Use of reducing expression of crept in treatment of chronic cholestatic liver diseases

Abstract

The present invention relates to use of reducing the expression of CREPT in the treatment of chronic cholestatic liver diseases and particularly to reducing the expression of the CREPT gene or attenuating or inactivating the function of the CREPT protein to treat chronic cholestatic liver diseases. The present application finds for the first time that CREPT is related to chronic cholestatic liver diseases, and that by reducing the expression of CREPT, both systemic and topical administration can effectively treat cholestatic liver diseases.

Description

Application of reducing CREPT expression in the treatment of chronic cholestatic liver disease Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to drugs, pharmaceutical compositions, pharmaceutical combination products, and applications for reducing CREPT expression in the treatment of chronic cholestatic liver disease. Background Technology

[0002] Cholestatic liver disease is a hepatobiliary disorder caused by various factors that disrupt the production, secretion, and excretion of bile. The bile cannot actively drain into the intestinal lumen through the bile ducts, instead accumulating in the liver and refluxing into the bloodstream, leading to a series of organic damage, metabolic disorders, and functional disturbances. This disease can affect any part of the entire biliary system, from the bile ducts formed by two adjacent hepatocytes to the duodenal ampulla, and can be divided into intrahepatic cholestasis and extrahepatic cholestasis. Common causes of intrahepatic cholestasis include viral hepatitis, drug-induced liver disease, primary biliary cirrhosis, and primary sclerosing cholangitis.

[0003] Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis, is an organ-specific chronic cholestatic autoimmune liver disease. Currently, only one first-line drug is available for PBC globally: ursodeoxycholic acid (UDCA). Cholestatic liver disease is associated with the accumulation of chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. These bile acids cause hepatocyte damage due to their detergent-like effects. UDCA is a non-toxic hydrophilic bile acid that competitively inhibits the absorption of toxic endogenous bile acids in the ileum. UDCA also increases bile secretion, exerting a choleretic effect, protecting hepatocytes and bile duct cells from the cytotoxicity of hydrophobic bile acids, and exerting anti-inflammatory and immunomodulatory effects. In addition to improving liver biochemical indicators in PBC patients, UDCA also has an anti-fibrotic effect. However, approximately one-third of patients do not respond completely and progress to cholestatic cirrhosis.

[0004] The U.S. FDA has granted accelerated approval this year for Iqirvo (elafibranor) 80 mg tablets in combination with ursodeoxycholic acid (UDCA) for the treatment of adult primary biliary cholangitis (PBC) with an inadequate response to UDCA, or as monotherapy for patients intolerant to UDCA. Iqirvo is reportedly the first new drug approved in nearly a decade for the treatment of the rare liver disease primary biliary cholangitis. Iqirvo is a first-in-class oral, once-daily peroxisome proliferation-activating receptor (PPAR) agonist.

[0005] The CREPT gene is expressed significantly higher in tissues of various cancers than in non-tumor cells and adjacent normal tissues. Its expression is associated with cancer development and also directly or indirectly promotes cell proliferation. However, the relationship between the CREPT gene and chronic cholestatic liver disease has not been reported in the current technology. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides the application of CREPT gene or protein as a target or reagent targeting CREPT gene or protein in the treatment of chronic cholestatic liver disease or in the preparation of drugs for the treatment of chronic cholestatic liver disease, thereby treating chronic cholestatic liver disease by reducing the expression of CREPT gene or reducing or inactivating the function of CREPT protein.

[0007] Chronic cholestatic liver disease is a hepatobiliary disorder caused by various factors that disrupt bile production, secretion, and excretion. Bile cannot actively drain into the intestinal lumen via the bile ducts, instead accumulating in the liver and refluxing into the bloodstream, leading to a series of organic damage, metabolic disorders, and functional disturbances. The main causes include genetics, immunity, degeneration, infection, gallstones, and tumors. This disease can affect any part of the entire biliary system, from the bile ducts formed by two adjacent hepatocytes to the duodenal ampulla, and can be divided into intrahepatic cholestasis and extrahepatic cholestasis.

[0008] The tumor-associated gene CREPT, also known as RPRD1B, C20ORF77, or Kub5-Hera, can promote cell cycle transition and tumorigenesis, making it a highly promising target for cancer therapy. CREPT is highly expressed in various tumor cell types; therefore, knocking out CREPT significantly inhibits tumor cell growth without affecting the normal cell state. CREPT is a target for anti-tumor drugs.

[0009] A target, also known as a biomolecule, is a biomolecule that performs a specific function in a living organism and can bind to an effective drug. A target must not only participate in the pathological process related to the disease and play a key role, but also not participate in the normal physiological process of tissues unrelated to the disease, and can specifically bind to the drug.

[0010] In some embodiments of this application, the use of the CREPT gene or protein as a target in the treatment of chronic cholestatic liver disease is provided. In some embodiments of this application, the use of agents targeting the CREPT gene or protein in the treatment of chronic cholestatic liver disease is provided. In some embodiments of this application, the use of the CREPT gene or protein as a target in the preparation of medicaments for the treatment of chronic cholestatic liver disease is provided. In some embodiments of this application, the use of agents targeting the CREPT gene or protein in the preparation of medicaments for the treatment of chronic cholestatic liver disease is provided. The above embodiments treat chronic cholestatic liver disease by reducing the expression of the CREPT gene or by reducing or inactivating the function of the CREPT protein, wherein the agents targeting the CREPT gene or protein reduce the expression of the CREPT gene or by reducing or inactivating the function of the CREPT protein.

[0011] As some embodiments of this application, the application has one or more of the following effects: slowing weight loss in individuals with chronic cholestatic liver disease; improving liver function in individuals with chronic cholestatic liver disease, wherein the improvement in liver function includes a decrease in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL; improving the pathological characteristics of chronic cholestatic liver disease, wherein the improvement in the pathological characteristics of chronic cholestatic liver disease includes a reduction in cholestasis and a reduction in bile duct reactivity; reducing the histological structural damage to the liver, wherein the reduction in the histological structural damage to the liver includes a reduction in bile duct hyperplasia; and reducing the level of immune cells, wherein the immune cells include CD4+. + and CD8 +Increases the number of anti-inflammatory neutrophils; reduces cytokine levels, including one or more of IL-1β, CXCL2, CXCL5, and CCL7; reduces fibrosis-related molecular levels, including one or more of TGFB1, CTGF, COL1A1, and COL3A1; reduces Plod2 expression; improves serum and liver discoloration caused by cholestasis; improves weight and liver weight loss in patients with chronic cholestatic liver disease; reduces liver tissue necrosis and inflammatory infiltration in patients with chronic cholestatic liver disease; chronic Reduced collagen deposition in patients with cholestatic liver disease; decreased levels of intrahepatic pro-inflammatory factors in patients with chronic cholestatic liver disease, including one or more of TGF-β, IL-6, and MIP-1α; treatment of liver injury by synergistically reducing liver damage, inflammation, and fibrosis; application in products that early block the progression of liver disease; treatment of fibrosis in patients with chronic cholestatic liver disease and improvement of fibrosis progression; and reduction of bile acid metabolites, including taurine-uric acid. It contains one or more of the following: α-TMCA, tauro-Beta-Muricholic Acid (β-TMCA), taurohyocholic Acid (THCA), glycodeoxycholic Acid (GHDCA), taurohyodexycholic Acid (THDCA), ursodeoxycholic Acid (UDCA), chenodeoxycholic Acid (CDCA), α-Muricholica Acid (α-MCA), β-Muricholica Acid (Beta-Muricholica Acid (β-MCA), ursodeoxycholic Acid (M-DCA), and allocholic Acid (ALCA).

[0012] As some embodiments of this application, the reagent targeting the CREPT gene or protein reduces CREPT gene expression or impairs or inactivates CREPT protein function.

[0013] As some embodiments of this application, the reagent targeting the CREPT gene or protein is selected from one or more of the following: an agent that inhibits CREPT protein expression, an agent that knocks out CREPT protein, an agent that alters CREPT protein, and an agent that degrades CREPT protein.

[0014] As some embodiments of this application, the formulation for inhibiting CREPT protein expression includes a nucleic acid reagent or a vector containing a nucleic acid fragment that inhibits CREPT protein expression.

[0015] As some embodiments of this application, the preparation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene.

[0016] As some embodiments of this application, the formulation for altering the CREPT protein includes agents that alter the structure of the CREPT protein.

[0017] As some embodiments of this application, the formulation for degrading CREPT protein includes one or more of the following: PROTAC, molecular glue, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, and AUTOTAC pathways.

[0018] PROTACs, also known as protein degradation chimeras, are heterobifunctional molecules composed of two ligands linked by a linker. One ligand binds to the target protein, while the other targets the E3 ligase. PROTACs can simultaneously bind to both the target protein and the E3 ligase, thus shortening the distance between them, inducing ubiquitination of the target protein, which is then recognized and degraded by the 26S proteasome. Traditional inhibitors employ a "site-driven" mechanism, requiring the drug molecule to bind tightly to the active site of the target protein to inhibit its activity and achieve a pharmacological effect. PROTACs, however, operate on an event-driven mechanism, binding to any site on the target protein without requiring high affinity to potentially induce protein degradation.

[0019] Molecular glue technology is one of the main technologies based on the ubiquitin-proteasome degradation system. In the field of targeted protein degradation, molecular glues are typically monovalent small molecules (molecular weight less than 500 Da). Their specific mechanism of action involves altering the surface of E3 ligases, thereby blocking the binding of E3 ligases to natural substrates, inducing the specific protein to be degraded to bind to the E3 ligase, further promoting the ubiquitination modification of the specific protein, and finally causing it to be degraded by the proteasome. Due to the complexity of their biological characteristics, some disease-related proteins may possess multiple domains or functional sites, making it difficult to form stable bindings with drug molecules. These proteins have not yet been effectively targeted using traditional drugs such as small molecule drugs or antibodies. The development of molecular glue technology offers a possibility for solving this problem.

[0020] Intracellular targeted protein degradation (iTPD) technologies, represented by PROTAC and molecular gels, have developed into an important new mode for small molecule drug development. While iTPD technology is rapidly developing, extracellular targeted protein degradation (eTPD) technology has also made several significant advances.

[0021] LYTAC is a bifunctional molecule with two binding domains. One end is an oligoglycopeptide group that binds to the cell surface transmembrane receptor CI-M6PR (cation-independent mannose-6-phosphate receptor), and the other end is an antibody or small molecule that binds to a target protein. These two binding domains are linked by a chemical linker. The trimer CI-M6PR–LYTAC–target protein complex formed on the plasma membrane is engulfed by the cell membrane, forming a transport vesicle. The vesicle transports the complex to the lysosome, where the target protein is degraded. LYTAC has the potential to degrade membrane proteins and soluble proteins.

[0022] The asialoglycoprotein receptor (ASGPR) is an endocytic cell surface receptor, highly expressed primarily on hepatocytes (up to 500,000 copies per cell), playing a crucial role in the natural process of endogenous protein internalization and degradation within hepatocytes. Multiple target proteins (such as EGFR) can be degraded by conjugating (multiple) targeting ligands of ASGPR, such as tri-GalNac, to target protein-binding antibodies. The bifunctional small-molecule extracellular targeted protein degradation (eTPD) technologies MoDE and ATAC (ASGPR Targeting Chimeras) utilize ASGPR. ATAC uses a high-affinity monodentate ASGPR targeting moiety that is three to four times smaller than tri-GalNAc. Because ASGPR is a liver-specific lysosomal targeted receptor, ATAC technology, compared to LYTAC (CI-M6PR, which is widely expressed in multiple cell types), can degrade extracellular proteins in a cell-type-restricted manner, offering a potential safety advantage.

[0023] Apt-LYTAC is a small aptamer (8–25 kDa) ASGPR adaptor that links tri-GalNac to the 5' end of the aptamer for the soluble growth factor PDGF and the membrane receptor PTK7, and has demonstrated that PDGF-labeled Apt-LYTAC is degraded in HepG2 cells. PDGF-bound Apt-LYTAC can be delivered to lysosomes.

[0024] Cytokine receptor-targeting chimera (KineTAC) technology is a novel degradative agent that utilizes the decoy circulating receptor CXCR7 to transport cell membrane and extracellular target proteins to lysosomes for degradation. For example, CXCL12 can be internalized after binding to the decoy receptor CXCR7. One end of KineTAC is the chemokine CXCL12, and the other end binds to the target protein. After the complex enters the lysosome, the target protein is degraded. Studies have confirmed that KineTAC technology can degrade membrane proteins such as PD-L1, HER2, and EGFR, as well as soluble proteins VEGF and TNF-α. The KineTAC platform expands the selectivity of eTPD by utilizing a series of circulating receptors with different tissue distributions and levels.

[0025] A novel integrin-facilitated lysosomal degradation (IFLD) strategy based on bifunctional compounds couples a target protein-binding ligand with an integrin-recognizing ligand. The resulting bifunctional compound induces the endocytosis and degradation of extracellular or cell membrane proteins in an integrin- and lysosome-dependent manner. Integrins are cell adhesion receptors expressed on the cell surface and play a crucial role in cell-matrix interactions. Because integrins can bind to ligands containing the Arg-Gly-Asp (RGD) motif and transport them to lysosomes, they represent an attractive degradation system.

[0026] As some embodiments of this application, the drug includes a small molecule inhibitor of the CREPT protein.

[0027] Small molecule inhibitors refer to a class of organic compound molecules with a molecular weight of less than 1,000 Daltons that can target proteins, reduce protein activity, or inhibit biochemical reactions. They are highly selective and have cell permeability, and are widely used in signaling pathway research.

[0028] As some embodiments of this application, the nucleic acid reagent is selected from one or more of siRNA, shRNA, microRNA, piRNA, and ASO.

[0029] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length, with many different uses in biology. siRNA is primarily involved in RNA interference (RNAi) to regulate gene expression in a specific manner.

[0030] Short hairpin RNA (shRNA), cloned into an shRNA expression vector, consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the pol III promoter. It is then followed by 5-6 T molecules as a transcription terminator for RNA polymerase III. Cloning the siRNA sequence as a "short hairpin" into a plasmid vector allows for the delivery of "small interfering RNA" (siRNA) in vivo. When introduced into an animal, the hairpin sequence is expressed, forming a "double-stranded RNA" (dsRNA), which is then processed by RNAi channels.

[0031] MicroRNAs (miRNAs) are non-coding RNAs approximately 22 nt in length, widely found in various organisms from viruses to humans. These small RNAs can bind to mRNA and block the expression of protein-coding genes, preventing them from being translated into proteins.

[0032] piRNA (Piwi-interacting RNA) is a class of small RNAs about 30 nt in length isolated from mammalian germ cells. It can specifically bind to PIWI, an analog of Argonaute protein in animal cells. This small RNA can only exert its regulatory role by binding to members of the PIWI protein family. It has the functions of silencing gene transcription, regulating translation and mRNA stability.

[0033] Antisense oligonucleotides (ASOs) are single-stranded oligonucleotide molecules that typically contain 15-25 nucleotides. After entering the cell, they bind to their complementary target mRNA through base pairing under the action of ribonuclease H1, thereby inhibiting the expression of the target gene.

[0034] As some embodiments of this application, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes one or more of adenovirus, adeno-associated virus, lentivirus, and retrovirus.

[0035] As some embodiments of this application, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

[0036] As some embodiments of this application, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene.

[0037] As some embodiments of this application, the nucleotide sequence of the sense strand of the siRNA targeting the mouse CREPT gene is shown in any one of SEQ ID No. 1 to 7, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 18 to 23. The siRNA with the sense strand shown in SEQ ID No. 7 does not have a corresponding antisense strand.

[0038] As some embodiments of this application, the sense strand nucleotide sequence of the siRNA targeting the human CREPT gene is shown in any one of SEQ ID No. 8 to 17, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 24 to 33.

[0039] The siRNA can have ≥75% homology with any of the nucleotide sequences shown in SEQ ID No. 1 to 17, for example, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0040] Homology refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules. The higher the homology, the more similar the nucleotide sequences of the two nucleic acid molecules are.

[0041] As some embodiments of this application, the chronic cholestatic liver disease includes one or more of the following: primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), primary biliary cholangitis, liver tumor, autoimmune hepatitis (AIH), primary biliary cirrhosis-autoimmune hepatitis overlap syndrome (PBC-AIH), primary sclerosing cholangitis-autoimmune hepatitis overlap syndrome (PSC-AIH), and Ig4-associated cholangitis (IAC).

[0042] This application also provides a medicament for treating chronic cholestatic liver disease, wherein the medicament reduces CREPT gene expression or inactivates CREPT protein function.

[0043] As some embodiments of this application, the drug is selected from one or more of the following: formulations that inhibit CREPT protein expression, formulations that knock out CREPT protein, formulations that alter CREPT protein, and formulations that degrade CREPT protein.

[0044] As some embodiments of this application, the formulation for inhibiting CREPT protein expression includes a nucleic acid reagent or a vector containing a nucleic acid fragment that inhibits CREPT protein expression.

[0045] As some embodiments of this application, the preparation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene.

[0046] As some embodiments of this application, the formulation for altering the CREPT protein includes agents that alter the structure of the CREPT protein.

[0047] As some embodiments of this application, the formulation for degrading CREPT protein includes one or more of the following: PROTAC, molecular glue, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, and AUTOTAC pathways.

[0048] As some embodiments of this application, the drug includes a small molecule inhibitor of the CREPT protein.

[0049] In some embodiments of this application, the nucleic acid reagent is selected from one or more of siRNA, shRNA, microRNA, piRNA, and ASO. In some embodiments of this application, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes one or more of adenovirus, adeno-associated virus, lentivirus, and retrovirus.

[0050] As some embodiments of this application, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

[0051] As some embodiments of this application, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene.

[0052] As some embodiments of this application, the nucleotide sequence of the sense strand of the siRNA targeting the mouse CREPT gene is shown in any one of SEQ ID No. 1 to 7, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 18 to 23. The siRNA with the sense strand shown in SEQ ID No. 7 does not have a corresponding antisense strand.

[0053] As some embodiments of this application, the sense strand nucleotide sequence of the siRNA targeting the human CREPT gene is shown in any one of SEQ ID No. 8 to 17, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 24 to 33.

[0054] This application also provides a pharmaceutical composition for treating chronic cholestatic liver disease, the pharmaceutical composition comprising a therapeutically effective amount of the drug and a pharmaceutically acceptable carrier.

[0055] As some embodiments of this application, the drug is delivered using a delivery system.

[0056] As some embodiments of this application, the delivery system includes GalNAc or LNP.

[0057] GalNAc is a high-affinity targeting ligand for the desialyl glycoprotein receptor (ASGPR), exhibiting highly specific binding to ASGPR, an endocytic receptor widely expressed on the hepatocyte membrane surface. Through ASGPR and clathrin-mediated endocytosis, GalNAc can be efficiently transported from the cell surface to the cytoplasm. Small interfering RNAs (siRNAs) bound to the GalNAc delivery system possess liver-specific delivery capabilities. In some embodiments, GalNAc is selected from Givosiran, Lumasilan, Inclisiran, or Vurisiran.

[0058] As some embodiments of this application, the pharmaceutical composition includes siRNA and the delivery system GalNAc.

[0059] As some embodiments of this application, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene.

[0060] As some embodiments of this application, the nucleotide sequence of the sense strand of the siRNA targeting the mouse CREPT gene is shown in any one of SEQ ID No. 1 to 7, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 18 to 23. The siRNA with the sense strand shown in SEQ ID No. 7 does not have a corresponding antisense strand.

[0061] As some embodiments of this application, the sense strand nucleotide sequence of the siRNA targeting the human CREPT gene is shown in any one of SEQ ID No. 8 to 17, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 24 to 33.

[0062] This application also provides a pharmaceutical combination product comprising a therapeutically effective amount of the aforementioned drug, and a second drug, for simultaneous or sequential administration.

[0063] As some embodiments of this application, the second drug includes one or more of ursodeoxycholic acid (UDCA), Iqirvo (elafibranor), choleretic acid, and S-adenosylmethionine (SAMe).

[0064] Ursodeoxycholic acid (UDCA), chemically known as 3α,7β-dihydroxy-5β-cholestan-24-acid, is an organic compound used medically to increase bile acid secretion. Cholestatic liver disease is associated with the accumulation of chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, which cause hepatocyte damage due to their detergent-like effects. UDCA is a non-toxic hydrophilic bile acid that competitively inhibits the absorption of toxic endogenous bile acids in the ileum. It enhances the secretory capacity of cholestatic hepatocytes by activating a signaling network composed of calcium ions and protein kinase C, and by activating mitogenic protein kinases, thereby reducing the concentration of endogenous hydrophobic bile acids in the blood and hepatocytes, achieving an anti-choleretic effect. UDCA can also competitively replace toxic bile acid molecules on cell membranes and organelles, preventing further damage to hepatocytes and bile duct cells from toxic bile acids.

[0065] Iqirvo (elafibranor) is a PPAR bi-isoform (α / δ) agonist that helps reduce bile acid production and alleviate fibrosis. After binding to its receptor, it reduces the production of inflammatory factors, alleviating inflammation in primary biliary cholangitis (PBC). Positive results from a Phase III clinical trial showed that 51% of patients in the Elafibranor treatment group achieved cholestasis, compared to only 4% in the placebo group, meeting the primary endpoint. After 78 weeks of treatment in PBC patients, disease progression remained slowed, and Elafibranor demonstrated a good safety profile. On June 11, 2024, the FDA granted accelerated approval to Ipsen and Genfit for the treatment of primary biliary cholangitis (PBC) in adults.

[0066] This application also provides the use of the said medicament, the said pharmaceutical composition, or the said pharmaceutical combination product in any of the following: a. use in the preparation of a product for alleviating chronic cholestatic liver injury; b. use in the preparation of a product for slowing weight loss in individuals with chronic cholestatic liver disease; c. use in the preparation of a product for inhibiting liver fibrosis; d. use in the preparation of a product for improving liver function in chronic cholestatic liver disease; e. use in the preparation of a product for reducing histological damage to the liver; f. use in the preparation of a product for reducing the level of immune cells, said immune cells including CD4. + CD8 +g. Application in the preparation of products that increase the number of anti-inflammatory neutrophils; h. Application in the preparation of products that reduce cytokine levels, wherein the cytokines include one or more of IL-1β, CXCL2, CXCL5, and CCL7; i. Application in the preparation of products that reduce the levels of fibrosis-related molecules, wherein the fibrosis-related molecules include one or more of TGFB1, CTGF, COL1A1, and COL3A1; j. Application in the preparation of products that reduce Plod2 expression; k. Application in the preparation of products that improve serum and liver discoloration caused by cholestasis; l. Application in the preparation of products that improve weight and liver weight loss in patients with chronic cholestatic liver disease; m. Application in the preparation of products that reduce liver tissue necrosis and inflammatory infiltration in patients with chronic cholestatic liver disease. Applications in the product: n. Application in the preparation of products for reducing collagen deposition in patients with chronic cholestatic liver disease; o. Application in the preparation of products for reducing the levels of intrahepatic pro-inflammatory factors in patients with chronic cholestatic liver disease, wherein the intrahepatic pro-inflammatory factors include one or more of TGF-β, IL-6, and MIP-1α; p. Application in the preparation of products for treating liver injury, wherein the treatment of liver injury is achieved by synergistically reducing the progression of liver injury, inflammation, and fibrosis; q. Application in the preparation of products for early blocking of liver disease progression; r. Application in the preparation of products for treating the occurrence of fibrosis and improving the progression of fibrosis in patients with chronic cholestatic liver disease; and s. Application in the preparation of products for reducing bile acid metabolites, wherein the bile acid metabolites include taurine-uric acid (Tauro-Alpha-Muricholic acid). It contains one or more of the following: α-TMCA, tauro-Beta-Muricholic Acid (β-TMCA), taurohyocholic Acid (THCA), glycodeoxycholic Acid (GHDCA), taurohyodexycholic Acid (THDCA), ursodeoxycholic Acid (UDCA), chenodeoxycholic Acid (CDCA), α-Muricholica Acid (α-MCA), β-Muricholica Acid (Beta-Muricholica Acid (β-MCA), ursodeoxycholic Acid (M-DCA), and allocholic Acid (ALCA).

[0067] As some embodiments of this application, the improvement in liver function after chronic cholestatic liver disease includes a reduction in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL.

[0068] As some embodiments of this application, the reduction in histological structural damage to the liver includes one or more of the following: reduced bile duct hyperplasia, reduced fibrosis, and reduced collagen fiber deposition.

[0069] This application also provides a method for treating chronic cholestatic liver disease, the method comprising administering to a patient a therapeutically effective amount of the drug, the pharmaceutical composition, or the pharmaceutical combination product.

[0070] As some embodiments of this application, the chronic cholestatic liver disease includes one or more of the following: primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), primary biliary cholangitis, liver tumor, autoimmune hepatitis (AIH), primary biliary cirrhosis-autoimmune hepatitis overlap syndrome (PBC-AIH), primary sclerosing cholangitis-autoimmune hepatitis overlap syndrome (PSC-AIH), and Ig4-associated cholangitis (IAC).

[0071] As some embodiments of this application, the drug, the drug combination product, or the drug combination product may be administered via systemic or local application.

[0072] In some embodiments of this application, the drug, the drug combination product, or the drug combination product is applied to mammals. In some embodiments of this application, the drug, the drug combination product, or the drug combination product is applied to humans or mice.

[0073] As some embodiments of this application, the method alleviates liver damage in chronic cholestatic liver disease.

[0074] As some embodiments of this application, the method slows down weight loss in individuals with chronic cholestatic liver disease.

[0075] As some embodiments of this application, the method inhibits liver fibrosis. As some embodiments of this application, the inhibition of liver fibrosis includes at least one of a significant reduction in inflammation, necrosis, and fibrosis.

[0076] As some embodiments of this application, the method improves liver function in patients with chronic cholestatic liver disease. As some embodiments of this application, the improvement in liver function after chronic cholestatic liver disease includes a reduction in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL.

[0077] As some embodiments of this application, the method improves the pathological characteristics of chronic cholestatic liver disease. As some embodiments of this application, the improvement in the pathological characteristics of chronic cholestatic liver disease includes one or more of reduced cholestasis and reduced bile duct reactivity.

[0078] As some embodiments of this application, the method reduces the histological structural damage of the liver. As some embodiments of this application, the reduction in histological structural damage of the liver includes one or more of the following: reduced bile duct hyperplasia, reduced fibrosis, and reduced collagen fiber deposition.

[0079] In some embodiments of this application, the method reduces the level of immune cells. In some embodiments of this application, the immune cells include CD4+. + CD8 + .

[0080] As some embodiments of this application, the method increases the number of anti-inflammatory neutrophils.

[0081] As some embodiments of this application, the method reduces cytokine levels, the cytokines including one or more of IL-1β, CXCL2, CXCL5, and CCL7.

[0082] As some embodiments of this application, the method reduces the level of fibrosis-related molecules, including one or more of TGFB1, CTGF, COL1A1, and COL3A1.

[0083] As some embodiments of this application, the method reduces Plod2 expression.

[0084] As some embodiments of this application, the method improves the darkening of serum and liver color caused by cholestatic liver disease.

[0085] As some embodiments of this application, the method improves weight loss and liver weight reduction in patients with chronic cholestatic liver disease.

[0086] As some embodiments of this application, the method reduces liver tissue necrosis and inflammatory infiltration in patients with chronic cholestatic disease.

[0087] As some embodiments of this application, the method reduces collagen deposition in patients with chronic cholestatic disease.

[0088] As some embodiments of this application, the method reduces the level of intrahepatic pro-inflammatory factors in patients with chronic cholestatic disease, said intrahepatic pro-inflammatory factors including one or more of TGF-β, IL-6, and MIP-1α.

[0089] As some embodiments of this application, the method treats liver injury in patients with chronic cholestatic disease by synergistically reducing the progression of liver injury, inflammation, and fibrosis.

[0090] As some embodiments of this application, the method blocks the progression of liver disease at an early stage.

[0091] As some embodiments of this application, the method treats the occurrence of fibrosis and improves the progression of fibrosis in patients with chronic cholestatic disease.

[0092] As some embodiments of this application, the method reduces bile acid metabolites, which include one or more of the following: tauro-alpha-muricholic acid (α-TMCA), tauro-beta-muricholic acid (β-TMCA), taurohyocholic acid (THCA), glycodeoxycholic acid (GHDCA), taurohyodexycholic acid (THDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), α-muricholica acid (α-MCA), β-muricholica acid (Beta-muricholica acid (β-MCA), ursodeoxycholic acid (M-DCA), and allocholic acid (ALCA).

[0093] This application also provides the use of reagents for detecting CREPT expression in the preparation of a kit, the kit having any of the following functions: a. assisting in the identification of damaged hepatocytes caused by cholestasis; b. assisting in the identification of diseased liver tissue caused by cholestasis; and c. assisting in the diagnosis of cholestasis.

[0094] As some embodiments of this application, the reagents for detecting CREPT expression include siRNA and antibodies.

[0095] As some embodiments of this application, the nucleotide sequence of the sense strand of siRNA targeting the mouse CREPT gene is shown in any of SEQ ID No. 1 to 7, and the corresponding nucleotide sequence of the antisense strand is shown in SEQ ID No. 18 to 23. The siRNA with the sense strand shown in SEQ ID No. 7 does not have a corresponding antisense strand.

[0096] As some embodiments of this application, the sense strand nucleotide sequence of siRNA targeting the human CREPT gene is shown in any one of SEQ ID No. 8 to 17, and its corresponding antisense strand nucleotide sequence is shown in SEQ ID No. 24 to 33.

[0097] As some embodiments of this application, the antibody includes polyclonal antibodies and monoclonal antibodies that bind to the CREPT protein.

[0098] As some embodiments of this application, the monoclonal antibody binding the CREPT protein includes a monoclonal antibody secreted by hybridoma cells with accession number CGMCC No. 5477.

[0099] Hybridoma cell line CREPT-Ab-4H1, accession number CGMCC No. 5477, published in CN102559601A.

[0100] This application also provides animal models of cholestasis, which are prepared by DDC diet induction or bile duct ligation in animals with specific knockout of CREPT in hepatocytes or whole-body CREPT knockout. This application also provides animal models of hepatobiliary injury and fibrosis caused by cholestasis, which are prepared by DDC diet induction or bile duct ligation in animals with specific knockout of CREPT in hepatocytes or whole-body CREPT knockout.

[0101] As some embodiments of this application, the animal includes a mouse.

[0102] As some embodiments of this application, hepatocyte-specific knockout of CREPT animals include CREPT hep- / - Mice.

[0103] As some embodiments of this application, animals with systemic CREPT knockout include ERT2-Cre + / -CREPT Flox / Flox Mice.

[0104] As some embodiments of this application, DDC diet-induced modeling includes adding 0.1 w / w% DDC to the diet.

[0105] As some embodiments of this application, bile duct ligation modeling involves ligating the bile ducts of animals two weeks prior to the experiment.

[0106] This application also provides the application of the aforementioned animal model of cholestasis in screening drugs for treating cholestasis, wherein the effect of treating cholestasis is evaluated by administering drugs for treating cholestasis to the animal model of cholestasis or animal models of liver and gallbladder damage and fibrosis caused by cholestasis, thereby screening drugs for treating cholestasis.

[0107] This application also provides the application of the animal models of hepatobiliary injury and fibrosis caused by cholestasis in screening drugs for the treatment of cholestasis. In this case, the efficacy of the treatment of cholestasis is evaluated by administering the drugs for the treatment of cholestasis to the animal models of cholestasis or the animal models of hepatobiliary injury and fibrosis caused by cholestasis, thereby screening drugs for the treatment of cholestasis.

[0108] As described above, the application of reducing CREPT expression in the treatment of chronic cholestatic liver disease according to the present invention has the following beneficial effects:

[0109] This application is the first to discover the correlation between CREPT and chronic cholestatic liver disease. It also clarifies that inhibiting CREPT expression can treat cholestatic liver disease, improve liver function biochemical indicators, reduce liver histological structural damage, reduce fibrotic septa, reduce necrosis and inflammation, and significantly reduce bile duct hyperplasia, inflammation, necrosis and fibrosis. It has a significant effect on reducing antibody weight and has a significant protective effect on the overall health of patients awaiting treatment.

[0110] This application found that both systemic knockout CREPT mice and liver knockout CREPT mice can alleviate or treat cholestatic liver disease, suggesting that systemic or local administration can be effective in treating cholestatic liver disease.

[0111] This application found that inhibition of CREPT can reduce immune cell levels, including reducing CD4. + and CD8 + Levels of cytokines can increase the number of anti-inflammatory neutrophils, reduce cytokine levels, including IL-1β, CXCL2, CXCL5, and CCL7, reduce fibrosis-related molecular levels, including TGFB1, CTGF, COL1A1, and COL3A1, and reduce Plod2 expression. Attached Figure Description

[0112] Figure 1 shows that CREPT is highly expressed in patients with cholestatic liver disease and co-localizes with bile duct reaction markers;

[0113] Figure 2 shows that whole-body CREPT knockout significantly reduced DDC diet-induced cholestasis;

[0114] Figure 3 shows that whole-body CREPT knockout significantly reduced DDC diet-induced cholestasis;

[0115] Figure 4 shows that CREPT liver-specific knockout significantly reduces BDL-induced cholestasis;

[0116] Figure 5 shows that CREPT liver-specific knockout significantly reduces BDL-induced cholestasis;

[0117] Figure 6 shows that liver-specific overexpression of CREPT significantly aggravates cholestasis induced by BDL;

[0118] Figure 7 illustrates GalNAc delivery of siCREPT for the treatment of diet-induced cholestasis in patients with DDC;

[0119] Figure 8 shows a comparison between GalNAc delivery of siCREPT and the drug seladelpar in the treatment of diet-induced cholestasis in patients with DDC.

[0120] Figure 9 illustrates the effects of LNP-siCREPT treatment on CCl4-induced liver injury and fibrosis;

[0121] Figure 10 shows the immunohistochemical results of CREPT in human liver fibrosis at different stages.

[0122] Figure 11 shows a Western blotting image of the inhibitory effect of seven siRNAs screened from the mouse hepatocellular carcinoma cell line Hepa1-6 on CREPT.

[0123] Figure 12 shows Western blotting images of the inhibitory effects of 10 siRNAs screened from human hepatocellular carcinoma cell line MHCC-97H and human highly metastatic breast cancer cell line LM2 on CREPT.

[0124] Figure 13 shows the 10×Genomics single-cell sequencing results of mice with whole-body CREPT knockout after DDC modeling, and the partial experimental results of single-cell transcriptome sequencing of mice with liver-specific CREPT knockout modeled by bile duct ligation.

[0125] Figure 14 illustrates the interaction between representative cytokines and fibrosis-related molecules and CREPT in liver-specific knockout mice after bile duct ligation modeling.

[0126] Figure 15 shows the significant reduction in toxic bile acid metabolites after GalNAc-siCREPT treatment. Detailed Implementation

[0127] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0128] [Experimental Materials]

[0129] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0130] Materials and methods

[0131] laboratory animals

[0132] All experiments involving mice in this study were conducted in accordance with the guidelines of the Tsinghua University Animal Care and Use Committee and approved by the Tsinghua University Ethics Committee (code AICUC-24-CZJ3). The procedure followed Guideline 2.0 (https: / / arriveguidelines.org / arrive-guidelines).

[0133] Five-week-old male C57BL / 6J mice were purchased from Viton Lever Biopharmaceuticals Ltd. Upon arrival, the mice were housed and acclimatized for one week. For the 3,5-diethoxycarbonyl-1,4-dihydropyridine (DDC)-induced cholestasis mouse model, CREPT... Flox / Flox Mice and ERT2-Cre + / - CREPT Flox / Flox Mice were divided into two groups: a control group (n=4 per genotype) and a 0.1% DDC diet group (n=4, CREPT). Flox / Flox n=4, ERT2-Cre + / - CREPT Flox / Flox 0.1% DDC (Beijing Translation Technology Co., Ltd.) diet for 4 weeks.

[0134] Bile duct ligation model (BDL): Mice were fasted for 8 hours preoperatively but allowed free access to water. They were weighed on an iron tray, anesthetized, and placed supine on a foam board for modeling. The board was disinfected with povidone-iodine. A longitudinal incision was made at the xiphoid process to expose the liver tissue. The liver lobe, stomach, and part of the intestine were dissected to the right, revealing the translucent bile duct originating from the porta hepatis. Further down, the bile duct was seen attached to a segment of the small intestine. A small segment of the bile duct was separated with ophthalmic scissors and ligated with 6-0 sutures. In the control group, only the common bile duct was freed. The incision was sutured layer by layer and disinfected with povidone-iodine. Postoperatively, mice had normal food and water intake, and received intraperitoneal injections of antibiotics (0.3 ml / mouse) daily for one week.

[0135] Hepatocyte-specific CREPT knockout mice (CREPT) hep- / - Hepatocyte-specific CREPT overexpression mice (TG-CREPT) are generated via the Cre / loxP system. Specifically, mice with Cre recombinase driven by the albumin promoter specifically express (Alb-Cre) in hepatocytes. + / -), with CREPT Flox / Flox Mouse hybridization was performed, where the third exon of the Crept gene is flanked by loxP sites. Alb-Cre was used. + / - ;CREPT Flox / Flox Genotype-specific hepatocyte knockout mice (denoted as CREPT) hep- / - For experimental use, using CREPT Flox / Flox Mice were used as a control.

[0136] TG-CREPT mice were generated using a similar method.

[0137] ERT2-Cre + / - CREPT Flox / Flox Mice, CREPT hep- / - TG-CREPT mice and their corresponding control mice underwent DDC-induced cholestasis and BDL-induced obstructive cholestasis.

[0138] Liver histological analysis

[0139] Liver histological evaluation was performed using hematoxylin and eosin (H&E) staining by different pathologists in a double-blind manner. Liver histological scoring was conducted by three experienced pathologists from Tsinghua University in Beijing, China. Scoring categories for inflammation, necrosis, and ductal reactions were established according to standard methods in the field.

[0140] Specifically, the liver inflammation score is 0, indicating no inflammation; 1, mild inflammation, with isolated inflammatory cells and no clear inflammatory focus; 2, moderate inflammation, characterized by the formation of inflammatory cells; and 3, severe inflammation, with a large number of inflammatory cells forming numerous or multilayered structures.

[0141] The liver necrosis assessment is 0, no necrosis; 1, isolated cell necrosis; 2, necrosis area <5%; 3, necrosis area in the range of 5-10%; 4, necrosis area >10%.

[0142] The bile duct reaction ranges from 0 (no bile duct dilation) to 5 (the highest degree of bile duct dilation, indicating liver damage).

[0143] Sirius red and Masson staining were used to assess the degree of collagen and liver fibrosis.

[0144] The slides were deparaffinized with xylene and rehydrated. Then, the slides were stained with 2% phosphomolybdic acid (Sigma-Aldrich) for 2 min and washed with dH₂O. Collagen was stained with 0.1% Sirius red (Gibco) for 3 h. Finally, the slides were incubated with 0.01N hydrogen chloride (hydrochloric acid) (Merck) for 2 min, dehydrated, mounted, and then scanned.

[0145] Immunohistochemistry (IHC) and image analysis

[0146] IHC analysis was performed on paraffin-embedded liver tissue sections.

[0147] Specifically, slides were dewaxed and heat-treated with citrate buffer (pH 6) (Zhongshan Jinqiao Biotechnology Co., Ltd.) for 30 min to extract antigen. Slides were then blocked with 0.3% H2O2 (Zhongshan Jinqiao Biotechnology Co., Ltd.) for 20 min at room temperature, followed by blocking with appropriate blocking serum (Zhongshan Jinqiao Biotechnology Co., Ltd.) for 30 min. Primary antibody was incubated overnight at 4°C (prepared in-house). Slides were washed with 1×PBST and incubated with the corresponding biotinylated secondary antibody. Signal amplification was achieved using ABC grade 3 reagents (Dako, Denmark). IHC was developed using 3,3'-diaminobenzidine (DAB) (Dako, Denmark). Representative images were analyzed under an Eclipse 80i (Nikon) microscope.

[0148] Immunofluorescence (IF) and multiple IF analysis

[0149] Paraffin-embedded liver tissue was rehydrated, and antigens were extracted using citrate buffer (using the same reagents as for immunohistochemistry). Slides were incubated with goat serum (Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 30 min, followed by overnight incubation with primary antibody at 4°C. As previously described, multiplex IF was performed on human or mouse liver sections using a four-color multiplex fluorescent immunohistochemical staining kit (Cat#abs50012 / abs50028; Absin, Shanghai, China) according to the manufacturer's instructions. CK19, CK7, and SOX9 were used as specific biomarkers for bile duct reactions. Slides were then incubated with fluorescently conjugated secondary antibody (Agilent Technologies, USA) and observed using a laser confocal microscope (Olympus, FV3000, Japan).

[0150] RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR) analysis

[0151] Total RNA was extracted from mouse liver tissue and cultured cells using TRIzol reagent (Sigma-Aldrich, USA). cDNA synthesis was performed using the PrimeScript reverse transcription kit (Tiangen, CA, China). Quantitative PCR was performed using the SYBR Premex ExTaq™ kit (Tiangen, CA, China) according to the manufacturer's instructions. Relative gene expression levels were determined using the 2^-ΔΔCT method and normalized to GAPDH expression levels.

[0152] Serological markers in mice were determined using a fully automated blood biochemistry analyzer.

[0153] Serological parameters of mice, including ALT, AST, TBiL, and DBiL, were analyzed using a fully automated blood biochemistry analyzer from the Laboratory for Laboratory Medicine at China Agricultural University, following the manufacturer's instructions.

[0154] Chemokines were measured using a biological reagent kit.

[0155] Chemokines and cytokines in mouse livers were analyzed using a custom-designed Bio-Plex assay kit (Bio-Rad) and the FACS-Canto II system (BD), following the manufacturer’s instructions.

[0156] Immunoblotting analysis

[0157] Tissue was lysed using RIPA lysis buffer (Beyotime). The lysis buffer was centrifuged at 12,000 rpm for 15 minutes at 4°C. Protein samples (20 μg per lane) were separated using 10% or 12% SDS-PAGE and transferred to PVDF membranes (Biorad). The membranes were blocked in 5% skim milk at room temperature for 1 hour. After blocking, the cell membranes were cut according to protein size markers. The membranes were then incubated overnight (12 hours) with primary antibody at 4°C, followed by incubation with HRP-conjugated secondary antibody at 37°C for 1 hour. The signals were visualized using an enhanced chemiluminescence (ECL) system, and images were captured using a gel recording system. Protein bands were analyzed using ImageJ software. The primary antibody used was (laboratory prepared, 1:500 dilution), PCNA (Aksomics, 307901, 1:500 dilution), GAPDH (Aksomics, KC-5G5, 1:3000 dilution), and HRP-conjugated secondary antibody (Aksomics, KC-RB-035, 1:5000 dilution).

[0158] RNAseq was performed by Meiji Biotechnology.

[0159] Single-cell sequencing of 10*genomics was performed by Ouyi Bio.

[0160] Statistical analysis

[0161] All data were statistically analyzed using GraphPad Prism 7.00 (GraphPad software). Comparisons between two groups were performed using independent samples t-tests or Mann-Whitney U tests, while comparisons among multiple groups were performed using one-way ANOVA or Kruskal-Wallis tests, depending on normality. Correlation analysis was performed using Pearson correlation tests (parametric) or Spearman correlation tests (non-parametric). Data are expressed as mean ± standard deviation (SD). All experiments were repeated at least three times. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 were considered statistically significant.

[0162] Example 1

[0163] The inventors discovered that CREPT is highly expressed in the livers of patients with typical cholestatic liver disease (PBC) and autoimmune hepatitis (AIH). To verify the correlation between CREPT and human cholestatic liver disease, they analyzed human clinical data.

[0164] This study involving human subjects was conducted in accordance with the Declaration of Helsinki (2013) and the Declaration of Istanbul (2018) of the World Medical Association. Clinical data and pathological information of human liver tissue were obtained from the Second Affiliated Hospital of Jiaxing, Zhejiang Province, China. Liver tissues were obtained by paraffin embedding and included patients with PBC (6), AIH (6), FNH (5 non-proliferating sections), and HCH (4). The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Tsinghua University. Immunohistochemistry and multiplex immunofluorescence methods are described in the experimental methods above. Figure 1A illustrates the HC staining results of liver tissue sections from patients with PBC, AIH, FNH, and HCH. The number of CREPT-positive cells in the liver tissues of various patients was detected. It can be seen that the proportion of CREPT-positive expression was significantly increased in patients with PBC and AIH, while the proportion of CREPT-positive expression was not significant in patients with FNH and HCH.

[0165] Multiplex immunofluorescence studies of CK7 and CREPT, one of the biomarkers of cholestatic liver disease, revealed co-expression of CREPT and CK7 in this region of the bile duct in cholestatic liver disease (see Figure 1C).

[0166] Example 2:

[0167] Tracing back to published public datasets, CREPT is primarily expressed in the liver parenchyma in normal livers. However, reports on cholestatic hepatocyte data show that CREPT is relatively highly expressed in bile duct cells and related hepatocyte parenchymal cells. Based on this, this study investigates the substantial role of CREPT in cholestatic liver disease.

[0168] CREPT Flox / Flox ERT2-Cre in mice and with systemic CREPT knockout + / - CREPT Flox / Flox The timeline for DDC modeling in mice is shown in Figure 2A. After week 12, various liver function indicators, including CREPT, were measured in the treated mice. Flox / Flox In mice, ALT, AST, ALP, TBIL, and DBiL were all significantly higher than those of ERT2-Cre. + / - CREPT Flox / Flox (See Figure 2B). Significant differences were also observed in the liver tissue staining images of DDC mice (see Figure 2C). Upon examination, the CREPT levels in the DDC model were... Flox / Flox Mice compared to ERT2-Cre + / - CREPT Flox / Flox Mice showed significant increases in bile duct hyperplasia, inflammation, necrosis, and fibrosis, which were also significantly increased compared to the group without DDC induction (see Figure 2D-2G).

[0169] CREPT for detecting DDC modeling Flox / Flox Mice and ERT2-Cre + / - CREPT Flox / Flox In mice, DDC-induced CREPT showed positive effects in α-SMA and Collegen III. Flox / Flox Mice also showed significantly higher levels of ERT2-Cre + / - CREPT Flox / Flox The mice showed significantly higher levels of [something] than the blank control group (see Figure 2H).

[0170] CREPT for detecting DDC modeling Flox / Flox Mice and ERT2-Cre + / - CREPT Flox / Flox CREPT expression in mouse liver, CREPT in DDC model Flox / Flox Mice also showed significantly higher levels of ERT2-Cre + / - CREPT Flox / Flox The mice showed significantly higher levels of [something] than the blank control group (see Figure 2I).

[0171] CREPT for detecting DDC modeling Flox / Flox Mice and ERT2-Cre + / - CREPT Flox / Flox Marking mouse bile duct epithelial cells with CK19, CK7, and SOX9 revealed CREPT. Flox / Flox Mice compared to ERT2-Cre + / - CREPT Flox / Flox Significantly increased, also significantly increased compared to the blank control (see Figure 3A). The levels of CK19, CK7, and SOX9 mRNA were measured using CREPT. Flox / Flox Mice compared to ERT2-Cre + / - CREPT Flox / Flox Significantly increased levels, also significantly higher than the blank control (see Figures 3B-3D). Multiplex immunofluorescence assays showed co-expression of CREPT and CK7 in the bile ducts (see Figure 3E). Immunohistochemistry showed significantly increased expression of PCNA (Proliferating Cell Nuclear Antigen) and CREPT in the bile ducts (see Figures 3F-3H).

[0172] Example 3:

[0173] CREPT Flox / Flox CREPT mice and hepatocyte-specific knockout mice hep- / - The procedure for constructing the mouse model and the time-consuming bile duct ligation model is shown in Figure 4A. CREPT of the BDL model was then measured. Flox / Flox Mice and CREPThep- / - CREPT expression in mice, showing CREPT Flox / Flox CREPT expression in mice was significantly higher than that of CREPT. hep- / - Mice (see Figures 4B-4C).

[0174] CREPT stained with H&E, Sirius Red, and Masson's red. Flox / Flox Mice and CREPT hep- / - The liver tissue sections from mice also differed, and the staining morphology of the livers also varied (see Figure 4D).

[0175] Testing revealed that the CREPT in BDL modeling... Flox / Flox Mice compared to CREPT hep- / - Significantly increased in bile duct hyperplasia, inflammation, necrosis, and fibrosis, and also significantly increased compared to the control group without bile duct ligation (see Figures 4E-4H).

[0176] Mice were treated after week 2, and various liver function indicators, including CREPT, were measured. Flox / Flox In mice, ALT and AST levels were significantly higher than those of hepatocyte-specific CREPT. hep- / - Mice (see Figures 4I-4J).

[0177] Immunohistochemistry showed a significant increase in the expression of PCNA (proliferating cell nuclear antigen) and CREPT in the bile ducts (see Figures 5A-5C).

[0178] Multiplex immunofluorescence assays showed co-expression of CREPT and CK7 in the bile ducts (see Figure 5D).

[0179] CREPT for detecting BDL modeling Flox / Flox Marking CK19, CK7, and SOX9 in bile duct epithelial cells of mice and CREPThep- / - mice revealed CREPT. Flox / Flox Mouse CK19 and CK7, as well as SOX9 positive cells compared to CREPT hep- / - The levels were significantly increased in mice, and also significantly increased compared to the blank control (see Figures 5E-5H).

[0180] Therefore, after liver-specific elimination of CREPT, the BDL model also significantly downregulated inflammation, fibrosis, and biliary responses caused by cholestasis. Furthermore, this biliary response focused on the regulation of cholestatic diseases, specifically the core responses of the biliary response and the regulation of fibrosis-related genes.

[0181] Example 4

[0182] To further validate the findings, the applicant constructed TG-CREPT mice, which specifically overexpress CREPT in the liver. The TG mice were tested using the same logic. It was found that CREPT overexpression, under modeling conditions, exacerbated inflammatory fibrosis and biliary reactions caused by cholestatic disease.

[0183] The construction and timing of the TG-CREPT mouse model, which specifically overexpresses CREPT in hepatocytes, are shown in Figure 6A. Two weeks later, the TG-CREPT mice were treated, and their liver tissue immunohistochemical staining was compared with that of the control Alb-Cre. + / - Comparison between mice (with the same CREPT gene copy number as wild-type and normal expression) showed enhanced CREPT expression (Fig. 6B), while Western blotting showed increased CREPT expression (Fig. 6C).

[0184] Various liver function indicators were tested. The ALT and AST levels in TG-CREPT mice were significantly higher than those in the control Alb-Cre... + / - The levels were significantly increased in all mice (see Figure 6D).

[0185] TG-CREPT mice stained with H&E, Sirius Red, and Masson staining, and control Alb-Cre + / - The liver tissue sections from mice also differed, and the staining morphology of the livers also varied (see Figure 6E).

[0186] Tests showed that the TG-CREPT mice induced by BDL model had better performance than the control Alb-Cre... + / - Mice showed significant increases in bile duct hyperplasia, inflammation, necrosis, and fibrosis, which were also significantly increased compared to the control group without bile duct ligation (see Figures 6F-6I).

[0187] TG-CREPT mice and control Alb-Cre mice were used to test BDL modeling. + / - In mice, the TG-CREPT mice modeled by BDL also showed significantly higher scores than Alb-Cre in α-SMA and Collegen III. + / - Mice (see Figures 6J-6K).

[0188] Example 5

[0189] GalNAc-siCREPT for the treatment of cholestasis mouse models

[0190] Given that CREPT is highly expressed in hepatocytes during cholestasis, the applicant further investigated whether targeting CREPT expression could treat cholestasis. To this end, in a mouse cholestasis model, the applicant used an n-acetylgalactosamine (GalNAc) delivery system, recently approved by the FDA for a rare liver disease, to specifically deliver siRNA targeting CREPT into hepatocytes. The applicant first screened for siRNAs with the strongest ability and specificity to inhibit CREPT expression; the sense and antisense strands of the siRNA sequences are shown in SEQ ID NO. 1 and 18, respectively. GalNAc was transfected (named GalNAc-siCREPT).

[0191] The applicant induced a cholestasis model in mice using a DDC diet and treated them with GalNAc-siCREPT. Starting with DDC diet challenge, GalNAc-siCREPT containing siRNA (10 mg / kg) was administered subcutaneously for 4 consecutive weeks. An equal amount of nonspecific siRNA (GalNAc-siNC) served as a control. The sequence of the nonspecific siRNA is shown in SEQ ID NO. 34.

[0192] Table 1 siCREPT and siNC sequences

[0193] To compare the effects of siRNA and delivery vectors, the applicant administered PBS to mice challenged with a DDC diet, and also used healthy mice as controls. To verify the distribution of GalNAc-siCREPT, the applicant performed fluorescence analysis using Cy5 labeling. The results showed that all mice treated with GalNAc-siCREPT or GalNAc-siNC exhibited strong fluorescence signals in the liver region 24 hours after sc injection (Figure 7D), and this signal remained intact for 28 days. Anatomical analysis of the organs at the end of the experiment indicated that GalNAc-siCREPT or GalNAc-siNC was primarily distributed in the liver, rather than in other organs (Figure 7E). These results demonstrate that GalNAc-siCREPT or GalNAc-siNC is specifically distributed and maintained in the liver. Notably, through quantitative analysis, the applicant observed no difference in radiation efficiency between GalNAc-siNC and GalNAc-siCREPT in the liver (p = 0.247), indicating that the amount of GalNAc was at a similar level between mice treated with GalNAc-siCREPT and GalNAc-siNC (Figure 7F). To detect changes in CREPT expression, the applicant performed Western blots on liver samples from different mice. The results showed that GalNAc-siCREPT significantly reduced CREPT expression in cholestatic mice. This result indicates that GalNAc-siCREPT can effectively inhibit CREPT expression in the liver of cholestatic mice (Figure 7G).

[0194] To investigate the efficacy of the treatment, the applicant examined changes in body weight during treatment. Results showed that mice treated with PBS and GalNAc-siNC experienced a significant decrease in body weight after a DDC diet compared to healthy (untreated) mice. Interestingly, mice treated with GalNAc-siCREPT showed an increase in body weight compared to mice treated with PBS or GalNAc-siNC (Figure 7B). The applicant observed that at the end of the experiment, the body weight of mice treated with GalNAc-siCREPT remained significantly higher than that of the control group (Figure 7C). These results indicate that GalNAc-siCREPT treatment has a significant effect on reducing body weight, which is commonly observed in cholestasis models. Since changes in body weight reflect changes in liver injury, the applicant attributed the recovery of body weight after GalNAc-siCREPT treatment to a reduction in liver damage. In stark contrast, the control group (PBS and GalNAc-siNC) exhibited significant weight loss, highlighting the systemic burden of liver dysfunction in untreated animals. The maintenance of body weight in the GalNAc-siCREPT group reflects the profound protective effect of this therapy on overall health during chronic liver injury. At the end of the experiment, the body weight of the GalNAc-siCREPT-treated cholestatic fibrosis mice was comparable to that of healthy mice.

[0195] Liver function was analyzed, and biochemical indicators were measured. Results showed that, compared to healthy mice, ALT and AST levels were significantly elevated after DDC diet (Figure 7H), indicating a successful establishment of the cholestasis model. Notably, GalNAc-siCREPT treatment significantly reduced both ALT and AST levels in mice. Simultaneously, the applicant observed decreases in other indicators, including TBiL, DBiL, LDH, and ALP (Figures 7J-7M).

[0196] In summary, all the results indicate that GalNAc-siCREPT treatment improved liver function following the DDC diet, suggesting an effective therapeutic effect against CREPT expression during cholestasis.

[0197] Mice were sacrificed after the experiment to observe the effect of GalNAc-siCREPT treatment on pathological changes in the liver. Macroscopic observation showed that the livers of healthy mice had a smooth and regular surface, while the livers of cholestatic mice had a rough and dark surface due to DDC-induced cholestasis. Interestingly, compared with the control group, the livers treated with GalNAc-siCREPT had a relatively smooth appearance and a more normal color (Figure 7I).

[0198] The applicant then underwent histopathological analysis. Hematoxylin-eosin (HE) staining showed that the healthy liver maintained normal histological structure, but the DDC diet induced extensive aggregation of bile duct reactive cells, neutrophil infiltration, and irregular fibrosis in the liver (Figure 7I).

[0199] Furthermore, the applicant observed extensive hepatocyte necrosis in DDC-induced mice. However, compared to livers from other mice, cholestatic mice treated with GalNAc-siCREPT showed reduced histological damage, less necrosis and inflammation, and less fibrosis in their livers. Liver sections were stained with Sirius red and Masson's stain to assess the degree of collagen deposition and fibrosis. The results showed that healthy livers maintained normal lobular structure and minimal collagen fibers. Extensive collagen deposition and fibrosis were observed in the DDC group, while the GalNAc-siCREPT group showed significantly reduced fibrosis and collagen deposition with mild collagen staining. Quantitative analysis indicated that cholestatic mice treated with GalNAc-siNC and PBS showed significantly reduced bile duct hyperplasia, inflammation, necrosis, and fibrosis. These results are consistent with observations from HE staining, suggesting that GalNAc-siCREPT treatment can prevent fibrosis during cholestasis.

[0200] Example 6

[0201] A comparison of the treatment effects of GalNAc-siCREPT and seladelpar / seladelpar on a mouse model of cholestasis.

[0202] To further investigate the effects of targeting CREPT expression in the treatment of cholestasis compared to existing clinical drugs, the applicant continued with the same experimental design as in Example 5, adding the positive control drug seladelpar / seladelpar. The results showed that, compared to healthy mice (untreated), macroscopic observation of liver tissue and serum color revealed that mice treated with GalNAc-siCREPT had more mice with normal serum color and more mice with normal liver color. Mice treated with PBS and GalNAc-siNC showed a significant decrease in body weight after a DDC diet. Interestingly, there was no significant difference in body weight between the GalNAc-siCREPT-treated group and the seladelpar / seladelpar-treated group (Figure 8A); however, there was no difference in liver weight between the GalNAc-siCREPT group and the seladelpar / seladelpar-treated group (Figures 8B-C); the liver-to-body weight ratio showed no difference between the groups (Figure 8D). Compared to the experimental control group, GalNAc-siCREPT treatment improved the decrease in body weight and liver weight in mice with chronic cholestasis. Further analysis using biochemical indicators TBiL, ALP, and CHOL revealed that the GalNAc-siCREPT-treated group showed better results (Figures 8E-G). Next, observation of HE-stained sections from each group showed no difference between the GalNAc-siCREPT-treated group and the positive drug group; in fact, the GalNAc-siCREPT-treated group showed better results than the positive drug group in both gross liver color and the amount of bile in HE-stained liver tissue (Figure 8H).

[0203] Example 7

[0204] Treatment of LNP-siCREPT in a mouse model of fibrosis

[0205] To evaluate the therapeutic effect of CREPT gene silencing on CCl4-induced liver fibrosis, we administered LNP-siCREPT intravenously to a CCl4-induced mouse fibrosis model (Figure 9A). Body weight change analysis showed a significant decrease in body weight in the CCl4-induced group compared to the healthy control group. Although the LNP-siCREPT treatment group showed a trend towards weight recovery, the difference was not statistically significant (Figures 9B-C), suggesting that this treatment can partially improve CCl4-induced weight loss. Fluorescence imaging confirmed that LNP-siCREPT could be efficiently delivered to the liver, with significantly higher signal intensity in the liver region compared to non-target organs such as the spleen, kidneys, lungs, and lymph nodes (Figures 9D-F). There was no statistically significant difference in liver fluorescence intensity between the LNP-siCREPT group and the control siRNA (LNP-siNC) group (Figures 9E-F), indicating that both lipid nanoparticles can achieve efficient targeted delivery to the liver, validating the liver tissue specificity of this delivery system.

[0206] Western blot analysis showed that LNP-siCREPT significantly reduced hepatic CREPT protein expression compared to the LNP-siNC control group (Figure 9G). Biochemical assays showed that serum ALT and AST levels (key markers of liver injury) in the LNP-siCREPT treatment group were significantly lower than those in the PBS and LNP-siNC groups (Figures 9H-I), suggesting that CREPT silencing can alleviate hepatocellular damage in the process of CCl4-induced liver fibrosis.

[0207] HE staining of liver tissue showed that the LNP-siCREPT treatment group had significantly less liver necrosis and inflammatory infiltration than the control group (Figure 9J). Sirius red and Masson trichrome staining further confirmed that collagen deposition was reduced in the LNP-siCREPT group, suggesting that the process of liver fibrosis was inhibited (Figure 9J). In addition, the detection of intrahepatic pro-inflammatory factors showed that the levels of TGF-β (Figure 9K), IL-6 (Figure 9L), and MIP-1α (Figure 9M) in the LNP-siCREPT group were significantly lower than those in the control group, indicating its anti-inflammatory effect.

[0208] In summary, LNP-siCREPT demonstrates therapeutic efficacy against liver injury by synergistically mitigating liver damage, inflammation, and fibrosis. These findings establish CREPT as an important therapeutic target for combating liver fibrosis and related lesions, suggesting that early targeted intervention can effectively halt the progression of liver disease.

[0209] Example 8

[0210] Expression of CREPT in human liver fibrosis and the therapeutic effect of GalNAc-siCREPT.

[0211] To evaluate the therapeutic effect of the CREPT gene on human liver fibrosis, the expression levels of CREPT in normal liver and fibrotic liver were first analyzed using a public database. The results showed that CREPT was significantly highly expressed in liver fibrosis specimens (Figure 10A). Immunohistochemistry was then used to stain CREPT in healthy human liver and liver fibrosis specimens at different stages. The results indicated that CREPT was significantly highly expressed in liver fibrosis specimens, and its expression became more pronounced with the progression of fibrosis, showing significant differences between different stages of fibrosis (Figures 10B-C).

[0212] In summary, targeting CREPT can effectively reduce cholestasis, alleviate bile duct reactions, decrease collagen deposition, and alleviate fibrosis, and is safe and effective. Strategies that reduce CREPT expression can be expected to be used to treat human cholestasis and induced or associated fibrosis.

[0213] To further screen siRNAs that target CREPT to inhibit CREPT expression and treat cholestasis, seven siRNAs were obtained by screening on the mouse hepatocellular carcinoma cell line Hepa1-6. Their inhibitory effect on CREPT is shown in the western bolting image in Figure 11. It can be seen that siRNAs with the positive strand sequences shown in SEQ ID No. 1 to 7 can effectively inhibit CREPT expression.

[0214] Similarly, screening was performed on the human hepatocellular carcinoma cell line MHCC-97H and the human highly metastatic breast cancer cell line LM2. The inhibitory effect on CREPT is shown in the western bolting results in Figure 12. All 10 siRNAs were found to effectively inhibit CREPT expression.

[0215] The above sequences are shown in Tables 2 and 3.

[0216] Table 2

[0217] Table 3

[0218] Example 9

[0219] To explore the mechanism by which reduced CREPT expression alleviates cholestatic liver disease in humans, this example first analyzed ERT2-Cre2 with systemic CREPT knockout. + / - CREPT Flox / Flox Changes in different cell populations in the liver of mice.

[0220] Figure 13A shows ERT2-Cre2 with systemic CREPT knockout. + / - CREPT Flox / Flox Liver samples from mice after DDC modeling were sequenced using a 10×Genomics single-cell sequencing method, including steps from enrichment to final separation.

[0221] Figure 13B shows the single-cell sequencing results of Figure 13A. The left side of Figure 13B shows the NC group after DDC modeling, and the right side shows the ERT2-Cre group with systemic CREPT knockout. + / - CREPT Flox / Flox In the mouse DDC model group, UMAP dimensionality reduction analysis was performed on the differences between different cell communities in the two groups based on the different cell communities in the markers. The significant differences in the numerical proportions of different cell types between the two groups are clearly visible in Figure 13C, for example, CD4 and CD8, in the KO group (systemic knockout of ERT2-Cre). + / - CREPT Flox / FloxThe proportion of mice with DDC (dual-induced dysplasia of the liver) was relatively low, while the proportion of mice with NC (non-cREPT knockout) was relatively high. These data are very consistent with the understanding of cholestatic liver disease: because it is an autoimmune liver disease, CD4 and CD8 immune cells are normally very active. The applicant has now found that knocking out CREPT significantly reduces these immune cells, which also proves from another perspective that targeting CREPT can regulate this disease.

[0222] Figure 13D shows a group of neutrophils marked in red in Figure 13B. Although the overall proportion of neutrophils did not change significantly between the NC and KO groups as shown in Figure 13C, the fluorescence image in Figure 13D reveals that the left side of the neutrophils in the NC group is brighter, indicating that this group of neutrophils on the left side is more abundant, while the right side of the KO group has more neutrophils. This novel finding shows that there are more pro-inflammatory neutrophils in the NC group (the left side of the neutrophils is brighter, indicating greater enrichment), while in the livers of CREPT knockout mice (KO group), these pro-inflammatory neutrophils are reduced, but the right side of the anti-inflammatory neutrophils is increased (the right side of the neutrophils is brighter, indicating greater enrichment).

[0223] Figures 13E-F show the CREPT mice with liver-specific knockout. hep- / - Single-cell transcriptome sequencing of mice with bile duct ligation model revealed upregulated and downregulated genes (see Figure 13E). After enrichment, these genes were found to mainly aggregate in cytokines. In Figure 13F, the most abundant aggregation was in the cytokine pathway, followed by the interaction between cytokines and cytokine receptors. It is speculated that the regulation of cholestasis by CREPT may be caused by this interaction between cytokines.

[0224] To verify that CREPT regulates cholestasis through cytokine pathways and the interaction between cytokine receptors, further research will be conducted on the relationship between cytokines and CREPT in regulating cholestasis.

[0225] As shown in Figure 14A, liver-specific knockout CREPT mice... hep- / - After bile duct ligation was performed in mice to establish a cholecystitis model, these representative cytokines were significantly downregulated. Figure 11B shows the effect of liver-specific knockout of CREPT mice. hep- / - In mice, fibrosis-related molecules significantly decreased after bile duct ligation modeling. This indicates that whether CREPT is knocked out systemically or specifically in the liver, the reduction in cytokines and inflammatory factors initially leads to a reduction in fibrosis symptoms.

[0226] Furthermore, Figures 14C-F show that after exogenous overexpression of CREPT, the expression of Plod2 with the added Flag tag also increased accordingly. Plod2 is a crucial enzyme in the fibrosis process, which can cause collagen molecules to covalently bind, leading to fiber aging and hardening. In other words, inhibiting CREPT can reduce Plod2 expression and weaken fibrosis. At the same time, CREPT can interact with Plod2, and reducing CREPT can also inhibit their interaction, preventing the occurrence and progression of fibrosis.

[0227] Since the downstream of fibrosis synthesized by Plod2 acts on the key proteins Smad2 / 3 in the TGF-β signaling pathway, which plays an important role in liver diseases from injury and inflammation to fibrosis [Dooley, S., &ten Dijke, P. (2012). TGF-β in progression of liver disease. Cell and Tissue Research, 347(1), 245–256.]. The applicant therefore verified the interaction between Smad2, Smad3, Smad4 and CREPT. Through in vitro interaction experiments, it was found that after overexpression of CREPT, Smad2 and Smad3 could interact with CREPT. When Co-Smad and Smad4 were increased, the interaction between CREPT and Smad2 and Smad3 was weakened, indicating that exogenous overexpression of CREPT increases the fibrotic process by competing with Smad4 to interact with Smad2 / 3.

[0228] Since bile acids (BAs) are a major cause of liver injury, and CREPT exacerbates liver damage, bile acid metabolomics were performed on the above samples. GalNAc-siCREPT treatment significantly reduced toxic bile acid metabolites (Figure 15A-L), including tauro-alpha-muricholic acid (α-TMCA), tauro-beta-muricholic acid (β-TMCA), taurohyocholic acid (THCA), glycodeoxycholic acid (GHDCA), taurohyodexycholic acid (THDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), alpha-muricholic acid (α-MCA), and beta-muricholic acid (β-TMCA). GalNAc-siCREPT treatment, which includes β-MCA, ursodeoxycholic acid (M-DCA), and allocholic acid (ALCA), effectively improves DDC diet-induced liver function damage.

[0229] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of the CREPT gene or protein as a target or an agent targeting the CREPT gene or protein in the treatment of chronic cholestatic liver disease or in the preparation of drugs for the treatment of chronic cholestatic liver disease, characterized in that, Chronic cholestatic liver disease can be treated by reducing the expression of the CREPT gene or by reducing or inactivating the function of the CREPT protein.

2. The application according to claim 1, characterized in that, The application has one or more of the following functions: Slowing down weight loss in individuals with chronic cholestatic liver disease; Improve liver function in patients with chronic cholestatic liver disease, wherein the improvement in liver function after chronic cholestatic liver disease includes a decrease in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL. Improvement in the pathological characteristics of chronic cholestatic liver disease, wherein the improvement in the pathological characteristics of chronic cholestatic liver disease includes one or more of the following: reduction in cholestasis and reduction in bile duct reactivity; The histological damage to the liver is reduced, including a reduction in bile duct hyperplasia; Reduce the level of immune cells, including CD4. + and CD8 + ; Increase the number of anti-inflammatory neutrophils; Reduce cytokine levels, wherein the cytokines include one or more of IL-1β, CXCL2, CXCL5, and CCL7; Reduce the level of fibrosis-related molecules, including one or more of TGFB1, CTGF, COL1A1, and COL3A1; Reduce Plod2 expression; It improves the darkening of serum and liver color caused by cholestasis; Improves weight loss and liver weight reduction in patients with chronic cholestatic liver disease; Patients with chronic cholestatic liver disease show reduced liver tissue necrosis and inflammatory infiltration; Patients with chronic cholestatic liver disease have reduced collagen deposition; To reduce the level of intrahepatic pro-inflammatory factors in patients with chronic cholestatic liver disease, wherein the intrahepatic pro-inflammatory factors include one or more of TGF-β, IL-6, and MIP-1α; Treatment of liver damage involves synergistically reducing the progression of liver damage, inflammation, and fibrosis. Application in products that prevent the early progression of liver disease; Treatment of fibrosis in patients with chronic cholestatic disease and improvement of fibrosis progression; And to reduce bile acid metabolites, wherein the bile acid metabolites include one or more of taurine-uric acid, taurine-β-mouse cholic acid, taurocholeric acid, glucodeoxycholic acid, taurocholeric acid, ursodeoxycholic acid, chenodeoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, ursodeoxycholic acid, and allochytic acid.

3. The application according to claim 1, characterized in that, The reagents targeting the CREPT gene or protein reduce CREPT gene expression or impair or inactivate CREPT protein function; The reagent targeting the CREPT gene or protein is selected from one or more of the following: agents that affect CREPT protein expression, agents that knock out CREPT protein, agents that alter CREPT protein, and agents that degrade CREPT protein. Preferably, the formulation affecting CREPT protein expression includes a nucleic acid reagent or a carrier containing a nucleic acid fragment that inhibits CREPT protein expression; Preferably, the formulation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene; Preferably, the formulation for altering the CREPT protein includes reagents that alter the structure of the CREPT protein; Preferably, the formulation for degrading CREPT protein includes one or more of the following: PROTAC, molecular glue, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, and AUTOTAC pathways. The drug includes a small molecule inhibitor of the CREPT protein.

4. The application according to claim 3, characterized in that, The nucleic acid reagent is selected from one or more of siRNA, shRNA, microRNA, piRNA, and ASO; The vector containing the nucleic acid fragment that inhibits CREPT protein expression includes one or more of adenovirus, adeno-associated virus, lentivirus, and retrovirus; The reagents used for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system; Preferably, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene; Preferably, the positive strand nucleotide sequence of the siRNA targeting the mouse CREPT gene is as shown in any one of SEQ ID No. 1 to 7; Preferably, the positive strand nucleotide sequence of the siRNA targeting the human CREPT gene is as shown in any one of SEQ ID No. 8 to 17.

5. The application according to claim 1, characterized in that, The chronic cholestatic liver disease includes one or more of the following: primary biliary cirrhosis, primary sclerosing cholangitis, primary biliary cholangitis, liver tumor, autoimmune hepatitis, primary biliary cirrhosis-autoimmune hepatitis overlap syndrome, primary sclerosing cholangitis-autoimmune hepatitis overlap syndrome, and Ig4-related cholangitis.

6. A drug for treating chronic cholestatic liver disease, characterized in that, The drug reduces CREPT gene expression or inactivates CREPT protein function. Preferably, the drug is selected from one or more of the following: formulations that affect CREPT protein expression, formulations that knock out CREPT protein, formulations that alter CREPT protein, and formulations that degrade CREPT protein.

7. The drug according to claim 6, characterized in that, The formulations that affect CREPT protein expression include nucleic acid reagents or vectors containing nucleic acid fragments that inhibit CREPT protein expression; Alternatively, the formulation for knocking out the CREPT protein may include reagents for homologous recombination or gene editing to eliminate the CREPT gene; Alternatively, the formulation that alters the CREPT protein may include agents that alter the structure of the CREPT protein; Alternatively, the formulation for degrading CREPT protein includes one or more of the following: PROTAC, molecular glue, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, and AUTOTAC pathways. The drug includes a small molecule inhibitor of the CREPT protein.

8. The medicament according to claim 7, characterized in that, The nucleic acid reagent is selected from one or more of siRNA, shRNA, microRNA, piRNA, and ASO; The vector containing the nucleic acid fragment that inhibits CREPT protein expression includes one or more of adenovirus, adeno-associated virus, lentivirus, and retrovirus; The reagents used for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system; Preferably, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene; Preferably, the positive strand nucleotide sequence of the siRNA targeting the mouse CREPT gene is as shown in any one of SEQ ID No. 1 to 7; Preferably, the positive strand nucleotide sequence of the siRNA targeting the human CREPT gene is as shown in any one of SEQ ID No. 8 to 17.

9. A pharmaceutical composition for treating chronic cholestatic liver disease, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the drug according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier; Preferably, the drug is delivered using a delivery system; Preferably, the delivery system includes GalNAc and LNP; Preferably, the pharmaceutical composition comprises siRNA and a delivery system GalNAc; Preferably, the siRNA includes siRNA targeting the mouse CREPT gene and siRNA targeting the human CREPT gene; Preferably, the positive strand nucleotide sequence of the siRNA targeting the mouse CREPT gene is as shown in any one of SEQ ID No. 1 to 7; Preferably, the positive strand nucleotide sequence of the siRNA targeting the human CREPT gene is as shown in any one of SEQ ID No. 8 to 17.

10. A pharmaceutical combination product, characterized in that, The drug combination product comprises a therapeutically effective amount of the drug according to any one of claims 6 to 8 and a second drug, for simultaneous or sequential administration.

11. The pharmaceutical combination product according to claim 10, characterized in that, The second drug includes one or more of ursodeoxycholic acid, Iqirvo, choleretic acid, and S-adenosylmethionine.

12. The use of the medicament according to any one of claims 6 to 8, the pharmaceutical composition according to claim 9, or the pharmaceutical combination product according to any one of claims 10 to 11 in any of the following; a. Application in the preparation of products that alleviate chronic cholestatic liver injury; b. Application in the preparation of products that slow down weight loss in individuals with chronic cholestatic liver disease; c. Application in the preparation of products that inhibit liver fibrosis; preferably, the inhibition of liver fibrosis includes one or more of the following: significant reduction in inflammation, necrosis, and fibrosis; d. Application in the preparation of products for improving liver function in patients with chronic cholestatic liver disease; preferably, the improvement in liver function after chronic cholestatic liver disease includes a reduction in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL; e. Application in the preparation of products that reduce liver histological structural damage; preferably, the reduction in liver histological structural damage includes one or more of the following: reduced bile duct hyperplasia, reduced fibrosis, and reduced collagen deposition; f. Application in the preparation of products that reduce the level of immune cells, said immune cells including CD4. + CD8 + ; g. Application in the preparation of products that increase the number of anti-inflammatory neutrophils; h. Application in the preparation of products that reduce cytokine levels, wherein the cytokines include one or more of IL-1β, CXCL2, CXCL5, and CCL7; i. Application in the preparation of products that reduce the level of fibrosis-related molecules, wherein the fibrosis-related molecules include one or more of TGFB1, CTGF, COL1A1, and COL3A1; j. Application in the preparation of products with reduced Plod2 expression; k. Application in the preparation of products that improve serum and liver color darkening caused by cholestasis; l. Application in the preparation of products that improve weight loss and liver weight reduction in patients with chronic cholestatic liver disease; m. Application in the preparation of products that reduce liver tissue necrosis and inflammatory infiltration in patients with chronic cholestatic liver disease; n. Application in the preparation of products for patients with chronic cholestatic liver disease who have reduced collagen deposition; o. Use in the preparation of products that reduce the levels of intrahepatic pro-inflammatory factors in patients with chronic cholestatic liver disease, wherein the intrahepatic pro-inflammatory factors include one or more of TGF-β, IL-6, and MIP-1α; p. Application in the preparation of products for the treatment of liver injury, wherein the treatment of liver injury is achieved by synergistically reducing the progression of liver injury, inflammation and fibrosis; q. Application in the preparation of products that prevent early progression of liver disease; r. Application in the preparation of products for treating the occurrence of fibrosis and improving the progression of fibrosis in patients with chronic cholestatic disease; And s. the use in the preparation of products that reduce bile acid metabolites, said bile acid metabolites including one or more of taurine-uric acid, taurine-β-mouse cholic acid, taurocholeric acid, glucodeoxycholic acid, taurocholeric acid, ursodeoxycholic acid, chenodeoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, ursodeoxycholic acid, and allochytic acid.

13. A method for treating chronic cholestatic liver disease, characterized in that, The method includes administering to a patient a therapeutically effective amount of the drug according to any one of claims 6 to 8, the pharmaceutical composition according to claim 9, or the pharmaceutical combination product according to any one of claims 10 to 11.

14. The method according to claim 13, characterized in that, The chronic cholestatic liver disease includes one or more of the following: primary biliary cirrhosis, primary sclerosing cholangitis, primary biliary cholangitis, liver tumor, autoimmune hepatitis, primary biliary cirrhosis-autoimmune hepatitis overlap syndrome, primary sclerosing cholangitis-autoimmune hepatitis overlap syndrome, and Ig4-related cholangitis.

15. The method according to claim 13, characterized in that, The administration methods of the drug, the drug combination product, or the drug combination product include systemic administration and local administration; Alternatively, the drug, the drug combination product, or the drug combination product may be administered to mammals; preferably, the drug, the drug combination product, or the drug combination product may be administered to humans or mice. Alternatively, the method may alleviate liver damage in patients with chronic cholestatic liver disease. Alternatively, the method can slow down weight loss in individuals with chronic cholestatic liver disease; Alternatively, the method inhibits liver fibrosis; preferably, the inhibition of liver fibrosis includes at least one of a significant reduction in inflammation, necrosis, and fibrosis; Alternatively, the method improves liver function in patients with chronic cholestatic liver disease; preferably, the improvement in liver function after chronic cholestatic liver disease includes a reduction in one or more of ALT, AST, TBiL, DBiL, LDH, ALP, and CHOL. Alternatively, the method improves the pathological characteristics of chronic cholestatic liver disease; preferably, the improvement of the pathological characteristics of chronic cholestatic liver disease includes one or more of cholestasis reduction and bile duct reactivity reduction. Alternatively, the method reduces the histological structural damage to the liver; preferably, the reduction in histological structural damage to the liver includes one or more of the following: reduced bile duct hyperplasia, reduced fibrosis, and reduced collagen deposition; Alternatively, the method reduces the level of immune cells, preferably, the immune cells including CD4. + CD8 + ; Alternatively, the method may increase the number of anti-inflammatory neutrophils; Alternatively, the method reduces cytokine levels, the cytokines including one or more of IL-1β, CXCL2, CXCL5, and CCL7; Alternatively, the method reduces the level of fibrosis-related molecules, including one or more of TGFB1, CTGF, COL1A1, and COL3A1. Alternatively, the method reduces Plod2 expression; Alternatively, the method can improve the darkening of serum and liver color caused by cholestasis; Alternatively, the method improves weight loss and liver weight reduction in patients with chronic cholestatic liver disease; Alternatively, the method reduces liver tissue necrosis and inflammatory infiltration in patients with chronic cholestatic disease; Alternatively, the method reduces collagen deposition in patients with chronic cholestatic disease; Alternatively, the method reduces the level of intrahepatic pro-inflammatory factors in patients with chronic cholestasis, including one or more of TGF-β, IL-6, and MIP-1α. Alternatively, the method can be used to treat liver damage in patients with chronic cholestatic disease, wherein the treatment of liver damage is achieved by synergistically reducing the progression of liver damage, inflammation, and fibrosis. Alternatively, the method may prevent the progression of liver disease in its early stages; Alternatively, the method can be used to treat the occurrence of fibrosis and improve the progression of fibrosis in patients with chronic cholestatic disease; Alternatively, the method reduces bile acid metabolites, which include one or more of taurine-uric acid, taurine-β-mouse cholic acid, taurocholic acid, glucodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, ursodeoxycholic acid, and allochytic acid.

16. The application of reagents for detecting CREPT expression in the preparation of a kit, characterized in that, The kit has any of the following functions: a. To aid in the identification of hepatocyte damage caused by cholestasis; b. To aid in the identification of liver tissue diseased by cholestasis; c. Auxiliary diagnosis of cholestasis.

17. The application according to claim 16, characterized in that, The reagents used to detect CREPT expression include siRNA and antibodies; Preferably, the positive strand nucleotide sequence of the siRNA targeting the mouse CREPT gene is as shown in any one of SEQ ID No. 1 to 7; Preferably, the positive strand nucleotide sequence of the siRNA targeting the human CREPT gene is as shown in any one of SEQ ID No. 8 to 17; Preferably, the antibody comprises a polyclonal antibody and a monoclonal antibody that bind to the CREPT protein; More preferably, the monoclonal antibody binding to the CREPT protein includes a monoclonal antibody secreted by hybridoma cells with accession number CGMCC No. 5477.

18. An animal model of cholestasis or an animal model of hepatobiliary injury and fibrosis caused by cholestasis, characterized in that, The animal models of hepatobiliary injury and fibrosis were prepared by DDC diet induction or bile duct ligation modeling in animals with specific knockout of CREPT in hepatocytes or animals with systemic knockout of CREPT. Preferably, the animal includes a mouse; Preferably, hepatocyte-specific knockout of CREPT animals includes CREPT hep- / - Mice; Preferably, animals with systemic CREPT knockout include ERT2-Cre + / - CREPT Flox / Flox Mice; Preferably, DDC diet-induced modeling involves adding 0.1 w / w% DDC to the diet; Preferably, bile duct ligation modeling involves ligating the bile ducts of the animals two weeks prior to the experiment.

19. The use of the animal model of cholestasis or the animal model of hepatobiliary injury and fibrosis caused by cholestasis as described in claim 18 in screening drugs for the treatment of cholestasis, wherein, By administering drugs for treating cholestasis to the aforementioned animal models of hepatobiliary injury and fibrosis, the efficacy of treatment for cholestasis was evaluated, thereby screening for drugs to treat cholestasis.

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