Use of an agent that inhibits ent4 in the manufacture of a medicament for treating liver fibrosis

Abstract

The present application relates to the technical field of biopharmaceuticals, and particularly relates to application of a preparation for inhibiting ENT4 in preparation of a medicine for treating liver fibrosis. The present application finds that in a CCl4-induced liver fibrosis mouse model, expression of ENT4 is significantly increased. Using an ENT4 inhibitor TCT-6000 can effectively reduce liver fibrosis, improve liver function indexes, reduce collagen deposition, and inhibit activation of hepatic stellate cells. Mechanism research shows that inhibiting ENT4 can up-regulate adenosine and inosine levels, and then down-regulate expression of a fibrosis-promoting factor PDGF-B. In addition, supplementing an adenosine analogue NECA or inosine can also alleviate liver fibrosis. The present application provides a new drug target and candidate drug for treatment of liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to the use of ENT4 inhibitors in the preparation of drugs for treating liver fibrosis. Background Technology

[0002] Chronic liver disease can lead to hepatocellular damage, ultimately resulting in liver fibrosis, cirrhosis, and liver failure. Liver fibrosis is a regenerative process that occurs after injury, characterized by fibroblast deposition in connective tissue accompanied by a proliferative response, a risk factor for hepatocellular carcinoma. The cellular population in liver tissue includes hepatic parenchymal cells and non-parenchymal cells. Parenchymal cells include epithelial cells and endothelial cells. Non-parenchymal cells include hepatic stellate cells and Kuffer cells. Activation of hepatic stellate cells is a key event in liver fibrosis. Various factors are involved in HSC activation, such as pathogens, toxicity, metabolism, and viral infections. The transdifferentiation of activated HSCs into proliferative and fibrotic myofibroblasts is the main mechanism driving liver fibrosis. Fibroblasts secrete large amounts of extracellular matrix (ECM), manifested by increased expression of α-SMA and col1α1, ultimately leading to pathological liver damage. Liver injury results in the infiltration of immune cells such as natural killer (NK) cells, T cells, Kuffer cells, and dendritic cells. Increased Kupffer cell activity leads to increased nuclear factor-κB activation, which in turn promotes the release of pro-inflammatory cytokines and further promotes HSC activation.

[0003] Currently, liver fibrosis is a pressing clinical problem that needs to be addressed, as there is no effective treatment available. Therefore, there is an urgent need to develop a new strategy for treating liver fibrosis. Summary of the Invention

[0004] To address the above problems, this invention provides the use of ENT4 inhibitors in the preparation of drugs for treating liver fibrosis.

[0005] This invention is achieved through the following technical solution: The application of an ENT4 inhibitor in the preparation of a drug for treating liver fibrosis, wherein the ENT4 gene has an NCBI Gene ID of 243328.

[0006] Preferably, the ENT4 inhibitor is TCT-6000.

[0007] Preferably, the drug has TCT-6000 as its sole active ingredient.

[0008] Preferably, the dosage of TCT-6000 is 4 mg / kg body weight.

[0009] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0010] Preferably, the pharmaceutically acceptable carrier is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

[0011] Preferably, the liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver disease, or non-alcoholic fatty liver disease.

[0012] Preferably, the ENT4 inhibitory formulation reduces liver fibrosis by upregulating adenosine and / or inosine levels and inhibiting the expression of platelet-derived growth factor-B.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of ENT4 inhibitors in the preparation of drugs for treating liver fibrosis and reveals their mechanism of action for the first time. The study found that ENT4 expression was significantly increased in CCl4-induced mice. TCT-6000 inhibition of ENT4 alleviated liver fibrosis and suppressed the activity of hepatic stellate cells (HSCs) in liver tissue. Given the important role of ENT4 in regulating adenosine and inosine levels in vivo, this invention further investigated the effects of NECA (an adenosine analog) and inosine supplementation on liver fibrosis. First, adenosine and inosine levels were significantly reduced in CCl4-induced mice, while ENT4 inhibition upregulated adenosine and inosine levels. Then, it was found that NECA and inosine could improve liver fibrosis. Furthermore, PDGF-B, a key pro-fibrotic cytokine, was elevated in CCl4-induced mice and could be significantly inhibited by TCT-6000, NECA, and inosine. PDGF-B protein treatment promoted HSC proliferation and activation, an effect that could be reversed by TCT-6000, NECA, and inosine.

[0014] In summary, this invention is the first to discover that inhibiting ENT4 can alleviate liver fibrosis by increasing adenosine and inosine levels, ultimately leading to a reduction in PDGF-B production. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1The diagram shows the results of this invention's method to alleviate CCl4-induced liver fibrosis in mice by inhibiting ENT4. Figure A shows the expression levels of ENT1, ENT2, ENT3, and ENT4 in mouse liver tissue; Figure B shows the expression level of ENT4 in control, CCl4-, and TCT-6000-treated mice analyzed by qRT-PCR; Figure C shows the serum ALT levels in control, CCl4-, and TCT-6000-treated mice; and Figure D shows the serum ALT levels in control, CCl4-, and TCT-6000-treated mice. T level results; E shows serum LDH levels in mice in the control, CCl4, and TCT-6000 treatment groups; F shows serum MAO levels in mice in the control, CCl4, and TCT-6000 treatment groups; G shows the results of HE, Masson, and Sirius red staining for detecting inflammatory infiltration and collagen deposition in the liver tissue of mice in the control, CCl4, and TCT-6000 treatment groups; Scale bar: 50 μm; Magnification: 20x; H shows the statistical chart of Masson staining positive area, using Image... Image J software was used for positive area statistics; Image I shows the positive area statistics of Sirius red staining, calculated using Image J software; Image J shows the expression level of F4 / 80 in the liver tissue of mice in the control, CCl4, and TCT-6000 treatment groups by immunohistochemical (IHC) staining analysis; scale bar, 50 μm; magnification: 20x; Image K shows the expression level statistics of α-SMA in the liver tissue of mice in the control, CCl4, and TCT-6000 treatment groups by qRT-PCR and immunohistochemical (IHC) staining analysis; Image L shows the expression level statistics of col1α1 in the liver tissue of mice in the control, CCl4, and TCT-6000 treatment groups by qRT-PCR and immunohistochemical (IHC) staining analysis; Image M shows the expression levels of α-SMA and col1α1 in the liver tissue of mice in the control, CCl4, and TCT-6000 treatment groups by qRT-PCR and immunohistochemical (IHC) staining analysis; scale bar, 50 μm; magnification: 20x. p <0.05,** p <0.01, **** p <0.0001, ns: no significant difference.

[0017] Figure 2The following figures illustrate the results of how NECA can alleviate CCl4-induced liver fibrosis in mice according to the present invention. A shows the serum adenosine level in patients with liver fibrosis; B shows the serum adenosine level in mice; C shows the serum ALT level in mice in the control, CCl4, and NECA treatment groups; D shows the serum AST level in mice in the control, CCl4, and NECA treatment groups; E shows the serum LDH level in mice in the control, CCl4, and NECA treatment groups; F shows the serum MAO level in mice in the control, CCl4, and NECA treatment groups; G shows the results of HE, Masson, and Sirius red staining to detect inflammatory infiltration and collagen deposition in the liver tissue of mice in the control, CCl4, and NECA treatment groups; scale bar: 50 μm; magnification: 20x; H shows the statistical chart of the positive area of ​​Masson staining, calculated using ImageJ software; I shows the statistical chart of the positive area of ​​Sirius red staining, calculated using ImageJ software. J software was used for positive area statistics; J is a graph showing the expression level of F4 / 80 in the liver tissue of mice in the control, CCl4 and NECA treatment groups by immunohistochemical (IHC) staining analysis; scale bar, 50μm; magnification 20x; K is a graph showing the expression level of α-SMA in the liver tissue of mice treated with CCl4 and NECA by qRT-PCR and IHC staining analysis; L is a graph showing the expression level of col1α1 in the liver tissue of mice treated with CCl4 and NECA by qRT-PCR and IHC staining analysis; M is a graph showing the expression levels of α-SMA and col1α1 in the liver tissue of mice treated with CCl4 and NECA by qRT-PCR and IHC staining analysis; scale bar, 50μm; magnification 20x;* p <0.05,** p <0.01, *** p <0.0005, **** p <0.0001, ns: no significant difference.

[0018] Figure 3The following figures illustrate the results of how inosine can alleviate CCl4-induced liver fibrosis in mice according to the present invention. A shows the serum adenosine level in patients with liver fibrosis; B shows the serum adenosine level in mice; C shows the serum ALT level in mice in the control, CCl4, and inosine treatment groups; D shows the serum AST level in mice in the control, CCl4, and inosine treatment groups; E shows the serum LDH level in mice in the control, CCl4, and inosine treatment groups; F shows the serum MAO level in mice in the control, CCl4, and inosine treatment groups; G shows the results of HE, Masson, and Sirius red staining to detect inflammatory infiltration and collagen deposition in the liver tissue of mice in the control, CCl4, and inosine treatment groups; scale bar: 50 μm; magnification: 20x; H shows the statistical chart of positive areas stained with Masson staining, calculated using ImageJ software; I shows the statistical chart of positive areas stained with Sirius red staining, calculated using ImageJ software. J software was used for positive area statistics; J is a graph showing the expression level of F4 / 80 in the liver tissue of mice in the control and CCl4 and inosine treatment groups by immunohistochemical (IHC) staining analysis; scale bar, 50μm; magnification 20x; K is a graph showing the expression level of α-SMA in the liver tissue of mice treated with CCl4 and inosine by qRT-PCR and IHC staining analysis; L is a graph showing the expression level of col1α1 in the liver tissue of mice treated with CCl4 and inosine by qRT-PCR and IHC staining analysis; M is a graph showing the expression levels of α-SMA and col1α1 in the liver tissue of mice treated with CCl4 and inosine by qRT-PCR and IHC staining analysis; scale bar, 50μm; magnification 20x;* p <0.05,** p <0.01, *** p <0.0005, **** p <0.0001, ns: no significant difference.

[0019] Figure 4 The diagram shows the results of TCT-6000, NECA, and inosine inhibiting HSC activation and proliferation by downregulating PDGF-B expression. In this invention, A shows the PDGF-B level in the serum of liver fibrosis patients detected by ELISA; B shows the PDGF-B level in the serum of mice in the control, CCl4, and TCT-6000 treatment groups detected by ELISA; C shows the PDGF-B level in the serum of mice in the control, CCl4, and NECA treatment groups detected by ELISA; and D shows the PDGF-B level in the serum of mice in the control, CCl4, and inosine treatment groups detected by ELISA. E shows the expression levels of α-SMA in LX2 cells treated with control, PDGF-B, and TCT-6000; F shows the expression levels of col1α1 in LX2 cells treated with control, PDGF-B, and TCT-6000; G shows the expression levels of α-SMA in LX2 cells treated with control, PDGF-B, and NECA; H shows the expression levels of col1α1 in LX2 cells treated with control, PDGF-B, and NECA; I shows the expression levels of α-SMA in LX2 cells treated with control, PDGF-B, and inosine; J shows the expression levels of α-SMA in LX2 cells treated with control, PDGF-B, and inosine. The graph shows the expression level of col1α1 in LX2 cells; K represents the effect of TCT-6000 treatment on PDGF-B-mediated LX2 proliferation and migration detected by cell proliferation assay; L represents the effect of NECA treatment on PDGF-B-mediated LX2 proliferation and migration detected by cell proliferation assay; M represents the effect of inosine treatment on PDGF-B-mediated LX2 proliferation and migration detected by cell proliferation assay; N represents the effect of TCT-6000, NECA, and inosine treatment on PDGF-B-mediated LX2 proliferation and migration detected by scratch assay; O is the statistical graph corresponding to N; * p <0.05,** p <0.01, *** p <0.0005, **** p <0.0001. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] The inventive concept of this invention is as follows: Liver fibrosis is a pressing clinical problem with no effective treatment currently available. This invention reveals that inhibiting ENT4 can alleviate CCl4-induced liver fibrosis in mice, manifested by reduced histopathological damage and improved liver function indicators. More importantly, this invention demonstrates that the liver fibrosis-alleviating effect of ENT4 inhibitors promotes the production of adenosine and inosine. Supplementation with adenosine and inosine can improve CCl4-induced liver fibrosis in mice.

[0023] ENTs are members of the SLC29 family and play a regulatory role in maintaining the homeostasis of nucleoside transport. The ENT family includes ENT1, ENT2, ENT3, and ENT4. ENT1 and ENT2 are primarily located on the plasma membrane, but can also be detected in the nuclear membrane. Additionally, ENT1 has been found on the mitochondrial membrane. ENT3 is expressed only on endosomes / lysosomes and mitochondrial membranes, while ENT4 is expressed only on the plasma membrane. ENT4 was initially characterized as a low-affinity, high-capacity monoamine transporter, rather than a nucleoside transporter. Its nucleoside transport capabilities were first demonstrated in 2006. Compared to other ENTs, ENT4 exhibits higher specificity for adenosine.

[0024] In this invention, we first observed that ENT4 expression was significantly elevated in the liver tissue of mice with CCl4-induced liver fibrosis, while the expression levels of ENT1, ENT2, and ENT3 remained unchanged. An ENT4 inhibitor (TCT-6000) alleviated liver fibrosis. Secondly, since ENT4 can affect adenosine levels in vivo, we speculated whether the allergic effect of the ENT4 inhibitor was related to adenosine. Serum adenosine was significantly decreased in patients and mice with liver fibrosis. Intraperitoneal injection of TCT-6000 increased adenosine concentration. More importantly, NECA supplementation (a metabolically stable adenosine analogue) alleviated CCl4-induced liver fibrosis in mice, both pathologically and functionally. These results suggest that increased ENT4 expression may participate in the development of liver fibrosis by regulating adenosine and inosine concentrations.

[0025] Hepatic stellate cells (HSCs) are the main cell population leading to liver fibrosis. Under homeostatic conditions, these cells remain quiescent and help regulate blood supply. However, following liver injury, various injury-associated inflammatory and metabolic signals can trigger the activation of HSCs and their differentiation into a myofibroblast-like phenotype. This phenotypic shift is characterized by enhanced cell proliferation, contraction, and migration, as well as increased production of inflammatory mediators and increased expression of ECM components, ultimately leading to excessive ECM deposition and liver fibrosis. In this study, it was found that inhibiting ENT4, NECA, and inosine can suppress the proliferation and migration of HSCs.

[0026] PDGF-B has been shown to be a key pro-fibrotic molecule. In this invention, PDGF-B levels were found to be significantly elevated in patients with liver fibrosis and in CCl4-induced mice, while PDGF-B levels in CCl4-induced mice were downregulated by treatment with TCT-6000, NECA, and inosine. More importantly, PDGF-B promotes the proliferation and migration of hepatic stellate cells (HSCs) and the production of α-SMA and col1α1, and these effects can be reversed by TCT-6000, NECA, and inosine. Existing studies have shown that PDGF-B secretion promotes the proliferation of hepatic stellate cells and enhances angiogenesis, while inhibiting PDGF-B signaling in HSCs can eliminate its pro-fibrotic effects. Therefore, these results indicate that ENT4 inhibitors, adenosine, and inosine mitigate its effects by inhibiting PDGF-B expression.

[0027] In summary, this invention reveals that ENT4 plays a crucial role in the CCl4-induced liver fibrosis process by regulating extracellular adenosine and inosine levels. ENT4 inhibitors may be potential drugs for the treatment of liver fibrosis.

[0028] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0029] Example 1 1. Materials and Methods 1.1 Mouse model of liver fibrosis Intraperitoneal injection (ip) of CCl4 (MACLIN, 56-23-5, China) to induce liver fibrosis in C57 / BL6 mice is a classic animal model of liver fibrosis. Male C57 / BL6 mice (8 weeks old, weighing 22g) were purchased from the Experimental Animal Center of the Fourth Military Medical University. All mice were housed in a standard environment (65% humidity, 24°C, 12-hour light-dark cycle). Mice were randomly divided into 5 groups: control group [n=8, olive oil (MACLIN, 8001-25-0, China), 1 mL / kg], CCl4 group [n=8, 25% CCl4 (CCl4: olive oil = 1:3), 1 mL / kg], TCT-6000 (ENT4 inhibitor, GLPBIO, GC50098, USA) group [n=8, 25% CCl4, 1 mL / kg intraperitoneal injection + TCT-6000, 4 mg / kg intraperitoneal injection], NECA (metabolic stable adenosine analogue, GLPBIO, GC15304, USA) group [n=8, 25% CCl4, 1 mL / kg intraperitoneal injection + NECA, 0.1 mg / kg intraperitoneal injection], and inosine (GLPBIO, GC17902, USA) group [n=8, 25% CCl4, 1 mL / kg intraperitoneal injection + inosine, 300 mg / kg intraperitoneal injection]. CCl4 was administered intraperitoneally twice a week for 6 weeks. Other drugs were administered intraperitoneally twice a week starting from the third week. The animal experimental study period was 6 weeks. After the last injection, all mice were fasted overnight. The following day, in accordance with the regulations of the American Veterinary Medical Association, the mice were anesthetized and euthanized, and blood and liver tissue were collected. This animal research protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Air Force Medical University (No. 20250036). All experiments were conducted in accordance with the ARRIVE Guidelines and the Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T35892-2018) promulgated in China.

[0030] 1.2 Liver function tests Serum alanine aminotransferase (ALT, Kehua Biotechnology, 202506120, China), aspartate aminotransferase (AST, Kehua Biotechnology, 202505120, China), lactate dehydrogenase (LDH, Kehua Biotechnology, 202503120, China) and monoamine oxidase (MAO, BJ•Xinchuangyuan Biotechnology, 20250401, China) levels were detected using a fully automated biochemical analyzer (Beckman Coulter, AU5800, Germany).

[0031] 1.3 Histopathological Analysis Liver tissue was collected, washed with PBS, and fixed overnight at room temperature with 4% (w / v) paraformaldehyde. Formalin-fixed paraffin-embedded (FFPE) liver tissue sections (5µm) were prepared. HE staining was used to evaluate necrosis, degeneration, and inflammatory infiltration; Masson's red and Sirius red staining were used to assess the content and extent of collagen fibers in the portal areas; panoramic and CaseViewer 2.4 software (3DHISTECH, Hungary) were used for image acquisition and analysis.

[0032] 1.4 Immunohistochemical analysis Immunohistochemical analysis was performed on paraffin-embedded sections of liver tissue to detect the expression of F4 / 80, α-SMA, and col1α1. In short, anti-F4 / 80 antibody (1:1000 dilution; Servicebio, GB113373), anti-α-SMA antibody (1:1000 dilution; Abcam, ab124964), and anti-col1α1 antibody (1:100 dilution; Servicebio, GB11022-3) were used as primary antibodies. The primary antibodies were incubated at 4°C for 12 h. HRP-labeled anti-rabbit secondary antibody (1:200 dilution, Servicebio, GB23303) was used as the secondary antibody and incubated at room temperature for 1 h. Images were acquired and analyzed using Panorama and Case Viewer 2.4 software (3DHISTECH, Hungary).

[0033] 1.5 Quantitative Real-time PCR Analysis (qRT-PCR) Total RNA was extracted from cells and liver tissues using Trizol reagent (TAKARA, Japan, Cat: 9109). cDNA synthesis was performed using PrimeScript™ RT Master Mix (ACCUBATE BIOLOGY, China, AG11728) software. qRT-PCR reaction systems were prepared using BlasTaq™ 2X qPCR Master Mix (abm, Canada, Cat: G891). Reactions were performed on Qiagen Amplifer (Rotor-gene QMDX 5, Germany). All procedures were performed according to the manufacturer's instructions. mRNA expression levels were calculated for each group using GAPDH as an internal standard.

[0034] 1.6 Cell Culture Human hepatic stellate cell line LX2 was cultured in a humidified incubator at 37°C with 5% CO2 using Dulbecco modified Eagle medium (DMEM, Gibco, Cat: 12800-017) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, Cat: FSS500).

[0035] 1.7 Cell proliferation experiment The effects of PDGF-B (Novoprotein, C199, China, 20 ng / mL), TCT-6000 (10 µM), NECA (10 µM), and inosine (3 mM) on cell proliferation were detected using a cell counting kit (CCK8, GLPBIO, GSK10001, USA). Cells were divided into a control group and a treatment group. Cells were evenly seeded in 96-well plates at 2500 cells per well. Cells were treated for 72 h, and cell viability was assessed at different time points using the CCK8 assay.

[0036] 1.8 Scratch Test The effects of PDGF-B, TCT-6000, NECA, and inosine on the migration ability of LX2 cells were detected using a scratch assay. Cells were cultured in six-well plates. Scratching was performed using the tip of a pipette. After scratching, cells were gently washed twice with PBS and then cultured in serum-free medium. Images were acquired immediately after scratching and at 48 h. Cell migration ability was expressed as relative migration rate (starting distance - ending distance) / starting distance.

[0037] 1.9 Detection of adenosine and inosine Serum adenosine and inosine levels were detected by high-performance liquid chromatography-mass spectrometry (LC-MS) in positive (ESI+) mode. A 100×2.1 mm Shim-pack GIST-HP column (Shimadzu, Japan) with a particle size of 3.5 µm was used. Mobile phase A was 0.1% (v / v) formic acid aqueous solution. Mobile phase B was 0.1% (v / v) formic acid-methanol, with a flow rate of 0.2 mL / min; column temperature was 40 °C, injection volume was 2 µL, and sample chamber temperature was 4 °C. Ion source parameters were: nitrogen flow rate 3.0 L / min, nitrogen flow rate 15 L / min, desolvation temperature 250 °C, and ion source temperature 400 °C. The transition of the m / z 268 precursor ion to m / z 136 was monitored to produce adenosine (268.1 > 136.1), 268.9 > 137.1 for inosine, and 230 > 112 for lamivudine (internal standard). The data was processed using LabSolution 5.81 (Shimadzu, Japan).

[0038] Serum samples were collected from patients who visited Tangdu Hospital between January and June 2025. All samples were collected as part of routine procedures. This invention has been approved by the Ethics Committee of Tangdu Hospital, Fourth Military Medical University (K-HG-202505-18) and complies with the Declaration of Helsinki. This document does not contain any personally identifiable information about patients and does not infringe on any individual rights. Therefore, informed consent for this invention has been waived by the Ethics Committee of Tangdu Hospital, Fourth Military Medical University.

[0039] 1.10 Determination of serum platelet-derived growth factor (PDGF-B) According to the instructions, serum PDGF-B levels were detected using the ELISA method (human: cat: CHE0073-096; mouse: cat: CSPE-0010-096, 4ABiotech, China). First, 100 µL of serum (diluted 2-fold using the sample dilution method) was added to each well and incubated at 37°C for 90 min. Second, 100 µL of biotinylated antibody was added and incubated at 37°C for 60 min. Third, 100 µL of HRP enzyme conjugate working solution was added and incubated at 37°C for 30 min. Fourth, 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at 37°C for 15 min. Finally, 100 µL of stop solution was added, and the absorbance at 450 nm was immediately measured.

[0040] 1.11 Statistical Analysis All quantitative results are expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 8.0 software. t-tests were used to analyze differences between two independent samples. p <0.05 indicates a statistically significant difference.

[0041] 0. Results 2.1 ENT4 inhibitors can alleviate CCl4-induced liver fibrosis in mice. CCl4-induced mice were used as a model of liver fibrosis. First, the expression levels of ENT1, ENT2, ENT3, and ENT4 in liver tissue were investigated. Compared with the control group, the expression level of ENT4 was significantly increased in CCl4-induced mice, while the expression levels of ENT1, ENT2, and ENT3 remained unchanged. The effect of ENT4 inhibitors on liver fibrosis was then observed. Intraperitoneal injection of TCT-6000 significantly inhibited ENT4 expression. Compared with CCl4-induced mice, TCT-6000 treatment significantly improved serum liver function indicators, including ALT, AST, LDH, and MAO. HE staining showed that TCT-6000 treatment reduced liver injury and inflammatory cell infiltration. Masson and Sirius-red staining indicated that ENT4 inhibition reduced collagen deposition in liver tissue. Furthermore, a significant reduction in macrophage infiltration was observed in TCT-6000-treated mice. The effect of intraperitoneal injection of TCT-6000 on HSC activation markers in liver tissue was further investigated. Compared with CCl4 mice, TCT-6000 treatment significantly reduced the mRNA and protein levels of α-SMA and col1α1 in mice, such as Figure 1 As shown.

[0042] 2.2 NECA alleviates CCl4-induced liver fibrosis in mice Given the crucial role of ENT4 in regulating adenosine levels, further investigation was conducted into the role of adenosine in CCl4-induced mice to explore the potential mechanisms of ENT4 in liver fibrosis. First, serum adenosine levels were observed to be decreased in both patients and mice with liver fibrosis compared to the control group. In CCl4-induced mice, ENT4 inhibition increased serum adenosine levels. Intraperitoneal injection of NECA significantly improved the performance of serum liver function indicators (i.e., ALT, AST, LDH, and MAO). HE staining, Masson's staining, and Sirius-red staining showed that NECA alleviated liver damage, inflammatory cell infiltration, and collagen deposition in the liver tissue of CCl4-induced mice. F4 / 80 staining revealed increased macrophage infiltration in the liver tissue of CCl4-induced mice, which was inhibited by NECA treatment. Furthermore, compared to CCl4-induced mice, NECA-treated mice showed significantly reduced mRNA and protein expression levels of α-SMA and col1α1, such as... Figure 2 As shown.

[0043] 2.3 Inosine alleviates CCl4-induced liver fibrosis in mice Inosine, a degradation product of adenosine, has been used as a hepatoprotective and anti-inflammatory drug. Due to the significant effect of ENT4 inhibition on adenosine levels, the changes and roles of inosine in liver fibrosis were further investigated. First, decreased serum inosine levels were observed in fibrotic patients and CCl4-induced mice. In CCl4-induced mice, TCT-6000 inhibition of ENT4 enhanced serum inosine levels. Compared to the CCl4-treated mouse group, inosine-treated mice showed decreased serum ALT, AST, LDH, and MAO levels. Histopathological staining of liver tissue sections showed that inosine treatment significantly reduced liver inflammation and collagen deposition. F4 / 80 staining showed that inosine treatment significantly reduced macrophage infiltration. Furthermore, compared to CCl4-treated mice, inosine-treated mice showed significantly reduced mRNA and protein levels of α-SMA and col1α1, such as... Figure 3 As shown.

[0044] 2.4 TCT-6000, NECA, and inosine inhibit the activation and proliferation of HSCs by downregulating PDGF-B. Previous studies have shown that PDGF-B promotes the development of liver fibrosis. In this invention, serum PDGF-B levels were observed to be elevated in fibrotic patients and CCl4- mice compared to the control group, while treatment with TCT-6000, NECA, and inosine in CCl4- mice decreased serum PDGF-B levels. Further observation revealed that PDGF-B treatment significantly increased the expression of α-SMA and col1α1 compared to the control group, while TCT-6000, NECA, and inosine treatment reversed this effect. Furthermore, PDGF-B was found to significantly promote the proliferation and migration of LX2 cells, while TCT-6000, NECA, and inosine treatment could reverse the effects of PDGF-B on LX2 cells. Figure 4 As shown.

[0045] In summary, this invention reveals that ENT4 plays a crucial role in the CCl4-induced liver fibrosis process by regulating extracellular adenosine and inosine levels. ENT4 inhibitors may be potential drugs for the treatment of liver fibrosis.

[0046] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. The application of an ENT4 inhibitor in the preparation of a drug for treating liver fibrosis, characterized in that, The NCBI Gene ID of the ENT4 gene is 243328.

2. The application according to claim 1, characterized in that, The ENT4 inhibitor is TCT-6000.

3. The application according to claim 2, characterized in that, The drug has TCT-6000 as its sole active ingredient.

4. The application according to claim 3, characterized in that, The dosage of TCT-6000 is 4 mg / kg body weight.

5. The application according to claim 3, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable carrier is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

7. The application according to claim 1, characterized in that, The liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver disease, and non-alcoholic fatty liver disease.

8. The application according to claim 1, characterized in that, The ENT4 inhibitory agents reduce liver fibrosis by upregulating adenosine and / or inosine levels and inhibiting the expression of platelet-derived growth factor-B.

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