Diagnostic markers for nonalcoholic fatty liver disease

Abstract

The present invention relates to a diagnostic marker for non-alcoholic fatty liver disease, and more specifically, to a composition and kit that can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy by including a preparation for measuring the level of exosome-derived ApoA-1 protein, and a composition and kit that can accurately predict the prognosis after administration of a therapeutic agent for non-alcoholic steatohepatitis. The composition and kit of the present invention can be used in various ways as a preparation for diagnosing non-alcoholic fatty liver disease, predicting the prognosis of non-alcoholic fatty liver disease, and screening for a therapeutic agent for non-alcoholic fatty liver disease.

Description

[Technical field]

[0001] The present invention relates to a diagnostic marker for non-alcoholic fatty liver disease, and more specifically, to a composition that includes a preparation for measuring the level of exosome-derived ApoA-1 protein and can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy, and a kit including the same. [Background technology]

[0002] Mesenchymal stem cells are pluripotent stromal cells that can differentiate into various cells, including osteoblasts, chondrocytes, muscle cells, and adipocytes. Mesenchymal stem cells can differentiate into various connective tissues, such as cartilage, bone tissue, ligaments, and bone marrow stroma, and are therefore being studied for use in treating various diseases, such as arthritis and soft tissue defects caused by trauma or burns.

[0003] On the other hand, non-alcoholic fatty liver is characterized by the accumulation of triglycerides, a type of neutral fat, in liver cells without excessive alcohol intake. Non-alcoholic fatty liver is becoming more and more prevalent due to overnutrition associated with high-fat and high-carbohydrate intake in modern people. Non-alcoholic fatty liver is often observed in obesity and diabetes, and various factors are thought to be associated with non-alcoholic fatty liver. It has been reported that 80% of adults with non-alcoholic fatty liver develop metabolic disorders such as insulin-resistant diabetes and heart disease.

[0004] Nonalcoholic fatty liver is classified into nonalcoholic simple steatosis and nonalcoholic steatohepatitis (NASH), which is accompanied by inflammation. If left untreated for a long period of time, it can progress to serious liver diseases such as hepatitis, liver fibrosis, and cirrhosis. Nonalcoholic fatty liver is characterized by the accumulation of fat (fatty infiltration) in liver cells (hepatocytes).

[0005] Nonalcoholic simple fatty liver can progress to nonalcoholic steatohepatitis, in which fat accumulation is associated with various levels of liver inflammation and scarring, and is often associated with insulin resistance, dyslipidemia, and hypertension. Nonalcoholic steatohepatitis often occurs in people who are overweight, have high cholesterol and triglyceride levels, and / or have insulin resistance.

[0006] However, in recent years, the number of non-alcoholic steatohepatitis patients has increased with the increase in the obese population, but the development of safe and long-term administrable non-alcoholic steatohepatitis treatment agents has only progressed slightly, and the development of preparations and kits for diagnosing non-alcoholic steatohepatitis is not satisfactory. Therefore, there is an increasing demand for the development of a composition or kit for diagnosing non-alcoholic steatohepatitis that is suitable for diagnosing chronic disease non-alcoholic steatohepatitis, which is accurate and rapid, and for a prognostic diagnostic composition after administration of a non-alcoholic steatohepatitis treatment agent. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present inventors confirmed that non-alcoholic fatty liver disease (NAFLD) can be diagnosed rapidly and accurately using exosome-derived ApoA-1 (Exosomal ApoA-1) protein as a diagnostic marker for non-alcoholic fatty liver disease (NAFLD).

[0008] Therefore, an object of the present invention is to provide a composition for diagnosing non-alcoholic fatty liver disease.

[0009] Another object of the present invention is to provide a kit for diagnosing non-alcoholic fatty liver disease.

[0010] It is yet another object of the present invention to provide a method for providing information for the diagnosis of non-alcoholic fatty liver disease.

[0011] It is still another object of the present invention to provide a method for screening a therapeutic agent for non-alcoholic fatty liver disease.

[0012] It is yet another object of the present invention to provide a composition for predicting the prognosis of non-alcoholic fatty liver disease.

[0013] It is still another object of the present invention to provide a kit for predicting the prognosis of non-alcoholic fatty liver disease.

[0014] It is still another object of the present invention to provide a method for predicting the prognosis of non-alcoholic fatty liver disease. [Means for solving the problem]

[0015] The present invention relates to a diagnostic marker for non-alcoholic fatty liver disease, and the diagnostic composition for non-alcoholic fatty liver disease, the prognosis predicting composition, or the kit thereof according to the present invention can rapidly and accurately diagnose non-alcoholic fatty liver disease and can accurately predict the prognosis after administration of a therapeutic agent for non-alcoholic fatty liver disease.

[0016] Therefore, the present inventors confirmed that the preparation for measuring the level of exosome-derived ApoA-1 protein according to the present invention can diagnose non-alcoholic fatty liver disease and predict the prognosis more accurately than existing methods.

[0017] The present invention will now be described in more detail.

[0018] One aspect of the present invention is a composition for diagnosing non-alcoholic fatty liver disease (NAFLD), comprising a preparation for measuring a level of exosome-derived ApoA-1 protein in an individual suffering from or suspected of suffering from non-alcoholic fatty liver disease (NAFLD).

[0019] The term "marker" as used herein refers to a substance that can diagnose individuals with non-alcoholic fatty liver disease by distinguishing them from normal individuals, and may include any organic biomolecule such as a polypeptide, protein, nucleic acid, lipid, glycolipid, glycoprotein, or sugar that shows an increase or decrease in individuals with non-alcoholic fatty liver disease, for example, a protein whose level is decreased in individuals with non-alcoholic fatty liver disease, but is not limited thereto.

[0020] As used herein, the term "exosome" refers to a membrane vesicle with a lipid bilayer structure that is secreted by cells or present within cells, and is present in the body fluids of almost all eukaryotic organisms. Exosomes have a diameter of about 30 to 1000 nm, and are released from cells when multivesicular bodies fuse with the cell membrane, or are immediately released from the cell membrane. It is well known that exosomes play a role in transporting intracellular biomolecules such as proteins, bioactive lipids, and RNA (miRNA) to mediate coagulation, cell-cell communication, and cellular immunity.

[0021] Exosomes are a concept that encompasses microvesicles. Known marker proteins for exosomes include CD63 and CD81, as well as cell surface receptors such as EGFR, signal transduction-related molecules, cell adhesion-related proteins, MSC-associated antigens, heat shock proteins, and Alix, which are related to vesicle formation.

[0022] The term "ApoA-1 protein" as used herein refers to a protein encoded by the ApoA-1 gene, which is known to play a specific role in lipid metabolism as a major protein component of HDL particles. ApoA-1 is often known to be used as a biomarker for predicting cardiovascular disease, but the use of exosome-derived ApoA-1 protein as a biomarker has not been known. The inventors of the present invention have confirmed that the presence or absence of non-alcoholic fatty liver disease in individuals who have or are suspected of having non-alcoholic fatty liver disease can be accurately diagnosed by measuring the protein level of exosome-derived ApoA-1 in the plasma of the individual.

[0023] As used herein, the term "non-alcoholic fatty liver disease" refers to the most common chronic liver disease, which is known to be closely related to type 2 diabetes, obesity, and metabolic syndrome, and includes diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and even cirrhosis.

[0024] In the present invention, the term "diagnosis" means to confirm the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to confirm the presence or absence of the onset of non-alcoholic fatty liver disease.

[0025] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be non-alcoholic steatohepatitis (NASH).

[0026] The term "non-alcoholic steatohepatitis" as used herein refers to one type of non-alcoholic fatty liver disease, a progressive liver disease characterized by fatty liver accompanied by inflammation or fibrosis, and anterior segment disease that can lead to liver cirrhosis or liver cancer.

[0027] The term "preparation for measuring the level of a protein" as used herein refers to a preparation used in a method for measuring the level of a target protein contained in a sample. The preparation for measuring the level of a protein may include a protein detection preparation known in the art, and may include, but is not limited to, antibodies used in methods such as western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence activated cell sorter analysis (FACS), and protein chip technology assay.

[0028] In one embodiment of the present invention, the agent for measuring the level of a protein may include an antibody or an aptamer specific for the protein.

[0029] In one embodiment of the present invention, the antibody may be any one or more selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, and a recombinant antibody.

[0030] The term "antibody" as used herein means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The antibody may be produced by cloning each gene into an expression vector by a conventional method to obtain a protein encoded by a marker gene, and then producing the antibody by a conventional method from the obtained protein. The form of the antibody is not particularly limited, and any part of a polyclonal antibody, a monoclonal antibody, an antibody fragment, or a recombinant antibody, etc., that has antigen-binding ability is included in the antibody of the present invention. In the present invention, the antibody includes all immunoglobulin antibodies, as well as special antibodies such as humanized antibodies. The antibody includes a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of an antibody molecule. The functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc.

[0031] The term "aptamer" as used herein means a single-stranded oligonucleotide, a nucleic acid molecule that has binding activity to a specific target molecule. Aptamers may have various three-dimensional structures depending on their base sequence, and may have high affinity to a specific substance, such as an antigen-antibody reaction. Aptamers can bind to a specific target molecule and inhibit the activity of the specific target molecule.

[0032] The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or cyclic in form, but is not limited thereto. The aptamer can be easily prepared by a person having ordinary skill in the art by a known method with reference to each base sequence.

[0033] Another aspect of the present invention is a kit for diagnosing non-alcoholic fatty liver disease, comprising a preparation for measuring a level of exosome-derived ApoA-1 protein in an individual suffering from or suspected of suffering from non-alcoholic fatty liver disease.

[0034] In the present invention, the kit may contain a base material, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like for immunological detection of an antibody.

[0035] The substrate may be, but is not limited to, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, or a glass slide, and the like, the color-developing enzyme may be, but is not limited to, peroxidase or alkaline phosphatase, the fluorescent substance may be, but is not limited to, FITC or RITC, and the color-developing substrate solution may be, but is not limited to, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethylbenzidine).

[0036] In the present invention, the kit may further include reagents for use in diagnosing non-alcoholic fatty liver disease in an individual. The reagents may include, but are not limited to, a buffer, an indicator, or a combination thereof.

[0037] In one embodiment of the present invention, the kit may be an enzyme-linked immunosorbent assay (ELISA) kit.

[0038] Yet another aspect of the present invention is a method for providing information for diagnosing non-alcoholic fatty liver disease in an individual, comprising a measuring step of measuring a level of exosome-derived ApoA-1 protein from a biological sample isolated from an individual suffering from or suspected of suffering from non-alcoholic fatty liver disease.

[0039] In one embodiment of the present invention, the method may further include a comparison step of comparing the measured protein levels with values ​​measured from normal control samples.

[0040] In one embodiment of the present invention, the measuring step may include a contacting step of contacting the sample with an antibody or aptamer that specifically binds to ApoA-1 protein.

[0041] The term "individual" as used herein can refer to any animal, including humans, that suffers from or may suffer from nonalcoholic fatty liver disease, including, but not limited to, mammals such as humans, cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, and the like, that require treatment for similar conditions.

[0042] As used herein, the term "normal control group" refers to individuals who do not suffer from or are not suspected of suffering from non-alcoholic fatty liver disease.

[0043] As used herein, the term "sample" refers to a direct subject isolated from a patient suffering from non-alcoholic fatty liver disease and for measuring the expression level of exosome-derived ApoA-1 protein.

[0044] In one embodiment of the present invention, the sample may be any one or more selected from the group consisting of tissue, cells, whole blood, serum, and plasma isolated from an individual.

[0045] In an embodiment of the present invention, the method may further include a step of determining whether or not the patient has non-alcoholic fatty liver disease when the value measured from the biological sample is lower than the value measured from a normal control sample.

[0046] Yet another aspect of the present invention is a method for screening a therapeutic agent for non-alcoholic fatty liver disease, comprising: a measuring step of treating a biological sample isolated from an individual suffering from the disease with a candidate substance for treating non-alcoholic fatty liver disease, and measuring an expression level of exosome-derived ApoA-1 protein; and a comparing step of comparing the measured protein expression level with an expression level of a control group not treated with the candidate substance.

[0047] As used herein, the term "candidate therapeutic agent" refers to a substance that increases the level of ApoA-1 protein in exosomes measured from an individual's sample and has the potential to treat non-alcoholic fatty liver disease, and includes, without limitation, oligonucleotides, proteins, compounds, etc.

[0048] As used herein, the term "oligonucleotide" refers to a polymer formed by polymerization of several to several tens of nucleotides via phosphodiester bonds.

[0049] In the comparison step of the screening method of the present invention, if the exosome-derived ApoA-1 protein level in the biological sample isolated from an individual is significantly increased compared to the control group, this means that the candidate substance has potential as a therapeutic agent for non-alcoholic fatty liver disease.

[0050] Yet another aspect of the present invention is a composition for predicting the prognosis of non-alcoholic fatty liver disease, comprising a preparation for measuring the level of exosome-derived ApoA-1 protein in an individual administered a therapeutic agent for non-alcoholic fatty liver disease.

[0051] As used herein, the term "therapeutic agent for non-alcoholic fatty liver disease" refers to any substance that can treat non-alcoholic fatty liver disease or improve or alleviate the severity or related parameters of non-alcoholic fatty liver disease. For example, the present inventors have confirmed that exosomes isolated from induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSC) are more effective in treating and alleviating non-alcoholic fatty liver disease than existing therapeutic agents used in clinical practice.

[0052] The term "prognosis" as used herein refers to the act of predicting the course of disease and the outcome of death or survival. More specifically, prognosis or prognosis prediction may be interpreted as any act of predicting the course of disease before / after treatment by comprehensively considering the state of the patient, since the course of disease may vary depending on the physiological or environmental condition of the patient. For the purposes of the present invention, prognosis may be interpreted as the act of predicting the disease-free survival rate or survival rate of patients with non-alcoholic fatty liver disease by predicting the course of disease and whether or not it will be cured before / after treatment of non-alcoholic fatty liver disease.

[0053] For example, predicting "good prognosis" means that the patient with non-alcoholic fatty liver disease has a high survival rate or an improved level of indicators associated with fatty liver disease, regardless of whether or not they are treated, and that the patient with non-alcoholic fatty liver disease is likely to be treated, while predicting "poor prognosis" means that the patient has a low survival rate or a worsening level of indicators associated with fatty liver disease after treatment for non-alcoholic fatty liver disease.

[0054] As an example, the present inventors confirmed that measuring the level of exosome-derived ApoA-1 protein can more accurately determine whether or not non-alcoholic fatty liver disease has improved, compared to measuring the level of ApoA-1 protein in serum, which is currently used in clinical practice.

[0055] In one embodiment of the present invention, the therapeutic agent for non-alcoholic fatty liver disease may be a composition comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells as an active ingredient.

[0056] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to self-renew and differentiate into two or more different types of cells. The stem cells of the present invention may be autologous or allogeneic stem cells.

[0057] As used herein, the term "induced pluripotent stem cells" refers to cells that have been induced to dedifferentiate already differentiated cells, such as somatic cells, to return to their initial undifferentiated state and acquire full pluripotency.

[0058] The dedifferentiation may be induced by introducing and expressing a specific gene (e.g., Sox2, c-Myc, Klf4, Oct-4, etc.) or by injecting a dedifferentiation-inducing protein produced in cells into which the specific gene has been introduced.

[0059] The term "total differentiation potential" refers to the ability to differentiate into tissues or organs originating from the three germ layers that constitute the living body, namely, the endoderm, mesoderm, and ectoderm.

[0060] As used herein, the term "mesenchymal stem cells" refers to cells that have multipotency and can differentiate into various cells including osteoblasts, chondrocytes, muscle cells, adipocytes, etc. As for the mesenchymal stem cells, bone marrow-derived mesenchymal stem cells are most commonly used, but they can also be derived from the umbilical cord or umbilical cord blood, adipose tissue, amniotic fluid, and the tooth bud of the molar, in addition to bone marrow. Mesenchymal stem cells are also called stromal cells.

[0061] The term "induced pluripotent stem cells" refers to cells that have been induced to have pluripotent differentiation ability through an artificial dedifferentiation process from differentiated cells, and are also called dedifferentiated stem cells.

[0062] In the present invention, the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells may not express stage-specific embryonic antigen 4 (SSEA-4) protein.

[0063] As used herein, the term "precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells" refers to cells that are at the stage immediately prior to complete differentiation from induced pluripotent stem cells into mesenchymal stem cells, can be considered a type of mesenchymal stem cell derived from induced pluripotent stem cells, do not express SSEA-4 protein, and will acquire the properties of a complete mesenchymal stem cell upon further culture.

[0064] In the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may be differentiated from precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 protein.

[0065] The artificial dedifferentiation process can be carried out by using viral-mediated or non-viral vectors using retroviruses, lentiviruses and Sendai viruses, by introducing non-viral-mediated dedifferentiation factors using proteins and cell extracts, etc., or by introducing dedifferentiation processes using stem cell extracts, compounds, etc.

[0066] The induced pluripotent stem cells have almost the same characteristics as embryonic stem cells; specifically, they show similar cell morphology, have similar gene and protein expression patterns, have full differentiation potential in vitro and in vivo, form teratomas, form chimera mice when inserted into mouse blastocysts, and are capable of germline transmission of genes.

[0067] The induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all mammals, such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, but are preferably induced pluripotent stem cells derived from humans.

[0068] Furthermore, the somatic cells before dedifferentiation of the induced pluripotent stem cells of the present invention may be somatic cells derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, amniotic fluid, placenta, etc., but are not limited thereto.

[0069] In the present invention, exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells refer to exosomes present within the above-mentioned induced pluripotent stem cell-derived mesenchymal stem cells (BxC) or secreted from BxC.

[0070] As used herein, the term "containing as an active ingredient" means that the exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells contain a sufficient amount of exosomes to achieve preventive or therapeutic activity against non-alcoholic fatty liver disease.

[0071] In one embodiment of the present invention, the therapeutic agent for non-alcoholic fatty liver disease may be a composition comprising, as an active ingredient, exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with a pretreatment substance.

[0072] As used herein, the term "pretreatment" refers to a process of contacting a cell culture medium containing a pretreatment substance with a progenitor cell of an induced pluripotent stem cell-derived mesenchymal stem cell during the culture process of the progenitor cell of an induced pluripotent stem cell-derived mesenchymal stem cell.

[0073] In one embodiment of the present invention, the pretreatment material may be 1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid or Exendin-4.

[0074] 1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid, which may go by the name "Lanifibranor", is an agonist of peroxisome proliferator-activated receptors (PPARs).

[0075] In one embodiment of the present invention, the therapeutic agent for non-alcoholic fatty liver disease may be a composition (BxC-V37e) containing, as an active ingredient, exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with lanifibranol.

[0076] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be non-alcoholic steatohepatitis.

[0077] In one embodiment of the present invention, the preparation for measuring the level of a protein may comprise an antibody or an aptamer specific to the protein.

[0078] In one embodiment of the present invention, the antibody may be any one or more selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, and a recombinant antibody.

[0079] Yet another embodiment of the present invention is a kit for predicting the prognosis of non-alcoholic fatty liver disease, comprising a preparation for measuring the level of exosome-derived ApoA-1 protein in an individual administered a therapeutic agent for non-alcoholic fatty liver disease.

[0080] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be non-alcoholic steatohepatitis.

[0081] In one embodiment of the present invention, the preparation for measuring the level of a protein may comprise an antibody or an aptamer specific to the protein.

[0082] In one embodiment of the present invention, the kit may be an enzyme-linked immunosorbent assay (ELISA) kit.

[0083] Yet another aspect of the present invention is a method for providing information for predicting the prognosis of non-alcoholic fatty liver disease, comprising a measuring step of measuring a level of exosome-derived ApoA-1 protein from a biological sample isolated from an individual to which a therapeutic agent for non-alcoholic fatty liver disease has been administered.

[0084] In one embodiment of the present invention, the method may further include a comparison step of comparing the measured protein levels with values ​​measured from normal control samples.

[0085] In one embodiment of the present invention, the measuring step may include a contacting step of contacting the sample with an antibody or an aptamer that specifically binds to the protein.

[0086] In one embodiment of the present invention, the biological sample may be any one or more selected from the group consisting of tissue, cells, whole blood, serum, and plasma isolated from an individual.

[0087] In one embodiment of the present invention, the method may determine that the risk of developing non-alcoholic fatty liver disease is high or the prognosis of non-alcoholic fatty liver disease is poor when the level of exosome-derived ApoA-1 protein measured from a sample isolated from an individual suspected of developing non-alcoholic fatty liver disease is lower than the level measured from a sample isolated from a normal control group.

[0088] In an embodiment of the present invention, the method may further include a step of determining that the risk of developing non-alcoholic fatty liver disease is high or the prognosis of non-alcoholic fatty liver disease is poor when the level of exosome-derived ApoA-1 protein measured from a sample isolated from an individual suspected of developing non-alcoholic fatty liver disease is lower than the level measured from a sample isolated from a normal control group.

[0089] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be non-alcoholic steatohepatitis (NASH). Effect of the Invention

[0090] The present invention relates to a diagnostic marker for non-alcoholic fatty liver disease, and more specifically, to a composition and kit that can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy by including a preparation for measuring the level of exosome-derived ApoA-1 protein, and a composition and kit that can accurately predict the prognosis after administration of a therapeutic agent for non-alcoholic steatohepatitis. The composition and kit of the present invention can be used in various ways as a preparation for diagnosing non-alcoholic fatty liver disease, predicting the prognosis of non-alcoholic fatty liver disease, and screening for a therapeutic agent for non-alcoholic fatty liver disease. [Brief description of the drawings]

[0091] [Figure 1] Heatmap showing the proteomic signatures of BxC-e (exosomes from non-pretreated BxC) and BxC-V37e (exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with lanifibranor).

[0092] [Diagram 2] 1 is a graph showing the drug signature of BxC-V37e exosomes according to one embodiment of the present invention.

[0093] [Diagram 3] KEGG pathway of BxC-V37e signature in HepG2. Red notation indicates BxC-V37e signature protein, blue notation indicates signature gene upregulated by BxC-V37e treatment, black notation indicates targets associated with BxC-V37e signature drug.

[0094] [Figure 4] 1 shows photographs of liver tissues from normal and methionine / choline deficient (MCD-diet)-induced NASH mice administered PBS or BxC-V37e according to an embodiment of the present invention.

[0095] [Diagram 5] 1 is a graph showing ALT levels measured from serum of NASH mice administered with PBS or BxC-V37e according to one embodiment of the present invention (normal; n=6, MCD-diet; n=5).

[0096] [Figure 6] 1 is a graph showing AST levels measured from serum of NASH mice administered with PBS or BxC-V37e according to one embodiment of the present invention (normal; n=6, MCD-diet; n=5).

[0097] [Figure 7] Photographs showing the results of hematoxylin and eosin staining in liver tissue of MCD diet-induced mice administered PBS or BxC-V37e according to one embodiment of the present invention (GLP-1R agonist was used as a positive control group, scale bar 50 μm).

[0098] [Figure 8] FIG. 1 is a graph showing the results of analyzing the NAFLD activity score (NAS) using indices of inflammation, hypertrophy, and adiposity (data are shown as mean±SEM, *p<0.05; ***p<0.001).

[0099] [Figure 9] FIG. 1 shows the adipogenesis-suppressing effect of exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cell precursor cells (BxC).

[0100] [Figure 10] 1 is a photograph showing the results of immunoblot analysis (Western blot) of exosome-derived ApoA-1 obtained from the serum of NASH mice administered PBS, a GLP-1R agonist, or BxC-V37e according to one embodiment of the present invention.

[0101] [Figure 11]1 is a graph showing the results of immunoblot analysis (Western blot) of exosomal ApoA-1 obtained from the serum of NASH mice administered PBS, a GLP-1R agonist, or BxC-V37e according to one embodiment of the present invention.

[0102] [Figure 12] 1 is a graph showing the results of measuring human exosome-derived ApoA-1 levels in serum from a control group (n=20) and obese NAFLD subjects with or without diabetes (n=16) or without diabetes (n=19) according to one embodiment of the present invention.

[0103] [Figure 13] 13 to 24 are graphs showing the correlation between exosome-derived ApoA-1 protein levels in human serum and metabolic parameters [BMI, ALT, AST, SBP (systolic blood pressure), DBP (diastolic blood pressure), insulin, HOMA-IR, Hs-CRP, TG, TC, HIS, and NAFLD liver fat score] according to one embodiment of the present invention. Figure 13 shows the correlation between exosome-derived ApoA-1 protein levels in human serum and BMI. [Figure 14] 1 shows the correlation between exosome-derived ApoA-1 protein levels in human serum and ALT according to one embodiment of the present invention. [Figure 15] 1 shows a correlation between exosome-derived ApoA-1 protein level in human serum and AST according to one embodiment of the present invention. [Figure 16] 1 shows the correlation between exosome-derived ApoA-1 protein level in human serum and SBP according to one embodiment of the present invention. [Figure 17] 1 shows a correlation between exosome-derived ApoA-1 protein levels in human serum and DBP according to one embodiment of the present invention. [Figure 18] 1 shows the correlation between exosome-derived ApoA-1 protein level in human serum and insulin according to one embodiment of the present invention. [Figure 19] 1 shows a correlation between exosome-derived ApoA-1 protein levels in human serum and HOMA-IR according to one embodiment of the present invention. [Figure 20] 1 shows the correlation between exosome-derived ApoA-1 protein level in human serum and Hs-CRP according to one embodiment of the present invention. [Figure 21] 1 shows the correlation between exosome-derived ApoA-1 protein level in human serum and TG according to one embodiment of the present invention. [Figure 22] 1 shows the correlation between exosome-derived ApoA-1 protein level in human serum and TC according to one embodiment of the present invention. [Figure 23] 1 shows a correlation between exosome-derived ApoA-1 protein level in human serum and HIS according to one embodiment of the present invention. [Figure 24] 1 shows a correlation between exosome-derived ApoA-1 protein levels in human serum and NAFLD liver fat score according to one embodiment of the present invention.

[0104] [Diagram 25] Figures 25 to 36 are graphs showing the correlation between ApoA-1 protein levels in human serum and metabolic parameters [BMI, ALT, AST, SBP (Systolic blood pressure), DBP (Diastolic blood pressure), insulin, HOMA-IR, Hs-CRP, TG, TC, HIS, and NAFLD liver fat score] according to one embodiment of the present invention. Figure 25 shows the correlation between ApoA-1 protein levels in human serum and BMI. [Figure 26] 1 shows the correlation between ApoA-1 protein levels in human serum and ALT according to one embodiment of the present invention. [Figure 27] 1 shows the correlation between ApoA-1 protein levels in human serum and AST according to one embodiment of the present invention. [Figure 28] 1 shows the correlation between ApoA-1 protein levels in human serum and SBP according to one embodiment of the present invention. [Figure 29] 1 shows the correlation between ApoA-1 protein levels in human serum and DBP according to one embodiment of the present invention. [Diagram 30] 1 shows the correlation between ApoA-1 protein levels in human serum and insulin according to one embodiment of the present invention. [Diagram 31] 1 shows the correlation between ApoA-1 protein levels in human serum and HOMA-IR according to one embodiment of the present invention. [Diagram 32] 1 shows the correlation between ApoA-1 protein levels in human serum and Hs-CRP according to one embodiment of the present invention. [Diagram 33] 1 shows the correlation between ApoA-1 protein level in human serum and TG according to one embodiment of the present invention. [Diagram 34] 1 shows the correlation between ApoA-1 protein levels in human serum and TC according to one embodiment of the present invention. [Diagram 35] 1 shows the correlation between ApoA-1 protein levels in human serum and HIS according to one embodiment of the present invention. [Diagram 36] 1 shows the correlation between ApoA-1 protein levels in human serum and NAFLD liver fat scores according to one embodiment of the present invention.

[0105] [Figure 37] This graph shows the correlation between the rate of change in AST values, an existing NASH-related parameter, for serum ApoA-1 and exosome-derived ApoA-1 in MCD-fed mice administered dulaglutide, a known candidate substance for the treatment of NASH, compared to a PBS-administered group.

[0106] [Figure 38] This graph shows the correlation between the rate of change in hs-CRP values, an existing NASH-related parameter, for serum ApoA-1 and exosome-derived ApoA-1 when MCD-fed mice were administered dulaglutide, a known candidate substance for the treatment of NASH, compared to a PBS-administered group.

[0107] [Figure 39] This graph shows the correlation between the rate of change in ACC1 values, an existing NASH-related parameter, for serum ApoA-1 and exosome-derived ApoA-1 in MCD-fed mice administered dulaglutide, a known candidate substance for the treatment of NASH, compared to a PBS-administered group.

[0108] [Diagram 40] This graph shows the correlation between the rate of change in NRF2 values, an existing NASH-related parameter, for serum ApoA-1 and exosome-derived ApoA-1 in MCD-fed mice administered dulaglutide, a known candidate substance for the treatment of NASH, compared to a PBS-administered group.

[0109] [Diagram 41] 1 is a graph showing the correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in AST values, an existing NASH-related parameter, in MCD-fed mice administered BxC-V37e compared to a PBS-administered group.

[0110] [Diagram 42] This is a graph showing the correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in hs-CRP values, an existing NASH-related parameter, when MCD-fed mice were administered BxC-V37e compared to a PBS-administered group.

[0111] [Diagram 43] 1 is a graph showing the correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in ACC1 values, an existing NASH-related parameter, in MCD-fed mice administered BxC-V37e compared to a PBS-administered group.

[0112] [Diagram 44] 1 is a graph showing the correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in PCNA values, an existing NASH-related parameter, in MCD-fed mice administered BxC-V37e compared to a PBS-administered group.

[0113] [Diagram 45] 1 is a graph showing the correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in hs-CRP values, an existing NASH-related parameter, when MCD-fed mice were administered BxC-e compared to a PBS-administered group.

[0114] [Figure 46] This is a graph showing the correlation between changes in serum ApoA-1 and exosome-derived ApoA-1 staining results with Oil red O, an existing NASH-related parameter, in MCD-fed mice administered BxC-e compared to a PBS-fed group.

[0115] [Figure 47] 1 is a graph showing the correlation between the rate of change in Annexin A5 values, an existing NASH-related parameter, for serum ApoA-1 and exosome-derived ApoA-1 in MCD-fed mice administered BxC-e compared to a PBS-fed group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] The present invention relates to a composition for predicting the prognosis of non-alcoholic fatty liver disease (NAFLD), comprising a preparation for measuring the level of exosome-derived ApoA-1 protein in an individual administered a therapeutic agent for NAFLD. EXAMPLES

[0117] The present invention will now be described in more detail with reference to the following examples, although these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0118] Example 1: Materials and Methods

[0119] 1-1.Animal testing Six-week-old C57BL / 6 male wild-type (WT) mice were obtained from Koatech Co., Ltd. and were fed a Chow diet (n=6) or a methionine / choline-deficient diet (MCD-diet) (n=15) for 12 weeks. Animal care and procedures were approved by the Rodent Animal Facility Area of ​​Knotus Co., Ltd., Republic of Korea (approval number: 19-KE-265). Mice fed the MCD-diet were subcutaneously injected with 2 nmol / kg dulaglutide (GLP-1 receptor agonist) every other day for 4 weeks at 18 weeks, and intravenously injected with 400 μg of BxC-e and BxC-V37e per mouse once a day, three times a week for 4 weeks. At the end of the experiment, the mice were anesthetized, and serum and liver tissue were collected. Environmental conditions were set to maintain the following: temperature, 23±3°C; relative humidity, 55±15%; ventilation, 10-20 air changes per hour; light intensity, 150-300 Lux; and regulated with a 12-hour light / 12-hour dark cycle.

[0120] 1-2.Cell culture For cell maintenance, human primary hepatocytes (ScienCell, Carlsbad, CA, USA) were cultured in Hepatocyte Basal Medium with 5% fetal bovine serum (FBS), 1% penicillin antibiotic and growth supplements (ScienCell) and THP-1 mononuclear cells (ATCC, Manassas, VA, USA) and growth supplements (ScienCell) and THP-1 mononuclear cells (ATCC, Manassas, VA, USA) were cultured in RPMI medium (Gibco, Waltham, MA, USA) with 10% FBS (Hyclone, Chicago, IL, USA) and 1% antibiotic / antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). To generate a NASH in vitro model, human primary hepatocytes were treated with 100 mM FA (2 oleic acid:1 palmitic acid) in 2% FBS+DMEM for 48 h, and then fed with 100 μg / mL BxC-V37e in serum-free DMEM containing 100 mM FA. 500 nM thapsigargin was also treated simultaneously with BxC-V37e in serum-free DMEM for 24 h. Meanwhile, THP-1 mononuclear cells were stimulated with 200 ng / mL PMA, 100 ng / mL LPS, and 20 ng / mL IFNγ in 10% FBS+RPMI for 24 h. Then, 100 μg / mL BxC-V37e was treated with 100 ng / mL LPS and 20 ng / mL IFNγ in serum-free DMEM for 24 h. Both cells were incubated in 5% CO 2 The cells were grown at 37°C in a 95% humidified incubator. To assess phosphorylation levels, human primary hepatocytes and THP-1 macrophages were treated with 100 μg / mL BxC-V37e for 24 h. Then, 100 mM FA or 200 ng / mL PMA, 100 ng / mL LPS, and 20 ng / mL IFNγ were added to primary hepatocytes for 30 min (phospho-Akt and phospho-AMPK) or to THP-1 macrophages for 10 min (phospho-p65), respectively.

[0121] 1-3. Isolation and culture of mesenchymal stem cell precursors (BxC) derived from induced pluripotent stem cells (iPSC) Induced pluripotent stem cells (iPSCs) were cultured for 7 days in DMEM supplemented with 10% FBS and 10 ng / ml bFGF. Next, SSEA-4(-) cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein on the cell surface were isolated from the cultured induced pluripotent stem cells using FACS. The isolated SSEA-4(-) cells were passaged and further cultured for 7 days in the same medium as above to produce progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention. The present inventors named the progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells as BxC (brexogen stem cells).

[0122] The precursor cells of induced pluripotent stem cells-derived mesenchymal stem cells, designated BxC, were further cultured in culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100X) (Gibco, Cat no. 11140-050)].

[0123] 1-4. Isolation of exosomes (BxC-e) derived from induced pluripotent stem cell-derived mesenchymal stem cell precursor cells (BxC) The culture medium of the cultured induced pluripotent stem cell-derived mesenchymal stem cell precursor cells (hereinafter referred to as BxC) was collected and centrifuged at 300xg for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered through a 0.22μm filter, then centrifuged at 10,000xg at 4℃ for 70 minutes using a high speed centrifuge. The centrifuged supernatant was further taken and centrifuged at 100,000xg at 4℃ for 90 minutes using an ultracentrifuge to remove the supernatant. The exosomes remaining in the bottom layer were diluted in PBS (phosphate buffered saline) and used in the following experiments.

[0124] 1-5. Culture of pan-PPAR agonist-pretreated iMSCs and RNA-seq analysis iMSCs (passage 4) were cultured in T-75 flasks (Eppendorf, Hamburg, Germany) at 37°C and 5% CO 2 The cells were cultured in 15% fetal bovine serum (FBS), high glucose Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Chicago, IL, USA) and 1% antibiotic / antimycotic (Hyclone, Chicago, IL, USA) at 95% humidity. Upon reaching 90% confluency, the cells were detached using TryPLE Express (Thermo Fisher Science) and cultured at 10,000 cells / cm. 2 The cells were seeded into a 4-layer Cell Factory system (Thermo Fisher Science) at a density of 10 μM each. The next day, the cells were treated with 10 μM Lanifibranor (Cayman, Ann Arbor, MI, USA) for 24 h, after which the medium was aspirated and washed with Dulbecco's phosphate buffered saline (DPBS) (Hyclone).

[0125] RNA sequencing was performed using the RNA sequencing protocol by Macrogen's application program. Hierarchical clustering, a similarity measure to present patterns of differentially varied scripts that satisfied |fold changes| ≥ 2 and independent t-test p < 0.05, was analyzed using complete linkage and Euclidean distance algorithms. Gene enrichment and pathway analysis were performed on significant gene lists based on gProfiler (https: / / biit.cs.ut.ee / gprofiler / gost) and KEGG pathways (http: / / www.genome.jp / kegg / pathway).

[0126] 1-6. Isolation of exosomes from iMSCs pretreated with pan-PPAR agonists Exosomes (BxC-V37e) derived from lanifibranol-pretreated iMSCs were isolated as follows.

[0127] Lanifibranol-pretreated iMSC medium was replaced with serum-free, xono-free StemPro MSC medium (Gibco). After 3 days of culture, the cultures were harvested and centrifuged at 300g for 10 min, and the supernatant was centrifuged at 2,000g for 20 min. The supernatant was centrifuged at 10,000g for an additional 80 min. The supernatant was then filtered through a 0.2 μm vacuum filter (Merck Millipore, Burlington, MA, USA). Finally, exosomes were isolated by ultracentrifugation at 100,000g for 80 min, after which the pellet was washed with PBS and ultracentrifuged (Beckman Coulter, CA, USA), and the exosome pellet was resuspended in PBS.

[0128] 1-7. Cryogenic Transmission Electron Microscope (TEM) 200-mesh copper grids (MiTeGen, Ithaca, NY, USA) coated with formvar / carbon film were treated to make them hydrophilic. EV suspension (4 μL) was placed on the grid and blotted for 90 s at 100% humidity and 4 °C temperature, respectively. Extracellular vesicles on the grid were visualized at 36,000x magnification using a Talos L120C FEI TEM (Thermo Fisher Scientific) at 120 kv.

[0129] 1-8. Nanoparticle Tracking Analysis (NTA) Analysis Particle size distribution and concentration measurements for BxC-V37e were performed on a nanosight NS300 instrument (Malvern Panalytical, Malvern, UK) based on NTA. For analysis, BxC-V37e was diluted with sterile PBS (1:100) to reach the optimal volume of NTA. Measurements were performed with several replicates using a Blue 488 nm laser and sCMOS camera at room temperature ranging from 23.0 to 25.2 °C. Sample analysis was performed for 10 min with camera settings and processing conditions such as shutter 600, gain 250, camera level 10, NTA version 3.00064, and sensing threshold 10.

[0130] 1-9. BxC-V37e labeling with DiR and DiD and fluorescence imaging BxC-V37e was incubated with 1 μg / mL DiR buffer for 10 min at 37°C according to the protocol of Lipophilic Tracers (Invitrogen, Waltham, MA, USA). Then, DiR-labeled BxC-V37e was centrifuged at 100,000xg and 4°C for 80 min and washed with PBS (Gibco). Finally, 200 or 400 μg of DiR-labeled BxC-V37e was resuspended in 0.1 mL PBS and injected into C56BL / 6 mice via intravenous route. After 24 h, DiR-labeled BxC-V37e was detected by In Vivo Imaging System (IVIS) (Caliper Life Sciences, Waltham, MA, USA) at an excitation wavelength of 740 nm, an excitation wavelength of 790 nm, and an emission wavelength of 790 nm. The intensity of the region of interest (ROI) is expressed in maximum photons per second per square centimeter per steradian (p / s / cm 2 The concentration of DiD-labeled BxC-V37e was expressed in units of 100 / sr. The procedure for preparing DiD-labeled BxC-V37e was the same as that described above. DiD-labeled BxC-V37e was treated with human primary hepatocytes or THP-1 macrophages for 24 hours with or without stimulation. After 24 hours, DiD-labeled BxC-V37e was observed under a Nikon Eclipse Ti2-U fluorescence microscope (Nikon, Tokyo, Japan).

[0131] 1-10. Bioinformatics analysis Total RNA was collected from HepG2 cells 6 hours after treatment with 100 mM fatty acid (2 oleic acid: 2 palmitic acid) using the RNeasy mini kit (Qiagen, Hilden, Germany). The collected RNA was profiled using GeneChip® Human Gene 2.0 ST arrays (Affymetrix, Santa Clara, CA, USA). Fatty acid-induced differentially expressed genes (DEGs) were identified with a fold change cutoff of 1.5. DEGs underwent gene set enrichment analysis using KEGG with an FDR q-value cutoff of 0.05 (http: / / www.gsea-msigdb.org / gsea). BxC-V37e signatures were constructed by transcriptomic, proteomic, and connectivity map analysis. BxC-V37e was applied to fatty acid-treated HepG2 cells, and total RNA was profiled as previously described. DEGs induced by BxC-V37e were identified with a fold change cutoff of 1.5. BxC-V37e-enriched proteins were qualitatively and quantitatively identified using LC-MS / MS (Yonsei Proteomics Institute, Seoul). DEGs induced by BxC-V37e were identified as drugs with similar transcript profiles to BxC-V37e and their target genes by connectivity map analysis. DEGs induced by BxC-V37e, BxC-V37e proteins, and the target genes of confirmed BxC-V37e-like drugs were identified as BxC-V37e signature. The established BxC-V37e signature was subjected to protein-protein interaction network and functional enrichment analysis at an interaction confidence level of 0.9 with (https: / / string-db.org).

[0132] 1-11.Flow cell analysis The isolated exosomes were stained using the MACSPlex Exosome Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed with an Attune NxT flow cytometer (Thermo Fisher Scientific). To analyze the effect of exosomes on hepatocyte regeneration, hepatocytes were stained with anti-human CD90APC-Cy7 (BioLegend, San Diego, CA, USA) after exosome treatment and analyzed with an Attune NxT flow cytometer (Thermo Fisher Scientific). To confirm whether iMSCs express typical cell surface markers for MSCs, iMSCs were stained with CD73APC, CD105PE, CD45FITC, CD31PE, and CD34APC (eBioscience, Waltham, MA, USA) and CD90APC-Cy7 (BioLegend). Analysis was performed with an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0133] 1-12. Serum biochemistry test Serum samples were collected 4 weeks after BxC-V37e injection and the following parameters were examined using a blood biochemical analyzer (7180, Hitachi, Japan): ALT (alanine transaminase), AST (aspartate transaminase), TG (triglyceride), glucose, TC (total cholesterol), HDL_C (high-density lipoprotein cholesterol), LDL_C (low-density lipoprotein cholesterol), LDH (lactate dehydrogenase), and GGT (gamma-glutamyltransferase).

[0134] 1-13. Real-time qPCR Total RNA was isolated from liver tissues and various cell types using TRIzol® (Ambion, Waltham, MA, USA). cDNA was synthesized using 1 μg of total RNA with AccuPower® CycleScript RT PreMix dT20 (Bioneer, Daejeon, South Korea). Amplification reactions were performed using PowerSYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. Gene expression levels were measured by real-time qPCR using the QuantStudioTM 5 Real-Time PCR system (Applied Biosystems).

[0135] GAPDH was used as a reference gene to normalize the difference in mRNA abundance in each sample. Relative gene expression levels were calculated using a 2 -ΔΔ Analysis was performed using the Ct method, and each experiment was performed in triplicate.

[0136] 1-14. Western blot Cells or tissues were lysed with NP40 lysis buffer (Life Technologies, Carlsbad, CA, USA) supplemented with protease inhibitors (Thermo Fisher Scientific). Protein concentrations were determined using Bradford Assay™ reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Samples were diluted 3:1 with 4x Laemmli buffer (Bio-Rad Laboratories) and heated at 100°C for 10 min. Proteins were loaded and separated on precast polyacrylamide Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with EveryBlot blocking buffer (Bio-Rad Laboratories) for 5 min and then incubated with primary antibodies overnight at 4°C. All primary antibodies were diluted in EveryBlot blocking buffer. The primary antibodies used were as follows: anti-GM130, PCNA, AMPK, phospho-AMPK (Thr172), phospho-p65 (Ser536), Pan-Akt, Phospho-Akt (Thr308) (Cell Signaling Technology, Leiden, The Netherlands), anti-CD9, Calnexin, Abca1, IL-1β, p65, ACC1, Annexin5, β-actin, GAPDH (Abcam, Cambridge, UK), anti-ApoA-1 (LSBio, Seattle, WA, USA), anti-TSG101, CD81 (Invitrogen), anti-TNF-α, PGC-1α, NRF2, and CHOP (Novusbio, Centennial, CO, USA). All target proteins except CD81 were performed under reducing conditions. The membrane was washed five times for 10 min and then incubated with secondary antibodies for 1 h. The secondary antibodies used were anti-rabbit IgG and anti-mouse IgG (Abcam).After washing the membrane five times for 10 min, target proteins were detected using ECL Select™ Western blotting detection reagents (GE Healthcare, Little Chalfont, UK), and images were analyzed using a ChemiDoc imaging system (Bio-Rad Laboratories).

[0137] 1-15. Enzyme-linked immunosorbent assay (ELISA) ELISA assays were performed using commercially available mouse ELISA kits. Insulin (Novus bio), hs-CRP (R&D systems), FFA (free fatty acids), and ApoA-1 (Abcam) assays were performed according to the manufacturer's protocols. The analytical sensitivity for insulin was <0.19 ng / mL, with intra- and inter-assay coefficients of variance of <5.93% and <6.35%. The analytical sensitivity for hs-CRP was <0.015 ng / mL, with intra- and inter-assay coefficients of variance of <7.7% and <10.8%. The analytical sensitivity for ApoA-1 was 11.2 pg / mL, with intra- and inter-assay coefficients of variance of <4.7% and <5.6%, respectively.

[0138] 1-16.Histopathological analysis Liver tissues were fixed in 10% paraformaldehyde and subjected to general tissue processing such as sectioning, dehydration, and paraffin embedding. 5 μm liver tissue cross-sectional slices were attached to slides, and the specimens were deparaffinized using xylene. Ethanol-rehydrated tissues were stained with hematoxylin and eosin (H&E). For Oil red O staining, tissues were embedded using OCT compound (Sakura Finetek, Torrance, CA, USA) and then sectioned at 20 μm using a cryotome (Leica, Wetzlar, Germany). Histopathological samples were then analyzed using a Zen2.3 Blue Edition image analyzer (Carl Zeiss, Oberkochen, Germany) and normalized to percentage of stained area by total area. The NAFLD activity score (NAS) was determined by histological criteria, and the levels of macrovesicular steatosis, microvesicular steatosis, and hypertrophy were scored from 0 to 3 based on the area occupied by the total area. Steatosis and hypertrophy were both scored at a magnification of 40 to 100. Inflammation scores were scored from 0 to 3 by randomly selecting five areas.

[0139] 1-17. Immunohistochemical staining The slides with the liver tissue sections were placed in a dryer maintained at 60°C for 1 h, and then paraffin was removed using xylene. The tissues rehydrated with ethanol and water were incubated with 0.03% peroxidase for 15 min to block endogenous peroxidase. Antigen retrieval was performed by reacting with Pres-EDTA buffer (pH 9.0) at 121°C for 15 min using a press cooker. To prevent nonspecific reactions, 4% BSA + dextran was added for 30 min. Then, anti-TNF-α (Abcam) primary antibody was reacted for 1 h, and then anti-LAISTE IgG H&L (Abcam) secondary antibody was incubated for 30 min at room temperature with gentle agitation. The samples were then imaged with a BX53 biological microscope (Olympus, Tokyo, Japan), and representative images were captured for analysis.

[0140] 1-18.Reactive oxygen species (ROS) analysis Primary hepatocytes were incubated for 24 h in 400 μM H 2 O 2 Then, BxC-V37e was stimulated with H in serum-free DMEM culture medium. 2 O 2 Primary hepatocytes stimulated with β-lactam 1 (C10) were treated for 24 h and then washed with DPBS. CellROX® reagent (Life Technologies) was added to the cells at a final concentration of 5 μM in serum-free DMEM and incubated at 37°C for 30 min. After staining, cells were fixed in 4% paraformaldehyde (Fujifilm Wako Chemicals, Richmond, VA, USA) for 10 min and then washed three times with DPBS. Nuclei and cell bodies were counter-stained with NucBlue™ Fixed Cell stain or CellTracker™ (Life technologies), respectively. After this process, all samples were observed using a Nikon Eclipse Ti2-U (Nikon, Tokyo, Japan) and analyzed by the percentage of ROS-positive intensity according to nuclear intensity.

[0141] 1-19. Cell viability analysis Primary human hepatocytes (2 × 10 3 The cells were grown in 96-well plates (100 cells / mL) at 37°C and 5% CO 2 The cells were cultured in serum-free DMEM under the same conditions for 4 h. Optical density data at 450 nm (OD value) were measured using a multiplate reader (Thermo Fisher Scientific). The effect of BxC-V37e on the viability of primary hepatocytes was measured using the Cell Counting Kit-8 (CCK-8) assay (Enzo life sciences, Farmingdale, NY, USA) according to the manufacturer's protocol.

[0142] 1-20. Measurement of metabolic parameters and exosomal ApoA-1 concentration in human serum Under a study protocol approved by the Institutional Review Board of Asan Hospital, Seoul (approval number: 2008-0367) and Inha University Hospital (approval number: 08-115, 2016-06-015), metabolic characteristics of serum samples were confirmed from 20 healthy subjects, 19 obese NAFLD subjects, and 16 obese NAFLD subjects with type 2 diabetes. The healthy group underwent elective abdominal surgery due to benign disease at the Department of Obstetrics and Gynecology, Asan Hospital, Seoul. All obese groups underwent laparoscopic RYGB surgery. Subjects with malignant or severe liver or kidney disease and pregnant or lactating women were excluded, and all subjects provided written informed consent at the time of enrollment. The diagnosis of NAFLD was based on clinical characteristics and blood tests. All subjects discontinued diabetes and hypertension medications 3 days before blood collection, blood samples were collected after 12 hours of fasting, and plasma and serum were immediately separated by centrifugation. Circulating concentrations of ALT, AST, fasting glucose, insulin, HOMA-IR, cholesterol, triglycerides, hepatic steatosis index (HIS) and NAFLD liver fat score, and hs-CRP were measured as known in the art. Exosome-derived ApoA-1 concentrations were measured using a human ELISA kit (Abcam) according to the manufacturer's protocol. The assay sensitivity for ApoA-1 was <3 ng / mL, with intra- and inter-assay coefficients of variance of <4% and <10%, respectively.

[0143] 1-21.Statistical analysis Statistical analysis was performed using SPSS (version 18.0 for IBM, Chicago, IL, USA). For comparisons involving three or more groups, statistical analysis was performed using one-way ANOVA with Tukey's post-hoc test. When comparisons involved only two groups, a paired one-tailed Student's t test was used. Data are expressed as mean ± standard error (SE), and p values ​​less than 0.05 were considered statistically significant.

[0144] Example 2: Effect of BxC-V37e exosomes on NASH 2-1.BxC-V37e characteristics analysis for NASH

[0145] A total of 32 proteins enriched in BxC-V37e were identified by LC-MS / MS, including apolipoprotein A-1 (abundance = 191.3), CD81 antigen (abundance = 183.8), thrombospondin-1 (abundance = 155.7), collagen alpha-1 (abundance = 118.1), and alpha-2-antiplasmin (77.2) (Figure 1).

[0146] To functionally predict the pharmacological outcome of BxC-V37e, we performed a connectivity map analysis and identified 18 drugs associated with 59 target genes that were significantly similar to the transcript profile of BxC-V37e, including triciribine (AKT inhibitor, connectivity score = 99.75), EI-273 (PKC inhibitor, 99.59), 4,5-dianilinophthalamide (EGFR inhibitor, 98.63), BRD-A94297859 (XIAP inhibitor, 98.49), and GW-0742 (PPAR receptor agonist, 94.6) (Figure 2).Functional enrichment also showed that 110 upregulated genes by BxC-V37e were significantly enriched in 117 canonical signaling pathways (q < 0.05). Therefore, the BxC-V37e signature was confirmed to be composed of 108 genes confirmed by protein, transcript and linkage map analysis.

[0147] Protein-protein interaction network and functional enrichment analysis showed that the BxC-V37e signature was highly enriched in signaling pathways related to fat metabolism, fibrosis, inflammatory responses including focal adhesion (q=5.7E-04), chemokine signaling pathway (q=5.5E-03), non-alcoholic fatty liver disease (q=6.2E-03), NF-kappa B signaling pathway (q=8.7E-03), insulin signaling pathway (q=1.1E-02) and PPAR signaling pathway (q=4.2E-02) (Figure 3), suggesting the potential application of BxC-V37e in hepatic steatosis and inflammation.

[0148] 2-2. In vivo evaluation of BxC-V37e in NASH models The therapeutic function of BxC-V37e was investigated using a mouse model of NASH induced by an MC-deficient diet. The overall morphology of the liver was observed to turn paler in MCD-diet mice compared to normal mice (Figure 4). In contrast, the liver of NASH mice treated with BxC-V37e was observed to be darker, as seen in the control group. And no difference was observed in the liver of dulaglutide and PBS-treated animals. The total liver weight was not different in PBS- and BxC-V37e-treated NASH mice (data not shown; MCD+PBS, 5.78±0.12% vs. MCD+BxC-V37e, 5.33±0.23%). Serum analysis showed that the concentrations of liver function markers (ALT and AST) were significantly decreased in the serum of the MCD+BxC-V37e group compared to PBS-treated animals (Figures 5 and 6).

[0149] In addition, H&E staining and morphological changes after BxC-V37e treatment revealed fewer lipid droplets and reduced infiltration of inflammatory cells in the MCD+BxC-V37e group compared to the MCD+PBS group (Figure 7), and the NAS score, an index of the severity of NAFLD, was reduced in the MCD+BxC-V37e group (Figure 8). In summary, these data suggest that BxC-V37e generally improves liver function in NASH.

[0150] 2-3. Confirmation of the effect of exosomes (BxC-e) derived from induced pluripotent stem cells-derived mesenchymal stem cell precursor cells on inhibiting adipocyte differentiation Human adipocytes (primary human adipocytes, ATCC, USA) were distributed to a 6-well plate and incubated at 37°C in 5% CO using DMEM medium (Gibco, USA) supplemented with 1% penicillin-streptomycin and 10% CS. 2 The cells were cultured in an incubator until they became confluent (maximum 6 days).

[0151] After culturing the adipocytes for 6 days, they were cultured for 5 days in a basal medium containing 1% penicillin-streptomycin and 10% FBS in DMEM medium (Gibco, USA) and added with adipocyte differentiation medium [34 μM pantothenate (Sigma), 66 μM biotin (Sigma), 0.5 mM insulin (Sigma), 1 mM dexamethasone (Sigma), and 0.05 M IBMX (Sigma)], and then cultured for another 9 days in a medium containing 34 μM pantothenate (Sigma), 66 μM biotin (Sigma), 0.5 mM insulin (Sigma), and 1 mM dexamethasone (Sigma)] in DMEM medium (Gibco, USA).

[0152] In this case, the negative control group consisted of adipocytes cultured for 14 days in DMEM medium supplemented with 10% FBS, the vehicle control group consisted of adipocytes cultured for 14 days under adipocyte differentiation conditions without treatment with BxC-e, and the BxC-e treatment group consisted of adipocytes cultured for 14 days under adipocyte differentiation conditions containing BxC-e.

[0153] After completely removing the culture medium, the wells were washed twice with PBS and 10% formalin solution was added at 400 ul / well to fix the cells for 1 hour. After washing with PBS, Oil-red O working solution was added at 400 ul / well to stain the fat in differentiated adipocytes for 2 hours. After that, the Oil-red O working solution was removed and the well walls were completely removed with secondary distilled water. After drying for 5 minutes in a dryer, isopropyl alcohol was added to the wells at 500 ul / well.

[0154] The absorbance was measured at 490 nm using a microplate reader (Model 680 microplate reader, Bio-Rad, USA) and the amount of fat was compared.

[0155] As can be seen from Figure 9, the exosomes derived from BxC of the present invention (BxC-e) have excellent effect of suppressing lipogenesis in adipocytes.

[0156] 2-4. Accuracy assessment of non-alcoholic fatty liver disease diagnosis using exosome-derived ApoA-1 protein ApoA-1 protein levels were quantitatively compared in BxC-V37e using immunoblot analysis. The analysis showed that exosome-derived ApoA-1 protein was decreased in NASH mice, whereas it was increased in animals administered BxC-V37e (Figures 10 and 11). Furthermore, exosome-derived ApoA-1 in serum was decreased in obese patients with NAFLD compared to the control group (Figure 12).

[0157] The correlation between the human serum ApoA-1 and exosome-derived ApoA-1 values ​​obtained by ELISA analysis and metabolic parameters related to NAFLD was analyzed using the Spearman method with the SPSS statistical program. An R value of (+) indicates a positive correlation, and (-) indicates a negative correlation, and a p value of 0.05 or less was considered to have statistical significance.

[0158] As a result of correlation analysis, when the correlation between ApoA-1 and metabolic parameters in human serum was analyzed, exosome-derived ApoA-1 and almost all metabolic parameters related to NAFLD showed a strong negative correlation (Figures 13 to 24). Moreover, it was observed that the correlation between exosome-derived ApoA-1 and metabolic parameters related to NAFLD was stronger than the correlation between human serum ApoA-1 and metabolic parameters, except for the TC index (Figures 25 to 36).

[0159] These results indicate that diagnosing NAFLD using exosome-derived ApoA-1 protein can identify the disease with significantly higher accuracy than existing methods for diagnosing NAFLD using serum ApoA-1 protein as a marker, and suggest that serum exosome-derived ApoA-1 can be used as a new biomarker for NAFLD.

[0160] 2-5. Accuracy assessment of prognosis prediction of non-alcoholic fatty liver disease using exosome-derived ApoA-1 protein We tested how well serum ApoA-1 and exosome-derived ApoA-1 reflect changes in existing NASH-related parameters after administration of NASH therapeutics and evaluated the accuracy of prognostic analysis using exosome-derived ApoA-1.

[0161] MCD-fed mice were administered dulaglutide, BxC-e, and BxC-V37e, which are known to be candidate substances for the treatment of NASH. The correlation between serum ApoA-1 and exosome-derived ApoA-1 and the rate of change in existing NASH-related parameters such as AST, ACC1, hs-CRP, NRF2, PCNA, Annexin A5 values, and Oil Red O staining results compared to the PBS group was calculated, and statistical analysis was performed using simple regression analysis in the SPSS statistical program (P values ​​of 0.05 or less were considered to have statistical significance).

[0162] As can be seen from Figures 37 to 40, the change rates of AST, ACC1, hs-CRP and NRF2 values ​​in the dulaglutide-administered group of MCD-fed mice compared to the PBS group were confirmed to have a more significant correlation with the change rate of exosome-derived ApoA-1 between the PBS group and dulaglutide, compared to the change rate of serum ApoA-1 between the PBS group and dulaglutide-administered groups.

[0163] Furthermore, as can be seen from Figures 41 to 44, the change rates of AST, ACC1, hs-CRP and PCNA values ​​in the BxC-V37e-administered group of MCD-fed mice compared to the PBS group were confirmed to have a more significant correlation with the change rate of exosome-derived ApoA-1 between the PBS group and BxC-V37e, compared to the change rate of serum ApoA-1 between the PBS group and BxC-V37e-administered groups.

[0164] Furthermore, as can be seen from Figures 45 to 47, when BxC-e was administered, the rate of change in hs-CRP, Annexin A5 values, and Oil Red O staining results in the BxC-e administration group of MCD-fed mice compared to the PBS group was confirmed to have a more significant correlation with the rate of change in exosome-derived ApoA-1 between the PBS group and the BxC-e administration group compared to the rate of change in serum ApoA-1 between the PBS group and the BxC-e administration group.

[0165] These results indicate that exosome-derived ApoA-1 more accurately reflects the degree of change in liver function index, lipogenesis index, and inflammation index after administration of a therapeutic agent such as dulaglutide, BxC-e, or BxC-V37e compared to serum ApoA-1. Therefore, the composition for predicting the prognosis of non-alcoholic fatty liver disease according to the present invention can more accurately reflect the degree of improvement of non-alcoholic fatty liver disease compared to existing prognosis prediction compositions or prognosis prediction methods, suggesting that it can be used in various ways in connection with the diagnosis of non-alcoholic fatty liver disease. [Industrial Applicability]

[0166] The present invention relates to a diagnostic marker for non-alcoholic fatty liver disease, and more specifically, to a composition that includes a preparation for measuring the level of exosome-derived ApoA-1 protein and can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy, and a kit including the same.

Claims

1. A composition for predicting the prognosis of non-alcoholic fatty liver disease (NAFLD) comprising a preparation for measuring the level of exosome-derived ApoA-1 protein, the composition being used on a biological sample from an individual administered a therapeutic agent for non-alcoholic fatty liver disease (NAFLD).

2. The composition of claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

3. The composition of claim 1 , wherein the formulation for measuring the level of the protein comprises an antibody or an aptamer specific for the protein.

4. The composition according to claim 3 , wherein the antibody is at least one selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, and a recombinant antibody.

5. A kit for predicting the prognosis of non-alcoholic fatty liver disease (NAFLD) includes a preparation for measuring the level of exosome-derived ApoA-1 protein, and is used on a biological sample from an individual who has been administered a therapeutic agent for non-alcoholic fatty liver disease (NAFLD).

6. The kit according to claim 5, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

7. The kit of claim 5 , wherein the preparation for measuring the level of the protein comprises an antibody or an aptamer specific to the protein.

8. The kit according to claim 5, which is an enzyme-linked immunosorbent assay (ELISA) kit.

9. A method for providing information for predicting the prognosis of nonalcoholic fatty liver disease (NAFLD), comprising a measuring step of measuring a level of exosome-derived ApoA-1 (Exosomal ApoA-1) protein from a biological sample isolated from an individual to whom a therapeutic agent for nonalcoholic fatty liver disease (NAFLD) has been administered.

10. 10. The method for providing information according to claim 9, further comprising a comparison step of comparing the measured protein levels with values ​​measured from samples of a normal control group.

11. The method for providing information according to claim 10 , wherein the measuring step includes a contacting step of contacting a sample with an antibody or an aptamer that specifically binds to the protein.

12. 10. The method for providing information according to claim 9, wherein the biological sample is at least one selected from the group consisting of tissue, cells, whole blood, serum, and plasma separated from an individual.

13. The information providing method according to claim 9, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

14. The information providing method according to claim 9, wherein, when a level of exosome-derived ApoA-1 protein measured from a sample isolated from an individual suspected of having non-alcoholic fatty liver disease is lower than a level measured from a sample isolated from a normal control group, it is determined that the individual is at high risk of developing non-alcoholic fatty liver disease or has a poor prognosis for non-alcoholic fatty liver disease.

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