Figure 4 is a histological staining evaluation of GAA intervention on MASLD short-term mouse model in the embodiment of the present application, wherein, Figure 4 A is a representative graph of hematoxylin-eosin (H&E) staining, oil red staining and Sirius red staining of liver of MASLD short-term mouse, Figure 4 B is a comparison of liver NAS scores, Figure 4 C is a comparison of the proportion of positive areas of liver oil red staining, Figure 4 D is a comparison of the proportion of positive areas of Sirius red staining; data is represented as mean ± SEM (n = 6) compared with db / db-Veh group, n P < 0.05, * P < 0.05, ** P < 0.01 and *** P < 0.001;

[0025] Figure 5 is a schematic diagram of the effect of GAA intervention on lipid accumulation of MASLD short-term mouse model in the embodiment of the present application, Figure 5 A is a comparison of liver triglyceride concentration of MASLD short-term mouse, Figure 5 B is a comparison of liver total cholesterol concentration of MASLD short-term mouse; data is represented as mean ± SEM (n = 6) compared with db / db-Veh group, n P < 0.05, * P < 0.05, ** P < 0.01 and *** P < 0.001;

[0026] Figure 6 is a schematic diagram of the effect of GAA intervention on inflammation of MASLD short-term mouse model in the embodiment of the present application; wherein, Figure 6 A is a comparison of serum glutamic-oxalacetic transaminase of MASLD short-term mouse, Figure 6 B is a comparison of serum glutamic-pyruvic transaminase of MASLD short-term mouse; data is represented as mean ± SEM (n = 6) compared with db / db-Veh group, n P < 0.05, * P < 0.05, ** P < 0.01 and *** P < 0.001;

[0027] Figure 7 A is a schematic diagram of GAA intervention on MASLD long-term mouse model in the embodiment of the present application, Figure 7B represents a comparison of liver indices in a long-term mouse model of MASLD treated with GAA; data are expressed as mean ± SEM. n = 8), compared to the GAN-Veh group, * P<0.05, ** P<0.01 and *** P<0.001;

[0028] Figure 8 This is a histological staining evaluation of the GAA intervention in a long-term MASLD mouse model in this embodiment of the invention; wherein, Figure 8 A is a representative image of liver hematoxylin-eosin (H&E) staining, oil red staining, and Sirius red staining in MASLD long-term mice. Figure 8 B represents a comparison of the proportion of positive areas for oil red staining in the liver. Figure 8 C represents the proportion of Sirius red-stained positive areas; data are expressed as mean ± SEM. n = 8), compared to the GAN-Veh group, * P<0.05, ** P<0.01 and *** P<0.001;

[0029] Figure 9 This is a schematic diagram illustrating the effect of GAA intervention on lipid accumulation in a long-term MASLD mouse model, as described in this embodiment of the invention; wherein, Figure 9 A represents a comparison of liver triglyceride concentrations in MASLD-treated mice over a long period. Figure 9 B represents a comparison of total liver cholesterol concentrations in MASLD-treated mice over a long period; data are expressed as mean ± SEM. n = 8), compared to the GAN-Veh group, * P<0.05, ** P<0.01 and *** P<0.001;

[0030] Figure 10 This is a schematic diagram illustrating the effect of GAA intervention on lipid synthesis-related genes in a long-term MASLD mouse model, as described in this invention; wherein, Figure 10 A is a fatty acid synthase in the liver of long-term MASLD mice. Fasn Comparison of mRNA expression Figure 10 B is the receptor α (a type of peroxisome proliferator-activated receptor) in the liver of long-term MASLD mice. Ppara Comparison of mRNA expression Figure 10 C is a fatty acid-binding protein in the liver of long-term MASLD mice. Fabp4 Comparison of mRNA expression Figure 10 D is the liver acetyl-CoA carboxylase 1 in long-term MASLD mice. Acc1Comparison of mRNA expression Figure 10 E is a protein 1c, a transcriptional activator of endoplasmic reticulum stress-related protein in the liver of MASLD-severe mice. Srebp1c Comparison of mRNA expression Figure 10 F is the liver chemokine 2 in long-term MASLD mice. Ccl2 Comparison of mRNA expression Figure 10 G is the liver long-chain fatty acid elongase 6 in MASLD-treated mice. Elovl6 Comparison of mRNA expression Figure 10 H is the liver diacylglycerol acyltransferase 2 in long-term MASLD mice. Dgat2 mRNA expression comparison; data are expressed as mean ± SEM. n = 8), compared to the GAN-Veh group, * P<0.05, ** P<0.01 and *** P<0.001;

[0031] Figure 11 This is a schematic diagram illustrating the effect of GAA intervention on inflammation in a long-term mouse model of MASLD, as described in this embodiment of the invention. Figure 11 A represents a comparison of serum aspartate aminotransferase (AST) levels in long-term MASLD mice. Figure 11 B represents a comparison of serum alanine aminotransferase levels in MASLD-positive mice over a long period. Figure 11 C is the tumor necrosis factor in the liver of long-term MASLD mice ( Tnf mRNA expression comparison; data are expressed as mean ± SEM. n = 8), compared to the GAN-Veh group, * P<0.05, ** P<0.01 and *** P<0.001. Detailed Implementation

[0032] To make the technical problems, solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] In the following description, specific details, such as particular internal procedures and techniques, are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will appreciate that the invention may be practiced in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods have been omitted so as not to obscure the description of the invention with unnecessary detail.

[0034] In a preferred embodiment of the present application, there is provided a use of guanidinoacetic acid (GAA) in the preparation of a medicament for preventing and / or treating metabolic-associated liver disease. The metabolic-associated liver disease is metabolic dysfunction-associated steatohepatitis (MASH). The metabolic dysfunction-associated steatohepatitis includes metabolic dysfunction-associated fatty liver (MASL), metabolic dysfunction-associated steatohepatitis (MASH), and cirrhosis thereof. The guanidinoacetic acid is the only active ingredient in the medicament.

[0035] In another preferred embodiment of the present application, there is provided a use of guanidinoacetic acid in the preparation of a medicament for treating lipid accumulation, inflammation, and fibrosis disease. The disease is metabolic dysfunction-associated steatohepatitis (MASH). The guanidinoacetic acid exerts its effects by one or more mechanisms of regulating liver lipid metabolism, inhibiting liver inflammatory response, and / or reducing liver oxidative stress. The regulating liver lipid metabolism includes reducing liver tissue triglyceride and total cholesterol content, and / or down-regulating the expression of lipid synthesis-related genes SREBP-1c, FASN. The inhibiting liver inflammatory response includes reducing serum ALT, AST levels, and / or down-regulating the mRNA expression of liver tissue pro-inflammatory factors TNF-α, IL-1β.

[0036] In a preferred embodiment of the present application, a pharmaceutical composition for preventing and / or treating metabolic-associated liver disease comprises a therapeutically effective amount of an active ingredient selected from guanidinoacetic acid, a pharmaceutically acceptable salt thereof, or a solvate thereof, and at least one pharmaceutically acceptable carrier. The active ingredient consists of guanidinoacetic acid. The pharmaceutical composition is formulated as an oral preparation, an injection, or a sustained-release preparation. The oral preparation is selected from a tablet, a capsule, a granule, or an oral solution. The therapeutically effective amount corresponds to 0.5 mg to 100 mg of guanidinoacetic acid per kilogram of body weight per day. The therapeutically effective amount corresponds to 5 mg to 50 mg of guanidinoacetic acid per kilogram of body weight per day.

[0037] The pharmaceutical composition further comprises an auxiliary ingredient for enhancing the synthesis, absorption, or effect of guanidinoacetic acid. The auxiliary ingredient includes, but is not limited to, a substance that regulates AGAT or GAMT enzyme activity or expression.

[0038] In a preferred embodiment of the present application, guanidinoacetic acid is used in the preparation of a medicament for improving liver tissue pathology and / or serum biochemical indicators in patients with metabolic-associated liver disease. Improving liver tissue pathology includes reducing hepatocyte steatosis, reducing intralobular inflammatory cell infiltration, inhibiting hepatocyte ballooning, and / or reducing liver fibrosis. Improving serum biochemical indicators includes reducing serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels. The improvement in liver tissue pathology is evaluated by a decrease in the NAS scoring system and / or a decrease in the fibrosis stage.

[0039] The application provides application of guanidino acetic acid in preparation of medicines for preventing / treating metabolic related liver diseases. The application systematically establishes clinical application value of GAA in improving liver lipid deposition, inflammatory reaction and fibrosis through specific animal model construction, drug intervention design, curative effect evaluation system and molecular mechanism verification. The technical solution constituent elements, operation steps and scientific basis are described in detail in the following modules.

[0040] 1. Animal model construction and grouping design

[0041] Model selection basis: In order to solve the difficulty of synergistic regulation of liver fibrosis and inflammation in the progress of MASLD / MASH disease, two animal models with clear pathological characteristics are used in the application:

[0042] Short-term MASH model: 6-8-week-old male C57BLKs / J db / db mice (genetic obesity and diabetes phenotype) are selected, fed with GAN diet for 4 weeks, and the progress of liver fatty degeneration and inflammation is accelerated.

[0043] Long-term MASH model: 6-8-week-old male C57BL / 6J mice are selected, fed with Gubra-Amylin MASH (GAN) high-fat high-cholesterol diet (40% fat, 20% fructose, 2% cholesterol, formula number D09100310) for 20 weeks, to induce MASLD fibrosis and inflammation.

[0044] Grouping logic: In order to distinguish the effects of GAA on different disease stages, multiple control groups and intervention groups are set:

[0045] In the short-term model, db / m control group, db / db-Veh group, db / db-GAA-L (10 mg / kg), db / db-GAA-H (50 mg / kg) group are set, and oral administration is performed every day.

[0046] In the long-term model, low-fat diet (LFD) group, GAN-vehicle (GAN-Veh) group, GAN-GAA group (5.0 mg / mL drinking water administration) are set, and the intervention time is 8 weeks (from the 12th week to the 20th week).

[0047] 2. Drug intervention scheme and dose design

[0048] Administration route: oral gavage (short-term model) or water dilution (long-term model) is used to simulate the clinical administration method and ensure continuous drug exposure.

[0049] Dose setting basis:

[0050] Short-term model GAA dose (10 mg / kg or 50 mg / kg) was set based on relevant literature; long-term model GAA dose (5.0 mg / mL drinking water administration) was set according to short-term experimental results, and drug solutions were prepared with high-pressure sterilized drinking water, which was replaced twice a week to ensure stability and sterile conditions.

[0051] 3. Therapeutic evaluation index system

[0052] To solve the problem of single evaluation of liver disease treatment effect in the prior art, a multi-dimensional therapeutic verification system is established in the present scheme:

[0053] Serum biochemical indicators: After taking the orbital venous blood, centrifuging the serum, and using the Nanjing Jiancheng biochemical kit to detect triglyceride (TG), total cholesterol (TC), glutamic-oxalacetic transaminase (AST), and glutamic-pyruvic transaminase (ALT), the improvement degree of liver damage and lipid metabolism disorder is quantified.

[0054] Histopathological evaluation: After the liver tissue is fixed with 4% paraformaldehyde and paraffin-embedded, hematoxylin-eosin (H&E) staining is performed, and the degree of fatty degeneration, inflammatory cell infiltration, and ballooning is evaluated by a pathological scanning instrument.

[0055] 4. Molecular mechanism verification scheme

[0056] Gene expression analysis: Total RNA is extracted from liver tissue using the Trizol method, and after reverse transcription into cDNA, the expression changes of inflammatory factors (such as IL-1β, TNF-α) and lipid metabolism-related genes (such as PPARα, SREBP-1c) are detected by real-time fluorescent quantitative PCR (qPCR).

[0057] 5. Key innovation points of the technical scheme

[0058] Model adaptability: By combining long-term (20 weeks) and short-term (4 weeks) models, the intervention effect of GAA on chronic fibrosis and acute inflammation is verified respectively, overcoming the limitations of a single model.

[0059] Multiple endpoint synchronous evaluation: Integrating serum biochemistry and histology, a comprehensive therapeutic evidence chain for MASLD / MASH is formed.

[0060] The above technical scheme has been verified by experiments, and the specific data are shown in the examples section.

[0061] Example 1: Short-term model (4-week intervention)

[0062] Using db / db Short-term MASLD model induced by mouse superimposed GAN feed to evaluate the effect of GAA intervention on MASLD

[0063] 1.1 Experimental Methods

[0064] 1.1.1 Animals and Grouping

[0065] Use SPF level db / db Male mice, 8-10 weeks old, weighing 22-24g. After one week of acclimatization, the mice were randomly divided into the following groups (n=6 per group):

[0066] (1) Blank control group: fed a low-fat diet (LFD, 10% Kcal, Research Diet D12450J), and no drugs were added to the drinking water.

[0067] (2) Model control group (GAN-diet): fed with Gubra-Amylin MASH (GAN) diet (60% Kcal, Research Diet D12492), with no drugs added to drinking water.

[0068] (3) GAA low-dose group: GAN diet, GAA (low dose: 2.5 mg / mL) added to drinking water.

[0069] (4) High-dose GAA group: GAN diet, GAA (high dose: 5 mg / mL) added to drinking water.

[0070] All groups had free access to food and water, and the medication was administered for 4 weeks (e.g. Figure 3 (As shown in A).

[0071] 1.1.2 Weight and liver weight monitoring

[0072] Mouse weight was recorded weekly; at the end of the experiment, mice were sacrificed, liver weight was measured, and liver weight / body weight ratio was calculated.

[0073] 1.1.3 Serum biochemical indicators

[0074] Blood was collected from the fundus venous plexus, serum was separated by centrifugation, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) were detected using a kit (Nanjing Jiancheng Bioengineering Institute).

[0075] 1.1.4 Liver Histological Analysis

[0076] Liver tissues harvested from mice were immediately fixed with 4% paraformaldehyde, then embedded with paraffin, cut into 2-micrometer-thick sections, and stained with hematoxylin and eosin (H&E). Histological features of MASLD, including steatosis, lobular inflammation, and ballooning, were scored according to the NASH CRN scoring system. H&E staining, Oil Red O staining (lipid deposition), and Sirius Red staining (fibrosis) were performed.

[0077] 1.1.5 Data analysis

[0078] Statistical graphs in this study were generated using GraphPad Prism 9.4.1 software, and the measurement data were expressed as Mean ± SEM. The significant differences between groups were compared using a two-tailed T test (Student's t test), and p < 0.05 was considered to have statistical differences.

[0079] 1.2 Experimental results

[0080] 1.2.1 GAA improves liver / body ratio in MASLD short-term mouse model

[0081] The increase in liver weight / body weight ratio is a direct reflection of pathological changes such as steatosis, inflammation, and edema in the liver, indicating liver enlargement and disease severity. For example, Figure 3 As shown in Figure B, compared with the db / db-Veh model control group, the GAA intervention group, especially the high-dose group (50 mg / kg), showed a statistically significant decrease in liver / body ratio. This result clearly indicates that GAA intervention effectively alleviates the pathological enlargement of the liver induced by GAN diet. The decrease in liver / body ratio is consistent with the subsequent observation of reduced liver lipid deposition and histological improvement, directly demonstrating the improvement of GAA on MASLD at the whole organ level.

[0082] 1.2.2 GAA improves liver histological lesions in MASLD short-term mouse model

[0083] The liver pathology was comprehensively evaluated by multi-lineage histological staining techniques. Figure 4 H&E staining showed that db / db-Veh group mice had severe macrovesicular steatosis and hepatocyte ballooning in liver tissue, while after GAA intervention, the morphology of hepatocytes was significantly improved, and the fat vacuoles were significantly reduced. Figure 4 Oil Red O staining (specifically showing neutral lipids) further confirmed that GAA treatment dose-dependently reduced lipid deposition in liver tissue. Figure 4The results of Sirius Red staining (specifically showing collagen fibers, i.e. the degree of fibrosis) showed that GAA intervention effectively reduced the deposition of collagen in the extracellular matrix, suggesting its potential to delay or inhibit the progression of MASH-related liver fibrosis.

[0084] NAS scoring system analysis based on the above staining results showed that both low and high doses of GAA significantly reduced the total NAS score. This comprehensive histological evaluation system, from fatty degeneration, inflammation, ballooning to fibrosis, provided strong morphological evidence that GAA could effectively reverse the progression of MASH disease.

[0085] 1.2.3 GAA reduces liver lipid accumulation in a short-term mouse model of MASLD

[0086] To quantify the regulatory effect of GAA on liver lipid metabolism, we detected the lipid content in liver homogenate. Figure 5 As shown in A, the content of triglyceride (TG) in liver tissue was significantly reduced in both low and high dose GAA groups, indicating that GAA could effectively remove the excessive accumulation of fat in the liver. Figure 5 As shown in B, the content of total cholesterol (TC) in liver tissue was also significantly reduced in the high dose GAA group. This result is particularly important because cholesterol crystals themselves can activate inflammasomes and exacerbate liver inflammatory damage. GAA can simultaneously reduce TG and TC, indicating that it has a broad and benign regulatory effect on liver lipid metabolism, not only reducing fat storage, but also possibly improving lipotoxic stress by reducing toxic lipid species.

[0087] 1.2.4 GAA reduces inflammation in a short-term mouse model of MASLD

[0088] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum are classic and sensitive indicators of hepatocyte damage and membrane integrity. As shown in Figure 6 The serum ALT and AST levels of mice in the high dose GAA intervention group were significantly reduced compared to the db / db-Veh group. This result directly demonstrates that GAA treatment effectively reduces the degree of hepatocyte damage during the MASH disease process. Combined with the observed reduction in inflammatory cell infiltration in histology, it can be concluded that GAA not only improves fat accumulation in the liver, but more importantly, suppresses inflammatory damage driven by lipotoxicity and other factors, thereby protecting the function and integrity of hepatocytes.

[0089] Example 2: Long-term model (12 weeks of diet induction + 8 weeks of intervention)

[0090] C57BL / 6J mice were given GAN feed to induce a long-term MASLD model, and the effect of GAA intervention on MASLD was evaluated.

[0091] 2.1 Experimental methods

[0092] 2.1.1 Animals and modeling: SPF grade C57BL / 6J mice, male, 8 weeks old, were used. The mice were randomly divided into:

[0093] Blank control group: fed with LFD diet for 12 weeks, no drug added in drinking water.

[0094] Model control group (GAN-diet): fed with GAN diet for 12 weeks to induce MASLD model, followed by adding solvent in drinking water for 8 weeks.

[0095] GAA intervention group: after 12 weeks of GAN diet, GAA (dose: 5 mg / mL) was added in drinking water for 8 weeks.

[0096] The experimental period was 20 weeks (as shown in Figure 7 A).

[0097] 2.1.2 Body weight and liver weight monitoring: body weight was recorded every week; at the end of the experiment, the mice were sacrificed and the liver weight was weighed, and the liver weight / body weight ratio was calculated.

[0098] 2.1.3 Serum biochemical indicators:

[0099] Same as 1.2.3 in Example 1.

[0100] 2.1.4 Histological analysis of liver:

[0101] Same as 1.2.4 in Example 1.

[0102] 2.1.5 Quantitative real-time PCR: total RNA was extracted using RNAiso Plus. cDNA was synthesized from 1 microgram of total RNA using PrimeScript™ RT Replication Kit (Shanghai Yeseain Biotechnology Co., Ltd.). After normalization with the corresponding Actin mRNA as a reference, the relative content of each mRNA was calculated, and the results were expressed as fold change relative to the control group.

[0103] 2.1.6 Data analysis

[0104] The statistical graphs in this study were generated using GraphPad Prism 9.4.1 software, and the measurement data was expressed as Mean ± SEM. The significant differences between groups were compared using two-tailed T test (Student's t test), and p<0.05 was considered statistically different.

[0105] Experimental results

[0106] 1.2.1 GAA improves liver / body ratio in long-term mouse model of MASLD

[0107] In the long-term modeling and intervention model lasting for 20 weeks, the liver pathological changes were more chronic and severe. As shown in FIG. 14B, compared with the GAN-Veh model control group, the GAA intervention group still showed a significant decrease in liver weight / body weight ratio. This finding is of great significance in the long-term model, which indicates that the efficacy of GAA is not a short-term effect, but can continue to play a role in chronic and progressive disease course, effectively suppressing the pathological proliferation and hypertrophy of the liver caused by long-term metabolic disorders. This result provides strong macroscopic evidence for the long-term efficacy of GAA. Figure 7

[0108] 1.2.2 GAA improves liver histological lesions in long-term mouse model of MASLD

[0109] Long-term GAN diet-induced liver lesions are more similar to the pathological features of late-stage MASH in humans. Figure 8 A comprehensive histological analysis was performed to confirm. H&E staining revealed that GAA intervention can significantly improve hepatocyte steatosis and ballooning in the long-term model, indicating that it still has the ability to repair established and stable liver damage. Oil Red O staining showed a significant reduction in lipid deposition in liver tissue, confirming that GAA can still effectively promote the clearance and metabolism of liver fat under long-term action. Sirius Red staining showed that GAA intervention can reduce the degree of liver fibrosis that has already formed. This has very high clinical value in long-term models, because fibrosis is a key factor in determining the prognosis of MASH patients, and reversing fibrosis is a core difficulty in current drug development. This result strongly suggests that GAA not only has the potential to treat early-stage fatty liver, but also has the potential to intervene in late-stage, fibrotic MASH.

[0110] 1.2.3 GAA reduces liver lipid accumulation in long-term mouse model of MASLD

[0111] We further explored the mechanism of action of GAA through biochemical and molecular biology methods. Liver tissue lipid quantification ( Figure 9 ) showed that after GAA intervention, the liver triglyceride (TG) and total cholesterol (TC) contents were significantly reduced. This is consistent with the results of the short-term model and is consolidated in the long-term model, confirming that the effect of GAA on regulating liver lipid homeostasis is persistent and stable.

[0112] Real-time fluorescent quantitative PCR (qPCR) analysis ( Figure 10 ​Further research revealed its mechanism of action at the gene expression level: GAA significantly downregulated the expression of key lipid synthesis genes in the liver (such as SREBP-1c and FASN). This indicates that the lipid-lowering effect of GAA is at least partially achieved by inhibiting the liver's own de novo lipid synthesis pathway. This discovery links the efficacy of GAA to a specific molecular target pathway, moving from phenomenological description to mechanistic explanation, greatly enhancing the depth and scientific rigor of the invention.

[0113] 1.2.4 GAA reduces inflammation in a short-term mouse model of MASLD

[0114] The inflammation assessment of the long-term model combined serum biochemical markers with intra-tissue gene expression. Figure 11 As shown in AB, serum ALT and AST levels decreased significantly after GAA intervention, demonstrating that GAA can still effectively reduce hepatocellular damage and maintain the integrity of hepatocellular membranes during the long-term disease process.

[0115] Figure 11 C showed a significant downregulation of TNF-α (tumor necrosis factor-α) mRNA expression levels in liver tissue, providing molecular evidence that GAA directly inhibits local hepatic inflammatory pathways. TNF-α is a core pro-inflammatory factor in the MASH inflammatory cascade. This result indicates that GAA not only alleviates the consequences of liver injury (elevated ALT / AST) but also inhibits the production of key pro-inflammatory factors upstream, thereby reducing the inflammatory state of the liver at its source.

[0116] The guanidinoacetic acid (C3H7N3O2) of the present invention has the following structural formula: Figure 2 (As shown) Its application in the preparation of drugs for the prevention / treatment of metabolic-related liver diseases (MASH) has, for the first time, clearly demonstrated that GAA has a significant therapeutic effect on MASH, reducing hepatic steatosis, inflammation, and fibrosis. GAA has a multi-target mechanism of action, regulating lipid metabolism, inflammation, and oxidative stress, resulting in comprehensive therapeutic effects. GAA is a natural metabolite with a well-defined metabolic pathway, and its long-term safety has been verified in the field of feed additives. It has high safety and few potential toxic side effects. Repositioning GAA from a nutritional supplement as a therapeutic drug for MASH provides a novel treatment strategy and drug development direction for MASH.

[0117] 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. Use of guanidinoacetic acid in the preparation of a medicament for the prevention / treatment of metabolic-related liver diseases.

2. Use according to claim 1, wherein The metabolic-related liver diseases are set as metabolic dysfunction-related fatty liver diseases, including metabolic dysfunction-related fatty liver, metabolic dysfunction-related fatty hepatitis and its related cirrhosis.

3. The use according to claim 1, wherein Guanidinoacetic acid reduces the content of triglyceride and total cholesterol in liver tissue, and reduces the expression of genes related to lipid synthesis in MASLD.

4. The use according to claim 1, wherein The dose of guanidinoacetic acid is set as 5-50 mg / ml.

5. The use according to claim 1, wherein the compound is ###0002### The guanidinoacetic acid is the only active ingredient in the preparation of a medicament for the prevention / treatment of metabolic-related liver diseases.

6. Use of guanidinoacetic acid in the preparation of a medicament for the prevention / treatment of diseases of lipid accumulation, inflammation and fibrosis.

7. A pharmaceutical composition for the prevention / treatment of metabolic-related liver diseases, comprising guanidinoacetic acid or a salt thereof, and at least one pharmaceutically acceptable carrier.

8. The medicine for preventing / treating metabolic associated liver disease according to claim 7, wherein MASH is treated by regulating lipid metabolism, inhibiting inflammation and oxidative stress.

9. The medicine for preventing / treating metabolic associated liver disease according to claim 7, wherein The activity or expression of key enzymes in GAA synthesis is regulated to enhance the effect of guanidinoacetic acid.

10. The medicine for preventing / treating metabolic associated liver disease according to claim 7, wherein The administration route of the medicament is set as one of oral administration, drinking water dilution, injection or sustained-release preparation.

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