Diagnosis marker for non-alcoholic fat liver disease

Abstract

To provide a diagnosis marker for a non-alcoholic fat liver disease.SOLUTION: The present invention relates to a composition and a kit for accurately diagnosing hepatitis (Non-alcoholic steatohepatitis;NASH) by including a formulation for measuring the level of ApoA-1 (Exosomal ApoA-1) protein derived from exosome, and relates to a composition and a kit for accurately predicting the prognosis after a medicine for treating non-alcoholic fat hepatitis is provided.SELECTED DRAWING: Figure 10

Description

Technical Field

[0001] The present invention relates to a marker for diagnosing non-alcoholic fatty liver disease, and more particularly, to a composition containing a preparation for measuring the level of exosomal ApoA-1 protein, which can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy, and a kit containing the same.

Background Art

[0002] Mesenchymal stem cells are stromal cells with pluripotency, referring to cells that can differentiate into various cells including osteoblasts, chondrocytes, muscle cells, adipocytes, etc. Since mesenchymal stem cells can differentiate into various connective tissues such as cartilage, bone tissue, ligaments, and bone marrow stroma, they are being studied for various therapeutic uses such as treating arthritis and soft tissue defects caused by trauma, burns, etc.

[0003] On the other hand, non-alcoholic fatty liver is characterized by the accumulation of triglycerides, which are neutral fats, in hepatocytes without excessive alcohol intake. Non-alcoholic fatty liver is on the rise due to overnutrition associated with the high-fat and high-carbohydrate intake of modern people. Non-alcoholic fatty liver is often observed in obesity and diabetes, but various factors are said 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] Non-alcoholic fatty liver is classified into non-alcoholic simple steatosis and non-alcoholic steatohepatitis (NASH) with inflammation, but if left untreated for a long time, it may progress to serious liver diseases such as hepatitis, liver fibrosis, and cirrhosis. Non-alcoholic fatty liver is characterized by the accumulation of fat (fat infiltration) in hepatocytes.

[0005] Since non-alcoholic simple fatty liver may progress to non-alcoholic steatohepatitis, fat accumulation in non-alcoholic steatohepatitis is associated with various levels of liver inflammation and scars, and is often known to be associated with insulin resistance, dyslipidemia and hypertension. Non-alcoholic steatohepatitis often occurs in people with overweight, high cholesterol and triglyceride levels, and / or insulin resistance.

[0006] However, in recent years, despite the increasing number of patients with non-alcoholic steatohepatitis along with the increasing obese population, the development of safe and long-term administrable therapeutic agents for non-alcoholic steatohepatitis has progressed only slightly, and the development of formulations and kits for the diagnosis of non-alcoholic steatohepatitis is in an unsatisfactory situation. Therefore, there is a growing demand for a composition or kit for accurate and rapid diagnosis of non-alcoholic steatohepatitis while being suitable for the diagnosis of non-alcoholic steatohepatitis, a chronic disease, and for a prognostic diagnosis composition after administration of a therapeutic agent for non-alcoholic steatohepatitis.

Summary of the Invention

Problems to be Solved by the Invention

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

[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] Still another object of the present invention is to provide a method for providing information for diagnosing non-alcoholic fatty liver disease.

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

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

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

[0014] Another object of the present invention is to provide a method for predicting the prognosis of non-alcoholic fatty liver disease.

Means for Solving the Problems

[0015] The present invention relates to a marker for diagnosing non-alcoholic fatty liver disease. The composition for diagnosing non-alcoholic fatty liver disease, the composition for predicting prognosis, or the kit thereof according to the present invention can diagnose non-alcoholic fatty liver disease quickly and accurately 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 a preparation for measuring the level of exosomal ApoA-1 protein according to the present invention can diagnose non-alcoholic fatty liver disease more accurately and predict the prognosis compared with existing methods.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] One aspect of the present invention is a composition for diagnosing non-alcoholic fatty liver disease, which comprises a preparation for measuring the level of exosomal ApoA-1 protein from an individual who has developed or is suspected of developing non-alcoholic fatty liver disease (NAFLD).

[0019] As used herein, the term "marker" refers to a substance that can distinguish an individual with non-alcoholic fatty liver disease from a normal individual, 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 an individual with non-alcoholic fatty liver disease. For example, it may be a protein whose level decreases in an individual with non-alcoholic fatty liver disease, but is not limited thereto.

[0020] As used herein, the term "exosome" refers to a membrane vesicle having a lipid bilayer structure that is secreted extracellularly by cells or present intracellularly, and is present in the body fluids of almost all eukaryotes. The diameter of exosomes is about 30 to 1000 nm, and they 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) in order to play a functional role in mediating coagulation, cell-cell communication, and cellular immunity.

[0021] Exosome is a concept that includes microvesicles. CD63, CD81, etc. are known as exosome marker proteins. In addition, proteins such as cell surface receptors like EGFR, signal transduction-related molecules, cell adhesion-related proteins, MSC-related antigens, heat shock proteins, and Alix related to vesicle formation are known.

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

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

[0024] As used in the present invention, the term "diagnosis" means to confirm the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the presence or absence 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] As used herein, the term "non-alcoholic steatohepatitis" is one of the non-alcoholic fatty liver diseases and is a progressive liver disease characterized by inflammation or fibrosis along with fatty liver, and means a pre-stage disease that induces cirrhosis or liver cancer.

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

[0028] In one embodiment of the present invention, the agent for measuring protein level may contain an antibody or 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 monoclonal antibodies, polyclonal antibodies, antibody fragments, and recombinant antibodies.

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

[0031] As used herein, the term "aptamer" means a single-stranded oligonucleotide, and means a nucleic acid molecule having a binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures depending on its base sequence, and may have a high affinity for a specific substance like an antigen-antibody reaction. The aptamer can inhibit the activity of a predetermined target molecule by binding to the predetermined target molecule.

[0032] The aptamers of the present invention may be RNA, DNA, modified nucleic acids or mixtures thereof, and their forms may be linear or circular, but are not limited thereto. The aptamers can be easily prepared by methods known to those of ordinary skill in the art with reference to their respective base sequences.

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

[0034] In the present invention, the kit may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for the immunological detection of an antibody.

[0035] The substrate is not particularly limited, and for example, a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a slide glass made of glass, etc. may be used. The chromogenic enzyme is not particularly limited, and for example, peroxidase or Alkaline Phosphatase may be used. The fluorescent substance may be, but is not limited to, FITC, RITC, etc. The chromogenic substrate solution is not particularly limited, and for example, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethylbenzidine) may be used.

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

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

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

[0039] In one embodiment of the present invention, the method may further include a comparing step of comparing the measured protein level with the value measured from a sample of a normal control group.

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

[0041] As used herein, the term "individual" can mean all animals, including humans who have developed or are likely to develop non-alcoholic fatty liver disease. The animals may be mammals such as cows, horses, sheep, pigs, goats, camels, deer, dogs, cats, etc. that require treatment for similar symptoms, but are not limited thereto.

[0042] As used herein, the term "normal control group" means an individual in whom non-alcoholic fatty liver disease does not develop or is not suspected of developing.

[0043] As used herein, the term "sample" means a direct object separated from a patient with non-alcoholic fatty liver disease and used to measure 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 tissues, cells, whole blood, serum, or plasma separated from an individual.

[0045] In one embodiment of the present invention, the method may further include a determination step of determining non-alcoholic fatty liver disease when the value measured from a biological sample is lower than the value of a normal control group sample.

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

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

[0048] As used herein, the term "oligonucleotide" means a polymer formed by polymerization of several to dozens of nucleotides through phosphodiester bonds.

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

[0050] Still another aspect of the present invention is a composition for predicting the prognosis of non-alcoholic fatty liver disease, including a preparation for measuring the level of exosome-derived ApoA-1 protein from an individual administered with 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 all substances 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 inventors have confirmed that exosomes isolated from induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) are superior to existing clinically used therapeutic agents in the treatment, alleviation, and improvement of non-alcoholic fatty liver disease.

[0052] As used herein, the term "prognosis" refers to the act of predicting in advance the course of a disease and the outcome of death or survival. More specifically, prognosis or prognostic prediction may be interpreted as all acts of predicting the course of a disease before and after treatment by comprehensively considering the state of such a patient, as the course of a disease may vary depending on the physiological or environmental state 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 a patient with non-alcoholic fatty liver disease by anticipating in advance the course of the disease and the presence or absence of complete recovery before and after treatment of non-alcoholic fatty liver disease.

[0053] For example, predicting that the "prognosis is good" indicates that, regardless of whether the patient has been treated or not, the survival rate of a patient with non-alcoholic fatty liver disease is at a high level or the indicators associated with fatty liver disease have been improved, meaning that the patient with non-alcoholic fatty liver disease is likely to be treated. Predicting that the "prognosis is poor" means that the survival rate of a patient after treatment for non-alcoholic fatty liver disease is low or the indicators associated with fatty liver disease have deteriorated.

[0054] As an example, the inventors confirmed that when measuring the level of exosome-derived ApoA-1 protein, it is possible to more accurately grasp the presence or absence of improvement in non-alcoholic fatty liver disease compared to measuring the level of ApoA-1 protein in serum used in existing clinical practice.

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

[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-derived stem cells.

[0057] As used herein, the term "induced pluripotent stem cell" means a cell that has been induced to dedifferentiate into an initially undifferentiated state from a previously differentiated cell such as a somatic cell, and has acquired pluripotency.

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

[0059] The pluripotency means the ability to differentiate into tissues or organs derived from the three germ layers that make up a living body, namely, the endoderm, mesoderm, and ectoderm.

[0060] As used herein, the term "mesenchymal stem cell" refers to a stem cell having multipotency and capable of differentiating into various cells including osteoblasts, chondrocytes, muscle cells, adipocytes, etc. Among these, bone marrow-derived mesenchymal stem cells are most commonly used, but in addition to bone marrow, they can also be derived from umbilical cord or umbilical cord blood, adipose tissue, amniotic fluid, and tooth buds of deciduous teeth. Mesenchymal stem cells are also called stromal cells.

[0061] The "induced pluripotent stem cell" is a term referring to a cell induced to have pluripotent differentiation ability through an artificial dedifferentiation process from a differentiated cell, and is also called a dedifferentiated stem cell.

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

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

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

[0065] The artificial dedifferentiation process is carried out by viral-mediated using retrovirus, lentivirus and Sendai virus or by using non-viral vectors, or by introducing non-viral-mediated dedifferentiation factors such as proteins and cell extracts, or includes a dedifferentiation process by stem cell extracts, compounds, etc.

[0066] The induced pluripotent stem cells of the present invention have substantially the same characteristics as embryonic stem cells. Specifically, they show similar cell shapes, have similar gene and protein expression patterns, have the ability of total differentiation in vitro and in vivo, can form teratomas, can form chimeric mice when inserted into the blastocysts of mice, and gene germline transmission is possible.

[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, rabbits, etc., but preferably are induced pluripotent stem cells derived from humans.

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

[0069] In the present invention, the exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells mean the exosomes that were present in or secreted from the above-described induced pluripotent stem cell-derived mesenchymal stem cells (BxC).

[0070] As used herein, the term "comprising as an active ingredient" means that the exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells contain an amount sufficient to achieve the 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" means a process of bringing a cell culture medium supplemented with a pretreatment substance into contact with the progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells during the culture process of the progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells.

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

[0074] 1-(benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid may be used under the name "Lanifibranor" and 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 ranifibranor.

[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 agent for measuring the level of a protein may contain an antibody or aptamer specific for the protein.

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

[0079] Still another aspect of the present invention is a kit for predicting the prognosis of non-alcoholic fatty liver disease, which includes an agent for measuring the level of exosome-derived ApoA-1 protein from an individual administered with 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 agent for measuring the level of a protein may contain an antibody or aptamer specific for the protein.

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

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

[0084] In one embodiment of the present invention, the method may further include a comparison step of comparing the measured protein level with the value measured from a sample of a normal control group.

[0085] In one embodiment of the present invention, the measurement step may include a contact step of contacting the sample with an antibody or 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, or plasma separated from an individual.

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

[0088] In one embodiment of the present invention, the method may further include a determination 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 separated from an individual suspected of having non-alcoholic fatty liver disease is lower than the level measured from a sample separated from a sample of a normal control group.

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

Advantages of the Invention

[0090] The present invention relates to a marker for diagnosing non-alcoholic fatty liver disease. More specifically, by including a preparation for measuring the level of exosomal ApoA-1 protein, the present invention relates to a composition and a kit capable of diagnosing non-alcoholic steatohepatitis (NASH) with high accuracy, and a composition and a kit capable of accurately predicting the prognosis after administration of a therapeutic agent for non-alcoholic steatohepatitis. The composition and kit of the present invention can be variously utilized as a preparation for diagnosing non-alcoholic fatty liver disease, predicting the prognosis of non-alcoholic fatty liver disease, and screening a therapeutic agent for non-alcoholic fatty liver disease.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0116] Relates to a composition for predicting the prognosis of non-alcoholic fatty liver disease, comprising a preparation for measuring the level of exosomal ApoA-1 protein from an individual administered with a therapeutic agent for non-alcoholic fatty liver disease (NAFLD).

Examples

[0117] Hereinafter, the present invention will be described in more detail using the following examples. However, these examples are merely for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0118] Example 1: Materials and Methods

[0119] 1-1. Animal Experiment Six-week-old male C57BL / 6 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 management and procedures were approved by the rodent facility area of Knotus Co., Ltd. in the Republic of Korea (approval number: 19-KE-265). Mice on 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 400 μg of BxC-e and BxC-V37e per mouse were intravenously injected once a day, three times a week, for 4 weeks. At the end of the experiment, the mice were anesthetized and serum and liver tissues 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; regulated on a 12-hour light / 12-hour dark cycle.

[0120] 1-2. Cell culture For cell maintenance, human primary hepatocytes (human primary hepatocyte, ScienCell, Carlsbad, CA, USA) were cultured in hepatocyte basal medium together with 5% fetal bovine serum (FBS), 1% penicillin antibiotic and growth supplement (ScienCell) and THP-1 monocytes (ATCC). And the growth supplement (ScienCell) and THP-1 monocytes (ATCC, Manassas, VA, USA) were cultured in RPMI medium (Gibco, Waltham, MA, USA) with 10% FBS (Hyclone, Chicago, IL, USA) and 1% antibiotic / antifungal agent (Thermo Fisher Scientific, Waltham, MA, USA). To create an in vitro NASH model, human primary hepatocytes were treated with 100 mM FA (2 oleic acids: 1 palmitic acid) in 2% FBS + DMEM for 48 hours, and then supplied with 100 μg / mL BxC-V37e containing 100 mM FA in serum-free DMEM. Also, 500 nM thapsigargin was co-treated with BxC-V37e in serum-free DMEM for 24 hours. On the other hand, THP-1 monocytes were stimulated with 200 ng / mL PMA, 100 ng / mL LPS and 20 ng / mL IFNγ in 10% FBS + RPMI for 24 hours. Then, 100 μg / mL BxC-V37e was treated in serum-free DMEM with 100 ng / mL LPS and 20 ng / mL IFNγ for 24 hours. Both cells were grown at 37°C in a 5% CO 2 and 95% humidified incubator. To evaluate the phosphorylation levels, human primary hepatocytes and THP-1 macrophages were treated with 100 μg / mL BxC-V37e for 24 hours. 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 minutes (phospho-Akt and phospho-AMPK) or to THP-1 macrophages for 10 minutes (phospho-p65), respectively.

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

[0122] The precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells named BxC were additionally cultured in a 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 precursor cells (BxC) of induced pluripotent stem cell-derived mesenchymal stem cells The culture medium of the cultured precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells (hereinafter referred to as BxC) was collected and centrifuged at 300 x g for 10 minutes to remove the remaining cells and cell debris. The supernatant was taken and filtered through a 0.22 μm filter, and then centrifuged at 10,000 x g at 4°C for 70 minutes using a high speed centrifuge. The centrifuged supernatant was further taken and centrifuged at 100,000 x g at 4°C for 90 minutes using an ultracentrifuge to remove the supernatant. The exosomes remaining in the lower layer were diluted in phosphate buffered saline (PBS) 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, 5% CO 2 and 95% humidity in 15% fetal bovine serum (FBS), high glucose Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Chicago, IL, USA) and 1% antibiotic / antifungal (Hyclone, Chicago, IL, USA). When 90% confluency was reached, the cells were detached with TryPLE Express (Thermo Fisher Science) and seeded at a density of 10,000 cells / cm 2 into a 4-layer Cell Factory system (Thermo Fisher science). The next day, the cells were treated with 10 μM lanifibranor (Cayman, Ann Arbor, MI, USA) for 24 h, then the medium was aspirated and the cells were washed with Dulbecco's DPBS (phosphate buffered saline) (Hyclone).

[0125] RNA sequencing was performed by applying the RNA sequencing protocol with an application program from Macrogen. Hierarchical clustering is a similarity measure to present patterns of differentially expressed transcripts that satisfy |fold changes| ≥ 2 and independent t-test p < 0.05, and was analyzed using the complete linkage and Euclidean distance algorithms. Gene enrichment and pathway analysis for important gene catalogs were performed based on gProfiler (https: / / biit.cs.ut.ee / gprofiler / gost) and KEGG pathways (http: / / www.genome.jp / kegg / pathway).

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

[0127] The ranifibranor-pretreated iMSC medium was replaced with serum-free, xeno-free StemPro MSC medium (Gibco). After 3 days of culture, the culture medium was harvested and centrifuged at 300 g for 10 minutes, and then the supernatant was centrifuged at 2,000 g for 20 minutes. The supernatant was further centrifuged at 10,000 g for 80 minutes. Then, the supernatant was filtered through a 0.2 μm vacuum filter (Merck Millipore, Burlington, MA, USA). Finally, exosomes were separated by ultracentrifugation at 100,000 g for 80 minutes, and then 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 microscopy (TEM) A 200-mesh copper grid (MiTeGen, Ithaca, NY, USA) coated with a formvar / carbon film was hydrophilized. The EV suspension (4 μL) was placed on the grid and blotted for 90 seconds at 100% and 4 °C humidity and temperature, respectively. Extracellular vesicles on the grid were visualized at a magnification of 36,000 times using a Talos L120C FEI TEM (Thermo Fisher Scientific) at 120 kV.

[0129] 1-8. Nanoparticle tracking analysis (NTA) The particle size distribution and concentration measurement for BxC-V37e were performed using a Nanosight NS300 instrument (Malvern Panalytical, Malvern, UK) based on NTA. For the analysis, BxC-V37e was diluted with sterile PBS (1:100) to reach the optimal volume for NTA. The measurements were performed several times repeatedly at room temperature in the range of 23.0 - 25.2 °C with a Blue 488 nm laser and an sCMOS camera. The sample analysis was carried out for 10 minutes with camera settings and processing conditions such as shutter 600, gain 250, camera level 10, NTA version 3.00064, and detection threshold 10.

[0130] 1 - 9. Labeling of BxC-V37e with DiR and DiD and Fluorescent Imaging BxC-V37e was cultured with 1 μg / mL DiR buffer at 37 °C for 10 minutes according to the protocol of Lipophilic Tracers (Invitrogen, Waltham, MA, USA). Then, the DiR-labeled BxC-V37e was centrifuged at 100,000 xg and 4 °C for 80 minutes 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 the intravenous route. After 24 hours, the DiR-labeled BxC-V37e was detected by an 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) was expressed in units of the maximum number of photons per second per square centimeter per steradian (p / s / cm 2 / sr). The procedure for producing DiD-labeled BxC-V37e was the same as the method described above. The DiD-labeled BxC-V37e was treated with human primary hepatocytes or THP-1 macrophages for 24 hours regardless of the presence or absence of their respective stimuli. After 24 hours, the DiD-labeled BxC-V37e was observed with a Nikon Eclipse Ti2-U fluorescence microscope (Nikon, Tokyo, Japan).

[0131] 1-10. Bioinformatics analysis After treating HepG2 cells with 100 mM fatty acid (2 oleic acids: 2 palmitic acids) for 6 hours, total RNA was recovered using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The recovered RNA was profiled using the GeneChip® Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). Differentially expressed genes (DEGs) were confirmed with a fold change cutoff of 1.5. DEGs were subjected to enrichment analysis of gene sets using KEGG with an FDR q-value cutoff of 0.05 (http: / / www.gsea-msigdb.org / gsea). The BxC-V37e signature was constructed by transcriptomic, proteomic, and Connectivity Map analyses. BxC-V37e was applied to fatty acid-treated HepG2 cells, and total RNA was profiled as described above. DEGs induced by BxC-V37e were confirmed with a fold change cutoff of 1.5. Proteins rich in BxC-V37e were qualitatively and quantitatively confirmed using LC-MS / MS (Yonsei Proteome Institute, Seoul). DEGs induced by BxC-V37e were used to identify drugs and their target genes with transcript profiles similar to BxC-V37e by Connectivity Map analysis. DEGs induced by BxC-V37e, proteins of BxC-V37e, and target genes of the identified BxC-V37e-similar drugs were confirmed as the BxC-V37e signature. The established BxC-V37e signature was subjected to protein-protein interaction network and functional enrichment analysis at an interaction confidence of 0.9 with (https: / / string-db.org).

[0132] 1-11. Flow cytometry 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 exosome effect on hepatocyte regeneration, the hepatocytes were stained with anti-human CD90 APC-Cy7 (BioLegend, San Diego, CA, USA) after exosome treatment and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). To confirm whether iMSCs express typical cell surface markers for MSCs, the iMSCs were stained with CD73 APC, CD105 PE, CD45 FITC, CD31 PE, and CD34 APC (eBioscience, Waltham, MA, USA) and CD90 APC-Cy7 (BioLegend). The analysis was performed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0133] 1 - 12. Serum biochemical examination Serum samples were collected 4 weeks after BxC-V37e injection, and the following mediators 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), 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). The amplification reaction was 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 a QuantStudioTM 5 Real-Time RCR system (Applied Biosystems).

[0135] GAPDH was used as a reference gene to normalize the difference in mRNA amounts of each sample. Relative gene expression levels were analyzed using the 2 -ΔΔ −ΔΔCt method, and each experiment was performed in triplicate.

[0136] 1 - 14. Western blot Cells or tissues were lysed in NP40 lysis buffer (Life Technologies, Carlsbad, CA, USA) supplemented with protease inhibitor (Thermo Fisher Scientific). Protein concentration was determined using Bradford AssayTM 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 minutes. Proteins were loaded and separated on precast polyacrylamide Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a PVDF membrane (Bio-Rad Laboratories). The membrane was blocked with EveryBlot blocking buffer (Bio-Rad Laboratories) for 5 minutes and then incubated overnight at 4 °C with the primary antibody. All primary antibodies were diluted with 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. After the membrane was washed 5 times for 10 minutes, it was incubated with the secondary antibody for 1 hour. Anti-rabbit IgG and anti-mouse IgG (Abcam) were used as the secondary antibodies.After washing the membrane five times for 10 minutes, the target protein was detected using ECL SelectTM Western blotting detection reagent (GE Healthcare, Little Chalfont, UK), and the image was analyzed using a ChemiDoc imaging system (Bio-Rad Laboratories).

[0137] 1-15. Enzyme-linked immunosorbent assay (ELISA) ELISA analysis was performed using a commercially available mouse ELISA kit. Insulin (Novus bio), hs-CRP (R&D systems), FFA (free fatty acid), and ApoA-1 (Abcam) analyses were performed according to the manufacturer's protocol. The analytical sensitivity for insulin was <0.19 ng / mL, and the coefficients of variation within and between analyses were <5.93% and <6.35%, respectively. The analytical sensitivity for hs-CRP was <0.015 ng / mL, and the coefficients of variation within and between analyses were <7.7% and <10.8%, respectively. The analytical sensitivity for ApoA-1 was 11.2 pg / mL, and the coefficients of variation within and between analyses were <4.7% and <5.6%, respectively.

[0138] 1-16. Histopathological analysis The liver tissues were fixed in 10% paraformaldehyde and subjected to general tissue processing such as cutting, dehydration, and paraffin embedding. Liver tissue sections of 5 μm were attached to slides, and the paraffin of the specimens was removed using xylene. The rehydrated tissues with ethanol were stained with hematoxylin & eosin (H&E). In the case of Oil red O staining, the tissues were embedded using OCT compound (Sakura Finetek, Torrance, CA, USA) and then sectioned at 20 μm using a cryotome (Leica, Wetzlar, Germany). Subsequently, the histopathological samples were analyzed using a Zen 2.3 blue edition image analyzer (Carl Zeiss, Oberkochen, Germany) and normalized to the percentage of the stained area by total area. The examination of NAFLD activity score (NAS) was made according to histological criteria, and based on the observation of the occupied area by the total area, macrovesicular and microvesicular steatosis, and the hypertrophy level were evaluated from 0 to 3 points. Both steatosis and hypertrophy were evaluated at a magnification of 40 - 100. The inflammation score was evaluated from 0 to 3 points by randomly selecting 5 regions.

[0139] 1 - 17. Immunohistochemical staining The slides with liver tissue sections were placed in an incubator maintained at 60 °C and dried for 1 hour, and then the paraffin was removed using xylene. The rehydrated tissues with ethanol and water were cultured with 0.03% peroxidase for 15 minutes to block endogenous peroxidase. Antigen retrieval was performed by reacting Pres-EDTA buffer (pH 9.0) at 121 °C for 15 minutes using a pressure cooker. To prevent non-specific reactions, 4% BSA + dextran was added for 30 minutes. Subsequently, the primary antibody anti-TNF-α (Abcam) was reacted for 1 hour, and then cultured with the secondary antibody anti-LAISTE IgG H&L (Abcam) for 30 minutes with gentle stirring at room temperature. Subsequently, the samples were imaged using 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 stimulated with 400 μM H 2 O 2 for 24 hours. Then, BxC-V37e was treated with primary hepatocytes stimulated with H 2 O 2 in serum-free DMEM culture medium for 24 hours 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 cultured at 37 °C for 30 minutes. After staining, the cells were fixed with 4% paraformaldehyde (Fujifilm Wako Chemicals, Richmond, VA, USA) for 10 minutes and then washed three times with DPBS. The nuclei and cell bodies were counterstained with NucBlueTM Fixed Cell staining or CellTrackerTM (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 based on nuclear intensity.

[0141] 1-19. Cell viability analysis Primary human hepatocytes (2×10 3 cells / mL) were grown in 96-well plates and cultured in serum-free DMEM for 4 hours at 37 °C and 5% CO 2 conditions. Absorbance data (OD value) at 450 nm was measured using a multiplate reader (Thermo Fisher Scientific). The effect of BxC-V37e on the viability of primary hepatocytes was measured using a 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 the research protocols approved by the Institutional Review Board of Seoul Asan Medical Center (approval number: 2008-0367) and Inha University Hospital (approval numbers: 08-115, 2016-06-015), the metabolic characteristics of serum samples were identified 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 diseases in the Department of Obstetrics and Gynecology at Seoul Asan Medical Center. All obese groups underwent laparoscopic RYGB surgery. Subjects with malignant or severe liver or kidney diseases and women who were pregnant or lactating were excluded, and written consent was obtained from all subjects at the time of registration. The diagnosis of NAFLD was made based on clinical features and blood tests. Three days before blood sampling, all subjects discontinued diabetes and hypertension medications, and blood samples were collected after a 12-hour fast. Plasma and serum were immediately separated by centrifugation. The circulating concentrations of ALT, AST, fasting glucose, insulin, HOMA-IR, cholesterol, triglyceride, hepatic steatosis index (HIS), NAFLD liver fat index (NAFLD liver fat score), and hs-CRP were measured as is known in the art. Exosome-derived ApoA-1 concentration was measured using a human ELISA kit (Abcam) according to the manufacturer's protocol. The analytical sensitivity for ApoA-1 was <3 ng / mL, and the coefficients of variation within and between assays were <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 more than three groups, statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. When only two groups were included in the comparison, a paired one-sided Student's t-test was used. Data were expressed as mean ± standard error (SE), and p-values less than 0.05 were considered statistically significant.

[0144] Example 2: Confirmation of the Effect of BxC-V37e Exosomes on NASH 2-1. Characterization of BxC-V37e for NASH

[0145] A total of 32 proteins rich in BxC-V37e were identified by LC-MS / MS, and it was confirmed that they included 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] As a result of performing a connectivity map analysis to functionally predict the pharmacological results of BxC-V37e, it was confirmed that 18 drugs associated with 59 target genes were significantly similar to the transcript profile of BxC-V37e. Here, it was confirmed that they included triciribine (AKT inhibitor, Connectivity score = 99.75), EI-273 (PKC inhibitor, 99.59), 4,5-dianilinophthalimide (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 standard signaling pathways (q < 0.05). Therefore, it was confirmed that the BxC-V37e signature was composed of 108 genes confirmed by proteome, transcriptome, and connectivity map analyses.

[0147] Protein-protein interaction network and functional enrichment analysis showed that the BxC-V37e signature was highly enriched in signaling pathways related to lipid metabolism, fibrosis, inflammatory response 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), such results suggesting the potential of applying BxC-V37e to hepatic steatosis and inflammation.

[0148] 2-2. In vivo evaluation of BxC-V37e in the NASH model The therapeutic function of BxC-V37e was examined using a mouse model of NASH induced by a methionine-choline-deficient diet (MC-deficient diet). Examination of the overall liver morphology showed that the livers of MCD-diet mice changed to bluish-white compared to normal mice (Figure 4). In contrast, the livers of NASH mice treated with BxC-V37e were observed to be darker as seen in the control group. And no difference was observed in the livers of animals treated with Dulaglutide and PBS. There was no difference in the total liver weight between NASH mice treated with PBS and BxC-V37e (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 those of animals treated with PBS (Figure 5 and Figure 6).

[0149] In addition, as a result of observing the morphological changes after H&E staining and treatment with BxC-V37e, fewer lipid droplets and a decrease in infiltration of inflammatory cells were found in the MCD + BxC-V37e group compared to the MCD + PBS group (Figure 7), and the NAS score analysis, which is an index of NAFLD severity, decreased in the MCD + BxC-V37e group (Figure 8). In short, such data suggest that BxC-V37e generally improves liver function in NASH.

[0150] 2-3. Confirmation of the inhibitory effect of exosomes (BxC-e) derived from induced pluripotent stem cell-derived mesenchymal stem cell precursors on adipocyte differentiation Human adipocytes (primary human adipocyte, ATCC, USA) were dispensed into 6-well plates and cultured at 37 °C and 5% CO 2 in an incubator until they reached a confluent state (up to 6 days).

[0151] After culturing the adipocytes for 6 days, they were cultured for 5 days in a basic medium supplemented with an adipogenic medium [34 μM pantothenate (Sigma), 66 μM biotin (Sigma), 0.5 mM insulin (Sigma), 1 mM dexamethasone (Sigma) and 0.05 M IBMX (Sigma)] in DMEM medium (Gibco, USA) supplemented with 1% penicillin-streptomycin and 10% FBS. Thereafter, they were further cultured for 9 days in a medium supplemented with an adipogenic medium [34 μM pantothenate (Sigma), 66 μM biotin (Sigma), 0.5 mM insulin (Sigma) and 1 mM dexamethasone (Sigma)] in DMEM medium (Gibco, USA).

[0152] At this time, the negative control group was adipocytes cultured in DMEM medium supplemented with 10% FBS for 14 days, the vehicle control group was adipocytes cultured under adipogenic conditions for 14 days without treatment with BxC-e, and the BxC-e treatment group was adipocytes cultured under adipogenic conditions containing BxC-e for 14 days.

[0153] After completely removing the culture medium, the cells were washed twice with PBS, and 400 μl / well of 10% formalin solution was added to fix the cells for 1 hour. The cells were washed with PBS, 400 μl / well of Oil-red O working solution was added, and the fat in the differentiated adipocytes was stained for 2 hours. Then, the Oil-red O working solution was removed, and after completely removing the Oil-red O working solution adhering to the well walls using double-distilled water, the cells were placed in a dryer and dried for 5 minutes, and then 500 μl / well of isopropyl alcohol was added to the wells.

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

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

[0156] 2-4. Evaluation of the accuracy of non-alcoholic fatty liver disease diagnosis using exosome-derived ApoA-1 protein The ApoA-1 protein levels in BxC-V37e were quantitatively compared using immunoblot analysis. As a result of the analysis, exosome-derived ApoA-1 protein decreased in NASH mice, whereas it increased in animals administered with BxC-V37e (Figs. 10 and 11). Also, exosome-derived ApoA-1 in serum decreased in obese patients with NAFLD compared to the control group (Fig. 12).

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

[0158] As a result of the correlation analysis, when analyzing the correlation between ApoA-1 and metabolic parameters in human serum, exosome-derived ApoA-1 and almost all non-alcoholic fatty liver disease-related metabolic parameters showed a strong negative correlation (Figs. 13 to 24). And it was observed that the correlation between exosome-derived ApoA-1 and non-alcoholic fatty liver disease-related metabolic parameters was stronger than the correlation between human serum ApoA-1 and metabolic parameters in all cases except for the TC index (Figs. 25 to 36).

[0159] Such results mean that the diagnosis of non-alcoholic fatty liver disease using exosome-derived ApoA-1 protein can identify the disease with significantly higher accuracy compared to the method of diagnosing non-alcoholic fatty liver disease using the existing serum ApoA-1 protein as a marker, suggesting that exosome-derived ApoA-1 in serum can be used as a new biomarker for non-alcoholic fatty liver disease.

[0160] 2-5. Evaluation of the accuracy of predicting the prognosis of non-alcoholic fatty liver disease using exosome-derived ApoA-1 protein Tested how well serum ApoA-1 and exosome-derived ApoA-1 reflected changes in existing NASH-related parameters after administration of NASH therapeutic agents, and evaluated the prognostic analysis accuracy using exosome-derived ApoA-1.

[0161] Administered Dulaglutide, BxC-e, and BxC-V37e, known as NASH treatment candidate substances, to MCD diet mice, calculated the correlation of the change rates of existing NASH-related parameters of AST, ACC1, hs-CRP, NRF2, PCNA, Annexin A5 numerical values and Oil Red O staining results of serum ApoA-1 and exosome-derived ApoA-1 compared to the PBS group, and then performed statistical analysis using simple regression analysis of the SPSS statistical program (if the P value is 0.05 or less, it is considered to have statistical significance).

[0162] As a result of the experiment, as can be confirmed from Figures 37 to 40, the change rates of AST, ACC1, hs-CRP, and NRF2 numerical values in the Dulaglutide-administered group of MCD diet mice compared to the PBS group had a more significant correlation with the change rate of exosome-derived ApoA-1 between the PBS group and Dulaglutide than with the change rate of serum ApoA-1 between the PBS group and the Dulaglutide-administered group.

[0163] And, as can be confirmed from Figures 41 to 44, the change rates of AST, ACC1, hs-CRP, and PCNA numerical values in the BxC-V37e-administered group of MCD diet mice compared to the PBS group had a more significant correlation with the change rate of exosome-derived ApoA-1 between the PBS group and BxC-V37e than with the change rate of serum ApoA-1 between the PBS group and the BxC-V37e-administered group.

[0164] Furthermore, as can be confirmed from FIGS. 45 to 47, even when BxC-e was administered, the change rates of hs-CRP, Annexin A5 numerical values, and Oil Red O staining results in the BxC-e administration group of MCD diet mice compared to the PBS group were more significantly correlated with the change rate of exosome-derived ApoA-1 between the PBS group and the BxC-e administration group than with the change rate of serum ApoA-1 between the PBS group and the BxC-e administration group.

[0165] These results indicate that after administration of a therapeutic agent such as dulaglutide, BxC-e, or BxC-V37e, the degree of change in liver function indices, lipogenesis indices, and inflammation indices is more accurately reflected by exosome-derived ApoA-1 than by 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 than existing prognosis prediction compositions or prognosis prediction methods, suggesting that it can be variously used in relation to the diagnosis of non-alcoholic fatty liver disease.

Industrial Applicability

[0166] The present invention relates to a marker for diagnosing non-alcoholic fatty liver disease, and more particularly, to a composition containing a preparation for measuring the level of exosomal ApoA-1 protein, which can diagnose non-alcoholic steatohepatitis (NASH) with high accuracy, and a kit containing the same.

Claims

1. A composition for diagnosing 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 suffering from or suspected of suffering from non-alcoholic fatty liver disease (NAFLD).

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

3. The composition for diagnosing non-alcoholic fatty liver disease according to claim 1 , wherein the preparation for measuring the level of the protein comprises an antibody or an aptamer specific to the protein.

4. The composition for diagnosing non-alcoholic fatty liver disease 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 non-alcoholic fatty liver disease (NAFLD) diagnostic kit 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 developed or is suspected of developing non-alcoholic fatty liver disease (NAFLD).

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

7. The kit for diagnosing non-alcoholic fatty liver disease according to claim 5 , wherein the preparation for measuring the level of the protein comprises an antibody or an aptamer specific to the protein.

8. The non-alcoholic fatty liver disease diagnostic kit according to claim 5, which is an enzyme-linked immunosorbent assay (ELISA) kit.

9. A method for providing information for diagnosing nonalcoholic fatty liver disease (NAFLD) in an individual, the method comprising a step of measuring a level of exosome-derived ApoA-1 (Exosomal ApoA-1) protein from a biological sample isolated from an individual suffering from or suspected of suffering from nonalcoholic fatty liver disease (NAFLD).

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 method for providing information according to claim 10, further comprising a step of determining that 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.

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

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