Use of a substance that inhibits or down-regulates the expression and / or activity of the transcription factor Glis3 for the manufacture of a medicament for the prevention and / or treatment of a metabolic-related liver fibrosis disease

Abstract

The application relates to application of a substance for inhibiting or down-regulating expression and / or activity of a transcription factor Glis3 in preparation of a medicine for preventing and / or treating a metabolic related liver fibrosis disease, and application of a reagent for detecting expression level of Glis3 and / or CCL23 in preparation of a related diagnosis, staging evaluation, curative effect monitoring or prognosis evaluation product. The application first clarifies a key pathogenic role of Glis3 in liver fibrosis and a mechanism of activation of hepatic stellate cells by a Ccl6 / CCL23-CCR1 axis, and provides a new molecular basis for targeted intervention and non-invasive evaluation of the metabolic related liver fibrosis disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of substances that inhibit or downregulate the expression and / or activity of transcription factor Glis3 in the preparation of drugs for the prevention and / or treatment of metabolic-related liver fibrosis. Background Technology

[0002] Metabolic fatty liver disease (MASLD, formerly known as NAFLD) is currently the most common chronic liver disease. A significant proportion of MASLD patients will progress to metabolic steatohepatitis (MASH, formerly known as NASH), and further develop into liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Liver fibrosis is the core pathological event that determines the prognosis of the disease. The presence of fibrosis stage, rather than simply steatohepatitis, is the strongest predictor of liver-related mortality and all-cause mortality in NAFLD patients.

[0003] The development and progression of liver fibrosis is a complex process involving multiple cells. Hepatic stellate cells (HSCs) play a central role: stimulated by liver injury signals, resting HSCs activate into myofibroblasts, capable of proliferation, contraction, and extracellular matrix (ECM) production, becoming the primary source of ECM deposition. HSC activation is not a cell-autonomous process but is driven by paracrine signals (cytokines, chemokines, injury-related molecular patterns, etc.) from damaged hepatocytes. However, the specific molecular mediators and upstream regulatory mechanisms within the hepatocyte-HSC paracrine axis remain incompletely elucidated.

[0004] Glis3, a Krüppel-like zinc finger transcription factor belonging to the GLI-similar family, has been the subject of previous research focusing on its role in pancreatic β-cell development, insulin gene regulation, and susceptibility to diabetes. Genome-wide association studies (GWAS) have identified the Glis3 locus as a significant risk site for both type 1 and type 2 diabetes. Furthermore, Glis3 plays an important role in thyroid hormone biosynthesis and kidney development. However, no studies have yet reported Glis3's involvement in the development and progression of liver fibrosis, and its function and mechanisms in liver diseases remain completely unknown.

[0005] Current treatments for liver fibrosis are extremely limited. Although Resmetirom (a thyroid hormone receptor beta agonist) was recently conditionally approved by the FDA for adult patients with moderate to severe fibrosis but without cirrhosis, NASH / MASH, therapeutic targets targeting upstream profibrotic molecular drivers remain limited. This significant unmet clinical need underscores the urgent need to find novel molecular targets. Summary of the Invention

[0006] This invention provides the use of substances that inhibit or downregulate the expression and / or activity of transcription factor Glis3 in the preparation of drugs for the prevention and / or treatment of metabolic-related liver fibrosis, aiming to solve the following technical problems:

[0007] (1) To date, no studies have revealed the role and mechanism of transcription factor Glis3 in liver fibrosis, and its value as a target for anti-fibrotic therapy is completely unknown.

[0008] (2) The key pro-fibrotic paracrine signaling molecules sent by hepatocytes to HSCs have not been fully identified, and there is a lack of circulating biomarkers that can reflect active fibrotic signals of the Glis3-Ccl6 / CCL23-CCR1 axis.

[0009] (3) Existing non-invasive diagnostic tools for liver fibrosis (such as liver stiffness measurement) lack specific biomarkers that can reflect the upstream molecular driving mechanism;

[0010] (4) There are no effective therapeutic drugs and intervention strategies for MASLD / MASH-related liver fibrosis that directly target the core fibrosis driving mechanism.

[0011] This invention provides the use of substances that inhibit or downregulate the expression and / or activity of transcription factor Glis3 in the preparation of drugs for the prevention and / or treatment of metabolic-related liver fibrosis.

[0012] Preferably, the substance includes nucleic acid interference molecules, antisense oligonucleotides, gene editing systems, small molecule compounds, polypeptides, antibodies or their antigen-binding fragments, viral vectors, non-viral delivery vectors, and combinations thereof.

[0013] Preferably, the metabolic-related liver fibrosis includes liver fibrosis and cirrhosis caused by metabolic-related fatty liver disease / metabolic steatohepatitis.

[0014] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0015] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0016] This invention also provides the application of reagents for detecting Glis3 expression levels and / or CCL23 levels in the preparation of products for the diagnosis, staging assessment, efficacy monitoring, or prognostic assessment of metabolic-related liver fibrosis.

[0017] Preferably, the drug exerts its antifibrotic effect by inhibiting the Glis3-Ccl6 / CCL23-CCR1 signaling axis, reducing Ccl6 and / or CCL23 expression, inhibiting HSC activation, and reducing extracellular matrix deposition.

[0018] Preferably, the diagnostic assessment product includes at least one of RT-qPCR reagents, nucleic acid probes, sequencing reagents, immunoassay reagents, and ELISA kits.

[0019] Explanation of English abbreviations:

[0020] Glis3: GLI-similar 3, GLI-like family 3, Krüppel-like zinc finger transcription factor.

[0021] Ccl6 / CCL6: CC chemokine ligand 6 (mouse), whose human homolog is CCL23 (CC chemokine ligand 23).

[0022] CCR1: CC chemokine receptor 1, which is the receptor for CCL6 / CCL23.

[0023] HSC: Hepatic Stellate Cell.

[0024] CBP / p300: CREB binding protein / p300 protein, histone acetyltransferase, encoded by the CREBBP / EP300 genes respectively.

[0025] HDAC11: Class IV histone deacetylase 11.

[0026] H3K27Ac: Histone H3 lysine acetylation at position 27, an epigenetic marker of activity enhancers and promoters.

[0027] AAV: Adeno-Associated Virus; TBG promoter: Thyroxine-binding globulin promoter, hepatocyte-specific.

[0028] MCD diet: a methionine-choline deficient diet that induces a mouse model of hepatic steatosis and fibrosis.

[0029] MASLD / MASH: Metabolic fatty liver disease / metabolic steatohepatitis (formerly known as NAFLD / NASH).

[0030] Beneficial effects

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) Discovery of a novel key driver of liver fibrosis: The key pathogenic role of transcription factor Glis3 in liver fibrosis was elucidated for the first time, filling a gap in the field and providing a new theoretical framework for the study of the molecular mechanism of liver fibrosis.

[0033] (2) Providing novel therapeutic targets: Glis3 hepatocyte-specific knockdown (AAV-si-Glis3) showed significant therapeutic protective effects in a mouse model of MCD diet-induced liver fibrosis, confirming that Glis3 is an interventionable anti-fibrotic therapeutic target. The AAV8-mediated hepatocyte-specific delivery strategy provides a precise targeting technical approach.

[0034] (3) Discovery of a novel anti-fibrotic intervention node—CCL6 / CCL23-CCR1 axis: Both CCL6 neutralizing antibody and CCR1 antagonist BX471 can effectively block the paracrine effect of Glis3 overexpressing hepatocytes activating HSCs, providing an alternative route for targeted therapy, which is more precise than existing broad-spectrum anti-inflammatory treatments.

[0035] (4) Discovery of novel circulating biomarkers for liver fibrosis: The liver expression level of GLIS3 increased with the progression of fibrosis in two independent human cohorts and has a high diagnostic value for AUROC; the plasma level of CCL23 was significantly positively correlated with liver stiffness in NAFLD patients. Both can be used as non-invasive diagnostic biomarkers to reflect the active fibrosis process, which can help make up for the shortcomings of existing non-invasive assessment tools.

[0036] (5) Revealing the epigenetic-transcriptional regulation dual-layer mechanism: The CBP / p300 deletion elucidates the dual-layer regulatory mechanism that leads to the hyperacetylation of Ccl6 promoter H3K27 through the downregulation of HDAC11 and the upregulation of Glis3 to drive Ccl6 transcription. This provides a mechanistic basis for designing multi-target joint intervention strategies (such as HAT activators combined with Glis3 inhibitors). Attached Figure Description

[0037] Figure 1 This study presents spontaneous progressive liver fibrosis in hepatocyte-specific Crebbp / Ep300 double knockout (CBP / p300LivDKO) mice. A shows a comparison of CREBBP and EP300 mRNA expression levels in the livers of cirrhotic patients and healthy individuals from a public database. B presents a heatmap of inflammation and fibrosis-related genes in NASH patients grouped by CREBBP / EP300 expression levels in the GSE61260 dataset. C shows representative images (scale bar 50 μm) of the livers of mice in each group, including H&E, Masson staining, α-SMA, and Sirius red staining. D shows liver staining images at different postnatal time points (P4, P7, P14).

[0038] Figure 2The study aimed to demonstrate how CBP / p300 deficiency activates profibrotic and inflammatory transcriptional programs in hepatocytes. A represents IPA pathway analysis using RNA-seq; B represents a heatmap of differentially expressed genes (collagen and ECM-related genes); C represents GSEA enrichment analysis; D represents hepatic hydroxyproline content; EF represents the mRNA and protein levels of profibrotic markers; and GI represents the expression of inflammatory genes and immune cell infiltration.

[0039] Figure 3 To induce CCL6 expression and activate HSCs in CBP / p300-deficient hepatocytes; AB shows the schematic diagram of the in vitro conditioned medium / co-culture system and α-SMA immunofluorescence; CD shows RNA-seq identification of Ccl6 as the most significantly upregulated gene, and verification of plasma Ccl6 levels; E shows the correlation analysis between plasma CCL23 and liver stiffness in NAFLD patients; FI shows the localization and expression verification of CCL6 in hepatocytes; JN shows the recombinant CCL6 treatment experiment and BX471 / Ccl6-mAb blocking experiment.

[0040] Figure 4 Glis3 drives CCL6-mediated HSC activation; where AG represents Glis3 at CBP / p300 LivDKO Upregulation in mouse liver and various metabolic disease models, as well as expression and diagnostic value in human fibrosis cohorts (GSE193066, GSE225740); HJ: Mechanism experiment of Glis3 overexpression driving Ccl6 expression (mRNA, luciferase reporter, ChIP-qPCR); KP: Effect of Glis3 overexpression / knockdown on hepatocyte-HSC co-culture system and CCL6 dependence verification.

[0041] Figure 5 This study aimed to investigate how hepatocyte-specific Glis3 overexpression exacerbates liver fibrosis in vivo. A represents the experimental design (AAV-OE-Glis3 combined with an MCD diet for 8 weeks); BL represents a comprehensive analysis of serum ALT, AST, hydroxyproline, histopathological findings (H&E, Masson, F4 / 80, Ly6G, α-SMA), inflammation / fibrosis gene expression, Ccl6 expression, and the proportion of CCL6-positive cells.

[0042] Figure 6 This study aimed to reduce MCD-induced in vivo liver fibrosis by knocking down hepatocyte-specific Glis3. A represents the experimental design (AAV-si-Glis3 combined with an MCD diet for 8 weeks); B and K represent a comprehensive analysis of serum ALT, AST, hydroxyproline, histopathological findings, inflammation / fibrosis genes, Ccl6 expression, and the proportion of CCL6-positive cells. Detailed Implementation

[0043] 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. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0044] Example 1

[0045] Establishment and pathological validation of a model of spontaneous progressive liver fibrosis caused by CBP / p300 deficiency:

[0046] (1) Public database analysis: Liver tissue transcriptome data of patients with cirrhosis and healthy individuals were collected, and the expression levels of CREBBP and EP300 mRNA were compared. At the same time, NASH patients in the GSE61260 dataset were used as subjects, and they were grouped according to the expression levels of CREBBP / EP300. Inflammatory factors, chemokines, collagen and extracellular matrix-related genes were extracted, heatmaps were drawn, and the differences between the two groups were compared.

[0047] (2) Animal model establishment: Crebbp / Ep300 double knockout mice (CBP / p300) were constructed using the hepatocyte-specific Cre system. LivDKO Wild-type or Cre-negative mice from the same litter served as the control group. Liver tissues were collected at P4, P7, P14 and subsequent observation time points after birth for H&E staining, Masson staining, α-SMA immunostaining and Sirius red staining, and hepatocyte damage, inflammatory infiltration, collagen deposition and HSC activation were observed under a microscope.

[0048] (3) Experimental results: such as Figure 1 As shown in AD, the expression of CREBBP and EP300 in liver tissue of patients with cirrhosis was lower than that in healthy controls; in NASH samples with low CREBBP / EP300 expression, inflammation and fibrosis-related genes were generally upregulated. LivDKO As time progressed after birth, mice developed progressive liver pathological changes, including increased inflammatory cell infiltration, increased Masson and Sirius red positive collagen deposition, and an increase in α-SMA positive HSCs / myofibroblasts, indicating that the loss of hepatocyte CBP / p300 is sufficient to induce spontaneous and progressive liver fibrosis.

[0049] Example 2

[0050] CBP / p300 deficiency activates profibrotic and inflammatory transcriptional programs.

[0051] (1) Take the wild-type control group and CBP / p300 LivDKOMouse liver tissue or hepatocytes were collected, and total RNA was extracted and RNA-seq was performed. IPA pathway analysis, collagen / extracellular matrix-related gene heatmap analysis, and GSEA enrichment analysis were performed on differentially expressed genes to evaluate changes in pathways related to fibrosis, inflammation, immune cell recruitment, and extracellular matrix remodeling.

[0052] (2) Hydroxyproline content was detected in the livers of animals in the same batch to quantitatively evaluate collagen deposition; RT-qPCR was used to detect profibrotic markers such as Col1a1, Col3a1, Acta2 (α-SMA), and Timp1, as well as the expression of inflammatory / chemokine genes; Western blot or immunostaining was used to detect α-SMA, collagen-related proteins, and immune cell infiltration markers.

[0053] (3) Experimental results: such as Figure 2 As shown in AI, CBP / p300 LivDKO The liver showed significant enrichment of profibrosis and inflammation-related pathways, with collagen and ECM-related genes being upregulated overall. The liver also showed increased hydroxyproline content, increased levels of profibrosis mRNA and protein, accompanied by enhanced expression of inflammatory genes and increased infiltration of immune cells, demonstrating that the absence of CBP / p300 initiates a profibrosis- and pro-inflammatory transcriptional program in hepatocytes.

[0054] Example 3

[0055] CBP / p300-deficient hepatocytes induce CCL6 expression and activate HSCs via the CCL6 / CCR1 axis:

[0056] (1) Separation of wild-type control group and CBP / p300 LivDKO Primary mouse hepatocytes were collected, and conditioned medium was used to collect the hepatocytes. Alternatively, hepatocytes were co-cultured with primary HSCs in a Transwell system. After culture, the expression of pro-fibrotic genes such as α-SMA immunofluorescence, Col1a1, Col3a1, and Acta2, as well as cell activation status, were detected in HSCs.

[0057] (2) The RNA-seq results of CBP / p300-deficient hepatocytes were used to screen for secretory factors and chemokines, with a focus on analyzing changes in Ccl6 expression. RT-qPCR, ELISA, or immunofluorescence / in situ localization methods were used to verify Ccl6 / CCL6 expression in hepatocytes and plasma CCL6 levels. Plasma CCL23 levels in NAFLD patients were simultaneously detected and correlated with liver stiffness measurements to evaluate the clinical relevance of the human homologous molecule CCL23.

[0058] (3) To verify the causal relationship, primary HSCs were treated with recombinant CCL6, or CCR1 antagonist BX471 or CCL6 neutralizing antibody (Ccl6-mAb) was added to the CBP / p300-deficient hepatocyte-HSC co-culture system. Subsequently, HSC profibrosis genes, α-SMA immunofluorescence and proliferation / activation indicators were detected.

[0059] (4) Experimental results: such as Figure 3 As shown in the AN diagram, conditioned medium or co-culture of CBP / p300-deficient hepatocytes significantly induced HSC activation. Ccl6 is a hepatocyte-derived secretory factor that is significantly upregulated after CBP / p300 deficiency, with plasma CCL6 levels increasing synchronously. Plasma CCL23 levels in NAFLD patients were positively correlated with liver stiffness. Recombinant CCL6 can directly activate HSCs, while BX471 or Ccl6-mAb can block the pro-fibrotic paracrine effect of CBP / p300-deficient hepatocytes on HSCs, indicating that the CCL6 / CCL23-CCR1 axis is a key pathway connecting damaged hepatocytes and HSC activation.

[0060] Example 4

[0061] Glis3 upregulation and diagnostic value in CBP / p300 deficiency and metabolic-related liver disease / liver fibrosis models:

[0062] (1) Screening differentially expressed transcription factors in NASH fibrosis-related transcriptome data to identify upregulated transcription factors associated with fibrosis progression; and simultaneously detecting wild-type control group and CBP / p300. LivDKO The level of Glis3 mRNA in the liver of mice at different time points after birth (P1, P7, P28).

[0063] (2) Establish or collect various metabolic-related liver disease models, including MCD diet model, HFD diet model and db / db mouse model, extract total RNA from the liver and detect the expression level of Glis3 by RT-qPCR, and compare the differences between the model group and the corresponding control group.

[0064] (3) In the human liver fibrosis cohorts GSE193066 and GSE225740, the expression levels of GLIS3 / Glis3 mRNA were compared according to the fibrosis stage, and the diagnostic efficacy of GLIS3 / Glis3 in distinguishing fibrosis stage or fibrosis status was analyzed by ROC curve.

[0065] (4) Experimental results: such as Figure 4As shown in AG, GLIS3 / Glis3 was significantly upregulated among NASH fibrosis-related transcription factors; Glis3 mRNA in the liver of CBP / p300LivDKO mice was higher than that in the control group at P1, P7, and P28, and was also upregulated in metabolic-related liver disease models such as MCD, HFD, and db / db. Human cohorts GSE193066 and GSE225740 showed that GLIS3 / Glis3 expression increased with the severity of fibrosis stages; the AUROC of ROC analysis in the GSE193066 cohort was 0.9616 (p=0.0001), and the AUROC of ROC analysis in the GSE225740 cohort was 0.7159 (p=0.0014), suggesting that Glis3 has application value as a molecular marker and therapeutic target for liver fibrosis.

[0066] Example 5

[0067] Verification of the mechanism by which Glis3 overexpression drives Ccl6 transcription:

[0068] (1) Primary mouse hepatocytes were transfected with Glis3 overexpression plasmid (CV702-3xFlag-Glis3), and RNA was extracted 48 h later. Ccl6 mRNA level was detected by RT-qPCR.

[0069] (2) Luciferase reporter assay: HEK293T cells were co-transfected with Ccl6 promoter-firefly luciferase reporter plasmid (GV238-Ccl6-promoter), Glis3 overexpression plasmid and Renilla luciferase internal control plasmid (pRL-TK), and detected by dual luciferase method after 24 h.

[0070] (3) ChIP-qPCR: Chromatin immunoprecipitation was performed on hepatocytes overexpressing Flag-Glis3 using Flag antibody, and quantitative PCR was performed using specific primers designed for the Ccl6 promoter to verify the direct binding of Glis3 to the Ccl6 promoter.

[0071] The molecular mechanism by which Glis3 directly activates Ccl6 transcription

[0072] (4) Experimental results: From Figure 4 HJ indicates that overexpression of Glis3 in primary wild-type hepatocytes can directly drive Ccl6 mRNA expression; luciferase reporter gene assays have demonstrated that Glis3 can activate the transcriptional activity of the Ccl6 promoter; ChIP-qPCR analysis has confirmed that Glis3 directly binds to the Ccl6 gene promoter region.

[0073] Example 6

[0074] CCL6 / CCR1 axis blockade inhibits hepatocyte-HSC profibrotic paracrine signaling (in vitro experiment):

[0075] (1) Glis3-overexpressing primary mouse hepatocytes and primary HSCs were co-cultured in Transwell (upper chamber hepatocytes / lower chamber HSCs, 48h).

[0076] (2) Experimental group design: control + Vehicle, Glis3-OE + Vehicle, Glis3-OE + BX471 (CCR1 antagonist), Glis3-OE + Ccl6-mAb (CCL6 neutralizing antibody).

[0077] (3) Extract total RNA from HSCs and detect the expression of profibrotic genes such as Col1a1, Col3a1, and α-SMA by RT-qPCR;

[0078] (4) α-SMA and Ki67 immunofluorescence staining were used to assess the activation and proliferation of HSCs.

[0079] (5) Experimental results: From Figure 4 According to KP, when Glis3-overexpressing hepatocytes are co-cultured with HSCs, they can strongly induce HSCs to express profibrotic genes (Col1a1, Col3a1, α-SMA). This effect can be completely blocked by the CCL6 receptor antagonist BX471 (CCR1 antagonist) or the CCL6 neutralizing antibody (Ccl6-mAb).

[0080] Example 7

[0081] AAV-mediated hepatocyte-specific Glis3 knockdown therapy for liver fibrosis (in vivo experiment):

[0082] AAV8 vector was constructed (a hepatocyte-specific TBG promoter-driven shRNA / miRNA interference sequence targeting Glis3). Eight-week-old C57BL / 6 mice were injected via tail vein with either AAV-si-Glis3 or the control vector (5 × 10⁻⁶). 11 (gc / animal), followed by feeding with an MCD diet for 8 weeks to induce liver fibrosis.

[0083] Endpoint detection:

[0084] (a) Serum ALT and AST (biochemical method);

[0085] (b) Liver hydroxyproline content (hydroxyproline kit);

[0086] (c) Liver histopathology (H&E, Masson staining);

[0087] (d) Liver immunohistochemistry (α-SMA, F4 / 80, Ly6G);

[0088] (e) Glis3 and Ccl6 mRNA (RT-qPCR);

[0089] (f) Percentage of CCL6-positive hepatocytes (immunofluorescence);

[0090] (g) Full spectrum of inflammation / fibrosis genes (RT-qPCR);

[0091] (h) α-SMA protein (Western blot).

[0092] Depend on Figure 5 AL studies have shown that AAV-OE-Glis3 overexpression mediated by AAV8 (driven by the hepatocyte-specific TBG promoter) significantly aggravates liver damage (significantly increased serum ALT and AST), increases liver hydroxyproline content, expands Masson staining positive area, enhances immune cell infiltration (F4 / 80+, Ly6G+ cells) and α-SMA expression in a MCD diet-induced liver fibrosis model. At the same time, liver Ccl6 mRNA and the proportion of CCL6 positive cells are significantly increased.

[0093] Depend on Figure 6 According to AK, AAV-si-Glis3 knockdown mediated by AAV8 (hepatocyte-specific TBG promoter) can significantly alleviate MCD diet-induced liver injury and fibrosis, including reducing serum ALT and AST levels, reducing liver hydroxyproline content and Masson staining positive area, reducing inflammatory cell infiltration and α-SMA expression, and simultaneously reducing liver Ccl6 mRNA levels and the proportion of CCL6 positive cells, indicating that inhibiting Glis3 has a clear anti-fibrotic therapeutic effect.

Claims

1. The use of substances that inhibit or downregulate the expression and / or activity of transcription factor Glis3 in the preparation of drugs for the prevention and / or treatment of metabolic-related liver fibrosis.

2. The application according to claim 1, characterized in that, The substance is selected from nucleic acid molecules that inhibit or downregulate Glis3 expression, small molecule compounds that inhibit Glis3 transcriptional activity or protein function, peptides, antibodies or their antigen-binding fragments, gene editing systems, viral or non-viral vectors that deliver the above substances, and combinations thereof.

3. The application according to claim 1 or 2, characterized in that, The metabolic-related liver fibrosis diseases include liver fibrosis and cirrhosis caused by metabolic-related fatty liver disease / metabolic steatohepatitis.

4. The application according to claim 1 or 2, characterized in that, The drug exerts its antifibrotic effect through at least one of the following pathways: inhibiting the Glis3-Ccl6 / CCL23-CCR1 signaling axis, reducing Ccl6 and / or CCL23 expression, inhibiting hepatic stellate cell activation, and reducing extracellular matrix deposition.

5. A pharmaceutical composition for the prevention and / or treatment of metabolic-related liver fibrosis, characterized in that, The pharmaceutical composition comprises the substance of claim 1 or 2 and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

7. Application of reagents for detecting Glis3 expression levels and / or CCL23 levels in the preparation of products for the diagnosis, staging assessment, efficacy monitoring, or prognostic assessment of metabolic-related liver fibrosis.

8. The application according to claim 7, characterized in that, The products include at least one of RT-qPCR reagents, nucleic acid probes, sequencing reagents, immunoassay reagents, and ELISA kits.

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