Application of β-1,4-galactosyltransferase 1 and its inhibitors in the preparation of drugs for treating acute and chronic liver diseases

Abstract

This invention discloses the application of β-1,4-galactosyltransferase 1 and its inhibitors in liver diseases, particularly acute liver injury and liver failure. The application of β-1,4-galactosyltransferase 1 and its inhibitors in acute liver injury and liver failure provides the correlation between β-1,4-galactosyltransferase 1 and acute liver injury and liver failure, confirming that inhibiting the activity of β-1,4-galactosyltransferase 1 can alleviate acute liver injury and liver failure. β-1,4-galactosyltransferase 1 can serve as a drug target for screening acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis, and acute and chronic liver failure. This invention also confirms the alleviating effect of β-1,4-galactosyltransferase 1 inhibitors on acute liver failure. β-1,4-galactosyltransferase 1 inhibitors improve acute liver failure by reducing the enzyme activity or protein expression of β-1,4-galactosyltransferase 1.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to the discovery and application of drug targets, specifically the application of β-1,4-galactosyltransferase 1 and its inhibitors in the preparation of drugs for treating acute and chronic liver diseases. Background Technology

[0002] Liver disease is a general term for abnormal liver function, usually referring to liver tissue damage caused by various pathogenic factors such as viruses, drugs, bacteria, alcohol, and high-fat diet ((1) Cooke GS, et al. Liver disease: at the heart of public health challenges for Europe in the 21st century[J]. Lancet. 2022, 399(10319): 9-10), which leads to a series of health problems, including acute and chronic hepatitis, liver injury, fatty liver, and liver fibrosis. In severe cases, it can lead to liver failure or even induce liver cancer. It has a high incidence and mortality rate in my country ((2) GBD2019 Hepatitis B Collaborators. Global, regional, and national burden of hepatitis B, 1990-2019: a systematic analysis for the global burden of disease study 2019[J]. Lancet Gastroenterol Hepatol. 2022, 7(9): 796-829). As is well known, severe or persistent acute liver injury can lead to acute liver failure (ALF), which affects multiple organs and presents as a group of clinical syndromes with jaundice, coagulation dysfunction, hepatorenal syndrome, hepatic encephalopathy, ascites, etc. It is the third leading cause of death among liver disease patients in my country after liver cancer and cirrhosis, and seriously threatens human health ((3) Stravitz R, et al. Acute liver failure[J]. Lancet. 2019, 394(10201):869-881). In Europe and the United States, drug-induced liver injury is mainly caused by acetaminophen. In my country, hepatitis B virus is the most common cause (accounting for about 80-85%), and with the increasing incidence of drug-induced liver injury, the incidence of ALF is also rising ((4) Wang J, et al. Landscape of DILI-related adverse drug reaction in China Mainland[J]. Acta Pharm Sin B. 2022, 12(12): 4424-4431). Although great progress has been made in the pathogenesis and diagnosis of ALF, there is still no specific treatment. The current treatments for acute liver failure include liver transplantation, N-acetylcysteine ​​(NAC), glucocorticoids, nucleoside analogs, etc.However, liver transplantation carries high risks and is extremely expensive, with less than 10% of patients undergoing the procedure. While NAC (acetaminophen) has proven effective in antioxidation and anti-inflammation, overdose of NAC can cause hepatotoxicity due to acetaminophen, leading to adverse reactions such as acute renal failure, hemolysis, and thrombocytopenia, and even death. Therefore, in-depth exploration of the important pathophysiology and mechanisms of action in acute and chronic liver diseases, identification of new therapeutic targets, and active pharmacological intervention are of great significance for improving these conditions.

[0003] Galactosamine transferase (B4GALT1) is a member of the β-1,4-galactosyltransferase gene family that encodes type II membrane-bound glycoproteins. It is generally located in the Golgi apparatus and is also expressed in the cell membrane. It plays an important role in various life activities such as embryonic development, nervous system development, immune and inflammatory responses, and tumor development. (5) Wang P, et al. B4GalT1 regulates apoptosis and autophagy of glioblastoma in vitro and invivo[J]. Technol Cancer Res Treat, 2020, 19(1): 1533033820980104) Studies have confirmed that B4GALT1 plays a key role in the acute phase of osteoarthritis and rheumatoid arthritis. (6) Sun X, et al. Identification of key genes in osteoarthritis using bioinformatics, principal component analysis and meta-analysis[J]. Exp Ther Med. 2021, 21(1):18) Inflammation-related cytokines IL-6 and TNF-α have also been found to promote the expression of B4GALT1. (7) Garcı´a-Vallejo J, et al. Tumor necrosis factor-alpha up-regulates the expression of beta1,4-galactosyltransferase I in primary human endothelial cells by mRNAstabilization[J].J Biol Chem, 2005, 13(280): 12676–12682) B4GALT1 is strongly associated with autoimmune diseases such as systemic lupus erythematosus and Crohn's disease.(8) Wang L, et al. Human retrotransposon insertion polymorphisms are associated with health and disease via gene regulatory phenotypes[J]. Front Microbiol, 2017, 2(8): 1418) B4GALT1 is widely expressed and plays a key role in the processing of N-linked oligosaccharide moieties in glycoproteins, transferring galactose from uridine diphosphate galactose (UDP-Gal) to specific glycoprotein substrates. Currently, the role of B4GALT1 in acute and chronic liver diseases such as acute liver failure has not been reported. Summary of the Invention

[0004] The first objective of this invention is to provide the application of β-1,4-galactosyltransferase 1 as a drug target in screening drugs for the prevention, relief and / or treatment of acute and chronic liver diseases induced by multiple factors;

[0005] A second objective of this invention is to provide the use of an inhibitor of β-1,4-galactosyltransferase 1 in the preparation of drugs for treating acute and chronic liver diseases, but not limited to acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis, and acute and chronic liver failure.

[0006] A third object of the present invention is to provide a pharmaceutical formulation containing an inhibitor of β-1,4-galactosyltransferase and a pharmaceutically acceptable carrier.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] Application of β-1,4-galactosyltransferase 1 as a drug target in the preparation of drugs for the prevention or treatment of acute or chronic liver diseases.

[0009] The acute and chronic liver diseases mentioned include acute hepatitis, chronic hepatitis, liver injury, fatty liver, liver fibrosis, acute liver failure, or chronic liver failure.

[0010] Furthermore, acute liver failure is a common clinical cause, but it includes not only acute and chronic liver failure caused by viral and drug factors.

[0011] The use of inhibitors of β-1,4-galactosyltransferase 1 in the preparation of drugs for the prevention, relief and / or treatment of acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis and acute and chronic liver failure.

[0012] The use of a pharmaceutical composition in the prevention, relief and / or treatment of acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis and acute and chronic liver failure, including an inhibitor of β-1,4-galactosyltransferase 1 and a pharmaceutically acceptable carrier.

[0013] Furthermore, based on the application of the pharmaceutical preparation in the prevention, relief and / or treatment of acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis and acute and chronic liver failure, the acute and chronic liver diseases are liver diseases caused by common clinical causes.

[0014] Inhibitors of β-1,4-galactosyltransferase 1 were obtained through screening, as detailed below:

[0015] The β-1,4-galactosyltransferase 1 inhibitor is as follows:

[0016] Sort Listed drug name Sort Raw material name Sort Natural product name 1 Ruby Star 26 UDP-galactose 51 Coumarin 2 Pentosan polysulfate 27 5'-Guanine triphosphate 52 Gentian Violet 3 polymyxin 28 5'-Adenosine pentaphosphate 53 13-Acetylphorbol 4 Idabi Star 29 N-Sulfoflavin Mononucleotide 54 Phenylacetocoumarin 5 Cefnixib 30 Ureidine diphosphate glucose 55 Carotenoid acids 6 cefepime 31 UDP-α-D-glucuronic acid 56 vanillic acid 7 Linopril 32 guanosine aminophosphate 57 Isocitrate 8 Fisophenadine 33 Arginine succinate 58 Ricinoleic acid 9 Trimetine 34 4-Hydroxy-3-methylbutyl diphosphate 59 Canavonine 10 losartan 35 Oxidized acetylcatechol 60 Palmitoleic acid 11 Doxazosin 36 5-Deoxycyclohexanone 61 (+)-Rutinol 12 Treprostacyclin 37 4-Nitrophenyl-β-D-galactoside 62 α-Linolenic acid 13 Formotero 38 N-acetylglucosamine 63 Isoferrin 14 Methotrexate 39 β-D-arabinofuranose 5-phosphate 64 p-coumaric acid 15 Amlodipine 40 Glycine ribonucleotide 65 Anisidine 16 Aloxilin 41 7,8-Dihydroneopterin 66 Sulfocoumarin 17 Budneid 42 1,2,3,4-Tetrahydro-isoquinoline-7-sulfonamide 67 Betaine aldehyde 18 Omeprazole 43 9-(4-hydroxybutyl)-N2-phenylguanine 68 p-Anethole 19 Imipenem 44 γ-Glutamylcysteine 69 thymol 20 Oxyconazole 45 N-Ethylretinamide 21 Pyrboterol 46 4'-Hydroxyflavanone 22 Miglinel 47 6-Hydroxymethylpterin diphosphate 23 Baclofen 48 12-Hydroxydodecanoic acid 24 Itramonium bromide 49 2,3-Dihydroxybenzoic acid 25 Giscitabine 50 1-Thio-β-D-glucopyranose

[0017] The salts of the compounds in the table above also have inhibitory effects on β-1,4-galactosyltransferase 1.

[0018] Preferred acquisition of epirubicin:

[0019] Epirubicin is an organic compound, with epirubicin hydrochloride being the preferred pharmaceutically acceptable form. Its chemical formula is C2. 27 H 30 ClNO 11 Epirubicin is an antitumor antibiotic used to treat lung and ovarian cancer. Its mechanism of action involves direct insertion between DNA nucleobase pairs, interfering with transcription and preventing mRNA formation, thereby inhibiting DNA and RNA synthesis. Similar to doxorubicin, its side effects are generally milder, especially cardiotoxicity. Other side effects include bone marrow suppression, cardiotoxicity, hair loss, mucositis (generally manifesting as gastritis with erosion, inflammation of the bilateral sides of the tongue and sublingual glands), gastrointestinal disturbances (nausea, vomiting, diarrhea), high fever, and occasionally fever, chills, and urticaria. It is important to note that epirubicin is primarily cleared through the hepatobiliary system, and clinically used doses can cause liver damage. Therefore, serum total bilirubin and aspartate aminotransferase (AST) levels should be assessed before and during treatment. Patients with elevated bilirubin or AST may experience slower drug clearance and increased systemic toxicity; therefore, patients with abnormal biochemical indicators require dose reduction and should not use it concurrently with heparin. However, the present invention has surprisingly discovered that low doses of epirubicin have a hepatoprotective effect, namely, the dose for treating liver failure (0.1 mg / kg) is only 1 / 227 of the recommended starting dose for antitumor therapy (22.68 mg / kg).

[0020] Advantages of the present invention

[0021] 1. Acute liver failure refers to massive or submassive necrosis or bridging necrosis of hepatocytes, accompanied by severe degeneration of surviving hepatocytes and collapse or partial collapse of the hepatic sinusoidal reticular framework. Given the diverse causes of acute liver failure, including drugs, viral infections, and autoimmune disorders, treatment methods must be individualized based on the underlying cause. Drug metabolism requires the participation of hepatocytes; common anti-inflammatory drugs may exacerbate liver damage and even lead to liver failure. Therefore, extreme caution is needed when treating acute liver failure, and selecting appropriate drugs is crucial.

[0022] This invention employs a D-GalN / LPS-induced mouse liver failure model to simulate acute liver failure induced by microbial infection factors, a ConA-induced mouse liver failure model to simulate acute liver failure induced by clinical immune damage, and a high-fat diet-induced non-alcoholic fatty liver model to simulate chronic non-alcoholic fatty liver caused by high fat and high cholesterol. Severe or persistent liver injury can lead to liver failure. Through different liver injury models, the key role of B4GALT1 in acute and chronic liver diseases is investigated.

[0023] This invention is the first to discover the association between β-1,4-galactosyltransferase 1 inhibitors and liver diseases, particularly acute liver failure. It confirms that inhibiting the activity of β-1,4-galactosyltransferase 1 can alleviate acute liver failure. Therefore, β-1,4-galactosyltransferase 1 can serve as a target for screening drugs in the prevention, relief and / or treatment of acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis and acute and chronic liver failure.

[0024] 2. This invention confirms that β-1,4-galactosyltransferase 1 inhibitors have an effective alleviating effect on acute liver failure, the most severe clinical symptom in liver disease. β-1,4-galactosyltransferase 1 inhibitors improve acute liver failure by reducing the activity and protein expression of β-1,4-galactosyltransferase 1; the β-1,4-galactosyltransferase 1 inhibitors are various drugs such as epirubicin hydrochloride, 5-tert-butyl-4-methylthiazol-2-carboxylic acid, or 3-aminocoumarin.

[0025] 3. This invention discloses a pharmaceutical formulation containing a β-1,4-galactosyltransferase 1 inhibitor and a pharmaceutically acceptable carrier, which can be used to reduce the enzyme activity and / or protein expression of β-1,4-galactosyltransferase 1, thereby improving and / or treating acute and chronic hepatitis, liver injury, fatty liver, liver fibrosis, and acute and chronic liver failure. Attached Figure Description

[0026] Figure 1Changes in B4GALT1 expression in liver tissue of mice with hepatitis induced by different factors (Western blotting was used to detect the expression of B4GALT1 protein in liver tissue of mice with acute and chronic hepatitis induced by various models; A: Changes in B4GALT1 in liver tissue of D-GalN / LPS-ALF mice n=3; B: Changes in B4GALT1 in liver tissue of ConA-ALF mice n=6; C: Changes in B4GALT1 in liver tissue of mice with non-alcoholic fatty liver disease n=6; D: Time-series changes in B4GALT1 in liver tissue of mice with acute liver failure induced by D-GalN / LPS; n=6, expressed as mean ± SD). # P<0.05, ## (P<0.01 compared with the control group);

[0027] Figure 2 The docking results of the marketed drug epirubicin with B4GALT1 (AutoDock 4.1 software was used to dock epirubicin with B4GALT1, and Python software was used for visualization analysis. The binding free energy of epirubicin with B4GALT1 is -9.96 kcal / mol).

[0028] Figure 3 The docking results of the active pharmaceutical ingredient 5-tert-butyl-4-methylthiazol-2-carboxylic acid with B4GALT1 (AutoDock 4.1 software was used to dock 5-tert-butyl-4-methylthiazol-2-carboxylic acid with B4GALT1, and Python software was used for visualization analysis. The binding free energy of 5-tert-butyl-4-methylthiazol-2-carboxylic acid with B4GALT1 is -6.37 kcal / mol).

[0029] Figure 4 The docking results of the natural product coumarin with B4GALT1 (AutoDock 4.1 software was used to dock coumarin with B4GALT1, and Python software was used for visualization analysis. The binding free energy of coumarin with B4GALT1 is -7.45 kcal / mol).

[0030] Figure 5 In vitro assay of the inhibitory effect of EPI on β-1,4-galactosyltransferase 1 activity (epirarubicin hydrochloride inhibits the activity of β-1,4-galactosyltransferase 1, with an IC50 of 2.139 μmol / L).

[0031] Figure 6 MST assay was used to detect the interaction between EPI and β-1,4-galactosyltransferase 1 (epirarubicin hydrochloride can bind to β-1,4-galactosyltransferase 1, with a Kd value of 16.10 ± 2.43 μmol / L).

[0032] Figure 7 Preliminary screening was conducted on marketed drugs, active pharmaceutical ingredients (APIs), and natural products obtained from global drug databases. The selected drugs were omeprazole and gemcitabine; APIs were UDP-galactose-disodium salt and N-acetyl-D-glucosamine; and natural products were 3-aminocoumarin and thymol. Molecular docking technology was first used for validation, followed by MST technology to detect the affinity of β-1,4-galactosyltransferase 1 (β-1,4-galactosyltransferase 1) for omeprazole. (A: Affinity of β-1,4-galactosyltransferase 1 with omeprazole: -4.87 kcal / mol; B: Kd value of β-1,4-galactosyltransferase 1 with omeprazole: 0.33 ± 0.81 μmol / L; C: Affinity of β-1,4-galactosyltransferase 1 with gemcitabine: -7.0 kcal / mol; D: Kd value of β-1,4-galactosyltransferase 1 with gemcitabine: 0.22 ± 0.54 μmol / L). μmol / L; E: Affinity of β-1,4-galactosyltransferase 1 to UDP-galactose-disodium salt: -12.52 kcal / mol; F: Kd value of β-1,4-galactosyltransferase 1 to UDP-galactose-disodium salt: 10.30 ± 0.01 μmol / L; G: Affinity of β-1,4-galactosyltransferase 1 to N-acetyl-D-glucosamine: -6.83 kcal / mol; H: Kd value of β-1,4-galactosyltransferase 1 to N-acetyl-D-glucosamine: 13.97 ± 1.22 μmol / L; I: Affinity of β-1,4-galactosyltransferase 1 to 3-aminocoumarin: -5.73 kcal / mol; J: Kd value of β-1,4-galactosyltransferase 1 to 3-aminocoumarin: 0.06 ± 1.35 μmol / L; K: the affinity of β-1,4-galactosyltransferase 1 for thymol is -4.43 kcal / mol; L: the Kd value of β-1,4-galactosyltransferase 1 for thymol is 0.07 ± 0.97 μmol / L).

[0033] Figure 8 The effects of selected marketed drugs, active pharmaceutical ingredients, and natural products on alanine aminotransferase (ALT) were validated in vitro. An in vitro acute liver failure model was induced using 20 mmol / L acetaminophen. Thirty min before modeling, patients were pre-administered 20 μmol / L of UDP-galactose disodium salt, N-acetyl-D-glucosamine, omeprazole, gemcitabine, 3-aminocoumarin, and thymol. ALT levels in AML 12 hepatocytes were measured 24 h after modeling. (n=5, values ​​are expressed as mean ± SD). # P<0.05, ## Compared with the control group, P<0.01 * P<0.05,** (P<0.01 compared with the model group);

[0034] Figure 9 Effects of intravenous epirubicin hydrochloride on liver function-related indicators in mice with acetaminophen-induced acute liver injury (an acute liver injury model was induced in mice by intraperitoneal injection of 300 mg / kg acetaminophen (APAP) solution, followed by epirubicin hydrochloride EPI infusion (100, 50, 25 μg / kg) 30 min later; samples were collected 6 h after model establishment; A: Effect of intravenous epirubicin hydrochloride on liver appearance in APAP-induced acute liver injury mice; B: Effect of liver coefficient; C: Effect of alanine aminotransferase (ALT); D: Effect of aspartate aminotransferase (AST); E: Effect of total bilirubin; F: Effect of glutathione; n=8, expressed as mean ± SD, # P<0.05, ## Compared with the control group, P<0.01 * P<0.05, ** (P<0.01 compared with the model group);

[0035] Figure 10 Effects of intravenous epirubicin hydrochloride on liver function-related indicators in mice with D-GalN / LPS-induced acute liver failure (mice were intravenously injected for three consecutive days at EPI doses of 0.1, 0.3, and 1 mg / kg before modeling; acute liver failure model was induced by intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution; samples were collected 6 h after modeling; A: Effect of intravenous epirubicin hydrochloride on liver appearance in mice with D-GalN / LPS-induced acute liver failure; B: Effect of liver coefficient; C: Effect of alanine aminotransferase (ALT); D: Effect of aspartate aminotransferase (AST); E: Effect of total bilirubin; F: Effect of glutathione; n=8, expressed as mean ± SD, # P<0.05, ## Compared with the control group, P<0.01 * P<0.05, ** (P<0.01 compared with the model group);

[0036] Figure 11Effect of intravenous EPI on D-GalN / LPS-induced B4GALT1 protein levels. A shows Western blotting images, and B shows statistical results. (In mice, EPI was administered intravenously for three consecutive days at doses of 0.1, 0.3, and 1 mg / kg before modeling. Acute liver failure was induced by intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. Liver tissue samples were collected 6 h after modeling, and B4GALT1 protein levels were detected by Western blotting. n=6, expressed as mean ± SD.) ## Compared with the control group, P<0.01 ** P<0.01, *** (P<0.001 compared with the model group);

[0037] Figure 12 Effects of intravenous epirubicin hydrochloride on liver function-related indicators in mice with D-GalN / LPS-induced acute liver failure (mice were induced with an acute liver failure model by intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. Treatment was initiated 15 min after modeling. To maintain a certain blood drug concentration, maintenance treatment was performed by intravenous infusion of three different concentrations of epirubicin hydrochloride for 30 min via tail vein. Samples were collected 6 h after modeling. A: Effect of intravenous epirubicin hydrochloride on liver appearance in mice with D-GalN / LPS-induced acute liver failure; B: Effect of liver coefficient; C: Effect of alanine aminotransferase (ALT); D: Effect of aspartate aminotransferase (AST); E: Effect of total bilirubin; F: Effect of glutathione; n=8, expressed as mean ± SD.) ## Compared with the control group, P<0.01 ** P<0.01, *** (P<0.001 compared with the model group)

[0038] Figure 13 Effect of intravenous epirubicin hydrochloride on B4GALT1 protein levels induced by D-GalN / LPS. A shows Western blotting images, and B shows statistical results. (An acute liver failure mouse model was induced by intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. Treatment was initiated 15 min after modeling. To maintain a certain blood drug concentration, three different epirubicin hydrochloride injection solutions were administered via tail vein infusion for 30 min as maintenance treatment. Liver tissue samples were collected 6 h after modeling, and the B4GALT1 protein level in liver tissue was detected by Western blotting; n=6, expressed as mean ± SD.) ## Compared with the control group, P<0.01 * P<0.05, *** P<0.001 (compared with the model group). Detailed Implementation

[0039] The following examples further illustrate the substantive content of the present invention, but do not limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention. In the present invention, the test materials or test methods not described in detail or particularly emphasized are all conventional test materials or test methods in the art, and those skilled in the art can obtain such test materials or have the ability to conduct such tests.

[0040] Example 1: Correlation between β-1,4-galactosyltransferase 1 and acute and chronic liver diseases

[0041] I. Experimental Materials

[0042] 1. Instruments and reagents

[0043] Intravenous visual mouse tail fixation device (Jinan Yiyan Technology Development Co., Ltd.), Infinite 200Pro microplate reader (Tecan, Switzerland), electronic analytical balance (Mettler-Toledo, Shanghai), Milli-Q ultrapure water system (Millipore, USA), miniature vortex apparatus (Dalong Xingchuang Experimental Instruments Co., Ltd.), intelligent adjustable peristaltic pump (Kammer Fluid Technology (Shanghai) Co., Ltd.), BIO-RAD Chemi Doc™ XRS + Gel imaging system (Bio-Rad, USA), tissue scissors, surgical forceps (Shanghai Surgical Instrument Factory).

[0044] Lipopolysaccharide, D-galactosamine (D-GalN) (Sigma Aldrich), concanavalin A (ConA) (Sigma Aldrich), high-fat diet formula (Nantong Trofi Feed Technology Co., Ltd.), epirubicin hydrochloride injection (EPI) (Pfizer Pharmaceuticals Co., Ltd.), sodium chloride injection (Anhui Shuanghe Pharmaceutical Co., Ltd.), β-galactosyltransferase activity assay kit (R&D Company); B4GALT1 primary antibody (Abcam), GAPDH secondary antibody (BioWorld).

[0045] 2. Laboratory animals

[0046] Twelve male C57BL / 6J mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Experimental Animal Production License No. SYXK(Su)2022-0009) for the purpose of constructing the D-GalN / LPS-ALF model.

[0047] Twelve male Balb / c mice were purchased from the Comparative Medicine Center of Yangzhou University (Experimental Animal Production License No. SYXK(Su)2020-0020) for the purpose of constructing the ConA-ALF model.

[0048] Eighteen male C57BL / 6J mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Experimental Animal Production License No. SYXK(Su)2022-0009) to construct a non-alcoholic fatty liver model.

[0049] II. Experimental Methods

[0050] 1. D-GalN / LPS model of acute liver failure in mice: Male C57BL / 6J mice were randomly divided into two groups of 6 mice each, namely blank group and model group. The model group was induced by intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. The mice were harvested 6 h after D-GalN / LPS modeling. (9) Chai F, et al. Protective effect of coptisine from rhizoma coptidis on LPS / D-GalN-induced acute liver failure in mice through up-regulating expression of miR-122[J]. Biomed Pharmacother. 2018, 2(98): 180-190).

[0051] 2. ConA model of acute liver failure in mice: Male Balb / c mice were randomly divided into two groups of 6 each, namely the blank group and the model group. The model group was injected with 20 mg / kg ConA solution via the tail vein. The mice were sacrificed and their samples were collected 8 h after modeling. The specific method is as follows ((10) Gao Y, et al. Magnesium isoglycyrrhizinate amelioratesconcanavalin A-induced liver injury via the p38 and JNK MAPK pathway[J].Immunopharmacol Immunotoxicol. 2020, 42(5): 445-455).

[0052] 3. High-fat diet to establish a mouse model of fatty liver: Male C57BL / 6J mice were randomly divided into two groups: a control group of 6 mice and a model group of 12 mice. The control group was fed a normal diet, while the model group was fed a high-fat diet for 16 weeks to establish a non-alcoholic fatty liver model. The specific method is as follows ((11) Wan J, et al. Gastrodin improves nonalcoholic fattyliver disease through activation of the adenosine monophosphate-activated protein kinase signaling pathway.[J].Hepatology, 2021, 74(6): 3074-3090).

[0053] 4. Immunoblot analysis was performed to detect the expression of β-1,4-galactosyltransferase 1. Liver tissue protein was extracted, and BCA quantification was performed. Samples were added for electrophoresis, followed by membrane transfer. The membrane was blocked with BSA at room temperature for 2 h, and incubated with primary antibody overnight at 4°C. The primary antibody was recovered, the membrane was washed, and incubated with secondary antibody at room temperature for 2 h. The PVDF membrane was washed with TBST and contrast agent was added. The membrane was then analyzed using a BIO-RAD ChemiDoc™ XRS spectrometer. + After development in a gel imaging system, the gel is analyzed.

[0054] 5. Detection of β-1,4-galactosyltransferase 1 expression

[0055] The expression of β-1,4-galactosyltransferase 1 in various hepatitis models was detected by immunoblotting.

[0056] 6. Data Processing

[0057] All data were analyzed using Graphpad Prism 8 software and expressed as mean ± SD. Student's t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons of three or more groups. Dunnett's test was used for the test, and P < 0.05 was considered statistically significant.

[0058] III. Experimental Results

[0059] 1. Changes in B4GALT1 expression in liver tissue of mice with hepatitis induced by multiple factors

[0060] like Figure 1 As shown, the expression of protein B4GALT1 was significantly increased in the livers of mice with hepatitis induced by multiple factors. Figure 1A. The expression of B4GALT1 in the liver tissue of mice with D-GalN / LPS-induced acute liver failure was detected by immunoblotting. Compared with the blank group, the expression of B4GALT1 in the model group was increased. Figure 1 Compared with the control group, B4GALT1 expression was increased in the concanavalin A (ConA) induced acute liver failure mouse model group. Figure 1 Compared with the control group, B4GALT1 expression was increased in the high-fat diet-induced non-alcoholic fatty liver mouse model group; in addition, the time-dependent changes of B4GALT1 in liver tissue of D-GalN / LPS-induced acute liver failure mice were detected. Figure 1 D) Immunoblotting was used to detect the time-dependent changes of B4GALT1 in the liver tissue of D-GalN / LPS-induced acute liver failure mice. Compared with the control group, the protein expression of B4GALT1 in the liver of D-GalN / LPS-induced ALF mice was significantly increased. The expression of B4GALT1 in the liver increased with time, with a significant increase at 1 h and a peak at 6 h, suggesting that B4GALT1 protein may play a key role in hepatitis. # P<0.05, ## P<0.01).

[0061] Example 2: Selection and preliminary validation of β-1,4-galactosyltransferase 1 inhibitors

[0062] I. Experimental Materials

[0063] 1. Database and software

[0064] Global drug database, AutoDock 4.1 molecular docking software, Python 3.8 software, MonolithNTAffinity analysis software.

[0065] 2. Instruments and reagents

[0066] Infinite 200Pro microplate reader (Tecan, Switzerland), electronic analytical balance (Mettler-Toledo, Shanghai), Milli-Q ultrapure water system (Millipore, USA), miniature vortex apparatus (Dalong Xingchuang Experimental Instruments Co., Ltd.), MST intermolecular interaction analyzer (NANOTEMPER, Germany).

[0067] β-galactosyltransferase activity assay kit (R&D Company), epirubicin hydrochloride injection for injection (Pfizer Pharmaceuticals Ltd.), Monolith TMRED-NHS second-generation protein labeling kit (Nuotanpu Technology Co., Ltd.), B4GALT1 recombinant protein (Xinhe Biotechnology), capillary tubes for MST detection (NANOTEMPER, Germany); epirubicin hydrochloride standard (Aladdin), sodium chloride injection (Anhui Shuanghe Pharmaceutical Co., Ltd.), alanine aminotransferase kit (Nanjing Jiancheng Bioengineering Institute).

[0068] II. Experimental Methods

[0069] 1. Screening for B4GALT1 inhibitors based on global drug databases: Enter the keyword "B4GALT1 inhibitor" into the database and search three sub-databases (marketed drug database, active pharmaceutical ingredient database, and natural product database), and export the results.

[0070] 2. Binding Free Energy Analysis Based on Molecular Docking Technology: AutoDock 4.1 software was used to perform molecular docking between epirubicin, 5-tert-butyl-4-methylthiazol-2-carboxylic acid, 3-aminocoumarin, and the target B4GALT1 to verify the interaction between the small molecules and the target. First, the PDB format of the B4GALT1 protein was downloaded from the PDB database (https: / / www.rcsb.org / ). Then, the three-dimensional structures of epirubicin, 5-tert-butyl-4-methylthiazol-2-carboxylic acid, and 3-aminocoumarin were downloaded from the PubChem database (https: / / www.ncbi.nlm.nih.gov / ). The target proteins were dehydrated and hydrogenated using AutoDock 4.1 software. Epirubicin, 5-tert-butyl-4-methylthiazol-2-carboxylic acid, 3-aminocoumarin, and the target protein were imported into AutoDock 4.1 software for docking. Finally, Python software was used for visualization analysis.

[0071]

[0072] 3-Aminocoumarin epirubicin 5-tert-butyl-4-methylthiazol-2-carboxylic acid

[0073] 3. In vitro detection of the effect of epirubicin hydrochloride on β-1,4-galactosyltransferase 1 activity: Using diphosphate nucleotide sugars as donor substrates, the enzyme activity of glycosyltransferases was measured. In the glycosyltransferase reaction, phosphatase quantitatively removes inorganic phosphate from the remaining nucleotide diphosphates, such as UDP or GDP. The released inorganic phosphate was then detected using malachite green phosphate reagent. The amount of inorganic phosphate released by the coupled phosphatase was equal to the consumed nucleotide sugar or the generated glycoconjugate.

[0074] 4. MST technology for detecting the binding energy of EPI to β-1,4-galactosyltransferase 1: First, B4GALT1 recombinant protein was labeled using a Monolith™ RED-NHS second-generation protein labeling kit, preparing 200 μL (100 nM) for later use. Based on previous preliminary experiments and the expected Kd value, epirubicin standards were prepared in 16 different concentrations, diluted 2-fold, and mixed with 10 μL of protein at a 1:1 ratio. The sample was then aspirated into a capillary tube and placed in the capillary tray. The capillary tray was inserted into the Monolith™ NT.115 instrument, the door was closed, and capillary scanning was initiated. The Monolith NT.115 automatically scanned the sample tray by measuring fluorescence. After the capillary scan was completed, the MST measurement process was initiated.

[0075] 5. Based on the global drug database, a selection of marketed drugs, active pharmaceutical ingredients (APIs), and natural products were selected for molecular docking and MST binding energy verification: Marketed drugs, APIs, and natural products obtained from the global drug database were initially screened, and those with higher and / or lower scores, including UDP-galactose disodium salt, N-acetyl-D-glucosamine, omeprazole, gemcitabine, 3-aminocoumarin, and thymol, were selected. Molecular docking and MST techniques were used to detect their interaction with β-1,4-galactosyltransferase 1.

[0076] 6. In vitro validation of the effects of selected marketed drugs, active pharmaceutical ingredients and natural products on alanine aminotransferase (ALT): Using an in vitro acute liver failure model induced by acetaminophen at 20 mmol / L, we validated the effects of six β-1,4-galactosyltransferase 1 inhibitors (UDP-galactosyltransferase 1), including N-acetyl-D-glucosamine, omeprazole, gemcitabine, 3-aminocoumarin, and thymol, on ALT levels in an acetaminophen-induced AML 12 acute liver failure model.

[0077] 7. Data Processing

[0078] Open the Monolith NTAffinity analysis software, load the data, and select "Thermophoresis" analysis. Perform Kd fitting in this analysis mode. After completion, select the "Fit" button, and then select the "Fit Curve" button. The system will automatically display the calculated Kd values.

[0079] III. Experimental Results

[0080] 1. Screening for B4GALT1 inhibitors based on drug databases

[0081] Inhibitors related to B4GALT1 were identified from a global drug database. Preliminary predictions identified approximately 3,000 potential drugs with anti-B4GALT1 activity, including marketed drugs, active pharmaceutical ingredients (APIs), and natural products. Excluding drugs with reported hepatoprotective effects and significant side effects, the top 50 most representative marketed drugs, APIs, and natural products were listed in descending order of their scores, without limitation to drugs with similar structures. The screening results showed that the highest-scoring (highest negative value) marketed drugs were epirubicin hydrochloride (predicted score -17.9154), 5-tert-butyl-4-methylthiazol-2-carboxylic acid (predicted score -19.6889), and 3-aminocoumarin (predicted score -14.6841), suggesting they may be B4GALT1 inhibitors. In vitro and in vivo experiments are planned to verify these findings.

[0082] From the initially screened marketed drugs, active pharmaceutical ingredients, and natural products, the highest-scoring representatives were selected for molecular docking with the target protein B4GALT1. The intermolecular binding free energy and specific binding sites were calculated. The docking results of B4GALT1 with epirubicin are shown below. Figure 2 As shown, with a binding free energy of -9.96 kcal / mol, the docking results of B4GALT1 with 5-tert-butyl-4-methylthiazol-2-carboxylic acid are as follows. Figure 3 As shown, with a binding free energy of -6.37 kcal / mol, the docking results of B4GALT1 with 3-aminocoumarin are as follows. Figure 4 As shown, the binding free energy is -7.45 kcal / mol. The absolute values ​​of the binding energies of the marketed drugs, active pharmaceutical ingredients (APIs), and natural products screened for B4GALT1 are all greater than 5 kcal / mol, indicating that the inhibitors screened from the global drug database can bind to B4GALT1. Higher scores (absolute values) indicate more stable binding between the marketed drugs, APIs, and natural products and B4GALT1.

[0083] 3. In vitro detection of the effect of β-1,4-galactosyltransferase 1 inhibitor EPI on enzyme activity

[0084] Figure 5 The effect of epirubicin hydrochloride on β-1,4-galactosyltransferase 1 activity was investigated in vitro. The results showed that epirubicin hydrochloride directly inhibited the activity of β-1,4-galactosyltransferase 1, with an IC50 concentration of [missing value]. 50 The concentration was 2.139 μmol / L. This further suggests that epirubicin hydrochloride may be a direct inhibitor of β-1,4-galactosyltransferase 1.

[0085] 4. MST detection of the interaction between EPI and β-1,4-galactosyltransferase 1

[0086] First, use Monolith. TM The B4GALT1 recombinant protein was labeled using the RED-NHS second-generation protein labeling kit, and the binding affinity of EPI to β-1,4-galactosyltransferase 1 was examined using MST technology. The results are as follows: Figure 6 As shown, the Kd value is 16.10±2.43 μmol / L, indicating that EPI can interact with β-1,4-galactosyltransferase 1, further confirming that EPI can bind to β-1,4-galactosyltransferase 1.

[0087] 5. Based on a global drug database, select a number of marketed drugs, active pharmaceutical ingredients (APIs), and natural products, and perform molecular docking and MST binding energy verification.

[0088] We conducted a preliminary screening of marketed drugs, active pharmaceutical ingredients (APIs), and natural products obtained from global drug databases. We selected marketed drugs omeprazole and gemcitabine, APIs UDP-galactose-disodium salt and N-acetyl-D-glucosamine, and natural products 3-aminocoumarin and thymol. We first used molecular docking technology to detect their affinity, and then used MST technology for corroboration to verify their interaction with β-1,4-galactosyltransferase 1. The molecular docking results are shown in Figure A. The affinity of β-1,4-galactosyltransferase 1 for omeprazole is -4.87 kcal / mol; for gemcitabine, it is -7.0 kcal / mol (7C); for UDP-galactose-disodium salt, it is -12.52 kcal / mol (7E); for N-acetyl-D-glucosamine, it is -6.83 kcal / mol (7G); for 3-aminocoumarin, it is -5.73 kcal / mol (7I); and for thymol, it is -4.43 kcal / mol (7E). K); This indicates that the above-mentioned marketed drugs, active pharmaceutical ingredients, and natural products all have an affinity for β-1,4-galactosyltransferase 1, such as Figure 7As shown in B, the Kd values ​​of β-1,4-galactosyltransferase 1 with omeprazole were 0.33 ± 0.81 μmol / L, with gemcitabine 0.22 ± 0.54 μmol / L (7 D), with UDP-galactose-disodium salt 10.30 ± 0.01 μmol / L (7 F), with N-acetyl-D-glucosamine 13.97 ± 1.22 μmol / L (7 H), with 3-aminocoumarin 0.06 ± 1.35 μmol / L (7 J), and with thymol 0.07 ± 0.97 μmol / L (7 L), further indicating that marketed drugs, active pharmaceutical ingredients, and natural products obtained from global drug databases may be inhibitors of β-1,4-galactosyltransferase 1.

[0089] 6. In vitro validation of the effects of selected marketed drugs, active pharmaceutical ingredients, and natural products on alanine aminotransferase (ALT) levels.

[0090] An acute liver failure model of AML12 was induced using 20 mmol / L acetaminophen, and the effects of six β-1,4-galactosyltransferase 1 inhibitors (UDP-galactose disodium salt, N-acetyl-D-glucosamine, omeprazole, gemcitabine, 3-aminocoumarin, and thymol) on alanine aminotransferase (ALT) levels in the acetaminophen-induced acute liver failure model were verified in vitro. The results are as follows: Figure 8 As shown, UDP-galactose disodium salt, N-acetyl-D-glucosamine, omeprazole, gemcitabine, 3-aminocoumarin, and thymol all have significant effects on improving ALT levels.

[0091] Example 3: The effect of intravenous infusion of epirubicin hydrochloride, a β-1,4-galactosyltransferase 1 inhibitor, on the improvement of acute liver injury.

[0092] I. Experimental Materials

[0093] 1. Instruments and reagents

[0094] Intravenous visual mouse tail fixation device (Jinan Yiyan Technology Development Co., Ltd.), Infinite 200Pro microplate reader (Tecan, Switzerland), electronic analytical balance (Mettler-Toledo, Shanghai), Milli-Q ultrapure water system (Millipore, USA), miniature vortex apparatus (Dalong Xingchuang Experimental Instruments Co., Ltd.), intelligent adjustable peristaltic pump (Kammer Fluid Technology (Shanghai) Co., Ltd.), BIO-RAD Chemi Doc™ XRS + Gel imaging system (Bio-Rad, USA), tissue scissors, surgical forceps (Shanghai Surgical Instrument Factory).

[0095] Acetaminophen (Dalian Meilun Biotechnology Co., Ltd.), Epirubicin Hydrochloride Injection (Pfizer Pharmaceuticals Co., Ltd.), Sodium Chloride Injection (Anhui Shuanghe Pharmaceutical Co., Ltd.), Alanine Aminotransferase, Aspartate Aminotransferase, Total Bilirubin, and Glutathione Reagent Kit (Nanjing Jiancheng Bioengineering Institute).

[0096] 2. Laboratory animals

[0097] Sixty male C57BL / 6J mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Experimental Animal Production License No. SYXK(Su)2022-0009) for the purpose of constructing the APAP-ALI model.

[0098] II. Experimental Methods

[0099] 1. Male C57BL / 6J mice were randomly divided into 6 groups of 12 mice each: blank control group, model group, epirubicin hydrochloride (EPI) high, medium, and low dose groups (100, 50, and 25 μg / kg), and positive control group (NAC). The model group was induced with an acute liver injury model by intraperitoneal injection of 300 mg / kg acetaminophen (APAP) solution. 30 min later, EPI was infused. Blood and liver tissue were collected 6 h after APAP modeling for biochemical and molecular analysis.

[0100] 2. Observation Indicators and Methods

[0101] 2.1 Record the external morphology of the liver

[0102] After perfusion with PBS using a smart peristaltic pump, the livers were removed and placed in PBS to record the morphological appearance of each group of mice.

[0103] 2.2 Detection of biochemical indicators in serum

[0104] Serum was separated, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and glutathione in the serum were detected using a kit. The specific detection methods were strictly in accordance with the kit instructions. The levels of transaminases (ALT and AST), total bilirubin, and glutathione in the serum of mice in each group were detected.

[0105] 3. Data Processing

[0106] All data were analyzed using Graphpad Prism 8 software and expressed as mean ± SD. Student's t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons of three or more groups, and Dunnett's test was used for the test. P < 0.05 was considered statistically significant.

[0107] III. Experimental Results

[0108] 1. Effects of intravenous injection of different doses of EPI on liver function-related indicators in mice with APAP-induced acute injury.

[0109] After APAP modeling, the model group showed significant liver damage. Intravenous infusion of epirubicin hydrochloride effectively improved liver bleeding and showed a dose-dependent improvement in liver appearance. Figure 9 As shown in Figure A. Figure 9 The liver coefficient results showed that, compared with the blank group, the liver weight ratio increased in the model group (B). ## (P<0.01) indicates liver congestion, edema, or hyperplasia / hypertrophy, suggesting severe liver damage after APAP stimulation. Treatment with different doses of EPI significantly improved liver function, with the high-dose EPI group showing better improvement than acetylcysteine ​​(NAC). * P<0.05, ** P<0.01. After APAP stimulation, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased (P<0.01). ## (P<0.01) indicates severe liver damage after APAP stimulation. Treatment with different doses of EPI showed a dose-dependent reduction in ALT and AST levels, resulting in significant improvement in transaminase levels. The high-dose EPI group showed a similar effect to NAC in reducing ALT and AST levels. * P<0.05, ** P<0.01), the results are shown in 9C and D. 9E By detecting total bilirubin, it was found that the total bilirubin level in the model group mice was significantly increased (P<0.01). # (P<0.05) indicates severe liver damage after APAP stimulation. Treatment with different doses of EPI reduced total bilirubin levels, with the high-dose EPI group showing a better effect on reducing total bilirubin than the positive control drug NAC. * P<0.05, ** P<0.01). Figure 9 F detected serum glutathione levels. Compared with the blank group, the glutathione levels in the model group mice were significantly reduced after APAP stimulation. # (P<0.01) After treatment with different doses of EPI, glutathione levels increased slightly.

[0110] Example 4: The effect of intravenous injection of epirubicin hydrochloride, a β-1,4-galactosyltransferase 1 inhibitor, on the improvement of acute liver failure.

[0111] I. Experimental Materials

[0112] 1. Instruments and reagents

[0113] Intravenous visual mouse tail fixation device (Jinan Yiyan Technology Development Co., Ltd.), Infinite 200Pro microplate reader (Tecan, Switzerland), electronic analytical balance (Mettler-Toledo, Shanghai), Milli-Q ultrapure water system (Millipore, USA), miniature vortex apparatus (Dalong Xingchuang Experimental Instruments Co., Ltd.), intelligent adjustable peristaltic pump (Kammer Fluid Technology (Shanghai) Co., Ltd.), BIO-RAD Chemi Doc™ XRS + Gel imaging system (Bio-Rad, USA), tissue scissors, surgical forceps (Shanghai Surgical Instrument Factory).

[0114] Lipopolysaccharide, D-galactosamine (Sigma Aldrich), epirubicin hydrochloride injection (Pfizer Pharmaceuticals Co., Ltd.), sodium chloride injection (Anhui Shuanghe Pharmaceutical Co., Ltd.), alanine aminotransferase, aspartate aminotransferase, total bilirubin, glutathione reagent kit (Nanjing Jiancheng Bioengineering Institute), B4GALT1 primary antibody (Abcam), GAPDH primary antibody (BioWorld).

[0115] 2. Laboratory animals

[0116] Sixty male C57BL / 6J mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Experimental Animal Production License No. SYXK(Su)2022-0009) for the purpose of constructing the D-GalN / LPS-ALF model.

[0117] II. Experimental Methods

[0118] 1. D-GalN / LPS model of acute liver failure in mice: Male C57BL / 6J mice were randomly divided into 5 groups of 12 mice each: blank control group, model group, and high, medium, and low dose groups of epirubicin hydrochloride (EPI) (1, 0.3, and 0.1 mg / kg). The model group was induced with intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. Before modeling, the EPI administration groups were intravenously injected for three consecutive days at doses of 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg. Blood and liver tissue were collected 6 h after D-GalN / LPS modeling for biochemical and molecular detection.

[0119] 2. Immunoblot analysis was performed to detect the expression of β-1,4-galactosyltransferase 1. Liver tissue protein was extracted, and BCA quantification was performed. Samples were added for electrophoresis, followed by membrane transfer. The membrane was blocked with BSA at room temperature for 2 h, and incubated with primary antibody overnight at 4°C. The primary antibody was recovered, the membrane was washed, and incubated with secondary antibody at room temperature for 2 h. The PVDF membrane was washed with TBST and contrast agent was added. The membrane was then analyzed using a BIO-RAD ChemiDoc™ XRS spectrometer.+ After development in a gel imaging system, the gel is analyzed.

[0120] 3. Observation Indicators and Methods

[0121] 3.1 Record the external morphology of the liver

[0122] After perfusion with PBS using a smart peristaltic pump, the livers were removed and placed in PBS to record the morphological appearance of each group of mice.

[0123] 3.2 Detection of biochemical indicators in serum

[0124] Serum was separated, and the levels of transaminases (alanine aminotransferase and aspartate aminotransferase), total bilirubin, and glutathione in the serum of mice in each group were detected.

[0125] 3.3 Detection of β-1,4-galactosyltransferase 1 expression

[0126] The expression of β-1,4-galactosyltransferase 1 stimulated by D-GalN / LPS was detected by immunoblotting after intravenous injection of different doses of epirubicin hydrochloride.

[0127] 4. Data Processing

[0128] All data were analyzed using Graphpad Prism 8 software and expressed as mean ± SD. Student's t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons of three or more groups, and Dunnett's test was used for the test. P < 0.05 was considered statistically significant.

[0129] III. Experimental Results

[0130] 1. Effects of intravenous injection of different doses of EPI on liver function-related indicators in mice with D-GalN / LPS-induced acute liver failure

[0131] Figure 10 The liver morphology results shown in Figure A indicate that after modeling with D-GalN / LPS, the model group showed significant liver hemorrhage and severe liver damage. Epirubicin hydrochloride, an inhibitor of β-1,4-galactosyltransferase 1, effectively improved acute liver failure in a dose-dependent manner. Figure 10 B. The liver coefficient of mice with acute liver failure was detected. Compared with the blank group, the liver coefficient of mice in the model group was significantly increased after D-GalN / LPS stimulation. # P<0.05, indicating severe liver damage after D-GalN / LPS stimulation. Treatment with different doses of EPI showed improvement in the high-dose group. * P<0.05). Figure 10Figures C and D show that, compared with the control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased after D-GalN / LPS stimulation. ## P<0.01 indicates severe liver damage following D-GalN / LPS stimulation. Treatment with different doses of epirubicin hydrochloride significantly improved alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the high-dose group. * P<0.05, ** P<0.01). Figure 10 E. Serum total bilirubin levels were measured to assess the severity of hepatocyte damage in ALF mice. Results showed that, compared to the control group, D-GalN / LPS stimulation significantly increased the total bilirubin level in the model group mice. ## P<0.01 indicates severe liver damage following D-GalN / LPS stimulation. Treatment with different doses of epirubicin hydrochloride significantly reduced total bilirubin levels in the high-dose group. * P<0.05). Figure 10 The serum glutathione level was measured to assess the antioxidant capacity of epirubicin hydrochloride. Results showed that, compared with the control group, glutathione levels in the model group mice were significantly reduced after D-GalN / LPS stimulation. ## P<0.01, treatment with different doses of epirubicin hydrochloride significantly improved glutathione levels. ** P<0.01).

[0132] 2. Effects of β-1,4-galactosyltransferase 1 inhibitor EPI on B4GALT1 protein expression in liver tissue of D-GalN / LPS-induced ALF mice

[0133] The expression of B4GALT1 in liver tissue stimulated by D-GalN / LPS after intravenous injection of different doses of epirubicin hydrochloride was detected by immunoblotting. Figure 11 As shown, compared with the control group, the protein expression level of B4GALT1 in the liver of D-GalN / LPS-induced ALF mice was significantly increased. ## P<0.01, after treatment with different doses of epirubicin hydrochloride, the protein expression level of B4GALT1 decreased in a dose-dependent manner. ** P<0.01, *** P<0.001).

[0134] Example 5: The effect of intravenous infusion of epirubicin hydrochloride, a β-1,4-galactosyltransferase 1 inhibitor, on the improvement of acute liver failure.

[0135] Note: The administration method differs from that in Example 4, which involved intravenous injection, while in Example 5, it involved intravenous infusion. Further investigation of the efficacy is needed.

[0136] I. Experimental Materials

[0137] 1. Instruments and reagents

[0138] Constant flow syringe pump LSP04-1A (Baoding Lange Constant Flow Pump Co., Ltd.), intravenous visual mouse tail fixation device (Jinan Yiyan Technology Development Co., Ltd.), Infinite 200Pro microplate reader (Tecan, Switzerland), electronic analytical balance (Shanghai Mettler-Toledo), Milli-Q ultrapure water system (Millipore, USA), miniature vortex apparatus (Dalong Xingchuang Experimental Instrument Co., Ltd.), intelligent adjustable peristaltic pump (Kammer Fluid Technology (Shanghai) Co., Ltd.), BIO-RAD ChemiDoc™ XRS + Gel imaging system (Bio-Rad, USA), tissue scissors, surgical forceps (Shanghai Surgical Instrument Factory).

[0139] Lipopolysaccharide, D-galactosamine (Sigma Aldrich), epirubicin hydrochloride injection (Pfizer Pharmaceuticals Co., Ltd.), sodium chloride injection (Anhui Shuanghe Pharmaceutical Co., Ltd.), alanine aminotransferase, aspartate aminotransferase, total bilirubin, and glutathione reagent kits (Nanjing Jiancheng Bioengineering Institute); B4GALT1 primary antibody (Abcam), GAPDH primary antibody (BioWorld).

[0140] 2. Laboratory animals

[0141] Sixty male C57BL / 6J mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Experimental Animal Production License No. SYXK(Su)2022-0013).

[0142] II. Experimental Methods

[0143] 1. D-GalN / LPS model of acute liver failure in mice: Male C57BL / 6J mice were randomly divided into 5 groups of 12 mice each: blank group, model group, and high, medium, and low dose groups (1, 0.3, and 0.1 mg / kg, respectively). The model group was induced with intraperitoneal injection of 500 mg / kg D-GalN combined with 10 μg / kg LPS solution. Treatment was initiated 15 min after modeling. To maintain a certain blood drug concentration, epirubicin hydrochloride injection was administered via a constant flow infusion pump to simulate tail vein infusion for 30 min as maintenance treatment. Blood and liver tissue were collected 6 h after D-GalN / LPS modeling for biochemical and molecular analysis.

[0144] 2. The observation indicators and methods are the same as in Example 3.

[0145] 2.1 Record the appearance and morphology of the liver, as in Example 3.

[0146] 2.2 Detection of biochemical indicators in serum, same as in Example 3.

[0147] 2.3 Detection of β-1,4-galactosyltransferase 1 expression, same as in Example 3.

[0148] 3. Data processing, same as in Example 3.

[0149] III. Experimental Results

[0150] 1. Effects of intravenous epirubicin hydrochloride on liver function indicators in mice with D-GalN / LPS-induced acute liver failure

[0151] Figure 12 The liver morphology results shown in Figure A indicate that after modeling with D-GalN / LPS, the model group showed significant liver hemorrhage and severe liver damage. Epirubicin hydrochloride, an inhibitor of β-1,4-galactosyltransferase 1, effectively improved acute liver failure in a dose-dependent manner. Figure 12 B. Liver coefficient was measured in mice with acute liver failure. Compared with the control group, the liver coefficient of the model group mice was significantly increased after D-GalN / LPS stimulation. ## P<0.01 indicates that liver damage was severe after D-GalN / LPS stimulation, and all dose groups showed improvement after treatment with different doses of epirubicin hydrochloride. ** P<0.01). Figure 12 Figures C and D show that, compared with the control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased after D-GalN / LPS stimulation. ## (P<0.01) indicates severe liver damage following D-GalN / LPS stimulation. Treatment with different doses of epirubicin hydrochloride resulted in a dose-dependent reduction in transaminase levels. ** P<0.01, *** P<0.001). Figure 12 E. Serum total bilirubin levels were measured to assess the severity of hepatocyte damage in ALF mice. Results showed that, compared to the control group, D-GalN / LPS stimulation significantly increased the total bilirubin level in the model group mice. ## P<0.01 indicates severe liver damage after D-GalN / LPS stimulation. Treatment with different doses of epirubicin hydrochloride reduced total bilirubin levels at all concentrations, with the high-dose group showing the best improvement in total bilirubin levels. **P<0.01). Figure 12 The serum glutathione level was measured to assess the antioxidant capacity of epirubicin hydrochloride. Results showed that, compared with the control group, glutathione levels in the model group mice were significantly reduced after D-GalN / LPS stimulation. ## (P<0.01) After treatment with different doses of epirubicin hydrochloride, the high-dose group showed a relatively better effect on improving glutathione levels.

[0152] 2. Effects of β-1,4-galactosyltransferase 1 inhibitor EPI on B4GALT1 protein expression in liver tissue of D-GalN / LPS-induced ALF mice

[0153] The expression of B4GALT1 in liver tissue stimulated by D-GalN / LPS after intravenous infusion of different doses of epirubicin hydrochloride was detected by immunoblotting. Figure 13 As shown, compared with the control group, the protein expression level of B4GALT1 in the liver of D-GalN / LPS-induced ALF mice was significantly increased. ## P<0.01, after treatment with different doses of epirubicin hydrochloride, the protein expression level of B4GALT1 decreased in a dose-dependent manner. * P<0.05, *** P<0.001).

Claims

1. The application of β-1,4-galactosyltransferase 1 as a drug target in the preparation of drugs for the prevention or treatment of acute or chronic liver diseases, characterized in that, The acute and chronic liver diseases mentioned include acute hepatitis, chronic hepatitis, liver injury, acute liver failure, or chronic liver failure.

2. The use of β-1,4-galactosyltransferase 1 inhibitors in the preparation of drugs for the prevention or treatment of acute liver disease, characterized in that, The acute liver diseases include acute hepatitis, liver injury, and acute liver failure; the β-1,4-galactosyltransferase 1 inhibitor is UDP-galactose disodium salt, omeprazole, gemcitabine, epirubicin, or 3-aminocoumarin.

3. The use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of acute or chronic liver diseases, characterized in that, The pharmaceutical composition comprises a β-1,4-galactosyltransferase 1 inhibitor and a pharmaceutically acceptable carrier, wherein: the acute or chronic liver disease includes acute hepatitis, chronic hepatitis, liver injury, acute liver failure, or chronic liver failure; and the β-1,4-galactosyltransferase 1 inhibitor is UDP-galactose disodium salt, omeprazole, gemcitabine, epirubicin, or 3-aminocoumarin.

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