Application of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in preparation of medicine for preventing and / or treating metabolism-related fatty liver fibrosis

Abstract

The invention provides application of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in preparation of a medicine for preventing and / or treating metabolism-related fatty liver fibrosis, and belongs to the field of biological medicine. The invention provides new application of 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid. The 4-hydroxyphenylpyruvic acid and the 4-hydroxyphenylacetic acid can be used for preparing medicines for preventing and / or treating metabolism-related fatty liver fibrosis. Specifically, 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid is independently administered to inhibit activation of hepatic stellate cells and improve mouse hepatic tissue fibrosis and liver function impairment caused by CDAHFD diet. The 4-hydroxyphenylpyruvic acid and the 4-hydroxyphenylacetic acid are tyrosine metabolites derived from endogenous organisms and / or intestinal flora, and have no obvious toxicity to organisms; the invention provides a more effective and innovative prevention and treatment method for clinical treatment of metabolism-related fatty liver fibrosis, and has important clinical value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in the preparation of drugs for the prevention and / or treatment of metabolic-related fatty liver fibrosis. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a broad term encompassing both metabolic dysfunction-associated fatty liver (MASL, i.e., solitary hepatic steatosis, considered benign in terms of liver-related prognosis) and metabolic dysfunction-associated steatohepatitis (MASH), the latter potentially leading to progressive liver fibrosis and long-term clinical outcomes. MASLD is characterized by patients with very low or no alcohol consumption, pre-existing metabolic risk factors, and no other known causes of hepatic steatosis or liver disease, accompanied by hepatocellular steatosis. MASH is a progressive necrotizing inflammatory subtype of MASLD, histologically defined as the presence of lobular inflammation and ballooning hepatocellular changes (enlarged cells, 1.5-2 times the diameter of normal hepatocytes, with loosened cytoplasm), which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

[0003] Epidemiological data shows that more than one-third of adults worldwide are affected by MASLD. By 2030, it is estimated that approximately 350 million people globally will be affected by MASH. The pathological mechanisms and impacts of MASH extend far beyond the liver, affecting organs throughout the body, including the metabolic, cardiovascular, digestive, endocrine, immune, and renal systems. It is closely associated with obesity, type 2 diabetes, cardiovascular disease, gut microbiota dysbiosis, and genetic risk factors. The obesity epidemic and the rising prevalence of type 2 diabetes have significantly exacerbated the disease burden of MASLD. In March 2024, the first drug, the thyroid hormone receptor-β (THRβ) agonist rasmetirom, was approved for the treatment of non-cirrhotic MASH with moderate to severe fibrosis. In July 2025, data from a phase II clinical trial published in *Nature Medicine* showed that the glucagon-like peptide-1 receptor agonist semaglutide achieved a 59% remission rate in MASH after 72 weeks of treatment (0.4 mg daily). The mechanism of action depends on both weight loss (13% in patients vs. 1% in placebo) and improving hepatic steatosis, inflammation, fatty degeneration, and fibrosis through independent pathways. However, treatment options for fatty liver fibrosis remain limited, making the development of novel drugs for the prevention and treatment of liver fibrosis of significant clinical importance. From a market perspective, the potential for MASH drug development is enormous.

[0004] 4-Hydroxyphenylpyruvic acid (4-HPPA) and 4-hydroxyphenylacetic acid (4-HPAA) are both endogenous tyrosine metabolites that can be detected in the blood of humans and mice. 4-Hydroxyphenylpyruvic acid can be produced in the liver through tyrosine metabolism or in the gut through gut microbiota metabolism. 4-Hydroxyphenylacetic acid is primarily produced through gut microbiota metabolism. Previous studies have reported that 4-Hydroxyphenylacetic acid can improve obesity induced by a high-fat diet in mice; however, no studies have reported the role of 4-Hydroxyphenylpyruvic acid in metabolic diseases. In conclusion, the roles of 4-Hydroxyphenylpyruvic acid and 4-Hydroxyphenylacetic acid in metabolic-related fatty liver fibrosis have not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in inhibiting hepatic stellate cell activation and improving metabolic-related fatty liver fibrosis and its related symptoms. It aims to provide the application of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in the preparation of drugs for the prevention and / or treatment of metabolic-related fatty liver disease, which has a very broad application prospect.

[0006] The specific technical solution for achieving the objective of this invention is as follows:

[0007] The use of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in the preparation of a drug for the prevention and / or treatment of metabolic-related fatty liver fibrosis, wherein the structural formulas of 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid are as follows:

[0008] .

[0009] Furthermore, the drug comprises 4-hydroxyphenylpyruvic acid and / or 4-hydroxyphenylacetic acid as the first active ingredient and pharmaceutically acceptable excipients.

[0010] Furthermore, the drug includes methods for reducing serum ALT and / or AST levels, and alleviating collagen deposition, inflammatory cell infiltration, and fibrosis in liver tissue pathology.

[0011] Furthermore, the drug includes those for inhibiting hepatic stellate cell activation.

[0012] Furthermore, the dosage forms of the drug include oral liquid, tablets, granules, pills, powders, capsules, and injections.

[0013] The purpose of this invention is to investigate the role of 4-hydroxyphenylpyruvic acid (4-hydroxyphenylacetic acid) or 4-hydroxyphenylacetic acid (4-hydroxyphenylacetic acid) in metabolism-related fatty liver fibrosis. To this end, a mouse model of metabolism-related fatty liver fibrosis was constructed using a TGF-β-induced human hepatic stellate cell (LX-2) fibrosis model and a CDAHFD diet. LX-2 cells were activated by adding 10 ng / mL TGF-β, and after 6 h, 0, 10, 50, 200, and 500 μmol / L of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid were added, respectively. Cell activation was assessed by analyzing collagen COL1A1 mRNA expression. Male C57BL / 6 mice were fed a CDAHFD diet for 10 weeks at 8 weeks of age, and 0.5 mg / mL or 1.5 mg / mL of 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid was supplemented in drinking water 4 weeks after the start of feeding. Serum enzyme levels of ALT and AST were detected, liver histopathological staining was used to assess liver steatosis, inflammation and fibrosis, qPCR was used to analyze the expression of liver fibrosis marker genes Acta2, Col1a1, Col3a1 and Mmp2, and Western blot was used to analyze liver α-SMA protein expression.

[0014] This invention provides for the first time the role of 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid in metabolism-related fatty liver fibrosis. Both 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid are endogenous tyrosine metabolites derived from the host and / or intestinal flora, and have no significant toxicity to organisms. The 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid proposed in this invention have good solubility, are stable at room temperature, and can be conveniently and quickly administered orally. Commercial reagents are available and readily available, showing good prospects for clinical application. Attached Figure Description

[0015] Figure 1 : mRNA expression level of COL1A1 in human hepatic stellate cells LX-2;

[0016] Figure 2 Mouse body weight changes, liver weight ratio, and liver pathology photographs;

[0017] Figure 3 Serum ALT and AST enzyme activity levels in mice;

[0018] Figure 4 : Mouse eosin-hematoxylin staining and Sirius red staining;

[0019] Figure 5 : mRNA expression levels of Acta2, Col1a1, Col3a1 and Mmp2 in mouse liver;

[0020] Figure 6 : Protein expression level of α-SMA in mouse liver. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the materials and methods used in the embodiments are existing methods.

[0022] The data in the following charts are expressed as averages. + The standard error is calculated using a one-way ANOVA test to analyze the significance of two or more groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate a significant difference compared to the control group, while "ns" indicates no significant difference compared to the control group.

[0023] Example 1: 4-Hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid inhibits TGF-β-induced activation of LX-2 cells.

[0024] 1.1 Implementation Materials

[0025] Human hepatic stellate cells (LX-2) were purchased from the Cell Bank of the Chinese Academy of Sciences. Human TGF-β1 cytokine (MedChemExpress, HY-P7118) was purchased from Shanghai Yuda Biotechnology Co., Ltd. Tyrosine (014025815), 4-hydroxyphenylpyruvic acid (H138306), tyramine (T105543), 4-hydroxyphenyllactic acid (S160986), 4-hydroxyphenylacetic acid (H103972), 4-hydroxyphenylacrylic acid (C108514), and 4-hydroxyphenylpropionic acid (P106355) were all purchased from Shanghai Titan Technology Co., Ltd. Primers were synthesized by Shanghai Platinum Technology Co., Ltd.

[0026] 1.2 Implementation Methods

[0027] LX-2 cells were cultured at 37°C in a 5% CO2 incubator in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. LX2 cells were seeded in 12-well cell culture plates. When cell confluence reached 80%, 10 ng / mL TGF-β was added to the medium to induce cell activation. After 6 h, 0, 10, 50, 200, and 500 μmol / L of tyrosine, 4-hydroxyphenylpyruvic acid (4-HPPA), tyramine, 4-hydroxyphenyllactic acid (4-HPLA), 4-hydroxyphenylacetic acid (4-HPAA), 4-hydroxyphenylacrylic acid (4-HPA), and 4-hydroxyphenylpropionic acid (4-HPP) were added, respectively. Cell fibrosis was assessed by analyzing collagen COL1A1 mRNA expression using RT-qPCR.

[0028] Total RNA extraction from cells was performed according to the Trizol reagent (Invitrogen) manufacturer's instructions. RT-qPCR was performed on a real-time quantitative PCR system (EBI, 7900). A 10 μL reaction mixture consisted of 5 μL SYBR Premix, 4 μL DNA, 0.5 μL upstream primer, and 0.5 μL downstream primer. The program was: 95 °C for 3 min, followed by 40 cycles (95 °C 20 s, 60 °C 20 s, 72 °C 20 s + plate reading), followed by melting curve analysis (heating from 55 °C to 95 °C, reading every 0.5 °C for 5 s). GAPDH gene was used as an internal control for cross-sample calibration. Results were expressed as a 22 -ΔΔCt The calculations were performed using the following methods. All values ​​are expressed as mean ± standard error. One-way ANOVA was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate a significant difference from the control group.

[0029] Table 1: qPCR primer sequences

[0030]

[0031] 1.3 Implementation Results

[0032] like Figure 1 As shown, seven candidate molecules (tyrosine and six metabolites derived from it) were selected to investigate their roles in TGF-β-induced LX-2 cell activation. Figure 1 A). The results showed that 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid significantly inhibited TGF-β-induced activation of LX-2 cells, as evidenced by a significant downregulation of collagen gene COL1A1 expression at stimulation concentrations of 200 and 500 μmol / L. Figure 1 B).

[0033] Example 2: 4-Hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid improves metabolic-related fatty liver fibrosis in mice.

[0034] 2.1 Implementation Materials

[0035] Wild-type male C57BL / 6 mice were purchased from Shanghai Slack Laboratory Animal Center. 4-hydroxyphenylpyruvic acid (H138306) and 4-hydroxyphenylacetic acid (H103972) were purchased from Shanghai Titan Technology Co., Ltd. A choline-deficient diet (CDAHFD, A06071302) consisting of 60% high-fat and 0.1% methionine was synthesized by Wuxi Fanbo Biotechnology Co., Ltd. Alanine aminotransferase (ALT) assay kits (C009-2-1) and aspartate aminotransferase (AST) assay kits (C010-2-1) were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. α-SMA antibody (14395-1-AP, 1:1000 dilution) and HSP90 antibody (60318-1-Ig, 1:5000 dilution) were purchased from Wuhan Sanying Biotechnology Co., Ltd. Primers were synthesized by Shanghai Platinum Technology Co., Ltd.

[0036] 2.2 Experimental Methods

[0037] 2.2.1 Animal Experiment Design

[0038] Male C57BL / 6 mice were housed in a standard SPF barrier environment, maintained at a constant temperature (22±2℃) and humidity (50%±10%), with a 12-hour diurnal cycle and free access to food and water. After one week of acclimatization to a normal control diet, 46 seven-week-old mice were divided according to body weight into a healthy control group, a liver fibrosis model group, a 0.5 mg / mL 4-hydroxyphenylpyruvate treatment group, a 1.5 mg / mL 4-hydroxyphenylpyruvate treatment group, a 0.5 mg / mL 4-hydroxyphenylacetic acid treatment group, and a 1.5 mg / mL 4-hydroxyphenylacetic acid treatment group. A metabolic-related fatty liver fibrosis model was induced using a 60% high-fat, 0.1% methionine choline-deficient diet (CDAHFD, Research Diet A06071302). The healthy control group was fed the normal control diet, while the other groups were fed the CDAHFD diet for 10 consecutive weeks. Four weeks after starting the CDAHFD diet, the treatment groups were given 0.5 mg / mL and 1.5 mg / mL of 4-hydroxyphenylpyruvic acid (4-HPA) and 4-hydroxyphenylacetic acid (4-HPA) respectively in their drinking water. Both 4-HPA and 4-HPA were dissolved in 0.1% DMSO. The healthy control group and the liver fibrosis model group also received 0.1% DMSO in their diet. The weight of the mice in each group was measured and recorded weekly during the feeding process.

[0039] 2.2.2 Sample Processing

[0040] After the experiment, blood and tissue samples were taken from the mice. Blood was collected from the eyeballs of the mice, serum was separated, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum were detected. Fresh mouse liver tissue was taken, fixed in 4% paraformaldehyde solution, and embedded. Eosin and hematoxylin (H&E) staining and Sirius red staining were performed according to standard procedures, and histological analysis was performed. Then, the area of ​​collagen deposition in the liver was statistically analyzed using ImageJ software.

[0041] 2.2.3 RT-qPCR

[0042] Total RNA extraction from liver tissue was performed according to the Trizol reagent (Invitrogen) manufacturer's instructions. RT-qPCR was performed on a real-time quantitative PCR system (EBI, 7900). The 10 μL reaction mixture consisted of 5 μL SYBR Premix, 4 μL DNA, 0.5 μL upstream primer, and 0.5 μL downstream primer. The program was: 95 °C for 3 min, (95 °C 20 s, 60 °C 20 s, 72 °C 20 s + plate reading) for 40 cycles, followed by melting curve analysis (heating from 55 °C to 95 °C, reading every 0.5 °C for 5 s). qPCR analysis was performed on the expression of liver fibrosis marker genes Acta2, Col1a1, Col3a1, and Mmp2. Gapdh gene was used as an internal control for cross-sample calibration. Results were expressed as a 22 -ΔΔCt The method is used for calculation.

[0043] Table 2: qPCR primer sequences

[0044]

[0045] 2.2.4 Western blot

[0046] Liver tissue protein extraction: Take about 20 mg of liver tissue, add an appropriate volume of denaturing RIPA lysis buffer (150 mM NaCl, 10 mM Tris pH7.2, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate and 5 mM EDTA) containing protease inhibitors and phospholipase inhibitors, sonicate for 10 s / 10 s under ice bath, after 10 min, centrifuge at 12000 rmp at 4 ℃ for 20 min, collect the supernatant to obtain the protein solution.

[0047] Immunoblotting: Protein concentration was measured using the Pierce BCA Kit. After concentration measurement, 5×SDS loading buffer (200 mM Tris-Cl, pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol, and 400 mM DTT) was added, and the mixture was boiled at 100℃ for 10 minutes before immediate loading for immunoblotting. Anti-α-SMA (14395-1-AP, 1:1000 dilution) and anti-HSP90 (60318-1-Ig, 1:5000 dilution) primary antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd. ImageJ software was used to statistically analyze the grayscale values ​​of the protein bands, with HSP90 protein used as an internal control.

[0048] 2.2.5 Statistical Analysis

[0049] The data is represented as average. + The standard error is calculated using a one-way ANOVA test to analyze the significance of two or more groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate a significant difference compared to the control group, while "ns" indicates no significant difference compared to the control group.

[0050] 2.3 Implementation Results

[0051] 2.3.1 Body weight, fat-to-body weight ratio, and liver pathology photographs of mice in each group are shown below. Figure 2 As shown, the experimental groups and mouse treatment times are as follows: Figure 2 A, CDAHFD diet feeding led to decreased body weight and increased fat-to-body weight ratio in mice, while supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid had no significant effect on body weight and liver-to-body weight ratio. Figure 2 B and 2C); CDAHFD diet feeding induced hepatic steatosis, and supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid significantly improved steatosis (B and 2C). Figure 2 D).

[0052] 2.3.2 Serum liver enzyme activity levels in each group of mice as follows Figure 3 As shown, CDAHFD-induced diets significantly increased AST and ALT levels, while supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid significantly reduced the AST and ALT increases induced by CDAHFD diets. Figure 3 (A and 3B) indicate that 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid improved CDAHFD-induced liver function damage.

[0053] 2.3.3 Results of H&E staining and Sirius red staining in each group of mice are as follows: Figure 4As shown. Eosin-hematoxylin and Sirius red histological staining showed that CDAHFD diet feeding led to increased hepatic lipid droplets, increased immune cell infiltration, and increased collagen deposition. Supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid significantly reduced inflammatory infiltration and collagen deposition. Figure 4 A), and reduced the area of ​​liver fibrosis ( Figure 4 B).

[0054] 2.3.4 Expression levels of liver fibrosis marker genes in each group of mice as follows: Figure 5 As shown. qPCR results showed that CDAHFD diet feeding led to liver Acta2 ( Figure 5 A), Col1a1 ( Figure 5 B), Col3a1 ( Figure 5 C) and Mmp2 Figure 5 The mRNA expression level of D) was significantly increased, and the expression level of the above genes was significantly downregulated by supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid.

[0055] 2.3.5 Expression of α-SMA protein in the liver of mice in each group as follows: Figure 6 As shown in the figure. Western blot results showed that CDAHFD diet significantly increased α-SMA expression, while supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid reduced the content of α-SMA in mouse liver. Figure 6 A and 6B).

[0056] The above results indicate that supplementation with 4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid reduces serum AST and ALT levels in CDAHFD-fed mice and alleviates collagen deposition, inflammatory cell infiltration, and fibrosis in liver tissue pathology.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

The use of 1,4-hydroxyphenylpyruvic acid or 4-hydroxyphenylacetic acid in the preparation of drugs for the prevention and / or treatment of metabolic-related fatty liver fibrosis, characterized in that, The structural formulas of 4-hydroxyphenylpyruvic acid and 4-hydroxyphenylacetic acid are as follows: 。 2. The application as described in claim 1, characterized in that, The drug comprises 4-hydroxyphenylpyruvic acid and / or 4-hydroxyphenylacetic acid as the first active ingredient and pharmaceutically acceptable excipients.

3. The application as described in claim 2, characterized in that, The drugs include those used to lower serum ALT and / or AST levels and reduce collagen deposition, inflammatory cell infiltration, and fibrosis in liver tissue pathology.

4. The application as described in claim 2, characterized in that, The drug includes those for inhibiting hepatic stellate cell activation.

5. The application as described in claim 2, characterized in that, The drug is available in dosage forms including oral liquid, tablets, granules, pills, powders, capsules, and injections.

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