KR102204521B1 - A composition comprising Progranulin for preventing or treating liver disease - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of liver disease comprising a vector including a progrenulin protein or a polynucleotide encoding a progrenulin protein.
The composition of the present invention is excellent in the prevention or treatment of liver disease by suppressing liver fibrosis and greatly reducing liver toxicity, reducing the amount of fat, and reducing the expression level of fibrosis and inflammation-related genes without showing toxicity to the human body. Shows the effect.

Description

A composition comprising Progranulin for preventing or treating liver disease, comprising a protein of PrOＧＲＡNＵLIN

The present invention relates to a composition for preventing or treating liver disease, including a vector containing a progranulin protein or a polynucleotide encoding a progranulin protein.

The liver plays various roles in our body, such as metabolism of lipids, detoxification, excretion of bile, storage of various nutrients, hematopoiesis or blood clotting, and regulation of circulating blood volume, and is one of the essential organs for life maintenance. Looking at the function of the liver in more detail, first of all, it has the function of managing energy metabolism, so that all nutrients absorbed from food are metabolized into substances that can produce energy in the liver and supplied or stored throughout the body. Second, the liver has the function of synthesizing, storing, and distributing about 2,000 kinds of enzymes, albumin, serum proteins of coagulation factors, bile acids, phospholipids, and fats such as cholesterol. Third, as a function of detoxification and decomposition, it has a function of detoxifying drugs, alcohol, toxic substances, etc., a function of excreting various metabolites into the duodenum, and an immune function, which plays an important role in maintaining life.

In general, hepatitis, which causes inflammation of the liver, accounts for the majority of liver diseases, and can be divided into acute hepatitis and chronic infection according to the pattern, and viral hepatitis, alcoholic hepatitis, and drug hepatitis according to the cause. In addition, liver diseases caused by these abnormalities include fatty liver, hepatitis, and cirrhosis. The mechanism of the progression of liver disease has not been fully elucidated, but it is known that the development of advanced liver disease such as fatty liver and cirrhosis is accompanied by secondary cell damage after the first occurrence of fatty liver. In addition, since liver disease has no self-conscious symptoms in the early stage and is detected only after it has progressed considerably, it is the leading cause of death not only in Korea but also in the world.

Progrenulin is a secreted glycoprotein composed of seven halves of cysteine-rich granulin motif domains, and its mRNA is expressed in macrophages, neutrophils, adipocytes, epithelial cells, and sperm. Progrenulin was first discovered as a peptide secreted from leukocytes, and it was reported to act as a neuronal growth factor for its function, and it was known that it may be associated with frontotemporal dementia.

Under this background, the present inventors have made great efforts to develop a therapeutic agent for liver disease suitable for the human body, and as a result, confirmed excellent therapeutic effects on liver disease with a new function of progrenulin, and completed the present invention.

Republic of Korea Patent Publication No. 10-2016-0119284 Korean Patent Application Publication No. 10-2017-0121549

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of liver disease comprising as an active ingredient a progrenulin protein, which has an activity of inhibiting inflammation occurring in fatty liver, liver tissue, liver damage, liver fibrosis, etc. .

In addition, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of liver disease comprising a vector containing a polynucleotide encoding a progrenulin protein as an active ingredient.

In addition, an object of the present invention is to provide a method for preventing or treating liver disease comprising administering a progrenulin protein to a subject.

In addition, an object of the present invention is to provide a method for preventing or treating liver disease, comprising administering a vector containing a polynucleotide encoding a progrenulin protein to a subject.

In addition, an object of the present invention is to provide a use of a progrenulin protein in the manufacture of a medicament for the treatment of liver disease.

In addition, it is an object of the present invention to provide a use of a vector containing a polynucleotide encoding a progrenulin protein in the manufacture of a drug for the treatment of liver disease.

In addition, it is an object of the present invention to provide a composition comprising a progrenulin protein for use in the treatment of liver disease.

In addition, it is an object of the present invention to provide a composition comprising a vector containing a polynucleotide encoding a progrenulin protein for use in the treatment of liver disease.

In addition, it is an object of the present invention to provide a use of a vector containing a polynucleotide encoding a progrenulin protein for the treatment of liver disease.

In addition, an object of the present invention is to provide a use of a cell containing a progrenulin protein for the treatment of liver disease.

In order to achieve the above object, an aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of liver disease comprising progrenulin protein as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of liver disease comprising a vector containing a polynucleotide encoding a progrenulin protein as an active ingredient.

Another aspect of the present invention provides a method of preventing or treating liver disease comprising administering a progrenulin protein to a subject.

Another aspect of the present invention provides a method for preventing or treating liver disease, comprising administering to a subject a vector containing a polynucleotide encoding a progrenulin protein.

Another aspect of the present invention provides a use of a progrenulin protein in the manufacture of a medicament for the treatment of liver disease.

Another aspect of the present invention provides the use of a vector comprising a polynucleotide encoding a progrenulin protein in the manufacture of a medicament for the treatment of liver disease.

Another aspect of the present invention provides a composition comprising a progrenulin protein for use in the treatment of liver disease.

Another aspect of the present invention provides a composition comprising a vector comprising a polynucleotide encoding a progrenulin protein for use in the treatment of liver disease.

Another aspect of the present invention provides the use of a vector comprising a polynucleotide encoding a progrenulin protein for the treatment of liver disease.

Another aspect of the present invention provides the use of a cell containing a progrenulin protein for the treatment of liver disease.

The composition of the present invention exhibits no toxicity to the human body and exhibits excellent effects in preventing or treating liver disease by inhibiting liver fibrosis and greatly reducing liver toxicity. Specifically, the composition of the present invention has an effect of inhibiting fibrosis by reducing the expression of genes related to fibrosis in liver tissue, and has an effect of reducing the expression of genes related to various inflammatory reactions due to liver damage. In addition, the composition of the present invention can be used to prevent or treat fatty liver by reducing the lipid component of liver tissue, and has an excellent effect of inducing the recovery of damaged liver tissue.

1 is a diagram showing the administration of a recombinant adenovirus containing a polynucleotide sequence encoding progrenulin in a liver disease induction model administered with CCl ₄ (carbon tetrachloride) and a schedule for confirming prevention and treatment experiments. Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
Figure 2 is a graph showing the comparison of the weight, liver weight, and spleen weight confirmed 4 weeks after performing the experiment in the liver disease induction model administered with CCl ₄ (carbon tetrachloride). Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
FIG. 3 is a graph showing a picture of a liver for confirming liver fibrosis in a liver disease induction model administered with CCl ₄ (carbon tetrachloride), a micrograph of sirus red staining of the liver, and a stained area numerically. Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
Figure 4 is a graph showing the results of confirming the amount of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to confirm the effect of reducing hepatotoxicity in a liver disease induction model administered with CCl ₄ (carbon tetrachloride). Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
Figure 5a shows the degree of liver fibrosis, inflammation, and liver damage by administering progrenulin in the liver fibrosis induction model administered with CCl ₄ (carbon tetrachloride), and α-SMA, Col1a1, F4/80, and It is the result of dyeing of TUNEL. 5B is a graph showing the comparison of the expression levels of inflammatory genes measured in liver tissues of the liver fibrosis induction model, and FIG. 5C is a Western blotting result confirming NFκB phosphorylation in relation to the inflammatory protein signaling system. Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
6 is a graph comparing the expression levels of inflammatory cytokine genes measured by treatment with progrenulin in Raw 264.7 mouse macrophages inducing an inflammatory response and purely isolated macrophages of the mouse abdominal cavity. PGRN means progrenulin.
7 is a graph comparing the expression levels of inflammation-related genes measured by treatment with progrenulin after inducing liver damage in human hepatocyte cell lines HepG2 and Huh7. PGRN means progrenulin.
FIG. 8 is a graph comparing activation of human astrocytes by inducing progrenulin treatment to confirm activation of astrocytes, and measuring and measuring the expression level of fibrosis-related genes. PGRN means progrenulin.
9 is a graph showing the results of oil red O staining of liver tissue by administering progrenulin to a non-alcoholic steatohepatitis animal model and measuring the amount of triglycerides. The lower part of FIG. 9 shows the results of Western blotting measuring the amount of fat synthesis induction markers FAS (fatty acid synthase) and SREBP1 (sterol regulatory element-binding proteins). Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group. FAS stands for'fatty acid synthase' and SREBP1 stands for'sterol regulatory element-binding protein'.
10A is a result of comparing hepatic fibrosis markers α-SMA, Col1a1 and macrophage marker F4/80 in liver tissue by administering progrenulin to a non-alcoholic steatohepatitis animal model. 10B is a graph comparing the expression levels of inflammation-related genes and hepatic fibrosis marker genes in liver tissues of the non-alcoholic steatohepatitis animal model. Ad.PGRN is measured for the experimental group administered with progrenulin, and Ad.CON refers to the control group.
FIG. 11 is a graph comparing the expression levels of inflammatory cytokine genes measured by treatment with progrenulin in Raw 264.7 mouse macrophages inducing a non-alcoholic-derived inflammatory response and pure isolated macrophages of the mouse abdominal cavity. PA stands for palmitate, PRGN stands for progrenulin.
12 is a graph comparing the expression levels of inflammatory cytokine genes measured by treatment with progrenulin after inducing a non-alcoholic-derived inflammatory response in human hepatocyte cell lines HepG2 and Huh7. PA stands for palmitate, PRGN stands for progrenulin.

Hereinafter, the present invention will be described in detail.

Each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising a progrenulin protein as an active ingredient.

The progranulin protein is not toxic to the human body and inhibits liver fibrosis in liver disease and reduces the expression of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) related to liver toxicity, thereby providing excellent prevention and treatment. It works. The progrenulin has an effect of inhibiting fibrosis in liver tissue where hepatic fibrosis has occurred, thereby reducing α-SMA and Col1a1 proteins related to fibrosis, F4/80 protein, which is a macrophage marker, and reducing TUNEL staining, which is a cell damage marker. , IL-6, IL-1β, TGF-β, MCP-1, TNF-α, etc. by reducing the expression level of genes related to the inflammatory response or inhibiting the phosphorylation of NFκB in the signaling system of inflammatory proteins, thereby improving liver fibrosis Or have a curative effect. In addition, the progrenulin has the effect of reducing the expression of genes related to the inflammatory response in macrophages in which the inflammatory reaction is induced or damaged hepatocytes, and the astrocytes that play a major role in the development and progression of liver fibrosis. It is possible to inhibit the activation and reduce the amount of fibrotic proteins such as α-SMA and Col1a1, thereby recovering damage to liver tissue or inhibiting fibrosis, thereby preventing or treating liver disease. The progrenulin can reduce the amount of triglycerides or triglycerides from non-alcoholic steatohepatitis liver tissue, and the amount of FAS (fatty acid synthase) or SREBP1 (sterol regulatory element-binding proteins) protein that induces the synthesis of fat. It is effective in preventing or treating fatty liver, reducing the expression levels of α-SMA, Col1a1 protein, α-SMA, Col1a1 genes and macrophage marker F4/80 protein related to fibrosis, and IL- 6, MCP-1, TNF-α, etc. By reducing the expression level of inflammatory response-related genes, there is an excellent effect that can prevent or treat non-alcoholic steatohepatitis. Furthermore, the progrenulin can reduce the expression of inflammatory response-related genes such as IL-6, IL-1β, MCP-1, and TNF-α in macrophages or liver cells in which non-alcoholic-derived inflammatory reactions are induced. It works.

In the present invention, the term "liver disease" refers to a problem in which one or more functions among various functions performed by the liver cannot be performed normally. Preferably, the liver disease may be any one or more selected from the group consisting of hepatitis, hepatotoxicity, fatty liver, cirrhosis, liver fibrosis, and non-alcoholic liver disease. The non-alcoholic liver disease may be non-alcoholic steatohepatitis.

As used herein, the term "treatment" refers to any action that improves or changes the symptoms of liver disease by administration of the pharmaceutical composition, and "prevention" is to suppress the onset of liver disease by administration of the pharmaceutical composition. It means anything that does or delays.

In the present invention, the term "Progranulin" is an 88 kD glycoprotein composed of 7 halves of granulin (Grn) domains composed of 12 "cysteine-rich motifs", for example It may have SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto, and as long as it has homology with a known progrenulin protein, and has the same activity as identified in the present invention, some sequences are deleted, added, or substituted. Or, even if it is a progrenulin protein derived from humans or other animals, all may be included within the scope of the present invention. In the present invention, the sequence homology may correspond to a sequence of 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the wild-type progrenulin protein. In addition, in the case of a fusion protein in which a fragment of a domain is cleaved for activity in a wild-type progrenulin protein, and a known peptide to increase expression, isolation, purification, or activity of the protein, the wild-type progrenulin protein and As long as they have the same activity, they can be included within the scope of the progrenulin protein of the present invention. In the present invention, the progrenulin protein may be prepared through a method known in the art, for example, peptide synthesis or production of a protein using transformed cells, but is not limited thereto.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating liver disease comprising a vector containing a polynucleotide encoding a progrenulin protein as an active ingredient.

The polynucleotide encoding the progrenulin protein according to the present invention may preferably include a polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, but is not limited thereto, and has homology with a known progrenulin protein, As long as it has the same activity as identified in the present invention, a partial sequence is deleted, added, or substituted, or if it is possible to produce a human or other animal-derived progrenulin protein, this is included.

The vector is a vector including any one selected from the group consisting of linear DNA expressed in human or animal cells, plasmid vectors, viral expression vectors, and viral expression vectors. In addition, the viral expression vector is a recombinant retrovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus (AAV) vector, a recombinant herpes simplex virus vector and recombinant It is preferably a recombinant viral vector, which is any one selected from the group consisting of lentivirus vectors, but is not limited thereto.

Preferably, the recombinant viral vector is an adenovirus vector. The “adenovirus” refers to a regular icosahedral DNA virus having no envelope and having a diameter of 60 to 85 nm. In general, adenoviruses are known as excellent gene transfer vectors for animal cells due to their high gene transfer efficiency, gene transfer ability to undifferentiated cells, and ease of production of high titer virus stocks. The "recombinant adenovirus" or "recombinant adenovirus vector" includes a gene encoding prognulin in the adenovirus, preferably a gene having a nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, after introduction into a host cell It refers to a recombinant adenovirus or recombinant adenovirus vector capable of expressing progrenulin.

The cell line for producing the recombinant adenovirus of the present invention may be a cell line producing an adenovirus, and it is most preferable to use the adenovirus producing cell line 293 cells, but is not limited thereto.

It is preferred that the nucleotide sequence encoding the progrenulin protein of the present invention is present in a suitable expression construct.

The term “expression construct” as used herein refers to a minimum element for expression including a nucleotide sequence for expression and an expression sequence (eg, a promoter, a polyadenylation signal sequence, etc.) that induces the expression of the sequence. elements). As used herein, the term “promoter operable in eukaryotic cells” means a transcriptional control sequence capable of inducing transcription of a target gene in eukaryotic cells. Each subunit coding nucleotide sequence is operably linked to a promoter. In the present specification, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, thereby The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

In the present invention, the promoter is preferably capable of controlling the transcription of the progrenulin-encoding nucleotide sequence by operating in an animal cell, more preferably a mammalian cell, and a promoter derived from a mammalian virus and a mammalian cell. Includes promoters derived from genome, such as U6 promoter, H1 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter , Metallotionine promoter, beta-actin promoter, and the like, but are not limited thereto. Most preferably, the promoter is a CMV promoter.

In the present invention, since the recombinant adenovirus is produced based on a viral vector, it can be carried out according to a virus infection method known in the art.

In the present invention, the pharmaceutical composition may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. Compositions containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose in one or more compounds. (lactose), gelatin, etc. can be mixed to prepare. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

In addition, the pharmaceutical composition of the present invention is a group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. It may have any one formulation selected from, but is not limited thereto.

The daily dosage of the progrenulin protein is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and more preferably once to several times a day, divided and administered.

The daily dose of the vector containing the polynucleotide encoding the progrenulin protein is preferably 0.05 to 500 mg, more preferably 0.1 to 300 mg, and the polynucleotide encoding progrenulin In the case of a recombinant virus containing, 10 ⁸ PFU to 10 ¹² PFU. It preferably contains about 10 ¹⁰ PFU, but is not limited thereto.

In addition, it may be administered at different times or concurrently with other treatments for liver disease, or a known treatment method for liver disease may be applied together. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

In the present invention, the term “administration” refers to introducing the pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue.

The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, route of administration, and dosage used in the art, depending on the purpose or need. Examples of the route of administration may be oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, an appropriate dosage and number of administrations may be selected according to methods known in the art, and the amount and number of administrations of the pharmaceutical composition of the present invention to be actually administered are the type of symptom to be treated, route of administration, sex, health condition It can be appropriately determined by various factors such as diet, age and weight of the individual, and the severity of the disease.

The term "pharmaceutically effective amount" in the present invention means an amount sufficient to inhibit or alleviate the increase in vascular permeability at a reasonable benefit/risk ratio applicable to medical use, and the effective dose level is the type and severity of the subject, age, Sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

In the present invention, the term “individual” refers to all animals including humans who have or have developed liver disease of the present invention. By administering the pharmaceutical composition of the present invention to an individual, liver disease can be prevented or treated.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

CCl ₄ (carbon tetrachloride) administration of liver fibrosis induction model confirmed the effect of improving fibrosis and hepatotoxicity

To establish a liver cirrhosis induction model, an experimental mouse of about 8 weeks old was purchased and adapted for one week. During the experiment, diet and water were freely ingested, and the indoor temperature was 22 ± 2℃, the humidity was 55 ± 5%, and the indoor lighting was controlled at a 12-hour cycle. The model was divided into two groups, and the number of experimental animals in each group was 4, and the control group was 2 during the experiment period by adding carbon tetrachloride (CCl ₄ ), a substance inducing liver fibrosis, to olive oil in an amount of 0.5 mg/kg. Intraperitoneal injection was performed for 4 weeks at daily intervals.

The experimental group was injected with the same dose of carbon tetrachloride as in the control group.

Vecto biolab company used adenovirus (Cat. No: ADV-260799) containing mouse progrenulin polynucleotide (SEQ ID NO: 1, Gene ID: 14824) using human adenovirus type 5 as the backbone and CMV promoter in the experiment I did. The first day of injection, the recombinant adenovirus (PGRN) was administered at 4× ^{10 10} PFU, and 2 weeks later, the recombinant adenovirus was administered again at the same dose.

Based on the experimental animals, the treatment effect was confirmed by confirming changes in body weight, changes in organ shape and weight, and changes in hematology and histopathology.

Checking the toxicity of progrenulin in the body by checking the weight change

After inducing suffocation after 4 weeks of the experimental animals, the liver and spleen were removed and the weights were compared, and the results are shown in FIG. 2.

2(A) shows the total weight, (B) shows the liver weight, and (C) shows the spleen weight. As can be seen from the results of FIG. 2, it was shown that the group treated with progrenulin did not show a significant change in the weight of each tissue compared to the control group, from which the progrenulin according to the present invention has no toxicity in the body. It was confirmed that there was no.

Confirmation of the inhibitory effect of liver fibrosis through histopathological analysis

After obtaining a picture of the liver extracted from the animal model in which the asphyxiation was induced, it was sufficiently fixed in 10% formalin solution immediately. In addition, a paraffin sample was prepared using a sample that has undergone an automated process for a tissue sample using an automated tissue processing machine (Tissue-Tek VIP6).

To compare the degree of liver fibrosis, sirus red staining was performed through the following steps: xylen: 3 times, 3 minutes each, 100% Et-OH 3 times 3 minutes, 95% Et-OH 3 minutes, 90% Et-OH 3 Min, 80% Et-OH 3 min, 70% Et-OH 3 min, 50% Et-OH 2 min, tap water washing and Sirus red staining (Abcam). Then, a microscopic picture was obtained and the stained area was quantified through the image J program.

The results are shown in FIG. 3.

(A) of FIG. 3 shows a photograph of the extracted liver, (B) shows the result of Sirus red staining, and (C) shows the result of quantification.

As can be seen in Figure 3, it was confirmed visually and quantitatively that the progrenulin treatment significantly reduced liver fibrosis compared to the control group.

Confirmation of hepatotoxicity reduction effect through pathological analysis

In order to test for hepatotoxic ALT/AST, blood was extracted through a vein in the jaw before asphyxiation induction, left at room temperature for about 30 minutes, and centrifuged for about 10 minutes to separate the serum. This ALT/AST analysis was analyzed using BECKMAN COULTER's AU480 equipment, and the results are shown in FIG. 4.

4(A) shows alanine aminotransferase (ALT), and (B) shows aspartate aminotransferase (AST).

As can be seen in FIG. 4, it was confirmed that both AST and ALT were significantly reduced by administration of progrenulin, thereby eliminating liver toxicity.

Taking all the above results together, it can be seen that progrenulin does not exhibit human toxicity, and has excellent preventive and therapeutic effects on liver diseases by alleviating liver toxicity and inhibiting liver fibrosis. Therefore, a vector including a progrenulin protein or a polynucleotide encoding a progrenulin protein can be very usefully used for preventing or treating liver disease.

CCl ₄ (carbon tetrachloride) administration confirmed the effect of inhibiting the expression of genes related to inflammation and fibrosis in liver fibrosis induction model

In order to construct an animal model of liver fibrosis, an experimental mouse of about 8 weeks old was purchased, and diet and water were freely ingested during the experiment under the same temperature, humidity, and lighting conditions as in Example 1. CCl ₄ (Sigma, St. Louis, MO, USA) was injected into the peritoneal cavity of mice at a concentration of 0.5 ml/kg three times a week for 4 weeks, and in the case of the progrenulin administration group, at the start of the experiment and at the 2nd week Granulin adenovirus molecules (granuline adenovirus particles, VectorBiolabs, USA) were injected once each into the anterior tibialis muscle of the mouse.

In order to check the degree of liver fibrosis, inflammation, and liver damage, after 4 weeks, the liver tissue of the mouse was collected and fixed with 10% formalin solution, and then tissue immunochemical staining was performed. As a result, in mice treated with progrenulin, α-SMA and Col1a1, markers of hepatic fibrosis, F4/80, markers of macrophages, and TUNEL staining, markers of cell damage, decreased. In addition, RNA was extracted from the same tissue using TRIzol solution (ambion), and after cDNA was synthesized using BioFACT's RT (reverse transcription)-kit, real-time PCR (Applied Biosystems) using SYBR Green (Applied Biosystems) ), it was confirmed that the expression levels of inflammatory genes such as IL-6 and MCP-1 were reduced. Furthermore, as a result of confirming the signaling system of inflammatory proteins by extracting proteins from mouse liver tissue, it was confirmed that phosphorylation of NFκB was reduced (FIGS. 5A to 5C ).

From the above results, administration of progrenulin to mice with fibrosis in liver tissue by administration of CCl ₄ has the effect of reducing fibrosis and reducing the expression of inflammation-related genes and protein signaling. Could be confirmed.

Inhibitory effect of inflammatory gene expression in macrophage and hepatocyte cell lines that induced inflammatory response

Checking the expression level of inflammation-related genes in macrophages

First, the inflammatory response of macrophages was induced, and progrenulin was treated to confirm the change in the expression level of inflammation-related genes. For the experiment, the macrophages of Raw 264.7 mice and the macrophages purely isolated from the abdominal cavity of the mice were cultured in 24 wells until the cell density grew to about 80%. In order to confirm the effect of progrenulin on the inflammatory response of macrophages, LPS was treated with 100 ng/ml to induce the inflammatory response of macrophages. Progrenulin was pretreated with a mouse progrenulin recombinant protein (R&D Systems) at a concentration of 1 μg/ml for 30 minutes.

As a result of comparing the expression levels of inflammatory cytokine genes after reacting to cells for 1 hour, the expression levels of IL-1β and MCP-1 in the macrophage cell line of Raw 264.7 mice treated with progrenulin were observed. In phagocytes, it was confirmed that the expression levels of IL-1β, IL-6, MCP-1, and TNF-α were all significantly reduced (FIG. 6).

Confirmation of the expression level of inflammation-related genes in the hepatocyte cell line that induced damage

In addition, in order to determine how progrenulin influences the inflammatory response due to hepatocyte damage, the human hepatocyte cell lines HepG2 and Huh7 were targeted to induce damage to hepatocyte lines, and the expression level of inflammation-related genes was measured by treating progrenulin. I did. The HepG2 and Huh7 were cultured in 24 wells until the cell density reached about 80%, and then 40 ng/ml of TNF-α (R&D System) was treated to induce damage to the hepatocyte line. Progrenulin was pretreated with human granulin recombinant protein at a concentration of 0.5 μg/ml for 30 minutes.

After treating the cells with the damage-inducing substance and progrenulin as described above for 1 hour or 24 hours, the expression levels of inflammation-related genes were checked and compared. As a result, it was confirmed that the expression levels of the inflammatory cytokine genes IL-1β, IL-6, and MCP-1 were decreased in the group treated with progrenulin (FIG. 7).

Confirmation of the effect of activating astrocytes and inhibiting the expression of fibrotic markers

Astrocytes are cells that play a major role in liver fibrosis, and in order to determine the effect between progrenulin and astrocytes, human astrocytes are treated with progrenulin to check the activation of astrocytes and a fibrosis marker gene The expression level of was measured.

Astrocytes were cultured in 24 wells until the cell density reached about 80%, and TGF-β was treated with 1 ng/ml to induce activation of astrocytes. Progrenulin was pretreated with human granulin recombinant protein at a concentration of 0.5 μg/ml for 30 minutes.

After treatment for 24 hours, the result of comparing the activation of astrocytes and the expression level of fibrosis-related genes. As shown in FIG. 8, when progrenulin was treated, the activation of astrocytes was reduced, and α-SMA, a fibrosis-related gene. And it was confirmed that the expression level of Col1a1 was significantly reduced compared to the control group not treated with progrenulin.

Inhibitory effect of fatty liver, steatohepatitis and hepatic fibrosis in non-alcoholic steatohepatitis mouse model

Confirmation of fatty liver inhibitory effect

First, in order to construct a non-alcoholic steatohepatitis animal model, mice were subjected to a methionine-choline deficient (MCD) diet for 8 weeks. In the case of the progrenulin administration group, progrenulin was injected once each into the anterior tibialis muscle of the mouse at the start of the experiment and at the 4th week.

In order to check the degree of fatty liver, mice were raised for 8 weeks, and then liver tissue was collected to make frozen sections, stained with Oil Red O and the amount of triglyceride was measured. The amount of triglycerides was measured using the Triglycerides kit (Abcam, ab65336). In addition, the amount of the marker protein that induces fat synthesis was confirmed through Western blotting.

As a result, in mice administered with progrenulin, the degree of staining of triglycerides was less than that of the control group, and the amount of triglycerides was also measured to be less. It was confirmed that the amount of proteins in fat synthesis induction markers FAS (fatty acid synthase) and SREBP1 (Sterol regulatory element-binding protein) were decreased (FIG. 9).

Confirmation of fatty liver inhibitory effect

In order to confirm the degree of non-alcoholic-derived hepatic fibrosis and inflammation from the tissue of the non-alcoholic steatohepatitis mouse model used to perform the experiment confirming the reduction effect of the fatty liver, mouse liver tissue after 8 weeks was collected in the same manner as in the above experiment. And used. The collected liver tissue was fixed in a 10% formalin solution, and tissue immunochemical staining was performed, and RNA was extracted from the tissue to determine the expression level of inflammatory genes after cDNA synthesis.

As a result, it was confirmed that staining of α-SMA and Col1a1, which are markers of liver fibrosis, and F4/80, which is a macrophage marker, were reduced in liver tissues collected from the group treated with progrenulin (FIG. 10A). In addition, it was confirmed that the expression levels of IL-1β, MCP-1, and TNF-α genes were reduced compared to the control group to which progrenulin was not administered, and the expression levels of the liver fibrosis markers α-SMA and Col1a1 genes were also reduced. Could be (Fig. 10b).

From the results of the above examples, it was found that when progrenulin was treated in a non-alcoholic steatohepatitis animal, it was found that it has the effect of reducing the amount of fat accumulation and fibrosis, and the expression of genes related to fibrosis and inflammation. .

Fatty acid origin Checking the effect of inhibiting the expression of inflammatory genes

Confirmation of the effect of inhibiting expression in macrophages

First, a non-alcoholic-derived inflammatory response of macrophages was induced, and progrenulin was treated to confirm the change in the expression level of fatty acid-derived inflammation-related genes. For the experiment, the macrophages of Raw 264.7 mice and the macrophages purely isolated from the abdominal cavity of the mice were cultured in 24 wells until the cell density grew to about 80%. In order to confirm the effect of progrenulin on the fatty acid-derived inflammatory response of macrophages, 200 μM of palmitate was treated to induce a non-alcoholic-derived inflammatory response of macrophages. Progrenulin was pretreated with a mouse progrenulin recombinant protein (R&D Systems) at a concentration of 1 μg/ml for 30 minutes.

As a result of comparing the expression levels of inflammation-related genes after reacting to cells for 1 hour, the expression levels of IL-6 in the macrophage cell line of Raw 264.7 mice treated with progrenulin, and IL-6 in macrophages purely isolated from the peritoneal cavity. , It was confirmed that the expression levels of MCP-1 and TNF-α were significantly reduced (FIG. 11).

Confirmation of the effect of inhibiting expression in hepatocyte line

In addition, in order to determine how progrenulin affects the inflammatory response of hepatocytes, the human hepatocyte cell lines HepG2 and Huh7 induce a non-alcoholic-derived inflammatory response of the hepatocyte line, and the expression level of inflammation-related genes by treatment with progrenulin. Was measured. The HepG2 and Huh7 were cultured in 24 wells until the cell density reached about 80%, and then, palmitate was treated with 100 or 200 μM to induce an inflammatory response of the hepatocyte line. Progrenulin was pretreated with human granulin recombinant protein at a concentration of 0.5 μg/ml for 30 minutes.

After the cells were treated with the inflammatory reaction-inducing substance and progrenulin as described above for 1 hour or 24 hours, the expression levels of inflammation-related genes were checked and compared. As a result, it was confirmed that the expression levels of the inflammatory cytokine genes IL-1β, IL-6 and MCP-1 were decreased in the HepG2 cell line treated with progrenulin (FIG. 12).

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A composition comprising progranulin for preventing or treating liver disease <130> 2019DPA3260 <150> KR 10-2018-0044386 <151> 2018-04-17 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 602 <212> PRT <213> Artificial Sequence <220> <223> Mouse PGRN <400> 1 Met Pro Pro Arg Glu Pro Gly Pro Arg Arg Arg Gln Thr Met Trp Val 1 5 10 15 Leu Met Ser Trp Leu Ala Phe Ala Ala Gly Leu Val Ala Gly Thr Gln 20 25 30 Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu Asp Gln Gly 35 40 45 Gly Ala Asn Tyr Ser Cys Cys Asn Pro Leu Leu Asp Thr Trp Pro Arg 50 55 60 Ile Thr Ser His His Leu Asp Gly Ser Cys Gln Thr His Gly His Cys 65 70 75 80 Pro Ala Gly Tyr Ser Cys Leu Leu Thr Val Ser Gly Thr Ser Ser Cys 85 90 95 Cys Pro Phe Ser Lys Gly Val Ser Cys Gly Asp Gly Tyr His Cys Cys 100 105 110 Pro Gln Gly Phe His Cys Ser Ala Asp Gly Lys Ser Cys Phe Gln Met 115 120 125 Ser Asp Asn Pro Leu Gly Ala Val Gln Cys Pro Gly Ser Gln Phe Glu 130 135 140 Cys Pro Asp Ser Ala Thr Cys Cys Ile Met Val Asp Gly Ser Trp Gly 145 150 155 160 Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp Arg Val His Cys 165 170 175 Cys Pro His Gly Ala Ser Cys Asp Leu Val His Thr Arg Cys Val Ser 180 185 190 Pro Thr Gly Thr His Thr Leu Leu Lys Lys Phe Pro Ala Gln Lys Thr 195 200 205 Asn Arg Ala Val Ser Leu Pro Phe Ser Val Val Cys Pro Asp Ala Lys 210 215 220 Thr Gln Cys Pro Asp Asp Ser Thr Cys Cys Glu Leu Pro Thr Gly Lys 225 230 235 240 Tyr Gly Cys Cys Pro Met Pro Asn Ala Ile Cys Cys Ser Asp His Leu 245 250 255 His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile Gln Ser Lys Cys 260 265 270 Leu Ser Lys Asn Tyr Thr Thr Asp Leu Leu Thr Lys Leu Pro Gly Tyr 275 280 285 Pro Val Lys Glu Val Lys Cys Asp Met Glu Val Ser Cys Pro Glu Gly 290 295 300 Tyr Thr Cys Cys Arg Leu Asn Thr Gly Ala Trp Gly Cys Cys Pro Phe 305 310 315 320 Ala Lys Ala Val Cys Cys Glu Asp His Ile His Cys Cys Pro Ala Gly 325 330 335 Phe Gln Cys His Thr Glu Lys Gly Thr Cys Glu Met Gly Ile Leu Gln 340 345 350 Val Pro Trp Met Lys Lys Val Ile Ala Pro Leu Arg Leu Pro Asp Pro 355 360 365 Gln Ile Leu Lys Ser Asp Thr Pro Cys Asp Asp Phe Thr Arg Cys Pro 370 375 380 Thr Asn Asn Thr Cys Cys Lys Leu Asn Ser Gly Asp Trp Gly Cys Cys 385 390 395 400 Pro Ile Pro Glu Ala Val Cys Cys Ser Asp Asn Gln His Cys Cys Pro 405 410 415 Gln Gly Phe Thr Cys Leu Ala Gln Gly Tyr Cys Gln Lys Gly Asp Thr 420 425 430 Met Val Ala Gly Leu Glu Lys Ile Pro Ala Arg Gln Thr Thr Pro Leu 435 440 445 Gln Ile Gly Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly 450 455 460 Gln Thr Cys Cys Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu 465 470 475 480 Pro His Ala Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly 485 490 495 Tyr Thr Cys Asn Val Lys Ala Arg Thr Cys Glu Lys Asp Val Asp Phe 500 505 510 Ile Gln Pro Pro Val Leu Leu Thr Leu Gly Pro Lys Val Gly Asn Val 515 520 525 Glu Cys Gly Glu Gly His Phe Cys His Asp Asn Gln Thr Cys Cys Lys 530 535 540 Asp Ser Ala Gly Val Trp Ala Cys Cys Pro Tyr Leu Lys Gly Val Cys 545 550 555 560 Cys Arg Asp Gly Arg His Cys Cys Pro Gly Gly Phe His Cys Ser Ala 565 570 575 Arg Gly Thr Lys Cys Leu Arg Lys Lys Ile Pro Arg Trp Asp Met Phe 580 585 590 Leu Arg Asp Pro Val Pro Arg Pro Leu Leu 595 600 <210> 2 <211> 593 <212> PRT <213> Artificial Sequence <220> <223> Human PGRN <400> 2 Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala 1 5 10 15 Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu 20 25 30 Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp Lys 35 40 45 Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln Val Asp 50 55 60 Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser Gly Thr 65 70 75 80 Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly Asp Gly His 85 90 95 His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys 100 105 110 Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp 115 120 125 Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp 130 135 140 Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp 145 150 155 160 Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr 165 170 175 Arg Cys Ile Thr Pro Thr Gly Thr His Pro Leu Ala Lys Lys Leu Pro 180 185 190 Ala Gln Arg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser Val Met Cys 195 200 205 Pro Asp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys Cys Glu Leu 210 215 220 Pro Ser Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys 225 230 235 240 Ser Asp His Leu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile 245 250 255 Gln Ser Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr 260 265 270 Lys Leu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val 275 280 285 Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp 290 295 300 Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His 305 310 315 320 Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr Cys Glu 325 330 335 Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro Ala His Leu 340 345 350 Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro Cys Asp Asn 355 360 365 Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu Thr Ser Gly 370 375 380 Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp His 385 390 395 400 Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala Glu Gly Gln Cys 405 410 415 Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met Pro Ala Arg 420 425 430 Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp Gln His Thr 435 440 445 Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp 450 455 460 Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp Arg Gln His 465 470 475 480 Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu 485 490 495 Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser Pro 500 505 510 His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys His 515 520 525 Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys 530 535 540 Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro 545 550 555 560 Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu Arg Arg Glu 565 570 575 Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu Arg Gln Leu 580 585 590 Leu <210> 3 <211> 1809 <212> DNA <213> Artificial Sequence <220> <223> Mouse PGRN <400> 3 atgcctccca gggagcccgg accccgacgc aggcagacca tgtgggtcct gatgagctgg 60 ctggccttcg cggcagggct ggtagccgga acacagtgtc cagatgggca gttctgccct 120 gttgcctgct gccttgacca gggaggagcc aactacagct gctgtaaccc tcttctggac 180 acatggccta gaataacgag ccatcatcta gatggctcct gccagaccca tggccactgt 240 cctgctggct attcttgtct tctcactgtg tctgggactt ccagctgctg cccgttctct 300 aagggtgtgt cttgtggtga tggctaccac tgctgccccc agggcttcca ctgtagtgca 360 gatgggaaat cctgcttcca gatgtcagat aaccccttgg gtgctgtcca gtgtcctggg 420 agccagtttg aatgtcctga ctctgccacc tgctgcatta tggttgatgg ttcgtgggga 480 tgttgtccca tgccccaggc ctcttgctgt gaagacagag tgcattgctg tccccatggg 540 gcctcctgtg acctggttca cacacgatgc gtttcaccca cgggcaccca caccctacta 600 aagaagttcc ctgcacaaaa gaccaacagg gcagtgtctt tgcctttttc tgtcgtgtgc 660 cctgatgcta agacccagtg tcccgatgat tctacctgct gtgagctacc cactgggaag 720 tatggctgct gtccaatgcc caatgccatc tgctgttccg accacctgca ctgctgcccc 780 caggacactg tatgtgacct gatccagagt aagtgcctat ccaagaacta caccacggat 840 ctcctgacca agctgcctgg atacccagtg aaggaggtga agtgcgacat ggaggtgagc 900 tgccctgaag gatatacctg ctgccgcctc aacactgggg cctggggctg ctgtccattt 960 gccaaggccg tgtgttgtga ggatcacatt cattgctgcc cggcagggtt tcagtgtcac 1020 acagagaaag gaacctgcga aatgggtatc ctccaagtac cctggatgaa gaaggtcata 1080 gcccccctcc gcctgccaga cccacagatc ttgaagagtg atacaccttg tgatgacttc 1140 actaggtgtc ctacaaacaa tacctgctgc aaactcaatt ctggggactg gggctgctgt 1200 cccatcccag aggctgtctg ctgctcagac aaccagcatt gctgccctca gggcttcaca 1260 tgtctggctc aggggtactg tcagaaggga gacacaatgg tggctggcct ggagaagata 1320 cctgcccgcc agacaacccc gctccaaatt ggagatatcg gttgtgacca gcataccagc 1380 tgcccagtag ggcaaacctg ctgcccaagc ctcaagggaa gttgggcctg ctgccagctg 1440 ccccatgctg tgtgctgtga ggaccggcag cactgttgcc cggccgggta cacctgcaat 1500 gtgaaggcga ggacctgtga gaaggatgtc gattttatcc agcctcccgt gctcctgacc 1560 ctcggcccta aggttgggaa tgtggagtgt ggagaagggc atttctgcca tgataaccag 1620 acctgttgta aagacagtgc aggagtctgg gcctgctgtc cctacctaaa gggtgtctgc 1680 tgtagagatg gacgtcactg ttgccccggt ggcttccact gttcagccag gggaaccaag 1740 tgtttgcgaa agaagattcc tcgctgggac atgtttttga gggatccggt cccaagaccg 1800 ctactgtaa 1809 <210> 4 <211> 1782 <212> DNA <213> Artificial Sequence <220> <223> Human PGRN <400> 4 atgtggaccc tggtgagctg ggtggcctta acagcagggc tggtggctgg aacgcggtgc 60 ccagatggtc agttctgccc tgtggcctgc tgcctggacc ccggaggagc cagctacagc 120 tgctgccgtc cccttctgga caaatggccc acaacactga gcaggcatct gggtggcccc 180 tgccaggttg atgcccactg ctctgccggc cactcctgca tctttaccgt ctcagggact 240 tccagttgct gccccttccc agaggccgtg gcatgcgggg atggccatca ctgctgccca 300 cggggcttcc actgcagtgc agacgggcga tcctgcttcc aaagatcagg taacaactcc 360 gtgggtgcca tccagtgccc tgatagtcag ttcgaatgcc cggacttctc cacgtgctgt 420 gttatggtcg atggctcctg ggggtgctgc cccatgcccc aggcttcctg ctgtgaagac 480 agggtgcact gctgtccgca cggtgccttc tgcgacctgg ttcacacccg ctgcatcaca 540 cccacgggca cccaccccct ggcaaagaag ctccctgccc agaggactaa cagggcagtg 600 gccttgtcca gctcggtcat gtgtccggac gcacggtccc ggtgccctga tggttctacc 660 tgctgtgagc tgcccagtgg gaagtatggc tgctgcccaa tgcccaacgc cacctgctgc 720 tccgatcacc tgcactgctg cccccaagac actgtgtgtg acctgatcca gagtaagtgc 780 ctctccaagg agaacgctac cacggacctc ctcactaagc tgcctgcgca cacagtgggg 840 gatgtgaaat gtgacatgga ggtgagctgc ccagatggct atacctgctg ccgtctacag 900 tcgggggcct ggggctgctg cccttttacc caggctgtgt gctgtgagga ccacatacac 960 tgctgtcccg cggggtttac gtgtgacacg cagaagggta cctgtgaaca ggggccccac 1020 caggtgccct ggatggagaa ggccccagct cacctcagcc tgccagaccc acaagccttg 1080 aagagagatg tcccctgtga taatgtcagc agctgtccct cctccgatac ctgctgccaa 1140 ctcacgtctg gggagtgggg ctgctgtcca atcccagagg ctgtctgctg ctcggaccac 1200 cagcactgct gcccccaggg ctacacgtgt gtagctgagg ggcagtgtca gcgaggaagc 1260 gagatcgtgg ctggactgga gaagatgcct gcccgccggg cttccttatc ccaccccaga 1320 gacatcggct gtgaccagca caccagctgc ccggtggggc agacctgctg cccgagcctg 1380 ggtgggagct gggcctgctg ccagttgccc catgctgtgt gctgcgagga tcgccagcac 1440 tgctgcccgg ctggctacac ctgcaacgtg aaggctcgat cctgcgagaa ggaagtggtc 1500 tctgcccagc ctgccacctt cctggcccgt agccctcacg tgggtgtgaa ggacgtggag 1560 tgtggggaag gacacttctg ccatgataac cagacctgct gccgagacaa ccgacagggc 1620 tgggcctgct gtccctaccg ccagggcgtc tgttgtgctg atcggcgcca ctgctgtcct 1680 gctggcttcc gctgcgcagc caggggtacc aagtgtttgc gcagggaggc cccgcgctgg 1740 gacgcccctt tgagggaccc agccttgaga cagctgctgt ga 1782

Claims (11)

As a pharmaceutical composition for the prophylaxis or treatment of liver disease comprising progrenulin protein as an active ingredient,
The liver disease is any one or more selected from the group consisting of hepatitis, liver toxicity, fatty liver, cirrhosis, liver fibrosis and non-alcoholic liver disease, a pharmaceutical composition for preventing or treating liver disease. The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the progrenulin protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the liver disease is liver fibrosis. The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the liver disease is cirrhosis. As a pharmaceutical composition for preventing or treating liver disease, comprising a vector containing a polynucleotide encoding a progrenulin protein as an active ingredient,
The liver disease is any one or more selected from the group consisting of hepatitis, liver toxicity, fatty liver, cirrhosis, liver fibrosis and non-alcoholic liver disease, a pharmaceutical composition for preventing or treating liver disease. The pharmaceutical composition for preventing or treating liver disease according to claim 5, wherein the polynucleotide encoding the progrenulin protein comprises a polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. The pharmaceutical composition for preventing or treating liver disease according to claim 5, wherein the vector is any one selected from linear DNA, plasmid vector, non-viral expression vector, and viral expression vector. The method of claim 7, wherein the viral expression vector is any one selected from the group consisting of a recombinant retroviral vector, a recombinant adenovirus vector, a recombinant adenovirus vector, a recombinant herpes simplex virus vector, and a recombinant lentivirus vector. Pharmaceutical composition for the prevention or treatment of human liver disease. The pharmaceutical composition for preventing or treating liver disease according to claim 8, wherein the viral expression vector is a recombinant adenovirus vector. The pharmaceutical composition for preventing or treating liver disease according to claim 5, wherein the liver disease is liver fibrosis. The pharmaceutical composition for preventing or treating liver disease according to claim 5, wherein the liver disease is cirrhosis.

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