Use of mertk inhibitors in the preparation of a medicament for the prevention and / or treatment of cholestatic liver injury

Abstract

This invention relates to the use of Mertk inhibitors in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury. Specifically, this invention provides the use of Mertk inhibitors in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury. This invention reveals for the first time the crucial role of the cytotoxic receptor tyrosine kinase Mertk in cholestatic liver injury and its associated liver fibrosis, clarifying that Mertk can serve as a potential therapeutic target for cholestatic liver injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of Mertk inhibitors in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury. Background Technology

[0002] Cholestatic liver injury (CLI) is a common clinical liver pathology and a significant concern in drug development and safety evaluation. Induction models using α-naphthylisothiocyanate (ANIT), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced models, and Mdr2 gene knockout mice (multidrug resistance 2 knockout mice) have been developed. - / - These are all commonly used research models for cholestatic liver injury in this field. They are classic modeling tools for studying cholestatic liver injury and can highly simulate the cholestasis phenotype caused by bile duct injury in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Therefore, they are widely used in mechanism exploration and drug intervention research. ANIT, DDC, and Mdr2 are all commonly used models for studying cholestatic liver injury in this field. - / - The model can induce damage to bile duct epithelial cells and cholestasis within the bile ducts, triggering a strong inflammatory response and subsequently recruiting a large number of inflammatory cells, such as neutrophils and monocytes / macrophages, to the peri-biliary and hepatic sinusoidal regions. This process is further accompanied by the release of inflammatory mediators, increased oxidative stress, and secondary hepatocellular damage, ultimately leading to liver dysfunction. In recent years, the crucial role of inflammatory cell recruitment and activation in the pathogenesis of cholestatic liver injury has received increasing attention.

[0003] Currently, treatment options for cholestatic liver injury are limited, and the first-line drug ursodeoxycholic acid is ineffective and has side effects in many patients.

[0004] Therefore, there is an urgent need to find a safe and effective drug or preparation to prevent and / or treat cholestatic liver injury. Summary of the Invention

[0005] The purpose of this invention is to provide a safe and effective medicine or preparation for preventing and / or treating cholestatic liver injury.

[0006] In a first aspect of the invention, the use of a Mertk inhibitor in the preparation of a medicament for the prevention and / or treatment of cholestatic liver injury is provided, wherein the Mertk inhibitor inhibits ferroptosis in Mertk-positive macrophages.

[0007] In another preferred embodiment, the Mertk inhibitor is administered to a patient in whom elevated levels of ferroptosis in Mertk-positive macrophages were detected in liver tissue.

[0008] In another preferred embodiment, the cholestatic liver injury is liver injury mediated by bile acid accumulation and accompanied by portal inflammation and bile duct reaction.

[0009] In another preferred embodiment, the Mertk inhibitor is not used to treat liver fibrosis or liver injury induced by non-cholestatic factors.

[0010] In another preferred embodiment, the cholestatic liver injury includes: primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, or progressive familial intrahepatic cholestasis (PFIC).

[0011] In another preferred embodiment, the Mertk inhibitor is a molecule that specifically inhibits the activity of Mertk receptor tyrosine kinase.

[0012] In another preferred embodiment, the Mertk inhibitor includes: small molecule compounds, nucleic acid molecules, peptides, proteins, antibodies, or antigen-binding fragments thereof.

[0013] In another preferred embodiment, the small molecule compound is UNC2025 or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0014] In another preferred embodiment, the nucleic acid molecule is a siRNA, shRNA, antisense oligonucleotide, or guide RNA of the CRISPR-Cas9 system that targets the Mertk gene.

[0015] In another preferred embodiment, the drug exerts its preventive and / or therapeutic effects by means of the group selected from: reducing the area of ​​liver necrosis, reducing serum transaminase (ALT / AST) levels, reducing serum alkaline phosphatase (ALP) levels, reducing the infiltration of hepatic macrophages and / or neutrophils, reducing the expression of inflammation-related genes in liver tissue, or reducing liver fibrosis.

[0016] In another preferred embodiment, the drug is formulated for administration via a route selected from the group consisting of: oral, intraperitoneal, intravenous, intramuscular, or subcutaneous injection.

[0017] In another preferred embodiment, the effective dose of the Mertk inhibitor is 1-20 mg / kg / day, preferably 5 mg / kg / day.

[0018] In a second aspect of the invention, the use of a Mertk inhibitor in the preparation of a medicament for the prevention and / or treatment of cholestatic liver injury in subjects, wherein the subjects are individuals in whom the level of ferroptosis in Mertk-positive macrophages in liver tissue is elevated relative to healthy controls.

[0019] In another preferred embodiment, the elevated level of ferroptosis in Mertk-positive macrophages refers to: ultrastructural changes observed by transmission electron microscopy, such as shrinkage of macrophage mitochondria, increase in membrane density, or reduction in mitochondrial cristae, or statistically significant upregulation or downregulation of ferroptosis markers MDA, 4-HNE, GPX4, or ACSL4 compared to healthy controls.

[0020] In a third aspect of the invention, the use of Mertk inhibitors in the preparation of medicaments for alleviating liver inflammation and / or macrophage recruitment associated with cholestasis in mammalian subjects is provided.

[0021] In another preferred embodiment, the reduction of the inflammatory response is manifested by a decrease in the infiltration of F4 / 80 positive macrophages and / or Ly6G positive neutrophils in the liver tissue.

[0022] In another preferred embodiment, the mammalian object includes humans or non-human mammals.

[0023] In another preferred embodiment, the non-human mammal includes: mouse, rat, dog, monkey or rabbit.

[0024] In a fourth aspect of the invention, a pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury is provided, comprising a therapeutically effective amount of a Mertk inhibitor and a pharmaceutically acceptable carrier or excipient, said pharmaceutical composition for inhibiting ferroptosis in Mertk-positive macrophages.

[0025] In another preferred embodiment, the inhibition of ferroptosis is detected or characterized by means selected from the group consisting of: observing ultrastructural changes in macrophage mitochondria using transmission electron microscopy, detecting the expression levels of ferroptosis markers MDA, 4-HNE, GPX4 or ACSL4, or combinations thereof.

[0026] In another preferred embodiment, the Mertk inhibitor is UNC2025 or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0027] In another preferred embodiment, the pharmaceutical composition further comprises a second therapeutic agent selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, corticosteroids, or anti-inflammatory agents.

[0028] In another preferred embodiment, the pharmaceutical composition is formulated as an oral or injectable preparation.

[0029] In a fifth aspect of the invention, a kit for preventing and / or treating cholestatic liver injury is provided, comprising:

[0030] a) A container containing a therapeutically effective amount of a Mertk inhibitor, or a pharmaceutical composition thereof, and

[0031] b) Instruction manual;

[0032] The specification indicates that the Mertk inhibitor be administered to subjects in whom elevated levels of ferroptosis in Mertk-positive macrophages in liver tissue were detected.

[0033] In another preferred embodiment, the kit further comprises a detection reagent for detecting ferroptosis levels in Mertk-positive macrophages, the detection reagent being selected from: fixatives and / or staining agents for transmission electron microscopy, MDA for detecting lipid oxidation levels, and the C11-Bodipy kit.

[0034] In a sixth aspect of the invention, a method is provided for screening candidate drugs for the prevention and / or treatment of cholestatic liver injury, comprising detecting whether a candidate substance can simultaneously meet the following two conditions:

[0035] a) Inhibit Mertk kinase activity; and

[0036] b) Reduce ferroptosis levels in macrophages in a cholestasis model;

[0037] The macrophage ferroptosis levels were detected using a method selected from the group consisting of: transmission electron microscopy to observe mitochondrial ultrastructural changes, MDA for detecting lipid oxidation levels, and the C11-Bodipy kit.

[0038] In a seventh aspect of the invention, a method for screening candidate drugs for the prevention and / or treatment of cholestatic liver injury is provided, comprising the following steps:

[0039] (1) Provide an in vitro or in vivo model of cholestatic liver injury;

[0040] (2) Apply the candidate material to the model;

[0041] (3) Detect the level of ferroptosis in macrophages in the model; and

[0042] (4) Candidate substances that can downregulate the level of ferroptosis in macrophages are identified as candidate drugs for the prevention and / or treatment of cholestatic liver injury.

[0043] In another preferred embodiment, the method for detecting macrophage ferroptosis levels in step (3) is selected from the following group: transmission electron microscopy observation of mitochondrial ultrastructural changes, MDA and C11-Bodipy kits for detecting lipid oxidation levels.

[0044] In another preferred embodiment, the indicators associated with cholestatic liver injury further include: (i) serum ALT, AST or ALP levels; and / or (ii) the area of ​​liver tissue necrosis or fibrosis.

[0045] In another preferred embodiment, the in vivo model of cholestatic liver injury is selected from the group consisting of: ANIT-induced acute cholestasis model, DDC diet-induced cholestasis model, or Mdr2 gene knockout mouse model.

[0046] In an eighth aspect of the invention, the use of a Mertk inhibitor in the preparation of a medicament for reducing ferroptosis levels in cholestasis-related Mertk-positive macrophages in mammalian subjects is provided, wherein the reduction in ferroptosis levels is detected or characterized by means of a group selected from: transmission electron microscopy showing reduced mitochondrial ultrastructural damage in macrophages, and decreased MDA and C11-Bodipy levels used to detect lipid oxidation.

[0047] In another preferred embodiment, the mammalian object includes humans or non-human mammals.

[0048] In a ninth aspect of the invention, a method for preventing and / or treating cholestatic liver injury in a subject is provided, comprising:

[0049] (1) Detect the level of ferroptosis in Mertk-positive macrophages in liver tissue samples from the subjects; and

[0050] (2) Administer a therapeutically effective dose of Mertk inhibitors to subjects with elevated ferroptosis levels.

[0051] In another preferred embodiment, the detection method in step (1) is selected from: observing changes in mitochondrial ultrastructure using transmission electron microscopy, and detecting the levels of lipid oxidation markers MDA and C11-Bodipy.

[0052] In another preferred embodiment, the method further includes administering a second therapeutic agent to the subject, the second therapeutic agent being selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, corticosteroids, or anti-inflammatory agents.

[0053] In another preferred embodiment, the object includes a human or a non-human mammal.

[0054] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0055] Compared with the prior art, the present invention has the following technical effects:

[0056] (1) This invention reveals for the first time that in cholestatic liver injury, Mertk promotes ferroptosis of Mertk-positive macrophages through a mechanism independent of cell burial, thereby exacerbating liver injury and fibrosis. Inhibition of Mertk can specifically block this ferroptosis pathway, thereby exerting a protective effect.

[0057] (2) This invention utilizes ANIT, DDC, and Mdr2 - / - Multiple cholestatic liver injury models have confirmed that inhibiting Mertk can significantly reduce liver tissue damage, inflammatory cell infiltration, bile duct reaction, and the degree of liver fibrosis, indicating that targeting Mertk has a protective effect against different types of cholestatic liver injury.

[0058] (3) The present invention uses systemic Mertk knockout, myeloid cell-specific Mertk knockout and Mertk inhibitor UNC2025 drug intervention to verify the therapeutic effect of inhibiting Mertk from both genetic and pharmacological perspectives, thereby improving the reliability of this target as a drug development object.

[0059] (4) The present invention further discovered that inhibiting Mertk can reduce abnormal infiltration of macrophages and neutrophils in the liver, reduce inflammatory response, and inhibit macrophage ferroptosis and its mediated activation of hepatic stellate cells under cholestasis, thereby improving liver injury and fibrosis process, providing important experimental basis and application direction for the development of new drugs based on specific inhibition of Mertk. Attached Figure Description

[0060] Figure 1 This demonstrates that systemic knockout of Mertk in this embodiment of the invention significantly reduces liver injury, fibrosis, and inflammatory response in a cholestatic liver injury model mouse. Wherein, A represents WT-DDC and Mertk. - / - - HE staining of liver tissue from DDC mice, n=6; B represents WT-DDC and Mertk mice. - / - - Serum ALT levels in DDC mice, n=6; C represents WT-DDC and Mertk mice. - / - Multiple immunohistochemical staining of α-SMA, Col1a1, and CK19 in liver tissue of DDC mice and quantitative analysis of α-SMA and Col1a1 positive areas, n=4; D represents Mdr2. - / - and Mertk- / - -Mdr2 - / - Mouse liver tissue HE staining, n=6; E represents Mdr2- / - and Mertk. - / - -Mdr2 - / - Mouse serum ALT levels, n=6; F is Mdr2 - / - and Mertk - / - -Mdr2 - / - Multiple immunohistochemical staining of α-SMA, Col1a1, and CK19 in mouse liver tissue and quantification of α-SMA and Col1a1 positive areas, n=4; G represents WT-DDC and Mertk. - / - KEGG pathway enrichment analysis of differentially expressed genes in liver tissue transcriptomes of WT-DDC mice, n=4; H represents WT-DDC and Mertk. - / - Immunohistochemical staining and quantification of positive area of ​​F4 / 80 and MPO / Ly6G in liver tissue of DDC mice, n=3.

[0061] Figure 2 This demonstrates that myeloid cell-specific knockout of Mertk significantly reduces liver injury and fibrosis in a cholestatic liver injury model mouse, as shown in this embodiment of the invention. Wherein, A represents Mertk. fl / fl -DDC and Lyz2 cre Mertk fl / fl - HE staining of liver tissue from DDC mice, n=6; B represents Mertk. fl / fl -DDC and Lyz2 cre Mertk fl / fl -DDC mouse serum ALT levels, n=6; C represents Mertk. fl / fl -DDC and Lyz2 cre Mertk fl / fl Multiple immunohistochemical staining and quantification of α-SMA, Col1a1, and CK19 positive areas in liver tissue of DDC mice, n=4; D represents Mertk. fl / fl -ANIT and Lyz2 cre Mertk fl / fl HE staining and quantification of necrotic area in liver tissue of ANIT mice, n=6; E represents Mertk. fl / fl -ANIT and LLYZ2 cre Mertk fl / fl -ANIT mouse serum ALT levels, n=6.

[0062] Figure 3This illustrates that drug inhibition of Mertk significantly reduces liver injury and fibrosis in cholestatic liver injury model mice in the embodiments of this invention. Specifically, A shows HE staining of liver tissue from DDC model mice in the solvent control group and UNC2025 treatment group (n=4); B shows serum ALT levels in DDC model mice in the solvent control group and UNC2025 treatment group (n=4); C shows multiple immunohistochemical staining of α-SMA, Col1a1, and CK19 in the liver tissue of DDC model mice in the solvent control group and UNC2025 treatment group, and quantitative analysis of the positive area of ​​α-SMA and Col1a1 (n=4); D shows Mdr2 in the solvent control group and UNC2025 treatment group. - / - Mouse liver tissue HE staining, n=5; E represents the solvent control group and the UNC2025 treatment group Mdr2. - / - Mouse serum ALT levels, n=5; F represents the solvent control group and the UNC2025 treatment group (Mdr2). - / - Multiple immunohistochemical staining of α-SMA, Col1a1 and CK19 in mouse liver tissue and quantification of the positive area of ​​α-SMA and Col1a1, n=4.

[0063] Figure 4 This illustrates how Mertk participates in cholestatic liver injury and hepatic stellate cell activation by regulating macrophage ferroptosis in an embodiment of the present invention. Wherein, A represents Lyz2. cre Mertk fl / fl -DDC mice and Mertk fl / fl - GO functional enrichment analysis of differentially expressed genes in the DDC mouse cholestatic liver injury model; B represents the ferroptosis score of Mertk-negative and Mertk-positive macrophages in single-cell transcriptome analysis; C represents the Mertk... fl / fl and Lyz2 cre Mertk fl / fl Ferrocyte death score in mouse macrophages; D represents WT mice, Mdr2 - / - Mice, Mdr2 - / - Mice and Mertk - / - -Mdr2 - / -Transmission electron microscopy observation of macrophages in mouse liver tissue; E: HE staining of liver tissue from DDC model mice in solvent control group and Fer-1 treatment group, n=4; F: Serum ALT level of DDC model mice in solvent control group and Fer-1 treatment group, n=4; G: Multiple immunohistochemical staining of Col1a1, α-SMA and CK19 in liver tissue of DDC model mice in solvent control group and Fer-1 treatment group, and quantitative analysis of positive areas of α-SMA and Col1a1, n=4; H: qPCR detection results of α-SMA in hepatic stellate cells after different macrophage culture supernatants treatment, n=6; I: Immunofluorescence staining and fluorescence intensity quantification of α-SMA in hepatic stellate cells after different macrophage culture supernatants treatment, n=4.

[0064] Figure 5 This illustration shows the role of Mertk in regulating arachidonic acid metabolism, ferroptosis, and lipid peroxidation in macrophages under bile acid and arachidonic acid stimulation, as described in this embodiment of the invention. In this diagram, A represents the UMAP distribution of Mertk-MDM and Mertk+MDM in single-cell transcriptome analysis, with each point representing one cell, n=3 mice / group; B represents Lyz2... cre Mertk fl / fl Mice and Mertk fl / fl GSEA analysis of ferroptosis and arachidonic acid metabolism pathways in a mouse model of cholestatic liver injury, n=3 / group; C represents Mertk. fl / fl and Lyz2 cre Mertk fl / fl Expression levels of arachidonic acid metabolism and ferroptosis-related genes Pla2g4a, Ptges3, Tbxas1, Alox5ap, Cyba, Por, Hmox1, Lta4h, Alox12, Acsl4, Lpcat3, Ncf2, Tfrc, and Pebp1 in mouse macrophages (n=3 mice / group); D shows cell viability assays after BMDM treatment with arachidonic acid in combination with cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid, and MDA level assays after BMDM treatment with lithocholic acid in combination with arachidonic acid (n=8-10 / group), MDA assays (n=8 / group); E shows a heatmap of arachidonic acid metabolism-related gene expression after BMDM stimulation with lithocholic acid under lithocholic acid treatment (n=4 / group); F shows the expression levels of WT-BMDM and Mertk. - / - - Heatmap of arachidonic acid metabolism-related gene expression after BMDM treatment with lithocholic acid and arachidonic acid, n=4 / group; G represents WT-BMDM-LCA, WT-BMDM-LCA+AA and Mertk. - / -Lipid peroxidation fluorescence staining and Ox-BODIPY / non-Ox-BODIPY quantification in the BMDM-LCA+AA group (n=6 / group), scale bar: 10 μm. Detailed implementation method.

[0065] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] the term

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0069] Mertk, a receptor tyrosine kinase for cell closure

[0070] Existing research indicates that Mertk is involved in the liver fibrosis process in some chronic liver disease models. Cholestatic liver disease is an important type of liver fibrosis; however, cholestatic liver injury is not a simple extension of ordinary chronic liver disease. Its development is characterized by bile acid accumulation, bile duct epithelial cell damage, abnormal recruitment of immune cells, and bile duct reactions, accompanied by complex interactions between macrophages, neutrophils, bile duct cells, and hepatic stellate cells. Unlike general chronic liver disease, which mainly manifests as persistent hepatocellular damage and fibrotic deposition, cholestatic liver injury has a more pronounced immune cell-driven characteristic, and its pathological process and potential intervention targets are unique.

[0071] Currently, the role of Mertk in cholestatic liver injury remains unclear, and existing technologies do not disclose a technical solution for intervening in cholestatic liver injury and related fibrosis by targeting Mertk. This invention is the first to discover that Mertk plays an important role in the progression of cholestatic liver injury. Systemic knockout of Mertk, myeloid cell-specific knockout of Mertk, or pharmacological inhibition of Mertk can all affect ANIT, DDC, and Mdr2 levels. - / -In cholestatic liver injury models, it can reduce hepatocellular damage, inflammatory cell infiltration, bile duct reaction, and the degree of liver fibrosis.

[0072] In recent years, studies have reported that Mertk mediates the burial of apoptotic bile duct cells by macrophages. However, burial is only one aspect of macrophage function. Whether Mertk affects cholestatic liver injury through other mechanisms (such as regulating macrophage ferroptosis) has not yet been reported.

[0073] Therefore, this invention provides a novel use of Mertk as a therapeutic target for cholestatic liver injury and its associated fibrosis, and further provides a technical solution for alleviating cholestatic liver injury by inhibiting Mertk. This solution can intervene in the pathological characteristics of cholestatic liver injury driven by immune cells and involving multiple cell interactions, and has good therapeutic potential, providing a new direction for drug development in cholestatic liver disease.

[0074] application

[0075] This invention relates to the use of Mertk receptor inhibitors in the preparation of drugs for the prevention and / or treatment of cholestatic liver injury and related liver fibrosis. The inventors constructed ANIT, DDC, and Mdr2... - / - Multiple cholestatic liver injury models were investigated, and compared with wild-type mice, Mertk knockout mice (both systemic and myeloid cell-specific knockout mice) exhibited milder liver tissue damage, inflammatory cell infiltration, bile duct reaction, and liver fibrosis. Furthermore, treatment of cholestatic liver injury model mice with the Mertk inhibitor UNC2025 also significantly reduced liver damage and fibrosis phenotypes. Mechanistic studies showed that inhibiting Mertk reduces abnormal infiltration of macrophages and neutrophils in the liver, lowers the inflammatory response, and inhibits macrophage-related damage processes under cholestatic conditions, thereby exerting a protective effect against cholestatic liver injury. Therefore, Mertk can serve as an effective intervention target for cholestatic liver injury and related liver fibrosis, and targeted inhibition of Mertk holds promise for the development of drugs for the treatment of cholestatic liver diseases.

[0076] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0077] Experimental methods:

[0078] The experimental methods involved in this invention include: detecting the level of target proteins in serum or tissue homogenates by ELISA; extracting total RNA and reverse transcribing it to synthesize cDNA, and detecting the expression of target genes by real-time quantitative PCR (qPCR); constructing a transcriptome sequencing library for high-throughput sequencing, and analyzing differentially expressed genes and enriching the KEGG pathway; performing single-cell RNA sequencing on isolated cells, performing quality control, normalization, clustering, and cell type annotation, and performing KEGG pathway analysis on differentially expressed genes in specific cell populations; performing HE staining on tissue sections to observe tissue structure and pathological changes; performing multiplex immunohistochemistry (mIHC) to label target proteins and performing fluorescence microscopy imaging and quantitative analysis; fixing, dehydrating, embedding, and preparing ultrathin sections of tissue fragments, and observing changes in cell ultrastructure by transmission electron microscopy (TEM).

[0079] 1. ELISA or biochemical detection methods

[0080] Peripheral blood was collected from mice, and serum was separated by centrifugation after standing at room temperature. The target markers in serum or tissue homogenates were detected according to the instructions of the ELISA kit or biochemical assay kit. Standards, blank wells, and sample wells were set up for testing, and the concentration of the target marker in the sample was calculated based on the standard curve.

[0081] 2. qPCR detection method

[0082] Tissue or cell samples were collected, and total RNA was extracted using the Trizol method or an equivalent RNA extraction method. After determining the RNA concentration and purity, cDNA was synthesized via reverse transcription. The expression level of the target gene was detected using a real-time quantitative PCR system. After the reaction, the expression was normalized using an internal reference gene, and the difference in target gene expression was calculated using a relative quantification method.

[0083] 3. Transcriptome sequencing and KEGG analysis methods

[0084] Total RNA was extracted from tissues or cells, and after passing quality testing, a transcriptome sequencing library was constructed and high-throughput sequencing was performed. Sequencing data underwent quality control, adapter and low-quality sequence removal, reference genome alignment, gene expression quantification, and differential expression analysis. After obtaining differentially expressed genes, KEGG pathway enrichment analysis was further performed to evaluate the biological pathways involved.

[0085] 4. Single-cell transcriptome sequencing and KEGG analysis methods

[0086] Fresh tissue was used to prepare single-cell suspensions. After cell filtration, erythrocyte lysis, viable cell counting, and quality control, single-cell RNA sequencing libraries were constructed and high-throughput sequencing was performed. Sequencing data underwent quality control, expression matrix construction, normalization, dimensionality reduction, clustering, and cell type annotation to obtain the transcriptional expression characteristics of different cell populations. Differentially expressed genes were analyzed for specific cell populations, and KEGG pathway enrichment analysis was further performed.

[0087] 5. HE staining method

[0088] Tissue samples were collected, fixed, dehydrated, cleared, and embedded in paraffin to prepare tissue sections. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin, followed by dehydration, clearing, and mounting. Tissue structure, cell morphology, and pathological changes were observed using an optical microscope, and quantitative analysis of necrosis area, inflammatory infiltration, or degree of tissue damage was possible.

[0089] 6. Immunohistochemistry and multiplex immunohistochemistry methods

[0090] Paraffin-embedded tissue sections are dewaxed, rehydrated, antigen-retrieved, and blocked. They are then incubated with the appropriate primary antibody, followed by incubation with a secondary antibody or fluorescent labeling system for colorimetric or fluorescent detection. Multiplex immunohistochemistry allows for the simultaneous detection of multiple target proteins through multiple rounds of antibody incubation and signal amplification. After staining, images are acquired using a microscope or fluorescence microscope, and the positive area, number of positive cells, or fluorescence intensity are quantitatively analyzed.

[0091] 7. Transmission electron microscopy detection methods

[0092] Fresh tissue fragments were fixed with fixative, washed with buffer, post-fixed with osmium tetroxide, dehydrated with graded ethanol, and embedded in resin to prepare ultrathin sections. After staining with uranium and lead salts, the sections were observed using transmission electron microscopy to examine changes in organelles and cellular ultrastructure, including mitochondrial morphology, membrane structure, organelle integrity, and other subcellular structural alterations.

[0093] 8. Cell culture supernatant processing and immunofluorescence detection methods

[0094] Cell culture supernatants under different treatment conditions were collected, centrifuged to remove cell debris, and then applied to target cells. After treatment, cells were collected for qPCR detection, or the cells were fixed and subjected to immunofluorescence staining. Immunofluorescence detection included fixation, permeabilization, blocking, incubation with primary antibody, incubation with fluorescent secondary antibody, nuclear staining, and fluorescence microscopy imaging, and quantitative analysis of fluorescence intensity or the proportion of positive cells was performed.

[0095] C11-Bodipy detection method

[0096] C11-Bodipy (MCE, HY-D1301) was incubated at a final concentration of 1-5 μM at 37°C in the dark for 30 minutes. After incubation, cells were washed with PBS to remove free probes, and then fluorescence signals were detected using fluorescence microscopy. The fluorescence of C11-BODIPY changed from red to green upon oxidation. Therefore, the level of intracellular lipid peroxidation can be evaluated by detecting the green fluorescence signal of oxidized C11-BODIPY, the red fluorescence signal of non-oxidized C11-BODIPY, or by calculating the ratio of oxidized to non-oxidized fluorescence intensity.

[0097] 9. A method for inducing macrophage ferroptosis using bile acids combined with arachidonic acid.

[0098] Toxic bile acid lithocholic acid (LCA, 100 uM) combined with arachidonic acid (AA, 100 uM) was added to macrophage culture medium for 6 h to construct a cholestasis cell injury model.

[0099] 10. Cell Viability Assay Method: BMDM was seeded in 96-well plates and treated with bile acids, arachidonic acid, or a combination of both according to experimental groups. After treatment, cell viability assay reagents were added, and the cells were incubated according to the kit instructions. The absorbance values ​​were measured using a microplate reader. The changes in cell viability in each treatment group were calculated using the control group as a baseline.

[0100] 11. MDA Detection Method: Cell or tissue samples under different treatment conditions are collected, lysed thoroughly with lysis buffer, and the supernatant is collected after centrifugation. The reaction is performed according to the MDA detection kit instructions to detect the malondialdehyde (MDA) content. The results can be normalized according to the sample protein concentration and expressed as the MDA level corresponding to unit protein content.

[0101] Example 1: Mertk gene knockout reduces liver injury, fibrosis, and inflammatory response in a cholestatic liver injury model mouse.

[0102] 1. Transgenic mice, mouse disease model construction, and experimental grouping:

[0103] (1) C57BL / 6 mice and Mertk mice were used. - / - Mice, Mertk fl / fl Mice, Lyz2 cre Mertk fl / fl Mice, Mdr2 - / - Mice, Mertk - / - -Mdr2 - / - Mice and corresponding control mice. All mice were of the C57BL / 6 genetic background and were purchased from Shanghai Southern Model Biotechnology Co., Ltd.

[0104] (2) DDC model: Eight-week-old mice were fed a diet containing 0.1% DDC for 14 consecutive days to establish a DDC-induced cholestatic liver injury model. The normal control group was fed a normal diet. Serum and liver tissue samples were collected from mice on the 14th day after modeling.

[0105] (3) ANIT model: Eight-week-old mice were selected and administered ANIT at a dose of 75 mg / kg via a single gavage to establish an ANIT-induced acute cholestatic liver injury model. The control group was administered an equal volume of solvent via gavage. ANIT was administered as a single dose for one day. Serum and liver tissue samples were collected from mice 48 hours after gavage.

[0106] (4) Mdr2 - / - Model: Selected 10-week-old Mdr2 - / - Mice were used as a model of chronic cholestatic liver injury and fibrosis. This model, due to the deletion of the Mdr2 gene, spontaneously exhibits pathological changes such as cholestasis, bile duct reaction, inflammatory infiltration, and liver fibrosis. Serum and liver tissue samples were collected from mice at 10 weeks of age.

[0107] 2. Methods for confirming successful model building:

[0108] The success of establishing a cholestatic liver injury model is determined by a comprehensive assessment of serological indicators, histopathological indicators, and molecular markers. Specifically, this includes:

[0109] (1) Serum ALT, AST or ALP levels were higher than those in the control group, indicating liver cell damage and cholestasis-related abnormal liver function.

[0110] (2) HE staining showed liver tissue with hepatocellular damage, necrosis, inflammatory cell infiltration, perichondral pathological changes, or disordered tissue structure.

[0111] 3. Experimental Design:

[0112] (1) Serum and liver tissue were collected from mice in the ANIT model 48 h after modeling, and serum and liver tissue were collected from mice in the DDC model 14 days after modeling. Mdr2 - / - Serum and liver lobe of mice were collected at 12 weeks. The levels of ALT, AST and ALP were detected by ELISA or corresponding biochemical assay kits to assess the degree of liver damage.

[0113] (2) Prepare paraffin sections of liver tissue, perform HE staining, and observe liver tissue necrosis, inflammatory infiltration and changes in tissue structure;

[0114] (3) Immunohistochemistry was used to detect F4 / 80 and Ly6G to assess the degree of infiltration of macrophages and neutrophils in the liver;

[0115] (4) Multiple immunohistochemical detection of α-SMA, Col1a1 and CK19 was used to assess liver fibrosis and bile duct reaction;

[0116] (5) Collect liver tissue samples for transcriptome analysis, analyze differentially expressed genes, and perform KEGG pathway enrichment analysis.

[0117] The results showed that in the DDC model, compared with WT-DDC mice, Mertk - / - -DDC mice showed reduced pathological damage to liver tissue, decreased serum ALT levels, and a significant reduction in the positive area of ​​α-SMA and Col1a1 in liver tissue. Figure 1 AC). In Mdr2 - / - In the chronic cholestasis model, compared with Mdr2 - / - Compared to mice, Mertk - / - -Mdr2 - / - In mice, liver tissue damage was reduced, serum ALT levels decreased, and the area of ​​α-SMA and Col1a1 positivity was reduced. Figure 1 DF). Transcriptome KEGG enrichment analysis showed that Mertk deletion significantly altered inflammation-related pathways such as the NF-κB signaling pathway, chemokine signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway. Figure 1 G). Further immunohistochemical results showed that Mertk - / - -DDC mice showed reduced infiltration of F4 / 80 positive macrophages and Ly6G positive neutrophils in liver tissue. Figure 1 H).

[0118] Further validation was performed using myeloid cell-specific Mertk knockout mice. In the DDC model, compared with Mertk... fl / fl Compared to DDC mice, Lyz2 cre Mertk fl / fl -DDC mice showed reduced pathological damage to liver tissue, decreased serum ALT levels, and a significant reduction in the positive area of ​​α-SMA and Col1a1 in liver tissue. Figure 2 AC). In the ANIT model, with Mertk fl / fl Compared to ANIT mice, Lyz2 cre Mertk fl / fl -ANIT mice showed a significant reduction in liver tissue necrosis area and serum ALT levels ( Figure 2 DE).

[0119] In conclusion, both systemic knockout of Mertk and myeloid cell-specific knockout of Mertk significantly reduced liver injury, fibrosis, and inflammatory response in cholestatic liver injury model mice, indicating that Mertk, especially myeloid cell-derived Mertk, plays an important role in the progression of cholestatic liver injury.

[0120] Example 2: Drug inhibition of Mertk alleviates liver injury and fibrosis in a mouse model of cholestatic liver injury.

[0121] 1. Laboratory animals, model construction, and drug administration process:

[0122] (1) Eight-week-old C57BL / 6 mice and ten-week-old Mdr2 mice were used. - / - Mice. C57BL / 6 mice and Mdr2 - / - Mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd.

[0123] (2) DDC model: Eight-week-old C57BL / 6 mice were fed a diet containing 0.1% DDC for 14 consecutive days to establish a DDC-induced cholestatic liver injury model. The model mice were treated with the Mertk inhibitor UNC2025 or the corresponding solvent control. The dosage of UNC2025 was 5 mg / kg / day, administered intraperitoneally once daily for 14 consecutive days after the start of DDC modeling. Serum and liver tissue samples were collected on the 14th day of modeling.

[0124] (3) Mdr2 - / - Model: Selected 10-week-old Mdr2 - / - Mice were used as a model of chronic cholestatic liver injury and fibrosis. The model mice were treated with the Mertk inhibitor UNC2025 or a corresponding solvent control. UNC2025 was administered at a dose of 5 mg / kg / day via intraperitoneal injection once daily for consecutive days; serum and liver tissue samples were collected after the administration period. The solvent control group received an equal volume of solvent via intraperitoneal injection.

[0125] 2. Methods for confirming successful model building:

[0126] Whether the DDC model was successfully constructed was mainly determined by detecting elevated serum ALT, AST, or ALP levels after 14 days of continuous feeding with 0.1% DDC diet, HE staining showing hepatocellular damage, inflammatory cell infiltration, or periductal pathological changes, and elevated levels of fibrosis-related and bile duct reaction-related indicators such as α-SMA, Col1a1, and CK19. Mdr2 - / - Whether the model was successfully constructed was mainly determined by the presence of elevated serum liver injury markers, bile duct reaction, inflammatory infiltration, and liver fibrosis at 10 weeks of age.

[0127] 3. Experimental Design:

[0128] (1) Serum and liver tissue were collected from mice in the ANIT model 48 h after modeling, and serum and liver tissue were collected from mice in the DDC model 14 days after modeling. Mdr2 - / - Serum and liver lobes of mice were collected at 12 weeks to detect ALT levels and assess the degree of liver damage.

[0129] (2) Prepare paraffin sections of liver tissue, perform HE staining, and observe pathological damage in liver tissue;

[0130] (3) Multiple immunohistochemical assays were used to detect α-SMA, Col1a1 and CK19 to assess liver fibrosis and bile duct reaction.

[0131] The results showed that, compared with the solvent control group, the UNC2025 treatment group mice had reduced pathological damage to liver tissue, decreased serum ALT levels, and reduced α-SMA and Col1a1 positive areas in liver tissue. Figure 3 AC). In Mdr2 - / - In the model, compared with the solvent control group, the UNC2025 treatment group showed reduced liver tissue damage, decreased serum ALT levels, and reduced α-SMA and Col1a1 positive areas. Figure 3 DF).

[0132] In conclusion, drug inhibition of Mertk can significantly alleviate cholestatic liver injury and its associated fibrotic process, suggesting that Mertk may serve as a drug intervention target for cholestatic liver injury.

[0133] Example 3: Mertk promotes cholestatic liver injury and hepatic stellate cell activation by regulating macrophage ferroptosis.

[0134] 1. Laboratory animals, model construction, and drug administration process:

[0135] (1) Using Mertk fl / fl Mice, Lyz2 cre Mertk fl / fl Mice, Mdr2 - / - Mice, Mertk - / - -Mdr2 - / - Mice and corresponding control mice, the source and catalog number of which are the same as in Example 1.

[0136] (2) DDC model: Eight-week-old mice were fed a diet containing 0.1% DDC for 14 consecutive days to establish a DDC-induced cholestatic liver injury model. Serum and liver tissue samples were collected on the 14th day after modeling.

[0137] (3) Fer-1 administration: Some DDC model mice were given the ferroptosis inhibitor Ferrostatin-1, i.e., Fer-1, or the corresponding solvent control. The Fer-1 dosage was 5 mg / kg / day, administered by intraperitoneal injection once daily for 14 consecutive days after the start of DDC modeling. Serum and liver tissue samples were collected on the 14th day of modeling.

[0138] (4) Mdr2 - / - Model: Selected 10-week-old Mdr2 - / - Mice and Mertk - / - -Mdr2 - / - Mice were used as a model of chronic cholestatic liver injury and fibrosis, and liver tissue samples were collected at 10 weeks of age.

[0139] 2. Methods for confirming successful model building:

[0140] The criteria for a successful DDC model include elevated serum ALT, AST, or ALP levels after 14 days of continuous feeding with 0.1% DDC diet; HE staining showing liver tissue damage or inflammatory infiltration; increased infiltration of F4 / 80 positive macrophages and MPO / Ly6G positive neutrophils; and elevated levels of α-SMA, Col1a1, and CK19. Mdr2 - / - The success criteria for the model include the presence of spontaneous cholestasis, bile duct reaction, inflammatory infiltration, and liver fibrosis at 10 weeks of age.

[0141] 3. Experimental Design:

[0142] (1) Single-cell transcriptome analysis was performed on liver immune cells to compare the ferroptosis scores of Mertk-positive macrophages and Mertk-negative macrophages, and GO and KEGG pathway analysis was performed on differentially expressed genes in specific cell populations.

[0143] (2) For Mertk fl / fl and Lyz2 cre Mertk fl / fl Differential gene analysis was performed on mouse-derived macrophages, and GO and KEGG pathway enrichment analysis was conducted.

[0144] (3) Prepare ultrathin sections of liver tissue and observe the morphological and ultrastructural changes of mitochondria in macrophages in the liver by transmission electron microscopy;

[0145] (4) HE staining and serum ALT detection were performed on DDC model mice in the Fer-1 treatment group and solvent control group to assess the degree of liver damage;

[0146] (5) Multiple immunohistochemical detection of Col1a1, α-SMA and CK19 was used to assess liver fibrosis and bile duct reaction;

[0147] (6) Collect the culture supernatant of macrophages under different treatment conditions and apply it to hepatic stellate cells. α-SMA expression was detected by qPCR and immunofluorescence to assess the degree of activation of hepatic stellate cells.

[0148] The results showed that GO functional enrichment analysis revealed significant alterations in inflammatory responses, oxidative stress, prostaglandin metabolism, leukotriene metabolism, and ferroptosis-related pathways after Mertk loss in myeloid cells. Figure 4 A). Single-cell transcriptome analysis showed that Mertk-positive macrophages had higher ferroptosis scores; while Lyz2 cre Mertk fl / fl In mice, macrophage ferroptosis scores were reduced ( Figure 4 BC). Transmission electron microscopy revealed that Mdr2 - / - In model mice, hepatic macrophages exhibited ferroptosis-related ultrastructural changes such as mitochondrial shrinkage, while Mertk deficiency alleviated these changes. Figure 4 D).

[0149] Further inhibition of ferroptosis with Fer-1 reduced liver tissue damage, decreased serum ALT levels, and reduced α-SMA and Col1a1 positive areas in DDC model mice. Figure 4 EG). Furthermore, RSL3-induced macrophage culture supernatant can promote increased α-SMA expression in hepatic stellate cells (EG). Figure 4 HI).

[0150] In summary, Mertk can promote cholestatic liver injury and hepatic stellate cell activation by regulating macrophage ferroptosis. Targeting Mertk or inhibiting macrophage ferroptosis may be used to alleviate cholestatic liver injury and fibrosis.

[0151] Example 4: Mertk promotes bile acid and arachidonic acid-induced macrophage lipid metabolism remodeling and ferroptosis.

[0152] 1. Experimental materials, cell treatment, and experimental grouping:

[0153] (1) Using Mertk fl / fl Mice, Lyz2 cre Mertk fl / fl Mice, WT mice and Mertk - / - Experiments were conducted using mouse-derived samples or cells.

[0154] (2) Single-cell transcriptome analysis was used to analyze Mertk in a cholestatic liver injury model. - MDM and Mertk + Differences in the distribution and functions of MDM.

[0155] (3) Using WT mice and Mertk mice - / - Mouse bone marrow-derived macrophages, or BMDM, were used for in vitro stimulation experiments.

[0156] (4) Lithocholic acid (LCA) was used to simulate bile acid stimulation; arachidonic acid (AA) was used to simulate an increased state of arachidonic acid metabolic substrates. Experimental groups included a control group, a BMDM-LCA group, a BMDM-LCA+AA group, and a Mertk group. - / - -BMDM-LCA+AA group.

[0157] (5) BMDM was treated with arachidonic acid in combination with different bile acids, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA).

[0158] 2. Experimental Design:

[0159] ① The distribution of Mertk- MDM and Mertk+ MDM in macrophage populations was analyzed using single-cell transcriptome UMAP.

[0160] ② For Mertk fl / fl and Lyz2 cre Mertk fl / fl GSEA analysis was performed on macrophages in a mouse cholestatic liver injury model to evaluate changes in the ferroptosis pathway and arachidonic acid metabolism pathway.

[0161] ③ Detect the expression levels of arachidonic acid metabolism and ferroptosis-related genes Pla2g4a, Ptges3, Tbxas1, Alox5ap, Cyba, Por, Hmox1, Lta4h, Alox12, Acsl4, Lpcat3, Ncf2, Tfrc and Pebp1.

[0162] ④ The effects of AA combined with CA, CDCA, DCA, or LCA on the survival status of BMDM were evaluated using cell viability assays.

[0163] ⑤ The degree of lipid peroxidation damage in BMDM after LCA combined with AA treatment was evaluated using MDA detection;

[0164] ⑥ The changes in arachidonic acid metabolism-related genes in BMDM after LCA and AA treatment were analyzed by gene expression heatmap, and WT-BMDM was further compared with Mertk. - / - - Differences in BMDM after LCA+AA treatment;

[0165] ⑦ C11-BODIPY was used to detect WT-BMDM-LCA, WT-BMDM-LCA+AA and Mertk. - / - -Lipid peroxidation level in the BMDM-LCA+AA group.

[0166] The results showed that single-cell transcriptome analysis could distinguish between two macrophage populations: Mertk-MDM and Mertk+MDM. Figure 5 A). GSEA analysis shows that, compared with Mertk fl / fl Compared to mice, Lyz2 cre Mertk fl / fl In a mouse model of cholestatic liver injury, macrophage ferroptosis and arachidonic acid metabolism pathways showed a decreasing trend. Figure 5 B). Further analysis of arachidonic acid metabolism and ferroptosis-related genes revealed that after Mertk deletion in myeloid cells, the expression of related genes such as Pla2g4a, Ptges3, Alox5ap, Cyba, Hmox1, Lta4h, Acsl4, Lpcat3, and Ncf2 was altered. Figure 5 C).

[0167] In vitro experiments showed that, in the presence of arachidonic acid (AA), different bile acids could reduce the activity of macrophages to varying degrees, with LCA combined with AA treatment showing the most significant damage to macrophages. Simultaneously, LCA combined with AA treatment significantly increased the MDA level in macrophages, suggesting that bile acids and arachidonic acid jointly promote lipid peroxidation damage in macrophages. Figure 5 D). Gene expression heatmaps showed that under LCA stimulation, AA could induce upregulation of the expression of multiple arachidonic acid metabolism-related genes in BMDM ( Figure 5 E). Further comparison of WT-BMDM and Mertk - / - -BMDM found that under LCA+AA treatment, Mertk deletion reduced the expression of multiple arachidonic acid metabolism-related genes. Figure 5 F). C11-BODIPY assays further confirmed that LCA+AA significantly increased lipid peroxidation levels in WT-BMDM, while Mertk deficiency reduced the LCA+AA-induced Ox-BODIPY / non-Ox-BODIPY ratio. Figure 5 G).

[0168] In summary, Mertk can promote arachidonic acid metabolic remodeling and lipid peroxidation in macrophages stimulated by bile acids and arachidonic acid, thereby participating in the macrophage ferroptosis-related injury process. Inhibition or absence of Mertk can alleviate the aforementioned lipid metabolism abnormalities and ferroptosis-related changes, suggesting that Mertk is an important target for intervening in macrophage lipid peroxidation and ferroptosis in cholestatic liver injury.

[0169] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. The use of a Mertk inhibitor in the preparation of a medicament for the prevention and / or treatment of cholestatic liver injury; wherein the Mertk inhibitor inhibits ferroptosis in Mertk-positive macrophages.

2. The application according to claim 1, characterized in that, The Mertk inhibitors mentioned herein are not intended for the treatment of liver fibrosis or liver injury induced by non-cholestatic factors.

3. The application according to claim 1, characterized in that, The cholestatic liver injury mentioned includes: primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, or progressive familial intrahepatic cholestasis (PFIC).

4. The application according to claim 1, characterized in that, The Mertk inhibitors include: small molecule compounds, nucleic acid molecules, peptides, proteins, antibodies, or their antigen-binding fragments.

5. Use of Mertk inhibitors in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury in subjects, wherein the subjects are those with elevated levels of ferroptosis in Mertk-positive macrophages in liver tissue relative to healthy controls.

6. A pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury, comprising a therapeutically effective amount of a Mertk inhibitor and a pharmaceutically acceptable carrier or excipient, said pharmaceutical composition for inhibiting ferroptosis in Mertk-positive macrophages.

7. The pharmaceutical composition according to claim 6, characterized in that, The inhibition of ferroptosis is detected or characterized by means selected from the group consisting of: transmission electron microscopy to observe changes in the ultrastructure of macrophage mitochondria, detection of the expression levels of ferroptosis markers MDA, 4-HNE, GPX4 or ACSL4, or combinations thereof.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further comprises a second therapeutic agent selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, corticosteroids, or anti-inflammatory agents.

9. A kit for the prevention and / or treatment of cholestatic liver injury, comprising: a) A container containing a therapeutically effective amount of a Mertk inhibitor, or a pharmaceutical composition thereof, and b) Instruction manual; in, The instruction manual directs the administration of the Mertk inhibitor to subjects in whom elevated levels of ferroptosis in Mertk-positive macrophages in liver tissue were detected.

10. A method for screening candidate drugs for the prevention and / or treatment of cholestatic liver injury, comprising the following steps: (1) Provide an in vitro or in vivo model of cholestatic liver injury; (2) Apply the candidate material to the model; (3) Detect the level of ferroptosis in macrophages in the model; and (4) Candidate substances that can downregulate the level of ferroptosis in macrophages are identified as candidate drugs for the prevention and / or treatment of cholestatic liver injury.

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