KR20230137078A - Catecholamine/ADRB2-mediated screening method for the therapeutic agents in liver diseases - Google Patents

Abstract

The present invention relates to a method for inducing catecholamine/ADRB2-mediated inflammatory Kupffer cell death and protecting against liver disease. The present invention relates to a method in which intestinal-derived catecholamines enhance the production of GDF15 in hepatocytes through ADRB2 activation, and the produced GDF15 is activated by ADRB2 in inflammatory Kupffer cells. Since the mechanism that protects the liver by increasing the expression of and ultimately causing apoptosis of Kupffer cells by catecholamines has been confirmed, it can be usefully used in a screening method for a treatment for alcoholic liver disease.

Description

Catecholamine/ADRB2-mediated screening method for therapeutic agents in liver diseases}

The present invention relates to a screening method for catecholamine/ADRB2-mediated liver disease treatment.

The liver is capable of metabolizing and processing a myriad of components derived from the gut, protecting it from lethal damage from food toxins, pathogen/damage-associated molecular patterns (PAMPs/DAMPs), and gut/microbe-derived neurotransmitters such as serotonin, dopamine, and catecholamines. They are known to protect themselves. To handle these complex processes, the liver must be regulated by the sympathetic and parasympathetic nervous systems and must also express monoamine oxidase for catecholamine metabolism. However, transplanted livers have been observed to perform numerous functions without neural connections. To understand this paradoxical phenomenon, the liver must either accept nerve signals from other organs or produce its own neurotransmitters. However, the beneficial functions of gut-derived catecholamines, including norepinephrine (NE) and epinephrine (EPI), on liver homeostasis have not yet been reported.

Kupffer cells (KC) are known to express α and β adrenergic receptors (ADRA and ADRB). However, there is little information regarding the effects and roles of catecholamines and their receptors in Kupffer cells in mice and humans after chronic alcohol consumption.

GDF15 (Growth differentiation factor 15) is secreted by various cells and acts on at least two receptors such as GFRAL (GDNF family receptor α-like) and TGF-β receptor II, so it plays a role in lipid and energy metabolism, dietary intake, and β-cell survival. It can perform various functions.

As a prior art, Korean Patent Publication No. 10-2019-0020581 discloses a method for predicting or diagnosing liver fibrosis in non-alcoholic fatty liver disease using the level of GDF15 protein, and Korean Patent No. 10-1727506 discloses a method for predicting or diagnosing liver fibrosis in non-alcoholic fatty liver disease. By confirming that when GDF15 protein was administered to an obese animal model, weight gain was suppressed and adipogenesis (lipogenesis) was reduced, the use of GDF15 protein for preventing or treating fatty liver disease was disclosed. However, the correlation between the triple interaction between intestinal catecholamines, GDF15 in hepatocytes, and ADRB2 in Kupffer cells and liver disease has not been disclosed.

Accordingly, the present inventors confirmed that catecholamines derived from the intestine induce mitochondrial CYP2E1 (cytochrome P450 2E1)-mediated oxidative stress through ADRB2 activation in hepatocytes, which enhances the production of GDF15, and the produced GDF15 is stored in adjacent inflammatory Kupffer cells. The present invention was completed by confirming that it protects liver damage by inducing ADRB2 expression, resulting in apoptosis of inflammatory Kupffer cells in a catecholamine/ADBR2-dependent manner.

The purpose of the present invention is to provide a screening method for a therapeutic agent for alcoholic liver disease.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating alcoholic liver disease.

In order to achieve the above purpose,

The present invention includes the steps of 1) treating cells with a test substance; 2) measuring the expression level of the ADRB2 gene or the activity level of its protein in the cells of step 1); and 3) selecting a test substance in which the expression level of the ADRB2 gene or the activity level of its protein in step 2) is increased compared to the control group that did not treat the test substance; It provides a screening method for a treatment for alcoholic liver disease, including a.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating alcoholic liver disease containing an ADRB2 agonist as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating alcoholic liver disease containing catecholamine as an active ingredient.

The present invention relates to a method for inducing catecholamine/ADRB2-mediated Kupffer cell death and protecting against liver disease. The present invention relates to a method for inducing catecholamine/ADRB2-mediated Kupffer cell death and protecting against liver disease. The present invention provides that intestinal-derived catecholamine improves the production of GDF15 in hepatocytes through ADRB2 activation, and the produced GDF15 is an inhibitor of ADRB2 in inflammatory Kupffer cells. Since the mechanism that protects the liver by increasing expression and ultimately causing apoptosis of Kupffer cells has been confirmed, it can be usefully used in a screening method for a treatment for alcoholic liver disease.

Figure 1a shows norepinephrine and epinephrine concentrations in cecum lysates, portal blood, and liver lysates of WT mice fed pair or 4.5% ethanol for 8 weeks (n=for pair fed group). 9, This figure shows n=11 for the ethanol intake group.
Figure 1b is a diagram showing the correlation between liver catecholamines and cecal catecholamines by linear regression analysis (n = 9 for the pair intake group, n = 11 for the ethanol intake group).
Figure 1c is a diagram showing the relative mRNA expression levels of adrenergic receptors and Maoa and Maob by liver zonation analyzed by single cell RNA sequencing (scRNA-seq) of isolated mouse hepatocytes (HEP) (data accession number: GSE84498 ).
Figure 1d shows the mRNA expression of adrenergic receptors and Maoa and Maob analyzed by bulk RNA-seq in liver tissue of WT mice fed pair or ethanol for 8 weeks (n = 3/group) (data accession number: PRJNA494040).
Figure 1e shows the results of representative Western blot analysis (3 blots/group) of MAOA, MAOB, and ADRB2 protein expression in liver tissue of WT mice.
Figure 1f is a diagram showing representative immunofluorescence staining results of MAOB, MAOA, and ADRB2 in liver sections of WT mice (scale bar, 50 μm).
Figure 1g is a diagram showing the relative mRNA expression of Maoa, Maob, and Adrb2 in freshly isolated HEP, hepatic stellate cells (HSC), and Kupffer cells (KC) from WT mice (n=3/group).
Figure 2a is a diagram showing the serum GDF15 level of WT mice (n=9 for pair intake group, n=11 for ethanol intake group).
Figure 2b is a diagram showing the relative mRNA expression of Gdf15 (n=3/group) in HEP, HSC, and KC freshly isolated from WT mice.
Figure 2c is a diagram showing the correlation between serum GDF15 levels and cecal epinephrine, norepinephrine, or liver Adrb2 mRNA levels in WT mice by linear regression analysis (n=9 for pair intake group, n=11 for ethanol intake group) ).
Figure 2d is a diagram showing representative immunofluorescence staining results for GDF15 and CYP2E1 in liver sections of WT mice (central vein; CV, scale bar, 50 μm).
Figure 2e is a diagram showing the results of representative Western blot analysis (repeated three times) of CYP2E1, GDF15, ADRB2, and PKA-Cα protein expression in liver tissue of WT mice according to ethanol supply time.
Figure 2f is a diagram showing the results of a representative Western blot analysis (repeated three times) of CYP2E1 protein expression in whole liver lysate, cytoplasmic fraction, and mitochondrial fraction of WT mice.
In Figures 2G-2K, HEPs were freshly isolated from WT mice and treated with vehicle, 50 mM ethanol, or the indicated doses of clenbuterol (CBL) for 12 h (repeated three times).
Figure 2g shows representative Western blot analysis (left) and densitometric analysis (right) results of mitochondrial CYP2E1 protein expression in HEP.
Figure 2h is a diagram showing the results of representative Western blot analysis (2 blots/group) of ADRB2, PKA-Cα, and GDF15 protein expression in HEP.
Figure 2i is a diagram showing the concentration of GDF15 (n = 3/group) in the culture medium of HEP.
Figure 2j is a diagram showing a representative image of HEP stained with the mitochondrial oxidative stress indicator 'MitoSOX' (scale bar, 50 μm).
Figure 2k shows the relative mRNA of Gdf15 in HEPs treated with allyl sulfide (AS, 50 μM) or N-acetyl-L-cysteine (NAC, 100 ng/ml) for 1 hour followed by the indicated doses of ethanol and CBL for 12 hours. This is a diagram showing expression (repeated 3 times).
In Figures 3A to 3F, GDF15 ^f/f and GDF15 ^AlbCre mice were fed an ethanol diet for 8 weeks (n = 5/group, 3 experiments).
Figure 3a is a diagram showing portal venous levels of epinephrine and norepinephrine in GDF15 ^f/f and GDF15 ^AlbCre mice.
Figure 3b is a diagram showing serum levels of ALT, GDF15, and TG in GDF15 ^f/f and GDF15 ^AlbCre mice.
Figure 3c shows flow cytometry results (left) and bar graphs analyzing the frequencies of F4/80 ⁺ CD11b ⁺ macrophages and Ly6G ⁺ CD11b ⁺ neutrophils in isolated liver mononuclear cells (MNC) from GDF15 ^f/f and GDF15 ^AlbCre mice (left). This is a diagram showing (right).
Figure 3D is a diagram showing representative immunofluorescence staining of CYP2E1 and CLEC4F and the number of CLEC4F ⁺ KC in the CYP2E1 ⁺ region in liver sections of GDF15 ^f/f and GDF15 ^AlbCre mice (central vein; CV, scale bar, 50 μm).
Figure 3e is a diagram showing a representative histogram and graph for the mean fluorescence intensity (MFI) of Annexin V apoptosis analysis of Kupffer cells isolated from GDF15 ^f/f and GDF15 ^AlbCre mice.
Figure 3f shows the pro-apoptotic pathway ( Atf4, Ddit3, Bak1, Bax, Bcl2l11 and Casp3) and anti-apoptotic pathway ( Mcl1 ) of Kupffer cells isolated from GDF15 ^f/f and GDF15 ^AlbCre mice (n = 3/group). and Bcl2 ), inflammatory genes ( Il1b, Tnf , and Ccl2) , GDF15 receptor ( Tgfbr2 ), and ADRB2 signaling ( Adrb2, Prkar1a, and Prkacb ).
In Figures 3g to 3i, Kupffer cells were freshly isolated from WT mice and treated with lipopolysaccharide (LPS, 100 ng/ml) with or without 1 μM KT5720 for 6 h, followed by the indicated doses of GDF15 or 1 μM CBL for an additional 6 h. Processed for a while (repeated 3 times).
Figure 3g is a diagram showing the relative mRNA expression of adrenergic receptors in Kupffer cells isolated from WT mice treated with LPS and vehicle (Veh) or GDF15.
Figure 3h shows representative Western blot results of ADRB2, PKA-Cα, and Bcl-2 protein expression in Kupffer cells isolated from WT mice treated with LPS, GDF15, CBL, or KT5720 (repeated three times).
Representative phase contrast images of culture dishes after the treatments shown in Figure 3I (top image), histogram and MFI for Annexin V apoptosis assay (bottom left), and relative cell number of adherent (green) or floating (purple) Kupffer cells (bottom right). (Scale bar, 50 μm).
In Figures 4a to 4e, scRNA-seq analysis was performed using Kupffer cells isolated from WT mice fed pair or ethanol for 8 weeks.
Figure 4a is a diagram showing annotations by UMAP conditions (top) and KC clustering (bottom).
Figures 4B and 4C show the frequency of mitochondrial genes and number of genes per each cell (B) and KC markers ( Clec4f, Adgre1, Vsig4, Timd4, and Marco ), infiltrated macrophage markers ( Ccr2 and Cx3cr1) , GDF15 receptor ( Gfral ), This is a diagram showing the relative expression levels of genes encoding adrenergic receptors and PKA subunits ( Pkar1a and Prkacb) (C).
Figure 4D is a diagram showing the expression levels of genes related to the indicated pathways in each cluster.
Figure 4E shows differentially expressed genes (left) between the cluster enriched in pair (cluster 3) and the cluster enriched in ethanol (cluster 1) and the top 50 enriched canonical pathways selected from the cluster enriched in ethanol (right). am.
In Figures 4F to 4I, WT mice were fed a pair or ethanol diet for 8 weeks.
Figure 4f is a diagram showing representative histograms and bar graphs (n = 4/group, repeated 3 times) for MFI values of Kupffer cells in Annexin V apoptosis analysis.
Figure 4g is a diagram showing representative CLEC4F immunofluorescence staining of a liver section (central vein; CV, portal triad; PT, scale bar, 50 μm).
Figure 4h is a diagram analyzing the expression levels of CLEC2, TIM4, and MARCO in F4/80 ⁺ CD11b ⁺ infiltrated macrophages (blue) and F4/80 ^hi CD11b ⁺ resident Kupffer cells (red) in normal liver.
Figure 4i is a t-SNE plot generated by the expression of CLEC2, TIM4, MARCO, and F4/80 in Kupffer cells of mice fed Pair or ethanol (top). This diagram shows a representative flow cytometry plot (bottom) for the expression levels of CLEC2 and TIM4 or TIM4 and MARCO in F4/80 ^hi CD11b ⁺ Kupffer cells.
In Figures 5A to 5F, the in situ closed liver perfusion system was performed on WT mice treated with media containing vehicle (Veh) or LPS (100ng/ml), CBL (1 μM), and ethanol (50mM) for 2 hours. This was performed using WT and ADRB1/2 DKO mice (n = 3/group, 3 replicates) treated with media containing LCE).
Figure 5a is a diagram showing an in situ closed liver perfusion system.
Figure 5b is a diagram showing the results of measuring GDF15 and ALT levels in circulating media (n = 3/group).
Figure 5c is a diagram showing representative flow cytometry results for F4/80 ^hi CD11b ⁺ Kupffer cells (top) and Annexin V apoptosis analysis of Kupffer cells and MFI (bottom).
Figure 5d is a diagram showing representative immunofluorescence staining results of CLEC4F in liver sections (central vein; CV, portal triad; PT, scale bar, 50 μm).
Figure 5E is a representative Western blot analysis of ADRB2, PKA-Cα and GDF15 protein expression in whole liver lysate and CYP2E1 protein expression in mitochondrial fraction (2 blots/group).
Figure 5F is a heatmap showing the relative expression levels of genes involved in the indicated pathways in isolated Kupffer cells (n = 3/group).
In Figures 5G-5I, WT mice were subjected to an in situ closed liver perfusion system using media containing Veh or KT5720 (1 μM) for 30 min and then cycled LCE for 1.5 h (n = 3/group; (repeat 3 times).
Figure 5g is a diagram showing representative analysis results for flow cytometry analysis of F4/80 ^hi CD11b ⁺ Kupffer cells (top) and histogram and MFI (bottom) of Annexin V apoptosis analysis of Kupffer cells.
Figure 5h is a representative Western blot analysis of ADRB2, PKA-Cα and GDF15 protein expression in whole liver lysate and CYP2E1 protein expression in mitochondrial fraction (2 blots/group).
Figure 5i is a diagram showing the results of heatmap analysis showing the relative expression levels of genes related to the indicated pathways in isolated Kupffer cells (n = 3/group).
In Figure J and Figure 5K, in situ closed liver perfusion was performed on WT or ADRB1/2 DKO mice.
Figure 5j is a diagram showing representative flow cytometry results for the frequencies of Ly6G ⁺ CD11b ⁺ neutrophils and F4/80 ⁺ CD11b ⁺ macrophages in liver MNC.
Figure 5k is a diagram showing the relative mRNA expression of Cxcl1, Ccl2, Tnf , and Il1b in whole liver tissue (top) or isolated Kupffer cells (bottom) (n=3/group, repeated 3 times).
6A to 6F, to inhibit catecholamine production, WT mice were fed an ethanol diet for 2 weeks and divided into two groups with vehicle (Veh; PBS) or fusaric acid (FA; 10 mg/kg, i.p., every other day). ) were injected for another 2 weeks (n = 5/group, repeated 3 times) while receiving ethanol (Experiment 1). To inhibit CYP2E1-mediated hepatic metabolism, CYP2E1 ^f/f and CYP2E1 ^f/-AlbCre mice were fed an ethanol diet for 6 weeks (n = 4/group, repeated 3 times) (Experiment 2). To inhibit catecholamine receptors, WT mice and ADRB1/2 DKO mice were fed an ethanol diet for 8 weeks (n = 5/group, repeated 3 times) (Experiment 3).
Figure 6a is a diagram showing the results of analyzing the portal vein concentrations of epinephrine and norepinephrine.
Figure 6b is a diagram showing serum levels of GDF15, ALT, and AST.
Figure 6C is a representative Western blot analysis of ADRB2, PKA-Cα and GDF15 protein expression in whole liver lysates and CYP2E1 protein expression in mitochondrial fractions (2 blots/group).
Figure 6d is a diagram showing the relative mRNA expression (n = 3/group) of Adrb2, Cyp2e1, and Gdf15 in liver tissue (nd, not detected).
Figure 6e is a diagram showing representative immunofluorescence staining of CYP2E1 and CLEC4F and the average number of CLEC4F ⁺ Kupffer cells in the CYP2E1 ⁺ pericentral vein region of liver sections (central vein; CV, portal triad; PT, scale bar, 50 μm) .
Figure 6F shows a representative flow cytometry panel (top) of liver F4/80 ⁺ CD11b ⁺ macrophages, F4/80 ⁺ Ly6C ^hi or F4/80 ⁺ Ly6C ^low macrophages (top) and F4/80 ⁺ CD11b ⁺ macrophages, F4/80 ⁺ Bar graph (bottom) showing the frequency of Ly6C ^hi or F4/80 ⁺ Ly6C ^low macrophages and Ly6G ⁺ CD11b ⁺ neutrophils.
Figure 7A shows serum norepinephrine and epinephrine in healthy controls and alcoholic patients without liver disease (AWLD, n = 14), alcoholic fatty liver disease (AFL, n = 12), and alcoholic steatohepatitis (ASH; n = 14). And this is a diagram measuring the level of GDF15.
Figure 7b is a diagram measuring norepinephrine and epinephrine levels in stool samples from alcoholic patients without steatosis (n=6), alcoholic patients with mild steatosis (n=10), or alcoholic patients with severe steatosis (n=7).
Figure 7c is a diagram showing the results of correlation analysis between serum GDF15 level and serum norepinephrine or epinephrine level (left two graphs) and GGT level and serum GDF15, norepinephrine or epinephrine level. (n = 7 for healthy controls, n = 40 for alcohol patients).
Figure 7d shows representative Western blot analysis results (2 blots/group) of ADRB2, PKA-Cα, GDF15, MAOA and MAOB protein expression.
Figure 7e shows the results of qRT-PCR analysis of ADRB2 , GDF15 , MAOA and MAOB mRNA expression levels in liver tissues of healthy controls and AWLD and AFL patients (n = 3/group).
In Figures 7f and 7g, isolated human hepatocytes were treated with vehicle, ethanol (50mM), or ethanol containing CBL (1μM) for 12 hours (repeated 3 times).
Figure 7f is a diagram showing representative Western blot results of ADRB2, PKA-Cα, and GDF15 protein expression.
Figure 7g is a diagram showing the level of GDF15 in culture medium (repeated 3 times).
Figure 7h shows representative H&E and immunostaining results of liver sections for MAOA, MAOB, ADRB2, CD68, CYP2E1, and GDF15 in healthy controls and AFL patients.
Figure 7i is a diagram showing the number of CD68 ⁺ cells.
Figure 7j shows the results of Annexin V apoptosis assay after treating human Kupffer cells freshly isolated from a healthy donor with LPS (100ng/ml), GDF15 (100ng/ml), and CBL (1μM) for 6 hours.
Figure 7k shows the results of qRT-PCR analysis after treating human Kupffer cells freshly isolated from a healthy donor with LPS (100ng/ml), GDF15 (100ng/ml), and CBL (1μM) for 6 hours.
Figure 7l shows the results of microarray analysis of the relative mRNA expression levels of GDF15, ADRB2, MAOA , and MAOB in liver tissues of healthy (n = 7) and alcoholic hepatitis patients (AH; n = 15) (data accession) Number: GSE28619).
Figure 7m shows representative immunostaining results for CYP2E1, GDF15, and CD68 in liver sections from healthy controls and patients with alcoholic cirrhosis (ALC).
Figure 7n is a schematic diagram of the 'triangular' interaction caused by alcohol-induced intestinal bacterial imbalance.
Figure 8 is a diagram showing that liver damage is protected by the death of inflammatory Kupffer cells by catecholamines derived from the intestine during chronic alcohol consumption.

Hereinafter, the present invention will be described in detail.

The present invention includes the steps of 1) treating cells with a test substance; 2) measuring the expression level of the ADRB2 gene or the activity level of its protein in the cells of step 1); and 3) selecting a test substance in which the expression level of the ADRB2 gene or the expression level of its protein in step 2) is increased compared to the control group that did not treat the test substance; It provides a screening method for a treatment for alcoholic liver disease, including a.

The cells may be Kupffer cells. Kupffer cells are cells that make up the liver and are macrophages that produce antibodies against bacteria and viruses and process foreign substances entering the body.

GDF-15 is also called macrophage inhibitory cytokine 1 (MIC-1), and other names include placental TGF-β prostate-derived factor (PDF), nonsteroidal anti-inflammatory drug-activator gene (NAG- 1) It is also called etc. Under normal conditions, GDF-15 is expressed at low levels in most organs and is known to be upregulated due to damage to organs such as the liver, kidneys, heart, and lungs.

The test substance in step 1) may be one or more selected from the group consisting of peptides, proteins, non-peptide compounds, active compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.

The expression level of the gene in step 2) is determined by real-time polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction, RT-PCR), quantified real time PCR, competitive RT-PCR, real-time It can be measured by any one or more methods selected from the group consisting of real time quantitative RT-PCR (RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chip analysis.

The expression level of the protein in step 2) was determined by enzyme-linked immunosorbent assay (ELISA), radioimmunoassays, sandwich enzyme-linked immunosorbent assay, immunoprecipitation, western blot, and immunosorbent assay. It can be measured by any one or more methods selected from the group consisting of histochemical staining (immunohistochemistry), fluoroimmunoassay, and flow cytometry (Fluorescence-activated cell sorting (FACS)).

The adrenergic receptor of the present invention is a receptor that binds to norepinephrine, a neurotransmitter secreted from the terminal part of an adrenergic neuron, or epinephrine secreted from the adrenal medulla. Adrenergic receptors include α1, α2, β1, β2, and β3. These receptors generally display their functions through activation of the sympathetic nervous system, and each function is different depending on the organ or tissue on which it acts.

The alcoholic liver disease includes alcoholic fatty liver, alcoholic steatohepatitis, or alcoholic hepatitis.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating alcoholic liver disease containing an ADRB2 agonist as an active ingredient.

In the present invention, agonists refer to substances that act to increase the expression of a target gene or protein or the activity of a target protein, that is, a gene, protein, compound, etc. A receptor agonist is a substance that acts on receptor molecules in the body and performs functions similar to those of neurotransmitters or hormones or assists such functions.

The ADRB2 agonist acts on the ADRB2 receptor to help produce neurotransmitters (e.g., epinephrine), maintain neurotransmitters by preventing them from being decomposed or reabsorbed, or increase secretion of neurotransmitters, etc. If they play a role, they can all be considered ADRB2 agonists.

ADRB2 agonists are well known to those skilled in the art and include, for example, clenbuterol, levosalbutamol, Isoproterenol (β1 and β2), Metaproterenol, and terbutaline. (Terbutaline), Isoetarine, pirbuterol, procaterol, ritodrine, epinephrine, fenoterol, butoxamine, salbuta It may include salbutamol, formoterol, or salmeterol. In the present invention, the preferred ADRB2 agonist in the experimental examples is clenbuterol. Clenbuterol is a highly selective ADRB2 agonist and will be known to those skilled in the art.

The pharmaceutical composition of the present invention may additionally include solvents, excipients, etc. The solvent includes physiological saline, distilled water, etc., and the excipient includes, but is not limited to, aluminum phosphate, aluminum hydroxide, or aluminum potassium sulfate, and is commonly used in the production of pharmaceutical compositions in the field to which the present invention pertains. Additional substances used may be included.

The pharmaceutical composition of the present invention can be prepared by methods commonly used in the technical field to which the present invention pertains. The pharmaceutical composition of the present invention can be prepared as an oral or parenteral preparation, and is preferably prepared as an injection solution, which is a parenteral preparation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural. It can be administered by the (eidural) route.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. Specifically, the effective amount of the compound according to the present invention may vary depending on the patient's age, gender, and body weight, and is generally administered at 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg, per kg of body weight every day or every other day. Alternatively, it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The alcoholic liver disease is as described above.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating alcoholic liver disease containing catecholamine as an active ingredient.

The catecholamine is a general term for monoamine-based neurotransmitters or hormones derived from catechol. Catecholamines in vivo include dopamine, norepinephrine, and epinephrine. They are all made from tyrosine, a type of amino acid. Tyrosine is created when phenylalanine is hydroxylated by phenylalanine hydroxylase. Alternatively, it can be consumed as external food. Nerve cells or neuroendocrine cells that secrete catecholamines convert this tyrosine into dopa (L-DOPA) through a series of biochemical processes. Afterwards, dopa is converted into dopamine, norepinephrine, and epinephrine in that order by each enzyme and stored in vesicles. Afterwards, when exocytosis occurs due to a specific nerve stimulus, the cell secretes catecholamines into the blood or postsynaptic neurons.

The pharmaceutical composition is as described above.

The alcoholic liver disease is as described above.

In a specific experimental example of the present invention, mice chronically fed ethanol had higher levels of norepinephrine and epinephrine in the cecum, portal blood, and liver tissue than mice fed a Lieber-DeCarli ethanol diet (pair) containing the same amount of maltose-dextrin. The concentration was significantly high (see Figure 1a), and it was confirmed that the liver catecholamine level was positively correlated with the cecal catecholamine level (see Figure 1b). Additionally, in healthy mice, the expression of genes related to adrenergic receptors differed depending on the liver region, and in particular, Adrb2 was confirmed to be expressed around the central vein (see Figure 1c), and Maoa, Maoa, and It was confirmed that Maob and Adrb2 mRNA and protein levels were high (see Figures 1d and 1e). In addition, compared to mice fed Pair, ADRB2 protein expression was confirmed to increase in the periphery of the central vein (CV) in mice fed ethanol, and Adrb2 mRNA was confirmed to increase in both hepatocytes and Kupffer cells (see Figure 1g). . The above results show that intestinal catecholamines are associated with increased MAOA and ADRB2 in venous lesions during chronic alcohol consumption.

In addition, it was confirmed that the level of GDF15 increased in the serum and liver cells of mice fed ethanol compared to mice fed pair (see Figures 2a and 2b), and the level of serum GDF15 was positively correlated with catecholamines in the cecum and Adrb2 mRNA levels in the liver. It was confirmed that there was (see Figure 2c). In addition, it was confirmed that the expression of CYP2E1 and GDF15 was increased in hepatocytes around the central vein where ADRB2 was expressed (see Figure 2d), and the levels of CYP2E1, GDF15, ADRB2 and their downstream PKA-Cα in the liver were increased. It was confirmed that it gradually increased as the ethanol intake time increased (see Figure 2e). It was confirmed that the level of CYP2E1 was higher in the mitochondria than in the cytoplasm when ethanol was fed (see Figure 2f), and the level of mitochondrial CYP2E1 in hepatocytes was confirmed to increase by treatment with ethanol or ADRB2 agonist (CBL) (see Figure 2g). At this time, it was confirmed that stimulation of ADRB2 using CBL increased the protein level of PKA-Cα, a regulator of CYP2E1 delivery to mitochondria (Figure 2h). In addition, it was confirmed that when hepatocytes were treated with ethanol alone or with CBL after ethanol treatment, the production of GDF15 increased in a CBL concentration-dependent manner (see Figure 2i). When CBL was treated with ethanol, mitochondrial oxidative stress increased (see Figure 2j), and treatment with CYP2E1 inhibitors or antioxidants suppressed Gdf15 mRNA expression (see Figure 2k). The above results show that liver ADRB2 stimulation increases GDF15 production through mitochondrial CYP2E1-mediated oxidative stress.

In addition, an increase in ALT and a decrease in GDF15, which are liver damage values, were confirmed in the serum of GDF15 ^AlbCre mice compared to GDF15 ^f/f mice, and there was no difference in serum triglyceride levels and hepatic portal epinephrine and norepinephrine levels (Figure 3a and 3b). At this time, there was no difference in the ratio of Ly6G ⁺ CD11b ⁺ neutrophils in the liver of GDF15 ^Albcre mice, but the ratio of F4/80 ⁺ CD11b ⁺ macrophages was confirmed to be increased (see Figure 3c). In addition, unlike GDF15 ^Albcre mice, Kupffer cells around the central vein disappeared in GDF15 ^f/f mice (see Figure 3d), and it was confirmed that there was less death of Kupffer cells in GDF15 ^Albcre mice (see Figure 3e). In addition, it was confirmed that the expression of genes related to apoptosis was decreased and the expression of genes related to inflammatory response was increased in Kupffer cells of GDF15 ^Albcre mice compared to GDF15 ^f/f mice (see Figure 3f). In addition, it was confirmed that the expression of Adrb2 was significantly increased by GDF15 treatment in inflammatory Kupffer cells pretreated with LPS (see Figure 3g), and it was confirmed that ADRB2 stimulation by CBL induced apoptosis of inflammatory Kupffer cells (Figure 3g). 3h and Figure 3i). The above results show that GDF15 prevents alcoholic liver damage by inducing apoptosis of inflammatory Kupffer cells.

In addition, compared to pair-fed mice in ethanol-fed mice, the expression of ADRB2 and PKA-related genes in Kupffer cells, expression of genes related to apoptosis, ER stress and inflammation, TGF-β receptor expression, and negative regulation of cell growth or proliferation were related. It was confirmed that gene expression increased (see FIGS. 4A to 4E). In addition, compared to pair-fed mice, more Kupffer cells died in the pericentral area of the liver of ethanol-fed mice (see Figures 4f and 4g), and all Kupffer cells expressed TIM4, a resident Kupffer cell marker. It was confirmed that there was (see Figures 4h and 4i). The above results indicate that Kupffer cells undergo ADRB2/PKA-mediated apoptosis in alcoholic fatty liver disease, but are not replaced by bone marrow-derived TIM4- macrophages.

In addition, ADRB2 activation during LCE circulation in in situ obstructive liver perfusion significantly increased GDF15 levels and apoptosis of inflammatory Kupffer cells in WT mice, but decreased it in ADRB1/2 DKO mice, and thus WT in ADRB1/2 DKO mice. Compared to mice, the ALT level, which is a serum level of liver damage, was found to be higher (see FIGS. 5A to 5C). In addition, unlike WT mice, it was confirmed that many Kupffer cells still remained in the pericentral vein area in ADRB1/2 DKO mice (see Figure 5d). In addition, ADRB2 activation through LCE circulation induced the expression of molecules related to ADRB/PKA signaling and apoptosis in WT mice, but was confirmed to be decreased in Kupffer cells in ADRB1/2 DKO mice (see Figures 5e and 5f). . In addition, inhibition of PKA through KT5720, a PKA-specific inhibitor, reduced the apoptosis of inflammatory Kupffer cells compared to the control group after LCE circulation, similar to the results in ADRB1/2 DKO mice, especially ADRB/PKA signaling and cell death. The expression of molecules related to apoptosis was reduced (see FIGS. 5G to 5I). Compared to WT mice, the frequency of neutrophils and macrophages and the expression of Tnf and Il1b mRNA in the liver were increased in ADRB1/2 DKO (see Figures 5j and 5k), which indicates that Cxcl1 and Cxcl1 were increased in Kupffer cells by stimulation with ethanol and LPS. associated with upregulated Ccl2 mRNA (see Figure 5K). The above results indicate that activation of ADRB2 through in situ occlusive liver perfusion stimulates apoptosis of inflammatory Kupffer cells in the pericentral region.

In addition, catecholamine production can be pharmacologically inhibited by treatment with FA (fusaric acid), a dopamine β-hydroxylase inhibitor, or by using hepatic CYP2E1 heterozygous KO mice (CYP2E1 ^f/-AlbCre ) or ADRB1/2 DKO mice. As a result of genetically suppressing the expression of CYP2E1 and ADRB1/2, the concentration of catecholamines in the portal vein decreased only in FA-treated mice (see Figure 6a), but the concentration of GDF15 in the serum of all three mice decreased, and the concentration of ALT and AST in the serum. increased (see Figure 6b). Additionally, liver ADRB2 and PKA-Cα protein levels were reduced compared to controls in two groups except for CYP2E1 ^f/-AlbCre mice, and mitochondrial CYP2E1 and hepatic GDF15 expression were reduced in all three groups (see Fig. 6c). The expression of Gdf15 mRNA in the liver was significantly decreased in all three mice, Adrb2 mRNA was significantly decreased in both groups except CYP2E1 ^f/-AlbCre mice, and Cyp2e1 mRNA was significantly decreased in CYP2E1 ^f/-AlbCre mice compared to the control group (see Figure 6d). ). In addition, CYP2E1 ⁺ CLEC4F ⁺ Kupffer cells around one central vein were significantly decreased in all three groups compared to the control group (see Figure 6e). In addition, the proportion of F4/80 ⁺ CD11b ⁺ macrophages and Ly6G ⁺ CD11b ⁺ neutrophils, as well as the proportion of F4/80 ⁺ Ly6C ^hi inflammatory macrophages were increased (see Figure 6f). The above results indicate that pharmacological and genetic inhibition of catecholamines, CYP2E1, and ADRB worsens alcoholic liver damage in mice through survival of inflammatory Kupffer cells.

Additionally, serum levels of catecholamines and GDF15 were significantly increased in patients with alcoholic liver disease (see Figure 7a), and there were no significant differences in fecal NE and EPI levels according to severity in steatotic patients compared with non-steatotic patients (Figure 7a). 7b), serum GDF15 levels were positively correlated with serum catecholamines (see Figure 7c). Protein and mRNA expression of ADRB2, PKA-Cα, GDF15, MAOA, and MAOB were increased in liver tissues of alcoholic patients without liver disease compared with healthy controls (see Figures 7d and 7e), and hepatocytes co-treated with ethanol and CBL Protein levels of ADRB2, PKA-Cα and GDF15 were significantly increased (see Figures 7f and 7g). In patients with alcoholic fatty liver disease, the expression of MAOA, ADRB2, CYP2E1, and GDF15 expanded from the central vein to the periportal hepatocytes (see Figure 7h), and the number of CD68 ⁺ macrophages was reduced compared to healthy controls (see Figure 7i). Additionally, LPS/GDF15/CBL treatment induced more apoptosis in ADRB2-activated inflammatory human Kupffer cells (see Figures 7j and 7k). GDF15 mRNA expression was high in the liver tissue of alcoholic hepatitis patients, but ADRB2 , MAOA , and MAOB mRNA expression was observed to be low compared to healthy controls (see Figure 7l). Increased CD68 ⁺ cells in the pericentral area and increased expression of CYP2E1 and GDF15 in the liver were observed in patients with alcoholic cirrhosis (see Figure 7m). The above results indicate activation of the catecholamine/GDF15/ADRB2 signaling pathway in patients with early alcoholic liver disease (see Figure 7n).

To summarize the above results, gut-derived catecholamines improved GDF15 production by increasing the mitochondrial translocation of CYP2E1 in pericentral hepatocytes expressing ADRB2 receptors, and GDF15 significantly increased ADRB2 expression in adjacent inflammatory Kupffer cells, thereby increasing catecholamine/ADRB2 levels. Promoted agonist-mediated apoptosis pathway. The above results reveal a novel catecholamine-sensitive Kupffer cell regulated by hepatic GDF15 and confirm a unique neurometabolic-immune communication between the intestine and liver that induces hepatoprotection (see Figure 8).

Hereinafter, the present invention will be described in detail through experimental methods and experimental examples.

However, the experimental methods and examples described below only specifically illustrate the present invention from one aspect, and the present invention is not limited thereto.

<Experiment method>

mouse

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST, Korea). Mice were maintained on a standard 12-hour light/dark cycle in a specific-pathogen-free (SPF) facility at KAIST. Male mice aged 8 to 10 weeks on a C57BL/6J background were fed the Lieber-DeCarli ethanol diet containing isocaloric maltose-dextrin (#710260, Dyets Inc) (hereafter referred to as Pair) or 4.5% ethanol (EtOH). fed. ADRB1/2 DKO (double KO) and albumin-Cre mice were purchased from Jackson Laboratories (stock numbers 003810 and 003574, respectively). GDF15 ^f/f and CYP2E1 ^f/f mice were purchased from the EMMA mouse repository (stock numbers EM10460 and EM06364, respectively). Flippase mice were provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Laboratory Animal Resource Center (LARC) (stock number PX00003369). In all experiments using genetically modified mice, littermates were used as controls. Weight changes and food intake were measured daily. For pharmacological inhibition of dopamine β-hydroxylase (DBH), mice were treated with fusaric acid (FA) (#F6513, Sigma-Aldrich; 10 mg kg ^-1 body weight, i.p., every other day). It was treated for the last two weeks.

human patient

Human stool, serum, and liver tissue from various stages of alcoholic liver disease (ALD) patients were collected at Seoul National University Boramae Hospital (Seoul, South Korea). All procedures were approved by the Institutional Review Board of Seoul National University Boramae Medical Center (IRB No. 16-2013-45). Patients who consumed excessive amounts of alcohol (40 g/day for men, 20 g/day for women) were eligible, and patients with hepatitis B or C virus infection, autoimmune-related liver disease, or cancer were excluded. For serum and liver tissue samples, patients collected from separate cohorts were divided into four subgroups according to histological characteristics (alcohol-consuming patients without liver disease; AWLD, alcoholic fatty liver; AFL, alcoholic steatohepatitis; ASH, alcoholic cirrhosis; ALC). A total of 23 patients from previous studies participated in the analysis of stool samples. Patients using antibiotics, proton pump inhibitors, immunosuppressants, and patients with advanced liver fibrosis or cirrhosis were excluded. Patients were divided into three subgroups (no steatosis, mild steatosis, moderate steatosis) according to the severity of steatosis as determined by histology. For the isolation of primary human hepatocytes or Kupffer cells, fresh liver tissue from alcohol-naive non-tumor sites among patients with hepatitis B virus-related hepatocellular carcinoma was obtained from a separate cohort of the Department of Surgery at Chungnam National University Hospital (Daejeon, Korea) and collagenase-treated. perfused. The Chungnam National University Hospital Institutional Review Board (IRB No. 2016-03-020-011) approved the use of liver tissue for research purposes, and informed consent was obtained from the patient.

Primary Cell Cultures

Hepatocytes, hepatic stellate cells, Kupffer cells, and liver mononuclear cells (MNC) were isolated from mice. Briefly, EGTA solution (5.4mM KCl, 0.44mM KH ₂ PO ₄ , 140mM NaCl, 0.34mM Na ₂ HPO ₄ , 0.5mM EGTA, 25mM Tricine, pH 7.2), collagenase solution (0.075% of collagenase type) The liver was sequentially perfused with HBSS buffer diluted with I (containing 0.2% DNase I). After two-step cycling, liver tissue was further digested in a shaking incubator (90 rpm, 20 min) with digestion buffer (0.2% DNase I in HBSS buffer diluted with 0.009% collagenase type I) at 37°C; It was filtered through a cell strainer (70 μm) to remove liver connective tissue or undigested liver tissue. To isolate hepatocytes, the suspension was centrifuged at 50 × g for 5 min and further purified with a 50% Percoll gradient solution. To isolate non-parenchymal cells, the supernatant was centrifuged at 650 xg and the pellet was divided into two tubes. For Kupffer cells and hepatic stellate cell isolation, the pellet was separated into 11.5% and 20% Opti-prep (Sigma-Aldrich) gradient solutions and centrifuged at 1800 xg for 17 min at 4°C. Cells were further purified by flow cytometry-based cell sorting (DAPI ⁺ for HSCs and F4/80 ^hi CD11b ⁺ for KCs) for further experiments. For liver MNC isolation, the pellet was suspended in 40% Percoll (GE Healthcare) gradient solution and centrifuged at 1800 × g for 20 min at 4°C. The supernatant was carefully removed, and the liver MNC was resuspended in saline solution for use. Non-tumor areas (approximately 10 g) of liver specimens were obtained from hepatitis B virus-related hepatocellular carcinoma resections for isolation of human primary hepatocytes and Kupffer cells. Two-stage collagenase liver perfusion was performed with two visible liver vessels. The remaining procedure was the same as the isolation method of mouse hepatocytes. For in vitro experiments using hepatocytes and Kupffer cells, DMEM (#LM001-11, Welgene) or RPMI (#LM-011-01) with 10% FBS (Welgene) and 1% penicillin-streptomycin (Thermo Fisher Scientific) , Welgene) and incubated for 5 hours at 37°C in a 5% CO ₂ incubator for stabilization. The cells were then washed with saline and replenished with fresh medium. In some experiments, cells were treated with ethanol (50mM, Millipore), clenbuterol (10nM or 1μM, Sigma-Aldrich), allyl sulfide (50μM, Sigma-Aldrich), or N-acetyl sulfide according to the corresponding experimental protocol. -L-cysteine (N-Acetyl-L-cysteine) (100ng/ml, Sigma-Aldrich), lipopolysaccharide (100ng/ml, Sigma-Aldrich), or GDF15 (50 or 100μM, R&D Systems).

In situ Obstructed liver perfusion ( In situ closed liver perfusion)

In situ liver perfusion was performed with wild-type and ADRB1/2 DKO mice. Specifically, a cannula was inserted into the portal vein and the IVC (infrahepatic inferior vena cava) was cut. Preheated saline solution was circulated for 5 minutes to wash away the blood, and the intrahepatic IVC was fixed. The chest was opened and a cannula was inserted into the right atrium of the heart with another catheter to access the suprahepatic IVC. A 1 ml syringe body was then connected to the catheter in the upper IVC as a reservoir. Finally, closed circulation was established and maintained for up to 2 hours. Low-glucose DMEM containing vehicle (#LM001-11, Welgene), KT5720 (1 μM), lipopolysaccharide (100 ng/ml), clenbuterol (1 μM), or ethanol (50 mM) was used as the experimental medium.

GDF15 ELISA

Mouse or human serum samples were collected from peripheral blood and performed with the mouse or human GDF15 quantikine ELISA kit (#MGD150 for mouse and #DGD150 for human, R&D Systems) according to the manufacturer's instructions. Plates were read and analyzed at 450 nm with an iMark™ microplate absorbance reader (Bio-Rad).

Catecholamine ELISA

Epinephrine/norepinephrine ELISA kit (#KA3768, Abnova) was applied to mouse portal blood, liver tissue lysate, cecal lysate, or human stool and serum samples according to the manufacturer's instructions. Plates were read and analyzed at 450 nm with an iMark™ microplate absorbance reader (Bio-Rad).

Clinical Chemistry Measurements

Serum samples were collected from mouse peripheral blood and analyzed. Serum aspartate transaminase (AST), alanine transaminase (ALT), triglyceride (TG), and total cholesterol (TC) levels were analyzed with a VetTest Chemistry Analyzer (IDEXX Laboratories) according to the manufacturer's protocol.

Histological Analysis

To obtain consistent results, similar areas of the left and medial lobes of mouse liver were used for histological analysis. Liver tissues were fixed with 10% neutral buffered formalin (Sigma-Aldrich) and subjected to H&E or Oil-Red O staining. For H&E staining, paraffin-embedded liver tissue was cut to a thickness of approximately 4 μm. Images were captured with an optical microscope (Olympus BX51) and analyzed with DP2-BSW software.

Immunostaining

For immunostaining, 4-μm-thick paraffin-embedded liver sections were used. After deparaffinization and rehydration, sections were transferred to 10mM citrate buffer (pH 6.0) and antigen retrieval was performed in the microwave for 5 min. For isolated hepatocytes, cells were fixed with 4% paraformaldehyde for 20 min, blocked with 10% normal donkey serum for 1 h, and incubated with primary antibody (1:50 to 1:200 dilution in 0.1% TWEEN-20). were incubated at 37 °C for 3 hours or at 4 °C overnight. For immunofluorescence staining, Alexa Fluor® 488- or Alexa Fluor® 594-conjugated secondary antibodies were incubated for 1 h at room temperature, and samples were covered with DAPI mounting solution (Abcam). For immunohistochemical analysis, slides were incubated with anti-mouse IgG (Vector Laboratories) for 1 h at room temperature, a DAB substrate kit (Vector Laboratories) was used for development, and covered with Balsam (Sigma-Aldrich). All images were acquired using an Olympus BX51 microscope equipped with a CCD camera (Olympus, Tokyo, Japan), and computer-assisted image analysis was performed with DP2-BSW.

Flow Cytometry Analysis

Mouse liver leukocytes and mouse or human Kupffer cells were isolated and flow cytometric analysis was performed. For mouse liver leukocytes and Kupffer cells, cells were incubated with anti-mouse CD16/CD32 (mouse Fc blocker) (BD Biosciences) and LIVE/DEAD™ fixable aqua dead cell stain kit (Thermo Fisher) for 405 nm excitation. Scientific) was used to distinguish living cells. Afterwards, cells were incubated with anti-mouse eFluor 450- or PE-Cy7-conjugated CD45 (clone 30-F11) and FITC- or PE-Cy7-conjugated F4/80 (clone BM8) (Thermo Fisher Scientific), PE-Cy7-conjugated or APC-conjugated Ly-6G (clone 1A8), Alexa Fluor® 647-conjugated Siglec-F, APC-Cy7-conjugated CD11b (clone M1/70), PE-conjugated Ly-6C (clone AL-21), PE-conjugated CD146 (clone ME-9F1), FITC-conjugated Annexin V (BD Biosciences), Alexa Fluor® 647-conjugated TIM-4 (clone RMT4-54), Alexa Fluor® 647-conjugated rat IgG2a, k isotype control (clone RTK2758) , PE-conjugated CLEC2 (clone 17D9) (BioLegend) and FITC-conjugated MARCO (clone 579511) (R&D Systems). Eosinophils (CD11b ⁺ SiglecF ⁺ ), neutrophils (CD11b ⁺ Ly6G ⁺ ), macrophages (F4/80 ⁺ CD11b ⁺ ), Kupffer cells (F4/80 ^hi CD11b ⁺ ) and Ly6C ^hi or Ly6C ^low monocytes were identified using FlowJo software (version 10). ; FlowJo LLC). For isolated human Kupffer cells, cells were incubated with human BD Fc Block™ (BD Biosciences), and viable cells were determined with the LIVE/DEAD™ fixable aqua dead cell stain kit (Thermo Fisher Scientific) for 405 nm excitation. This was followed by anti-human V450-conjugated CD45 (clone HI30), BV786-conjugated CD15 (clone HI98), PE-conjugated CD14 (clone MΦP9), FITC-conjugated Annexin V (BD Biosciences), APC-conjugated LYVE1 (20-238). amino acids) (LS Bio) and PE-Cy7-conjugated CD68 (Y1/82A) (Thermo Fisher Scientific). Kupffer cells (CD45 ⁺ LYVE1 ^- CD15 ^- CD14 ⁺ CD68 ⁺ ) were analyzed in FlowJo software (version 10; FlowJo LLC).

Mitochondrial fraction isolation

Mitochondrial and cytosolic fractions were isolated from fresh liver tissue or isolated hepatocytes using a mitochondrial isolation kit (#89801, Thermo Fisher Scientific) according to the manufacturer's instructions. After fractionation, protein samples were immediately separated and used for Western blot analysis.

Mitochondrial superoxide detection

Hepatocytes isolated from WT mice were treated with vehicle, ethanol (50mM), or ethanol containing clenbuterol (1μM) for 12 hours and washed three times with warm HBSS containing calcium and magnesium. MitoSOX™ Red Mitochondrial Superoxide Indicator (#M36008, Thermo Fisher Scientific) stock solution (5mM) was diluted in warm HBSS/Ca/Mg to make a working solution (5μM), and 1.5ml of the working solution was applied to hepatocytes. The cells were then incubated at 37°C for 30 min in the dark. After 30 min, cells were gently washed three times with warm HBSS/Ca/Mg and counterstained with Hoechst 33342 solution (#H3570, Thermo Fisher Scientific) for 15 min at 37°C. Finally, cells were washed again and fluorescence images were captured within 1 hour.

Western blot analysis

Total protein samples from frozen liver tissue or isolated cells were lysed in RIPA lysis buffer (30mM Tris, pH 7.5, 150mM NaCl, 1mM PMSF, 1mM Na3VO4, 10% SDS, 10% SDS) with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). glycerol). Afterwards, the same amount of protein (approximately 50 to 100 μg) was separated by 10% SDS-polyacrylamide gel electrophoresis, and the protein was transferred using a nitrocellulose membrane (#88018, Thermo Fisher Scientific). After transfer, the membrane was blocked with 5% skim milk solution for 1 h at room temperature and then incubated with primary antibody (1:500 to 1:2000 dilution in 0.1% TWEEN-20) overnight at 4°C. The next day, the corresponding secondary antibodies (1:2000 dilution in 0.1% TWEEN-20) were incubated for 1 hour at room temperature. Immunoreactive bands were acquired by SuperSignal™ West Pico PLUS chemiluminescent substrate (#34577, Thermo Fisher Scientific) and captured by ImageQuant™ LAS 4000 (GE Healthcare). Protein expression levels were normalized to VDAC levels for the mitochondrial fraction and to β-actin levels for other fractions, which were used as loading controls.

Quantitative PCR

Total RNA was isolated from liver tissues or cells using TRIzol reagent (Thermo Fisher Scientific) and reverse transcribed into cDNA using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Japan) according to the manufacturer's instructions. qRT-PCR was performed with SYBR Green Real-time PCR Master Mix (Toyobo, Japan). The expression levels of genes of interest were normalized using the mRNA expression levels of 18s rRNA (for mouse) and ACTB (for humans).

Single-cell RNA sequencing of isolated Kupffer cells

WT mice were fed a pair (n=1) or ethanol (n=2) diet for 8 weeks, and their livers were digested by two-step collagenase perfusion. The hepatocyte fraction was removed by centrifugation at 50 xg for 5 minutes. Afterwards, liver immune cells were purified with 40% Percoll gradient solution by centrifugation at 650 xg for 10 minutes. The supernatant was carefully removed by suction and the pellet was resuspended in BSA buffer solution (0.5% BSA and 0.05% sodium azide in Dulbecco's PBS). After incubation with anti-mouse CD16/CD32 (mouse Fc blocker) (BD Biosciences) and LIVE/DEAD™ fixable aqua dead cell stain kit (Thermo Fisher Scientific) for 405 nm excitation, cells were incubated with anti-mouse PE-Cy7- Conjugated CD45 (clone 30-F11) and FITC-conjugated F4/80 (clone BM8) (Thermo Fisher Scientific), APC-conjugated Ly-6G (clone 1A8), Alexa Fluor® 647-conjugated Siglec-F, APC-Cy7-conjugated Conjugated CD11b (clone M1/70) and PE-conjugated CD146 (clone ME-9F1) (BD Biosciences) and Kupffer cells (DAPI ^- CD45 ⁺ CD146 ^- SiglecF ^- Ly6G ^- F4/80 ^hi CD11b ⁺ ) were FACS (fluorescence-activated). classified by cell sorting. In the pair-fed mice, 55,075 Kupffer cells were classified, and in the ethanol-fed mice, 97,403 and 90,263 Kupffer cells were classified. Cell viability and density were measured on a Countess® II automatic cell counter (Thermo Fisher Scientific), and the viability and density of sorted cells were >80% and 800–1,000 cells/uL, respectively. Cells were loaded into each channel at a target output of 7,500 or 10,000 cells per sample in the 10X Genomics Chromium Controller and then bound to beads. cDNA libraries were constructed following the 10X Genomics Protocol using v3 chemistry and sequenced using Illumina HiSeq X to a median depth of approximately 60,000 reads per cell.

Filtering and Clustering of scRNA-seq Data

The obtained fastq file was processed through CellRanger 3.1.0 (10X genomics), referring to the mouse reference genome (refdata-cellranger-mm10-3.0.0). The resulting file was analyzed using the scanpy package (v.1.4.5). In each group, cells with a UMI value of less than 1,000, a number of detected genes of less than 500 or more than 7,000, or a mitochondrial gene ratio of more than 50% were removed. After the removal process, the results for each cell were normalized by log(CPM/100+1), and genes that appeared significantly different were identified. After scaling the expression levels of these genes, principal component analysis was performed. Neighborhood graph formation, clustering, and UMAP production were carried out in accordance with the scanpy format.

Quantification and statistical analysis

Statistical analysis was performed using Prism version 8.0 (GraphPad Software) and data were expressed as mean ± SEM. The unpaired Student t-test was used to assess statistical significance between two groups, and for more than two groups, One-way Analysis of Variance (ANOVA) with Tukey or Dunnett's test for multiple comparisons was used. did. For next-generation sequencing data, adjusted p-values were used. A p-value <0.05 was considered statistically significant (*p <0.05, **p <0.01, ***p <0.001).

<Experimental Example 1> Determination of the effect of chronic alcohol consumption on gut-derived catecholamine levels and MAOA and ADRB2 expression in central venous lesions

Tissue catecholamine levels, expression of catecholamine receptors in the liver, and degradative enzymes were analyzed in mice chronically fed ethanol or Pair.

As a result, as shown in Figure 1a, the concentrations of norepinephrine and epinephrine in the cecum, portal blood, and liver tissues were higher in mice fed ethanol than in mice fed Pair. was significantly higher.

Additionally, as shown in Figure 1b, it was confirmed that the catecholamine level in the liver was positively correlated with the catecholamine level in the cecum (however, there was no significant change in genes related to catecholamine production in the liver and brain).

The above results suggest that catecholamines derived from the intestine are delivered to the liver.

Experiments were performed to determine the fate of hepatic catecholamines.

As a result of performing scRNA-seq (single-cell RNA-sequencing), as shown in Figure 1c, the expression of genes encoding adrenergic receptors ( Adra1a, Adra1b, Adrb2, Adrb3 ) and degrading enzyme ( Maoa) in healthy mice While it was high in the central vein (CV), which is a hepatic vein, the expression of Adra2b and Maob was confirmed to be mainly high around the portal triad (PT).

The above results suggest that the response to catecholamines varies depending on the liver region.

In addition, the results of qRT-PCR and Western blot showed that As shown in Figures 1D and 1E, Maoa, Maob , and Adrb2 mRNA and protein levels were confirmed to be increased in mice fed ethanol compared to mice fed Pair.

In addition, as a result of immunostaining, as shown in Figure 1f, MAOB was mainly observed around the hepatic portal vein (PT) in mice fed ethanol compared to mice fed Pair, and MAOA and ADRB2 proteins were observed in the central hepatic vein (CV). They were located together.

The above results suggest that gut-derived catecholamines function in the pericentral hepatic region through ADRB2.

In addition, as a result of qRT-PCR, as shown in Figure 1g, Maoa and Maob mRNA increased only in hepatocytes (HEP), but Adrb2 mRNA increased in both hepatocytes and Kupffer cells.

The above results suggest that gut-derived catecholamines may be associated with and influence hepatic MAOA and ADBR2 expression, especially in the pericentral liver region.

<Experimental Example 2> Confirmation of the effect of liver ADRB2 stimulation on mitochondrial CYP2E1-mediated oxidative stress and GDF15 production

Chronic ethanol intake enhanced the expression of Nrf2 (nuclear factor erythroid 2-related factor 2), a regulator of oxidative stress, and its target genes in pericentral HEP, and Nrf2 , Gdf15, Adrb2 genes, and antioxidant-related genes in liver oxidative stress. is involved. Accordingly, an experiment was performed to check the level of GDF15.

As a result, as shown in Figures 2a and 2b, the level of GDF15 was confirmed to be significantly increased in the serum and liver cells of mice fed ethanol compared to mice fed pair.

Additionally, as shown in Figure 2c, serum GDF15 levels were confirmed to be positively correlated with cecal catecholamines (epinephrine and norepinephrine) and liver Adrb2 mRNA levels.

The above results suggest that Nrf2-mediated ADRB2 expression and catecholamine-mediated GDF15 production are related in liver tissue.

In addition, as a result of immunostaining, as shown in Figure 2d, it was confirmed that the expression of CYP2E1 and GDF15 was increased in hepatocytes around the central vein, and as a Western blot result, as shown in Figure 2e, CYP2E1, GDF15, ADRB2 and their expression in the liver It was confirmed that the level of PKA-Cα present downstream gradually increased as the ethanol intake time increased.

In addition, Western blot results confirmed that, as shown in Figure 2f, the level of CYP2E1 was higher in the mitochondria than in the cytoplasm of hepatocytes due to ethanol treatment, and as shown in Figure 2g, CYP2E1 level was increased by treatment with ethanol or CBL, an ADRB2 agonist. It was confirmed that the level increased.

The above results indicate that increased mitochondrial oxidative stress caused by CYP2E1 increases GDF15 production.

In addition, as shown in Figure 2h, Western blot results showed that stimulation of ADRB2 using CBL increased the protein level of PKA-Cα, a regulator of CYP2E1 delivery to mitochondria.

In addition, as a result of checking the concentration of GDF15 in the hepatocyte culture medium, as shown in Figure 2i, it was confirmed that when hepatocytes were treated with ethanol or CBL, the production of GDF15 increased depending on the treatment of ethanol or CBL concentration.

In addition, as shown in Figure 2j, when CBL was treated with ethanol, it was confirmed through MitoSOX labeling that mitochondrial oxidative stress increased.

However, as a result of confirming Gdf15 mRNA expression, as shown in Figure 2k, treatment with AS (allyl sulfide), a CYP2E1 inhibitor, or NAC (N-acetyl-L-cysteine), an antioxidant, suppressed Gdf15 mRNA expression.

The above results suggest that GDF15 production may be induced by ADRB signaling in pericentral hepatocytes after alcohol exposure.

<Experimental Example 3> Confirmation of the effect of GDF15 on Kupffer cell apoptosis and alcoholic liver protection

To confirm the effect of production of hepatocyte-derived GDF15 on alcohol-related liver damage, a mouse experiment was performed in which the GDF15 gene was specifically deleted from hepatocytes using GDF15 ^f/f and GDF15 ^AlbCre mice.

As a result, as shown in Figures 3a and 3b, the levels of epinephrine and norepinephrine in the hepatic portal vein did not differ between the GDF15 ^f/f and GDF15 ^AlbCre mouse groups, but serum aspartate aminotransferase (ALT) was significantly higher in GDF15 compared to GDF15 ^f/f mice. increased in ^AlbCre mice, serum GDF15 was decreased, and triglyceride content did not differ (food intake, body weight change, serum aspartate aminotransferase (AST), and total cholesterol also did not differ between the two groups).

In addition, flow cytometry results showed that, as shown in Figure 3c, the frequency of F4/80 ⁺ CD11b ⁺ macrophages increased in GDF15 ^Albcre mice compared to GDF15 ^f/f mice, but there was no difference in the frequency of Ly6G ⁺ CD11b ⁺ neutrophils. .

The above results suggest that as Kupffer cells in GDF15 ^Albcre mice were more activated than in GDF15 ^f/f mice, more CCR2 ⁺ macrophages were gathered and increased liver inflammation and damage were observed in GDF15 ^Albcre mice.

To explain the relationship between GDF15 and Kupffer cells in the liver ingested with ethanol, liver tissue from GDF15 ^f/f and GDF15 ^Albcre mice was stained with antibodies to CYP2E1 and CLEC4F, a specific marker for Kupffer cells.

As a result of immunostaining, as shown in Figure 3d, it was confirmed that Kupffer cells were significantly disappeared in the CYP2E1 ⁺ pericentral region of GDF15 ^f/f , but not in GDF15 ^Albcre mice.

In addition, as a result of flow cytometry, as shown in Figure 3e, it was confirmed that there was less death of Kupffer cells in GDF15 ^Albcre mice than in GDF15 ^f/f mice.

The above results suggest that GDF15 can directly or indirectly induce apoptosis of Kupffer cells.

To confirm the apoptosis mechanism of Kupffer cells, genetic analysis and in vitro experiments were performed using Kupffer cells newly isolated from healthy mice or the above two groups of mice.

As a result of qRT-PCR, as shown in Figure 3f, the expression of genes related to pro-apoptosis was significantly reduced in Kupffer cells of GDF15 ^Albcre than in GDF15 ^f/f mice, and inflammation ( The expression of genes related to the inflammation response was significantly increased. In addition, the expression of Tgfbr2 mRNA, which is presumed to encode the GDF15 receptor, as well as Adrb2, Prkar1a , and Prkacb mRNA were significantly decreased in GDF15 ^Albcre compared to GDF15 ^f/f mice.

The above results suggest that Kupffer cells can die through ADRB2 induction by GDF15.

An experiment was performed to confirm the expression of adrenergic receptors in response to GDF15 treatment in Kupffer cells sensitized by LPS in vitro.

As a result, as shown in Figure 3g, the expression of Adrb2 was superior to Adrb1 and Adra2 mRNA in Kupffer cells, and this was confirmed to be significantly increased by GDF15 treatment.

Additionally, as shown in Figures 3h and 3i, treatment with GDF15 alone does not induce apoptosis of LPS-sensitized Kupffer cells, but stimulation of ADRB2 by CBL increases the release of PKA-Cα and decreases Bcl-2 levels. This induced apoptosis of Kupffer cells, which was reduced by treatment with KT5720, a PKA inhibitor.

Based on the above results, it was possible to hypothesize that chronic alcohol consumption induces apoptosis of inflammatory Kupffer cells through ADRB2 activation by gut-derived catecholamines and GDF15-mediated ADRB2 induction.

<Experimental Example 4> Effect of chronic alcohol intake on Kupffer cell apoptosis and phenotype

To investigate the expression of ADRB2 and apoptosis signaling-related genes in Kupffer cells, scRNA-seq was performed using F4/80 ^high CD11b ⁺ Kupffer cells isolated from paired or ethanol-fed mice by the 10X Genomics Chromium platform.

As a result, as shown in Figure 4a, the changed characteristics of Kupffer cells were observed by ethanol and annotated into three clusters as ethanol (cluster 1), mixed (cluster 2), and pair (cluster 3) groups. As shown in Figure 4b, based on the function plot, even after removing dead cells in cluster 1, a high frequency of mitochondrial genes but a low number of genes per cell were observed, indicating the characteristics of highly stressed or apoptotic Kupffer cells. (Apoptosis of infiltrated F4/80 ⁺ CD11b ⁺ macrophages was not different in both groups).

Relative expression of Kupffer cell markers (C lec4f, Adgre1, Vsig4, Timd4, and Marco ) and infiltrated macrophage markers ( Ccr2, Cx3cr1) , GDF15 receptor ( Gfral ), adrenergic receptor, and PKA subunit ( Prkar1a , and Prkacb ) was measured. As a result of comparison, as shown in Figures 4c and 4d, all cells isolated from the three clusters were identified as Kupffer cells, and the expression of ADRB2 and PKA-related genes was confirmed to be specifically elevated in cluster 1 compared to cluster 3. ( Adra1, Adra2 and Adrb3 were barely expressed in all Kupffer cells).

Additionally, as shown in Figure 4d, the expression of genes related to apoptosis ( Casp3, Bak1, Bax, Bcl2l11, Mcl1, and Bcl2 ), ER stress ( Atf4, Ddit3 ), and inflammation ( Il1b, Nlrp3 ) were clustered in clusters 2 and 3. Compared to this, it was relatively higher in Kupffer cells in cluster 1 (ADRB2 expression was almost always observed in Kupffer cells, but its expression was suppressed in the non-alcoholic liver disease model, suggesting that ADRB2-mediated apoptosis of Kupffer cells is an ethanol-specific characteristic) . In addition, as shown in Figures 4c and 4d, with respect to the GDF15 binding receptor, Kupffer cells in cluster 1 expressed more TGF-β receptors ( Acvrl1, Tgfbr1, and Tgfbr2 ) than Kupffer cells in cluster 3, but all Kupffer cells Gfral mRNA was not expressed.

The scRNA-seq data were further analyzed to find the underlying mechanism for the altered properties of Kupffer cells.

As a result, as shown in Figure 4e, compared to Kupffer cells in cluster 3, Kupffer cells in cluster 1 were significantly enriched in gene sets related to negative regulation of cell growth or proliferation, regulation of extrinsic cell death signaling pathways, and misfolded proteins. It was revealed.

Therefore, as shown in Figures 4f and 4g, more dead Kupffer cells were identified when staining with Annexin V and CLEC4F antibodies in the pericentral vein area of mice fed ethanol compared to mice fed pair.

As TIM4+ Kupffer cells (yolk sac-derived) are replaced by TIM4- macrophages (bone marrow-derived) during the development of non-alcoholic steatohepatitis, to determine whether alcohol exposure can lead to replacement of Kupffer cells by macrophages compared to normal conditions. An experiment was performed to do this.

First, as shown in Figure 4h, yolk sac-derived F4/80 ^hi CD11b ⁺ Kupffer cells show a TIM4 ⁺ MARCO ⁺ phenotype, but bone marrow-derived F4/80 ⁺ CD11b ⁺ macrophages show a TIM4 ^- MARCO ^- phenotype. confirmed.

In addition, as a result of flow cytometry, as shown in Figure 4i, the expression levels of CLEC2 (CLEC4F alternative marker), TIM4, MARCO, and F4/80 were determined by t-SNE (t-distributed stochastic neighbor embedding) plot of Kupffer cells. When combining the populations, there were three clusters, consistent with scRNA-seq, and TIM4, MARCO, and F4/80 expression was slightly reduced in Kupffer cells from ethanol-fed mice compared to pair-fed mice, consistent with scRNA-seq. . However, Kupffer cells from ethanol-fed mice still expressed CLEC2 and TIM4.

The above results indicate that Kupffer cells undergo ADRB2/PKA-mediated apoptosis in alcoholic fatty liver disease but are not replaced by bone marrow-derived TIM4-macrophages.

<Experimental Example 5> Activation of ADRB2 ( In situ ) confirmed its effect on the apoptosis of inflammatory Kupffer cells in the pericentral venous area.

In situ liver perfusion was performed with medium containing LPS, CBL, and ethanol (hereinafter referred to as LCE) using WT and ADRB1/2 DKO mice (Figure 5a).

As a result, as shown in Figures 5b and 5c, ADRB2 activation in WT mice by LCE significantly increased GDF15 levels and Kupffer cell apoptosis, but in ADRB1/2 DKO mice, GDF15 was decreased compared to the control group even after LCE circulation. There were no differences in the expression and inflammatory Kupffer cell death, which induced liver damage and significantly higher ALT levels than in WT mice.

The above results reaffirm that the survival of inflammatory Kupffer cells in the above GDF15 ^Albcre mice has detrimental effects, such as inducing liver damage.

In addition, as a result of staining liver tissue with CLEC4F antibody, as shown in Figure 5d, it was found that many Kupffer cells were still in the pericentral vein area in ADRB1/2 DKO mice compared to WT mice.

In addition, as shown in Figures 5e and 5f, the protein levels of PKA-Cα, GDF15 in the liver and CYP2E1 in mitochondria and ADRB/PKA signaling ( Adrb1, Adrb2, Prkar1a and Prkacb) and apoptosis (Adrb1, Adrb2, Prkar1a and Prkacb ) in Kupffer cells were significantly affected. The expression of genes related to Atf4, Ddit3, Bak1, Bax, Bcl2l11, and Casp3 ) was upregulated by ADRB2 activation via circulating LCE in WT mice, but in ADRB1/2 DKO mice, defects in ADRB/PKA signaling resulted in increased activation of inflammatory Kupffer cells. It was confirmed that death was reduced and, conversely, the expression of the relevant genes was down-regulated and the expression of anti-apoptotic genes ( Mcl1 and Bcl2 ) was increased.

In addition, as shown in Figures 5g to 5i, inhibition of PKA by KT5720 reduced apoptosis of Kupffer cells and decreased hepatic GDF15 production, showing similar results to ADRB1/2 DKO mice.

Additionally, as shown in Figures 5J and 5K, the frequency of neutrophils (Ly6G ⁺ CD11b ⁺ ) and macrophages (F4/80 ⁺ CD11b ⁺ ) in the liver increased after LCE circulation in ADRB1/2 DKO compared to WT mice. , Tnf and Il1b mRNA expression was also increased in both whole liver tissue and isolated Kupffer cells. This may be due to the upregulated expression of Cxcl1 and Ccl2 mRNA in Kupffer cells by stimulation of ethanol and LPS, as shown in Figure 5k.

<Experimental Example 6> Confirmation of the effects of pharmacological and genetic inhibition of catecholamines, CYP2E1, and ADRB on Kupffer cell survival and alcoholic liver damage in mice

Catecholamine production can be inhibited pharmacologically by inhibiting dopamine β-hydroxylase activity through FA, or genetically inhibiting CYP2E1 and ADRB1 using hepatic CYP2E1 heterozygous KO mice (CYP2E1 ^f/-AlbCre ) or ADRB1/2 DKO mice. An experiment was performed to determine whether inhibiting the expression of /2 could worsen alcoholic liver damage.

As a result, as shown in Figure 6a, the concentration of catecholamines in the portal vein was decreased in FA-treated mice, but there was no difference from the control group in CYP2E1 ^f/-AlbCre and ADRB1/2 DKO mice.

However, as shown in Figure 6b, FA-treated mice, CYP2E1 ^f/-AlbCre mice, and ADRB1/2 DKO mice all had decreased GDF15 and increased serum levels of ALT and AST, indicators of liver damage, compared to the control group.

The above results indicate that catecholamines, CYP2E1 and ADRB1/2 protect against alcoholic liver damage.

Moreover, as shown in Figure 6c, ADRB2 and PKA-Cα protein levels in the liver were reduced in FA-treated mice and ADRB1/2 DKO mice, and hepatic GDF15 and mitochondrial CYP2E1 expression were decreased in all three experimental groups compared to the control group. did.

Additionally, as shown in Figure 6d, the expression of Gdf15 mRNA in the liver was significantly decreased in FA-treated mice, both CYP2E1 ^f/-AlbCre and ADRB1/2 DKO mice, whereas Adrb2 mRNA expression was significantly decreased in FA-treated mice. In mice and ADRB1/2 DKO mice, Cyp2e1 mRNA levels were significantly reduced only in CYP2E1 ^f/-AlbCre mice compared to controls.

Accordingly, as shown in Figure 6e, many CLEC4F ⁺ Kupffer cells in FA-treated mice, CYP2E1 ^f/-AlbCre, and ADRB1/2 DKO mice were observed in the pericentral venous area where CYP2E1 ⁺ hepatocytes were located compared to the control group.

Flow cytometry results showed that, as shown in Figure 6f, the proportions of F4/80 ⁺ CD11b ⁺ macrophages and Ly6G ⁺ CD11b ⁺ neutrophils as well as the proportions of FA-treated mice, CYP2E1 ^f/-AlbCre and ADRB1/2 DKO mice compared to controls. The proportion of inflammatory F4/80 ⁺ CD11b ⁺ Ly6C ^hi macrophages was also increased.

The above results reaffirmed that the apoptosis mechanism of inflammatory Kupffer cells through the catecholamine-CYP2E1-GDF15-ADRB2 signaling axis protects against alcoholic liver damage.

<Experimental Example 7> Confirmation of the catecholamine/GDF15/ADRB2 signaling pathway axis in patients with early alcoholic liver disease

An experiment was performed to measure serum levels of catecholamines and GDF15 in patients with alcoholic liver disease (ALD).

As a result, as shown in Figure 7a, serum concentrations of NE, EPI, and GDF15 were significantly increased in all alcohol patients compared to healthy controls. Specifically, the levels of NE, EPI, and GDF15 were significantly higher in alcohol-consuming patients without liver disease (AWLD) than in patients with alcoholic fatty liver (AFL) and alcoholic steatohepatitis (ASH). It was high.

However, as shown in Figure 7b, there was no significant difference in fecal NE and EPI levels according to severity in steatosis patients compared to non-steatosis patients.

Instead, as shown in Figure 7c, serum GDF15 levels are positively correlated with serum catecholamines, but gamma-glutamyl transpeptidase (GGT) levels, known as diagnostic markers of ALD, do not correlate with patients' GDF15 or catecholamines. It was confirmed that there was no correlation with the serum level of

The above results indicate that the early stages of ALD can be diagnosed using catecholamines and GDF15.

Additionally, as shown in Figures 7D and 7E, protein and mRNA expression of ADRB2, PKA-Cα, GDF15, MAOA, and MAOB were increased in the liver tissue of AWLD patients compared with healthy controls, but the levels were attenuated in AFL patients. , which appears to reflect a decrease in serum catecholamines.

Consistently, as shown in Figures 7f and 7g, co-treatment of ethanol and CBL significantly increased protein levels of ADRB2, PKA-Cα, and GDF15 compared to hepatocytes treated with vehicle and ethanol.

Additionally, we investigated the expression of MAOA, MAOB and ADRB2, CD68, CYP2E1 and GDF15 proteins in liver tissue of AFL patients.

As a result, as shown in Figure 7h, compared to healthy controls, the expression of MAOA, ADRB2, CYP2E1, and GDF15 in AFL patients expanded from around the central vein to hepatocytes around the portal vein, and in the case of MAOB, on the contrary, from around the portal vein to around the central vein. expanded. Additionally, CD68 ⁺ Kupffer cells were significantly decreased in the pericentral area of AFL patients compared to healthy controls (Figure 7i).

Additionally, in vitro ADRB2 activation by CBL induced apoptosis in Kupffer cells treated with LPS/GDF15/CBL compared to Kupffer cells treated with LPS or LPS/GDF15 (Figure 7j). As a result of qRT-PCR analysis, as shown in Figure 7k, genes related to ADRB2 signaling and apoptosis promotion were highly expressed in Kupffer cells treated with LPS/GDF15/CBL compared to Kupffer cells treated with LPS or LPS/GDF15. Anti-apoptotic genes were downregulated.

Additionally, as shown in Figure 7l, GDF15 mRNA was significantly upregulated in alcoholic hepatitis (AH) patients compared with healthy controls, but the expression of ADRB2, MAOA and MAOB was significantly decreased.

Additionally, as shown in Figure 7M, an increase in CD68 ⁺ cells in the pericentral area and an increase in the expression of GDF15 in the liver of alcoholic cirrhosis (ALC) patients were observed compared to healthy controls.

The above results suggest that the concentration of catecholamines, the expression of catecholamine degrading enzymes, and ADRB2-related signals can diagnose the severity of ALD patients, regardless of the concentration of GDF15 in the blood.

Claims (12)

1) treating cells with a test substance;
2) measuring the expression level of the ADRB2 gene or the activity level of its protein in the cells of step 1); and
3) selecting a test substance in which the expression level of the ADRB2 gene or its protein in step 2) is increased compared to the control group that did not treat the test substance;
A screening method for a treatment for alcoholic liver disease, comprising:
The method of screening for a therapeutic agent for alcoholic liver disease according to claim 1, wherein the cells are Kupffer cells.
The method of screening for a treatment for alcoholic liver disease according to claim 1, wherein expression of ADRB2 induces death of Kupffer cells.
The method of screening for a therapeutic agent for alcoholic liver disease according to claim 1, wherein expression of ADRB2 induces inhibition of inflammatory cytokines.
The method of screening for a therapeutic agent for alcoholic liver disease according to claim 1, wherein the alcoholic liver disease is alcoholic fatty liver, alcoholic steatohepatitis, or alcoholic hepatitis.
A pharmaceutical composition for preventing or treating alcoholic liver disease, comprising an ADRB2 agonist as an active ingredient.
The pharmaceutical composition for preventing or treating alcoholic liver disease according to claim 6, wherein the ADRB2 agonist increases mitochondrial CYP2E1-mediated oxidative stress.
The pharmaceutical composition for preventing or treating alcoholic liver disease according to claim 6, wherein the ADRB2 agonist increases the production of GDF15 in the liver.
The pharmaceutical composition for preventing or treating alcoholic liver disease according to claim 6, wherein the ADRB2 agonist induces death of Kupffer cells.
A pharmaceutical composition for preventing or treating alcoholic liver disease, comprising catecholamine as an active ingredient.
The pharmaceutical composition for preventing or treating alcoholic liver disease according to claim 10, wherein the catecholamine activates ADRB2.
The pharmaceutical composition for preventing or treating alcoholic liver disease according to claim 10, wherein the catecholamine is dopamine, norepinephrine, or epinephrine.