Composition for preventing or treating non-alcoholic steatohepatitis and screening method for non-alcoholic steatohepatitis therapeutic agent

Abstract

The present invention relates to a composition for the prevention or treatment of non-alcoholic fatty liver disease and a screening method for a non-alcoholic fatty liver disease treatment. More specifically, it was confirmed that FPR2 is expressed in the livers of female mice, whereas its expression is suppressed in the livers of male mice; it was confirmed that nodular and inflammatory responses increase in the livers of male mice fed a high-fat diet in which FPR2 expression is suppressed; and it was confirmed that liver fibrosis and inflammation increase in the livers of female mice fed a high-fat diet in which FPR2 expression is deficient. Accordingly, the FPR2 or an agent thereof may be provided as a composition for the prevention or treatment of non-alcoholic fatty liver disease.

Description

Technology Field

[0001] The present invention aims to provide a composition containing FPR2 (Formyl peptide receptor 2) or an agonist thereof as an active ingredient, to be provided as a preventive or therapeutic agent for non-alcoholic steatohepatitis (NASH). Background Technology

[0002] The liver is an organ that plays a central role in nutrient metabolism, and a normal human liver weighs about 1,500g. When liver disease develops and liver dysfunction occurs, problems arise in the body's nutrient metabolism; specifically, liver dysfunction occurs in areas such as converting glucose into glycogen, converting proteins into albumin, or breaking down unnecessary substances and delivering them to bile.

[0003] Among various liver diseases, nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and refers to fatty liver that primarily occurs in people who do not drink alcohol or rarely drink.

[0004] The symptoms of non-alcoholic fatty liver disease vary in severity, ranging from simple steatosis (nonalcoholic fatty liver, NAFL) to the chronic disease of nonalcoholic steatohepatitis (NASH). In the severe form, nonalcoholic steatohepatitis, necrosis and inflammation processes induce the progressive accumulation of fibrosis in the liver, which can progress to cirrhosis and liver cancer. The causes of this non-alcoholic fatty liver disease are presumed to include obesity, diseases such as hyperlipidemia (high blood lipid levels) or diabetes, medications, and severe nutritional deficiencies.

[0005] In addition, NAFL shows slow histological progression of simple steatosis, whereas NASH shows faster histological progression and can progress to liver cirrhosis, and about 5 to 10 percent of people diagnosed with NAFL are diagnosed with NASH.

[0006] However, effective diagnostic methods for NASH have not yet been developed, and there are currently no approved treatments for the condition. Consequently, medications for other metabolic syndromes—such as those for abdominal obesity, hyperlipidemia, and diabetes—are being used for treatment; for instance, insulin resistance improvers, antioxidants, dyslipidemia treatments, and hepatoprotective agents. However, these cannot be considered direct treatments for NASH. Prior art literature

[0007] Republic of Korea Published Patent No. 10-2020-0031745 (Published on March 25, 2020) The problem to be solved

[0008] The present invention provides a pharmaceutical composition and a health food useful for the prevention and treatment of non-alcoholic fatty liver disease comprising FPR2 or an agonist thereof as an active ingredient, and can provide a treatment for non-alcoholic fatty liver disease by screening candidate substances that induce an increase in FPR2 expression. means of solving the problem

[0009] The present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH) containing FPR2 or an agonist thereof as an active ingredient.

[0010] The present invention provides a health food for the prevention or improvement of non-alcoholic steatohepatitis (NASH) containing FPR2 or an agonist thereof as an active ingredient.

[0011] In addition, the present invention comprises the step of treating a candidate substance to a cell isolated from an organism;

[0012] The present invention provides a screening method for a non-alcoholic fatty liver disease (NASH) treatment, comprising the steps of: confirming the FPR2 expression level in cells treated with the candidate substance; and comparing the FPR2 expression level with the FPR2 expression level in cells not treated with the candidate substance. Effects of the invention

[0013] According to the present invention, it was confirmed that FPR2 is expressed in the liver of female mice, whereas its expression is suppressed in the liver of male mice; it was confirmed that nodular and inflammatory responses increase in the liver of male mice fed a high-fat diet with suppressed FPR2 expression, and liver fibrosis and inflammation increase in the liver of female mice fed a high-fat diet with a deficiency in FPR2 expression. Accordingly, the FPR2 or an agent thereof may be provided as a composition for the prevention or treatment of non-alcoholic fatty liver disease (NASH). Brief explanation of the drawing

[0014] Figure 1 shows the results of confirming the creation of a human-like NAFLD experimental animal model with a CDAHFD diet, and Figure 1a is a schematic diagram showing the animal experiment process mimicking human-like NAFLD, in which 7-week-old WT male and female mice were fed a normal diet (Chow-WT male and Chow-WT female) or CDAHFD (CDAHFD-WT male and CDAHFD-WT female) for 6, 12, and 36 weeks. Figure 1b shows the results of confirming the representative liver morphology of each group, where nodules indicated by yellow arrows were identified in the liver images of CDAHFD-treated mice, Figure 1c shows the results of counting the number of mice with nodules and measuring and plotting the diameter of the nodules of said mice (*p<0.05, **p<0.005 vs CDAHFD-WT male), and Figure 1d shows the results of confirming the serum AST and ALT levels in all WT mice, with the results expressed as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 2 shows the results of confirming liver damage in male and female mice fed a CDAHFD diet. Figure 2a is a representative image of H&E-stained liver tissue from WT male and female mice fed a normal diet or treated with CDAHFD, where white circles indicate nodular regions within the liver. Figure 2b shows the ratio of liver weight to body weight. Figure 2c shows the results of confirming the liver triglyceride (TG) content of the mice. Data are presented as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 2d is a representative image of active Caspase-3-immunohistochemically stained liver tissue from the mice, where all scale bars are 50 μm. Figure 3 shows the results of confirming the increased expression of fibrosis and pro-inflammatory factor markers in WT male and female mice fed either the feed or CDAHFD for 6, 12, and 36 weeks. Figure 3a shows the results of qRT-PCR analysis of the fibrosis markers, α-Sma and Col1α1, and Figure 3b shows the results of confirming the expression levels of pro-inflammatory markers including Tnf-α and Il-6. Data values ​​are expressed as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 4 shows the results confirming increased liver fibrosis and inflammation in males compared to females in the CDAHFD diet group. Figure 4a shows the Western blot analysis results of liver α-Sma and Col1α1 in WT male and female mice treated with the diet or CDAHFD, where each line contains protein lysates pooled from 3 mice per group at the same concentration, and the graph uses an internal control. Figure 4b shows the results confirming liver hydroxyproline content in liver tissues obtained from representative mice per group. Data are presented as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 4c shows representative images of liver tissues from the CDAHFD-WT group stained with Sirius Red (left) and F4 / 80 (right) (Scale bar, 50 μm), and Figure 4d shows the results of confirming serum Tnf-α and Il-6 levels in representative mice of each group. Data are presented as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 5 shows the results of confirming distinct expression of FPR2 in the livers of female mice. Figure 5a shows the qRT-PCR analysis results, and Figure 5b shows the Western blot analysis results. Data values ​​are expressed as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Each lane contained protein lysates pooled from 3 mice per group at the same concentration, and the graph used an internal control. The data is one of three experiments with similar results. Figure 5c shows a representative FPR2 immunostained liver image of the mice mentioned above. The right panel is an X100 (Scale bar, 50μm) magnified image of a WT female mouse fed the feed. Figure 6 shows the results confirming that FPR2 deficiency promotes severe liver damage in female mice in the CDAHFD diet group. Figure 6a is a schematic diagram showing the process of conducting animal experiments on 7-week-old KO male and female mice fed either feed or CDAHFD for 6, 12, and 36 weeks. Figure 6b shows the results confirming representative liver morphologies for each group, with yellow arrows indicating nodules. Figure 6c shows the results of counting the number of KO mice with nodules and measuring and plotting the diameter of the nodules in said mice. Figure 6d shows the results confirming the serum AST and ALT levels of said mice. Data values ​​are expressed as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Figure 7 shows the results of confirming liver damage in FPR2-deficient female mice, Figure 7a is a representative H&E-stained liver tissue image of KO male and female mice treated with feed or CDAHFD (Scale bar, 50μm), Figure 7b is a graph confirming liver weight versus body weight (LW / BW), Figure 7c is the results confirming liver TG levels in the mice, data are presented as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control), and Figure 7d is the immunohistochemistry result of staining active Caspase-3 in liver sections of representative mice from each group (Scale bar, 50μm). Figure 8 confirms that FPR2 deficiency increases liver fibrosis in female mice fed the CDAHFD diet, based on the results of examining α-Sma and Col1α1 in the livers of male and female KO mice fed or treated with CDAHFD for 6, 12, and 36 weeks. Figure 8a shows the results of qRT-PCR analysis, and Figure 8b shows the results of Western blot analysis. Data are presented as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control). Each line of the immunoblot contains a pooled protein lysate from three representative mice per group at the same concentration. The graph uses an internal control, and the data shown represents one of three experiments with similar results. Figure 8c shows the results of confirming the liver hydroxyproline content in the mouse, and Figure 8d is a representative image of a Sirius Red-Silver section of the mouse, magnified to ×40 (Scale bar, 50 μm) of a female mouse on the CDAHFD diet at 36 weeks. Figure 9 shows the results confirming that FPR2 deficiency increases inflammation in female mice during the CDAHFD diet; Figure 9a shows the qRT-PCR analysis results confirming inflammatory markers including Tnf-α and Il-6 in KO male and female mice fed or treated with CDAHFD for 6, 12, and 36 weeks, and Figure 9b shows the results confirming the serum levels of Tnf-α and Il-6 in the mice; all data are expressed as mean ± SEM (n ≥ 3 / group, *p<0.05, **p<0.005 vs own control), and Figure 9c is a representative image of F4 / 80 stained liver tissue (Scale bar, 50μm). Figure 10 shows the results confirming the protective effect of high FPR2 expression on hepatocytes in the livers of female mice. Figure 10a shows the qRT-PCP analysis results confirming FPR2 expression in primary hepatocytes (pHEPs) of WT and KO male and female mice; each experiment was repeated at least three times, and the results are expressed as mean ± SEM (*p<0.05, **p<0.005 vs WT male-pHEPs). Figure 10b shows the Western blot analysis results, and Figure 10c shows the confocal image confirming FPR2 (red) in the cells. The graph used an internal control, nuclear contrast staining was performed with DAPI (blue), and the indicated data represents one of three experiments with similar results (Scale bar, 20μm). Figure 10d shows the results of qRT-PCR analysis for FPR2 and G6pc, Figure 10e shows the results of confirming cell viability, and Figure 10f shows the results of Western blot analysis confirming activated Caspase-3 and pro-Caspase-3 in primary hepatocytes (pHEPs) isolated from WT and KO female mice treated with 250 μM of vehicle (Veh) or palmitate (PA). The data shown is one of three experiments with similar results, and the results obtained from the replicate experiment are expressed as mean ± SEM (*p<0.05, **p<0.005 vs WT-Veh). Figure 11 shows the results of confirming FPR2 expression in hepatocytes. Figure 11a shows the results of Western blot analysis confirming FPR2 in primary hepatocytes (pHEP), quiescient hepatic stellate cells (HSC D0), cultured activated HSCs (HSC D7), intrahepatic vascular endothelial cells (LSECs), and Kupffer cells. The graph uses an internal control, and the data is one of three experiments with similar results. Figure 11b is a representative confocal image of FPR2 (red) in primary Kupffer cells isolated from the above mice, with DAPI (blue) used for nuclear counterstaining (Scale bar, 20 μm). Figure 12 shows the results of confirming FPR2 expression in human livers according to sex and age during the progression of NAFLD. It is a microarray analysis result confirming intrahepatic FPR2 expression in the livers of healthy individuals and NAFLD patients among young people aged 13 to 19, and the data values ​​are displayed as a dot plot representing mean ± SEM (n ≥ 5 individuals / group, *p<0.05, **p<0.005 vs healthy female). Figure 13 is a schematic diagram illustrating the therapeutic potential of FPR2 for the treatment of NAFLD / NASH. Since FPR2 has been identified as alleviating liver inflammation and fibrosis and contributing to the progression of NAFLD / NASH, FPR2 has the potential to be a new therapeutic target. Figure 14 shows the results of analyzing the hepatoprotective effects of different ligand-activated Fpr2s on lipotoxicity in primary hepatocytes, where Figure 14a is a schematic diagram showing an in vitro experiment in which primary hepatocytes (pHEP) of WT male mice treated with LXA4 (20 nM) were exposed to PA (250 μM) for 24 hours, and Figure 14b is a diagram of formyl peptide receptor 2 ( Fpr2 ) and glucose-6-phosphatase( G6pcFigure 11c is a graph of qRT-PCR analysis for ), and a graph showing cell viability in each cell, with results obtained from three replicate experiments presented as mean ± SEM (*p<0.05, **p<0.005 vs own control). Figure 11d is the result of Western blot analysis for activated Caspase-3, pro-Caspase-3, and Fpr2 in each cell, and Gapdh was used as an internal control. The data represents one of three experiments with similar results. Figure 11e is the result of Oil-red O staining for lipid droplets in each cell, with a scale bar of 20 µm. Specific details for implementing the invention

[0015] The present invention will be described in more detail below.

[0017] The present invention may provide a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH) containing FPR2 or an agonist thereof as an active ingredient.

[0018] The above agent may be one or more selected from the group consisting of LXA4, Resolvin D1, MMK-1, Annexin I, TC-FPR42 and Quin-c1.

[0019] The above FPR2 or its agonist may inhibit hepatocyte fibrosis and inflammatory response caused by lipotoxicity.

[0020] Specifically, the above agent can protect cells by inducing Fpr2 expression and activation to inhibit hepatocyte death caused by lipotoxicity, thereby suppressing liver fibrosis and inflammatory responses.

[0021] The above FPR2 may show differences in expression depending on gender, and more specifically, the expression of the above FPR2 may be suppressed among men, whereas it may be expressed among women.

[0022] The FPR2 (Formyl peptide receptor 2) of the present invention may be NCBI accession number. 2358.

[0023] In one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease (NASH) containing the FPR2 or an agent thereof as an active ingredient may use any one formulation selected from the group consisting of an injection, a granule, a powder, a tablet, a pill, a capsule, a suppository, a gel, a suspension, an emulsion, a drop, or a liquid according to conventional methods.

[0024] In another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease (NASH) containing FPR2 or an agent thereof as an active ingredient may further include one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants that are commonly used in the manufacture of pharmaceutical compositions.

[0025] Specifically, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as the base for suppositories.

[0026] According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner through intravenous, intra-arterial, intra-abdominal, intramuscular, intrasternal, transdermal, nasal, inhalation, local, rectal, oral, ocular, or intradermal routes.

[0027] The preferred dosage of the above FPR2 or its agonist may vary depending on the subject's condition and body weight, the type and severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, and more specifically 0.1 to 100 mg / kg. The administration may be performed once a day or divided into several doses, and the scope of the present invention is not limited by this.

[0028] In the present invention, the 'object' may be a mammal including a human, but is not limited to these examples.

[0029] The present invention may provide a health food for the prevention or improvement of non-alcoholic steatohepatitis (NASH) containing FPR2 or an agonist thereof as an active ingredient.

[0030] The above-mentioned health food may be used in combination with other foods or food additives in addition to the above-mentioned FPR2 or its agent, and may be used appropriately according to conventional methods. The mixture of active ingredients may be appropriately determined according to the purpose of use, for example, prevention, health, or therapeutic treatment.

[0031] The effective dose of the compound contained in the above health food may be used in accordance with the effective dose of the above therapeutic agent, but in the case of long-term consumption for the purpose of health and hygiene or health control, it may be less than the above range, and it is certain that the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety.

[0032] There are no special restrictions on the types of health foods mentioned above, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0033] The present invention comprises the step of treating a candidate substance to a cell isolated from an individual;

[0034] A step of confirming the FPR2 expression level in cells treated with the above candidate substance; and

[0035] A screening method for a non-alcoholic fatty liver disease treatment can be provided, comprising the step of comparing the above FPR2 expression level with the FPR2 expression level of cells not treated with the candidate substance.

[0036] The above screening method for non-alcoholic fatty liver disease treatments may involve screening for a non-alcoholic fatty liver disease treatment if the FPR2 expression level in cells treated with the candidate substance is higher than the FPR2 expression level in cells not treated with the candidate substance.

[0037] The above sample may be selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

[0038] The above FPR2 expression level may be measured by one or more selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting, and flow cytometry (FACS), but is not limited thereto.

[0040] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0042] <Experimental Example>

[0043] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0045] 1. Animal testing

[0046] Male and female C57BL / 6 mice (wild-type; WT) were purchased from Hyochang (Daegu, Korea). FPR2 knockout (KO) mice induced by a 129S / SvEv × C57BL / 6 strain mixture were donated by Dr. Kim (Pusan ​​National University School of Medicine, Yangsan, Korea).

[0047] All mice were maintained in a 12-hour light / dark cycle and given free access to normal food and water. To mimic human-like NAFLD, 7-week-old male and female WT and KO mice were fed a normal diet or a choline-deficient, high-fat diet consisting of 60 kcal% and 0.1% methionine with L-amino acid definition (CDAHFD; Research diet, New Brunswick, NJ, USA) (n=5 / group) for 6, 12, and 36 weeks, respectively (n=4 / each chow group; n=5 / each CDAHFD group).

[0048] Mice were sacrificed to collect blood and liver samples at the end of each experiment.

[0050] 2. Isolation of pHEP and Cell Experiments

[0051] To isolate primary hepatocytes (pHEPs), two-step collagenase perfusion was performed using the previously reported method (Seglen PO., J. Toxicol. Environ. Health 5, 551-560, 1979).

[0052] Briefly, mice were anesthetized with isoflurane, secured in a supine position on a treatment table, and a cannula was inserted into the inferior vena cava under sterile conditions.

[0053] Cells were dispersed by perfusing liver with EGTA and collagenase (Sigma Aldrich, St. Louis, MO, USA). Primary hepatocytes were separated from destroyed cells using Percoll density gradient centrifugation, and cell viability was > 92% in all experiments, as confirmed by the trypan blue exclusion test.

[0054] Primary hepatocytes were placed in a collagenase-coated 6-well plate at a ratio of 1 × 10⁶ 5 4 × 10 cells / well or 60 mm plate 5 Cells were cultured at a cell / plate density using Williams' Medium E, which is phenol red-free and contains 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1 μM dexamethasone, penicillin / streptomycin cocktail solution (P / S), ITS+ (insulin, transferrin, selenium complex, BSA, and linoleic acid), GlutaMAX™, and HEPES (Gibco).

[0055] After attaching the cells for about 4 hours, the culture medium was replaced with serum-free Williams' Medium E containing 0.1 μM dexamethasone, P / S cocktail solution, ITS+, GlutaMAX™, and HEPES to maintain the hepatocytes.

[0056] To determine the effect of FPR2 in hepatocytes, pHEP isolated from WT and KO female mice was deprived of serum overnight and exposed to 250 μM palmitic acid (PA; P0500; Sigma Aldrich), a lipotoxin inducer, for 24 hours, and the experiment was repeated at least 3 times.

[0058] 3. Cell proliferation analysis

[0059] Cell proliferation was confirmed using the Cell Titer Proliferation Assay (MTS; Promega) according to the manufacturer's instructions. Briefly, pHEP was 4 × 10 3 Cells were seeded into a 96-well plate at a cell / well density and further exposed to 250 μM PA for 24 hours.

[0060] After treatment, 10 μl of MTS reagent was added to each well, and the plate was incubated in a CO2 incubator at 37°C until color developed.

[0061] Absorbance was measured at a wavelength of 490 nm using a Glomax multi-detection system (Promega).

[0063] 4. Liver Histology and Immunohistochemistry

[0064] To confirm liver morphology and evaluate liver fibrosis, liver specimens were fixed with 10% neutral buffered formalin, paraffin-embedded, and then cut into 4 μm sections. After removing the paraffin from the specimens, they were hydrated and stained with standard hematoxylin and eosin (H&E) and Sirius Red using standard methods.

[0065] For histochemical immunochemistry (IHC), compartments were incubated in 3% hydrogen peroxide for 10 minutes. Antigen recovery was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes or by incubating with 0.2% pepsin for 10 minutes. Compartments were blocked with protein blocking solution (X9090; Dako, Carpinteria, CA, USA) for 30 minutes and specifically stained by incubating overnight at 4°C with primary antibody-activated caspase-3 (AF835; R&D systems, Minneapolis, MN, USA), F4 / 80 (ab6640; Abcam, Cambridge, MA, USA), FPR2 (ab203129; Abcam), or non-immunosed serum.

[0066] Polymer horadish peroxide (HRP) anti-rabbit (K4003; Dako) or HRP anti-rat IgG (A110-105P; BETHYL, Montgomery, Texas, USA) was used as a secondary antibody, and 3,3'-Diaminobenzidine (DAB) (K3466; Dako) was used for the detection process.

[0068] 5. Immunofluorescence staining

[0069] For immunofluorescence staining, pHEP and Kupffer cells were collected from WT and KO male / female mice and deposited as a monolayer on a slide area designated by cytospin centrifugation (Cell spin; Hanil Scientific, Gimpo, Korea).

[0070] The separated pHEP was fixed and permeated with cold acetone and methanol, respectively.

[0071] Cells were washed with TBS and incubated with blocking solution for 30 minutes. pHEP was incubated with the primary antibody anti-FPR2 (ab203129; Abcam) overnight at 4°C. Cells were washed with TBS and incubated with the fluorescence-labeled secondary antibody Alexa Fluor 568 goat anti-rabbit IgG (diluted 1:100; Invitrogen) for 30 minutes at room temperature.

[0072] Slide antifed mounting medium containing 4′,6-diamidno-2-phenylinole (DAPI, VectaShield, Burlingame, CA, USA) was placed on the slide, and the slide was examined using a Zeiss LSM 800 confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA).

[0074] 6. Liver triglyceride analysis

[0075] Liver triglyceride (TG) levels were checked using the Triglyceride Colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.

[0076] Briefly, liver tissue was homogenized using NP40, and the tissue homogenate was centrifuged at 10,000 rcf for 10 minutes at 4°C. The supernatant containing TG was used for biochemical analysis. 10 μl of the supernatant and 150 μl of the enzyme solution were added to each well and incubated for 15 minutes, after which the absorbance was determined at a wavelength of 540 nm using a Glomax multi-detection system (Promega).

[0078] 7. Serum Biochemical Analysis and Enzyme-Linked Immunosorbent Assay (ELISA)

[0079] Serum aspartate aminotransferase (AST / GOT, glutamate-oxaloacetate transaminase) and alanine aminotransferase (ALT / GPT, glutamate pyruvate transaminase) were identified using GOT reagents (AM103-K; Asan Pharmaceutical, Seoul, Korea) and GPT reagents (AM102-K; Asan Pharmaceutical) according to the manufacturer's instructions.

[0080] Serum levels of 17β-estradiol, TNF-α, and IL-6 were determined using 17β-estradiol (ES180S-100; Calbiotech, El Cajon, CA, USA), IL-6 (M6000B; R&D systems), and TNF-α (MTA00B; R&D systems) mouse ELISA kits according to the manufacturer's instructions.

[0082] 8. Western Blot Analysis

[0083] Total protein was extracted from primary cells or freeze-fixed liver tissue samples and stored at -80°C. The samples were homogenized with Triton lysis buffer (TLB) containing a protease inhibitor (Complete mini; Roche, Indianapolis, IN, USA) and centrifuged at 13,000 rcf for 15 minutes at 4°C.

[0084] Subsequent biochemical analysis was performed using the supernatant containing the protein extract.

[0085] Protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Scientific). The expression of each protein was verified prior to pooling the protein lysates. Subsequently, equal amounts of protein lysates from representative mice (n=3) per treatment group were combined, and the protein concentration was determined.

[0086] To denature and reduce the protein sample, the protein was boiled at 100°C for 10 minutes in 5× sample buffer containing β-mercaptoethanol and sodium dodecyl sulfate (SDS).

[0087] The total 50 μg protein lysate was separated onto a 10 or 12% tris-glycine gel by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a 0.45 μm pore-sized polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany).

[0088] Primary antibodies were used against rabbit polyclonal anti-FPR2 (diluted 1:1000; NLS1878; Novus Biologicals), mouse monoclonal anti-α-Sma (diluted 1:1000; A5228; Sigma-Aldrich), rabbit polyclonal anti-Col1α1 (diluted 1:1000; NBP1-30054; Novus Biologicals, LLC, USA), rabbit polyclonal anti-cleaved caspase-3 (diluted 1:1000; 9661; Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-caspase-3 (diluted 1:1000; 9662; Cell Signaling), and mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Gapdh; diluted 1:1,000; AbD Serotec, Oxford, UK). HRP-conjugated anti-rabbit or anti-mouse IgG (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used as a secondary antibody.

[0089] Protein bands were detected using EzWestLumi ECL solution (ATTO Corporation, Tokyo, Japan) according to the manufacturer's instructions. Band intensity was calculated using the CS analyzer 4.0 program (Version 1.0.3, ATTO).

[0091] 9. RNA Analysis

[0092] Total RNA was extracted from liver tissue or pHEP using Trizol reagent (Invitrogen).

[0093] RNA concentration and purity were verified using a nanodrop. Complementary template DNA was synthesized using the SuperScript First-strand Synthesis System (Invitrogen) according to the manufacturer's instructions. Real-time qRT-PCR analysis was performed using the Power SYBR Green Master Mix (Applied Biosystem) according to the manufacturer's instructions. All reactions were repeated twice, and data analysis was performed using the ΔΔCt method. Expression values ​​were normalized to the level of mouse 40S ribosomal protein S9 mRNA. The sequences of all primers used are shown in Table 1, and all PCR products were directly sequenced for gene verification (Macrogen).

[0095] 10. Hydroxyproline Analysis

[0096] The hydroxyproline content of the liver was confirmed using the previously reported method (33).

[0097] Briefly, 50 mg of freeze-dried liver tissue was hydrolyzed with 6N HCl at 110 °C for 16 hours. The hydrolysate was evaporated under vacuum, and the precipitate was redissolved in 1 ml of distilled water. The solution was filtered at 14,000 rpm for 5 minutes using a 0.22 μm filter centrifuge tube (Corning Incorporated, Corning, NY, USA). 0.5 ml of a chloramine-T solution, prepared by dissolving 1.41 g of chloramine-T in 80 ml of acetate buffer and 20 ml of 50% isopropanol, was added, and the mixture was incubated at room temperature for 20 minutes. Subsequently, 13 ml of 60% perchloric acid and 0.5 ml of Ehrlich's solution, prepared by dissolving 7.5 g of dimethylaminobenzaldehyde in 30 ml of isopropanol, were added and mixed, then incubated at 65 °C for 15 minutes. After cooling to room temperature, the standard and sample were measured at 561 nm using a spectrophotometer.

[0098] The amount of hydroxyproline in each sample was determined using the regression curve of high-purity trans-4-hydroxy-L-proline (Sigma-Aldrich) as a standard. Total hydroxyproline was calculated based on the weight of each liver (mg hydroxyproline / mg liver).

[0100] 11. Analysis of Microarray Results of Inter-human Samples

[0101] Microarray datasets of human samples were obtained from the publicly available Gene Expression Omnibus (GEO). The samples used were healthy controls (males, n=5, females, n=8) and NAFLD patients (males, n=6, females, n=16) aged 13–19 years (GEO access number: GSE66676). For comparison between NAFLD patients and healthy controls, FPR2 expression differences were graphed after considering a p-value less than 0.05 as differential expression with a p-value greater than 2.05.

[0103] <Example 1> Confirmation of liver damage caused by human-like NAFLD / NASH induced by CDAHFD

[0104] To mimic the pathogenesis of human NAFLD progression with NASH-HCC and to confirm sex differences in NAFLD, mice of two sexes fed CDAHFD for 6, 12, and 36 weeks were used as an animal model, as shown in Figure 1a. When the livers of the male and female mice fed with food were examined visually, they showed a healthy liver condition during the feeding period, as shown in Figure 1b. However, the livers of the male mice fed with CDAHFD were enlarged and pale in color compared to the mice fed with food, whereas the livers of the female mice fed with CDAHFD showed a relatively smooth macroscopic appearance at 6 and 12 weeks.

[0105] In addition, as shown in Figure 1c, compound nodules were identified in all male mice at 36 weeks of CDAHFD treatment, whereas compound nodules were identified in only 2 out of 5 female mice. Furthermore, it was observed that the size of the nodules was significantly larger in male mice than in female mice.

[0106] Meanwhile, as shown in Figure 2a, fat accumulation was observed in mice supplied with CDAHFD at 6 weeks, and as shown in the H&E staining results, much larger and more numerous fat droplets were observed in male mice than in female mice.

[0107] At 12 weeks, hepatocyte death was first observed in male mice supplied with CDAHFD, and at 36 weeks, nodular lesions (circular markings) showing increased inflammation, hepatocyte swelling and nodular lesions, and atypical adenomatous proliferation were observed.

[0108] Female mice fed CDAHFD showed relatively less hepatocyte death and small nodular lesions compared to male mice fed CDAHFD, and no abnormal histomorphological changes were observed in the feed group.

[0109] As shown in Figures 2b and 1d, the ratio of liver weight to body weight (LW / BW) and serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased significantly during the CDAHFD diet, and among mice fed CDAHFD, the LW / BW ratio and AST levels were found to be significantly higher than those of female mice at 36 weeks.

[0110] In addition, as shown in Figure 2c, the level of hepatic triglycerides (TG) gradually increased in male mice during the CDAHFD diet and was found to be significantly higher than in female mice at 36 weeks after CDAHFD supply. Since massive hepatocyte death is a major process involved in the development of NASH, hepatocyte death was confirmed in the experimental model.

[0111] Immunohistochemical analysis of active caspase-3, the active form of a major regulator of apoptosis, revealed caspase-3-expressing cells in the livers of mice fed the CDAHFD diet, as shown in Figure 2d. These cells were found in greater numbers in male mice than in female mice at 12 and 36 weeks, and were rarely found in the livers of WT mice in the diet group.

[0112] From the above results, it was confirmed that CDAHFD produced an experimental animal model of NAFLD progression similar to human NAFLD / NASH, and that male mice were more susceptible to this damage than female mice.

[0114] <Example 2> Confirmation of Excessive Liver Fibrosis and Inflammation in Male Mice on CDAHFD Diet

[0115] Since inflammation and fibrosis are associated with the pathogenesis of NAFLD, liver fibrosis and inflammation were confirmed in a mouse model.

[0116] As a result, as shown in Figure 3a, it was confirmed that the fibrosis markers alpha-smooth muscle actin (α-Sma) and collagen 1 (Col1α1) were significantly increased in WT mice on the CDAHFD diet compared to mice on the feed group, and it was confirmed that the expression of the said genes was significantly increased in male mice on the CDAHFD diet compared to female mice on the CDAHFD diet at weeks 12 and 36.

[0117] In addition, RNA data was confirmed by Western blot analysis, and as shown in Figure 4a, higher expression of α-SMA and Col1α1 was observed in male mice of the CDAHFD diet group compared to female mice of the CDAHFD diet group. Furthermore, as shown in Figure 4b, an increase in collagen fibers was confirmed in the CDAHFD diet group compared to the diet groups at weeks 12 and 36 of the biochemical analysis, and the highest amount was observed in male mice at week 36 among all mice fed CDAHFD.

[0118] As shown in the Sirius Red staining analysis results, more collagen fibrils were deposited in the liver of the CDAHFD diet group (Fig. 4c, left panel) than in the liver of the feed diet group (Fig. 5, left panel).

[0119] In particular, fibrous septa were clearly identified in the livers of male mice treated with CDAHFD at 36 weeks. Referring to Figure 3b, consistent with the changes in fibers in the CDAHFD diet group, the pro-inflammatory markers tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were increased in the CDAHFD diet group compared to the feed group, and it was confirmed that the levels of these markers were more increased in male mice than in female mice in the CDAHFD diet group at 36 weeks.

[0120] In addition, as shown in Fig. 4d, serum levels of TNF-α and IL-6 were found to be significantly upregulated in the CDAHFD group and CDAHFD male mice compared to the diet group and CDAHFD female mice, respectively. Immunostaining results for F4 / 80, a marker for Kupffer cells, showed that F4 / 80-positive cells accumulated in the livers of CDAHFD diet group mice, as shown in Fig. 4 (right), and these cells were more clearly identified in male mice than in female mice.

[0121] From the above results, it was confirmed that liver fibrosis and inflammation were less pronounced in female mice than in male mice after CDAHFD injury.

[0123] < Examples 3> CDAHFD In female mice during diet FPR2's Increased Confirmation of alleviation of expression

[0124] Considering that inflammation is closely related to the progression of NAFLD and that FPR2 regulates inflammation, FPR2 expression in experimental animals was confirmed.

[0125] As a result of performing qRT-PCR and Western blot analysis, FPR2 expression was fundamentally very high in the livers of female mice compared to male mice, as shown in Figures 5a and 5b, and it was confirmed that the CDAHFD diet reduced FPR2 expression in both sexes. As a result of IHC analysis, FPR2-positive cells were more clearly identified in the livers of female mice in the diet group than in the livers of male mice, as shown in Figure 5c, and these cells were almost absent in the CDAHFD diet group.

[0126] From the above results, it can be suggested that distinct expression of FPR2 in female mice is associated with resistance to NAFLD-induced injury in women.

[0128] <Example 4> Confirmation of the effect of FPR2 deletion on liver damage in female mice

[0129] To determine whether FPR2 is associated with sex differences in CDAHFD-induced NAFLD, CDAHFD was administered to FPR2-deficient mice (KO).

[0130] After confirming that FPR2 expression did not appear in the livers of KO mice that had consumed feed or CDAHFD, the mice were treated with CDAHFD as shown in Fig. 6a, and the liver response was checked.

[0131] As a result, as shown in Fig. 6b, the livers of KO mice that consumed the feed were found to be normal upon visual inspection, whereas the livers of KO mice that consumed CDAHFD showed a rough surface at 12 weeks, and at 36 weeks, multiple nodules were found in both females and males.

[0132] In addition, the number of mice with nodules was significantly increased in KO female mice compared to WT female mice after injury, as shown in Fig. 1c (Fig. 6c and left table).

[0133] Along with increased nodule formation in female KO mice in the CDAHFD diet group, the size of the nodular lesions in these mice was larger than that of WT female mice in the CDAHFD diet group and was found to be similar to that of male KO mice exposed to CDAHFD (Fig. 6c and left graph). As a result of H&E staining, as shown in Fig. 7a, it was confirmed that KO mice on the diet exhibited a normal morphology, except for slight immune cell infiltration and the accumulation of fatty liver cells in the liver of female KO mice, whereas female and male KO mice on the CDAHFD diet showed severe liver tissue damage, such as excessive hepatocyte death, increased inflammation, and atypical adenomatous proliferation, at 36 weeks, and it was confirmed that they showed results similar to WT male mice treated with CDAHFD at 36 weeks.

[0134] In addition, as shown in Figures 6d, 7b, and 7c, it was confirmed that the LW / BW ratio, serum levels of AST / AST, and liver TG levels were significantly increased in the CDAHFD diet KO group. In particular, at 36 weeks, the LW / BW ratio and liver TG levels in the CDAHFD diet KO group were similar in female and male mice, while AST levels were found to be significantly higher in female mice than in male mice.

[0135] As a result of IHC analysis of caspase-3, as shown in Figure 7d, FPR2 deficiency increased the apoptosis threshold in the livers of female mice, and it was confirmed that CDAHFD promoted the accumulation of apoptotic cells in the livers of both male and female KO mice.

[0136] From the above results, it was confirmed that FPR2 deficiency caused more severe liver damage in female mice fed the CDAHFD diet, and that similar liver damage occurred in male and female KO mice, unlike the liver response of WT mice exposed to CDAHFD.

[0138] < Examples 5> During liver damage FPR2 In deficient female mice Increased Confirmation of fibrosis and inflammation

[0139] Since FPR2 deficiency exacerbated CDAHFD-induced liver damage in female mice as much as in male mice, we investigated whether FPR2 deficiency promotes liver fibrosis and inflammation in female mice.

[0140] As a result, as shown in Fig. 8a, CDAHFD increased the RNA levels of Col1α1 and α-SMA fibrosis markers in male and female mice fed the KO diet. Additionally, as shown in Fig. 3a, it was confirmed that the levels of the aforementioned factors were significantly increased in WT male mice compared to WT female mice at 12 and 36 weeks after CDAHFD treatment, and it was confirmed that the expression of fibrosis markers in the CDAHFD diet KO group was similar between male and female mice.

[0141] In addition, as shown in Figures 8b and 8c, the protein levels of Col1α1 and α-SMA and the hydroxyproline content were increased in male and female KO mice during liver injury, and in particular, it was confirmed that the hydroxyproline content was significantly increased in female KO mice compared to male KO mice at 36 weeks after CDAHFD treatment.

[0142] In addition, Sirius Red staining results, such as those in Fig. 8d, also confirmed excessive collagen in the CDAHFD diet KO group compared to the feed diet KO group. At 36 weeks, fibrous nodules were observed in both sexes of the CDAHFD diet KO group, and collagen fibers penetrating the parenchymal region were clearly observed in the livers of female mice.

[0143] Meanwhile, referring to Figures 9a and 9b, inflammatory markers TNF-α and IL-6 were upregulated in both CDAHFD diet KO mice compared to the diet KO group, consistent with the expression of fibrosis in damaged KO mice. RNA levels of the markers were significantly higher in female mice than in male KO mice, and even serum TNF-α was found to be significantly higher in female KO mice than in male KO mice at 36 weeks after the CDAHFD diet.

[0144] In addition, as shown in Fig. 9c, F4 / 8 positive Kupffer cells aggregated surrounded by dying hepatocytes in the CDAHFD diet KO group, and these cells were more clearly identified in CDAHFD diet KO female mice than in CDAHFD diet KO male mice at 36 weeks. Additionally, cells expressing F4 / 80 were identified in the diet KO female mice but not in the KO male mice.

[0145] From the above results, it was confirmed that FPR2 deficiency exacerbates liver fibrosis and inflammation in female mice during liver injury, and that FPR2 expression in the livers of female mice is associated with sex differences in fibrosis and inflammation.

[0147] < Examples 6> In the liver of female mice FPR2's Confirmation of hepatoprotective effect due to high expression

[0148] The protective effect of FPR2 against liver damage caused by lipotoxicity was further confirmed.

[0149] First, we confirmed what type of FPR2 cells in the liver express.

[0150] As shown in Figure 5c, since hepatocyte-like cells in the healthy liver of females expressed FPR2, primary hepatocytes (pHEPs) were isolated from WT and KO mice, and qRT-PCR and Western blot analysis were performed.

[0151] As a result, as shown in Figures 10a and 10b, it was confirmed that the level of FPR2 was higher in the hepatocytes of WT female mice than in the hepatocytes of WT male mice.

[0152] In addition, immunofluorescence staining results confirmed FPR2 expression in hepatocytes of WT females, as shown in Fig. 10c. As shown in Fig. 11, FPR2 was rarely expressed in other types of female mouse cells, such as hepatic stellate cells (HSCs), cultured activated HSCs, and liver sinusoidal endothelial cells (LSECs), whereas it was present in Kupffer cells. FPR2 was not expressed in KO males or KO females.

[0153] To determine whether FPR2 expression protects hepatocytes from lipotoxicity, pHEP expressing FPR2 was isolated from female mice and exposed to 250 μM palmitic acid (PA), a hepatotoxicity inducing agent, for 24 hours.

[0154] As a result, as shown in Figures 10d to 10f, PA induced a decrease in FPR2 expression, reduced levels and viability of glucose-6-phosphatase (G6pc), a hepatocyte function marker, and increased levels of active caspase 3 in pHEP of WT female mice.

[0155] Interestingly, FPR2-deficient hepatocytes had low levels of G6pc expression, low cell viability, and higher expression of activated caspase-3 at baseline compared to WT cells, and damage induced by PA exacerbated this damage in FPR2-deficient hepatocytes compared to FPR2-expressing cells.

[0157] <Example 6> FPR2 Microarray Analysis in NAFLD Patients and Control Groups

[0158] To confirm in humans that FPR2 expression in female mice is associated with the alleviation of non-alcoholic fatty liver disease, FPR2 expression was analyzed using publicly available microarray data (GEO access number: GSE66676). Analysis of FPR2 expression in healthy men and women aged 13 to 19, as well as in male and female NAFLD patients, revealed that FPR2 expression was significantly higher in the livers of healthy women than in men, as shown in Figure 12, while this difference in expression was not observed in NAFLD patients. The finding that FPR2, which is highly expressed in healthy women, decreases in the damaged liver suggests a correlation with the lower incidence of NAFLD in women.

[0160] < Examples 7> Ligand activation for lipotoxicity in primary hepatocytes Fpr2's liver protective effect

[0161] The following experiments were conducted to investigate whether FPR2 agonists protect hepatocytes from lipotoxicity and whether the action of FPR2 agonists is mediated by FPR2 in hepatocytes.

[0162] Primary hepatocytes (pHEP) isolated from WT male mice were cultured in a medium containing LXA4 (20 nM) for 24 hours to activate Fpr2, and then exposed to PA (250 μM) for an additional 24 hours to provide lipotoxicity to these cells. Controls were treated with LXA4 in the form of a vehicle, 0.1% ethanol, and distilled water (DW), respectively (Fig. 14a).

[0163] Referring to Figures 14b through d, in PA-treated pHEPs of WT male mice Fpr2 and glucose-6-phosphatase, a marker of hepatocyte function ( G6pc )RNA levels and cell viability decreased, and the amount of activated Caspase-3 increased. Compared to PA+vehicle-treated cells, LXA4 showed Fpr2 and in PA-treated cells and G6pc The expression and cell viability were significantly upregulated, while the amount of activated Caspase-3 was downregulated. Looking at Figures 14b and d, PA exposure reduced Fpr2 expression in pHEPs, but the expression level was significantly higher in the LXA4+PA group than in the Veh(EtOH)+PA treatment group. In the absence of PA damage, LXA4 had little effect on G6pc expression, cell viability, and apoptosis in pHEPs. Looking at Figure 14e, in the Oil-red O staining assay to examine lipid droplets, red lipid droplets accumulated in PA-treated pHEPs, but these lipid droplets were clearly reduced in LXA4-treated cells exposed to PA.

[0164] These results confirmed that LXA4 increased cell viability and reduced lipid accumulation, and induced Fpr2 activation, indicating that LXA4 is involved in Fpr2-mediated hepatocyte protection against lipotoxicity.

[0166] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 A screening method for a non-alcoholic fatty liver disease treatment, comprising the steps of: treating a sample isolated from a male individual with a candidate substance; confirming the FPR2 expression level in the sample treated with the candidate substance; and comparing the FPR2 expression level with the FPR2 expression level of a sample not treated with the candidate substance, wherein if the FPR2 expression level of the sample treated with the candidate substance is increased compared to the FPR2 expression level of the sample not treated with the candidate substance, the candidate substance is screened as a non-alcoholic fatty liver disease treatment. Claim 8 delete Claim 9 A screening method for a non-alcoholic fatty liver disease treatment according to claim 7, wherein the sample is selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

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