Therapeutic use of exosomes containing superrepressor-I [kappa] B (srI [kappa] B) for liver disease

Abstract

The present invention relates to the use of exosomes comprising a superrepressor-I [kappa] B (srI [kappa] B) for the treatment of liver disease. The present invention provides a pharmaceutical composition for preventing or treating a liver disease, the pharmaceutical composition comprising an exosome containing a superrepressor-I [kappa] B (srI [kappa] B) as an active ingredient, the liver disease being hepatic fibrosis, cirrhosis, fatty liver, alcoholic liver disease, cholestasis, or a combination thereof.

Description

Technical Field

[0001] The present invention relates to the therapeutic use of exosomes containing super repressor-IκB (srIκB) in liver diseases. Background Art

[0002] Liver diseases vary in type and severity, ranging from fatty liver to cirrhosis, and are often not discovered until the late stage because they do not have any initial subjective symptoms. The causes of liver disease are diverse, such as viral or bacterial infection, alcohol or toxic substances, excessive accumulation of fat or heavy metals, and abnormal immune response. Although various treatments are used for liver disease depending on the cause and type of the disease, in many cases, traditionally known therapeutic agents do not work, and they are therefore the leading cause of death in the world. Therefore, there is still a need to develop drugs for the treatment of liver disease. At the same time, exosomes have recently attracted considerable attention as a new type of biological carrier for gene / drug delivery. Exosomes are extracellular vesicles that play an important role in intercellular communication by delivering bioactive substances to receptor cells or affecting the signaling pathways of target cells.

[0003] The present inventors have developed exosomes containing physiologically active substances capable of preventing or treating liver diseases, and confirmed that the exosomes stably and effectively deliver physiologically active substances to liver cells and tissues, thereby providing excellent therapeutic effects for liver diseases, and therefore, aim to provide the exosomes of the present invention as a preventive or therapeutic agent for liver diseases. Summary of the invention

[0004] Technical issues

[0005] The present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising exosomes containing super repressor-IκB (srIκB) as an active ingredient.

[0006] Solution to the problem

[0007] One aspect for achieving the present invention relates to a pharmaceutical composition for preventing or treating liver disease, the pharmaceutical composition comprising exosomes containing super repressor-IκB (srIκB) as an active ingredient.

[0008] Another aspect for implementing the present invention relates to a method for preventing or treating liver disease, the method comprising administering exosome therapy containing super repressor-IκB (srIκB) protein to an individual at risk of liver disease.

[0009] Beneficial Effects

[0010] The exosomes containing super repressor-IκB (srIκB) according to the present invention can be effectively used for preventing or treating liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the preparation of an animal model induced with bile duct stricture according to one embodiment of the present invention.

[0012] Figure 2 is a graph showing the measurement of the relative weight (%) of the liver when the carrier and Exo-srIκB were administered to an animal model induced with bile duct stricture according to one embodiment of the present invention. In particular, Figure 2 b shows the measurement of the relative weight (%) of the liver according to the body weight (g) of the animal.

[0013] Figure 3 is a diagram confirming liver abscess when the cargo body and Exo-srIκB were administered to an animal model induced with bile duct stricture according to one embodiment of the present invention.

[0014] Figure 4 is a diagram confirming jaundice symptoms when a cargo body and Exo-srIκB are administered to an animal model induced with bile duct stricture according to one embodiment of the present invention.

[0015] Figure 5 2 is a graph showing the evaluation results of scoring the following four items by observing liver tissue according to one embodiment of the present invention when the carrier and Exo-srIκB are administered to an animal model induced with bile duct stricture: debris-like necrosis, lobular necrosis, histological activity score, and fibrosis.

[0016] Figure 6 : is a graph showing the results of measuring the fibrosis area in the stained liver tissue and calculating it as the mean value and standard deviation when the cargo body and Exo-srIκB were administered to the animal model induced with bile duct stricture according to one embodiment of the present invention.

[0017] Figure 7 : is a graph showing blood analysis on the day of necropsy and the results calculated as the mean value and standard deviation when a cargo body and Exo-srIκB were administered to an animal model induced with bile duct stricture according to one embodiment of the present invention.

[0018] Figure 8 It is a schematic diagram of the preparation of a TAA-induced liver fibrosis animal model according to one embodiment of the present invention.

[0019] Fig. 9 is a graph demonstrating the ICG concentration when the cargo body and Exo-srIκB were administered to the TAA-induced liver fibrosis animal model according to one embodiment of the present invention.

[0020] Fig.10: is a graph showing the results of measuring the blood concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) when the cargo body and Exo-srIκB were administered to the TAA-induced liver fibrosis animal model according to one embodiment of the present invention.

[0021] Fig.11 : is a graph showing the results of measuring changes in α-smooth muscle actin (a-SMA), a marker of fibrosis, by Western blotting when a cargo body and Exo-srIκB are administered to a TAA-induced liver fibrosis animal model according to one embodiment of the present invention.

[0022] Fig.12 is a diagram confirming liver necrosis when the cargo body and Exo-srIκB were administered to a TAA-induced liver fibrosis animal model according to one embodiment of the present invention.

[0023] Fig.13 It is a schematic diagram of the preparation of a DMN-induced liver injury animal model according to one embodiment of the present invention.

[0024] Fig.14 The expression levels of genes related to the exosome uptake process increased in mouse and human inflammatory Kupffer cells (KC) according to one embodiment of the present invention are shown. (ab) Single cell RNA sequencing (scRNA-seq) of normal mouse liver (GSE132042) was analyzed. Uniform manifold approximation and projection (UMAP) display of feature maps of cell type annotation (a) or gene expression levels (b). (ce) A large number of RNA sequences of mouse primary KC (GSE86397) were analyzed. (c) Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of upregulated genes in KC treated with lipopolysaccharide (LPS). (d) Gene ontology (GO) biological processes increased in LPS-treated KC were analyzed. (e) Heatmap shows the relative expression levels of genes associated with the pathways shown. (f, g) scRNA-seq from human liver (GSE136103) from healthy controls (Ctrl, n = 5) or patients with alcoholic cirrhosis (ALC, n = 2) was analyzed. (f) t-distributed stochastic neighbor embedding (t-SNE) display of feature plots shows annotation by group (top) and expression levels in MARCO (bottom). (g) Relative expression levels of genes associated with the indicated pathways were analyzed in MARCO+KC. For RNA-seq, corrected p-values ​​were used.

[0025] Fig.15 The results show that Exo-srIκB according to one embodiment of the present invention inhibited the expression level of inflammatory genes induced by LPS in KC. (ac) The indicated doses of DiI-labeled Exo-srIκB were used to treat the load (normal saline, VEH) or LPS (100 ng ml -1 ) isolated mouse KCs under the condition of (n = 5 per group). (a) Representative photos of cultured mouse KCs. Scale bar, 50 μm. (b, c) Frequency of DiI+ KCs in total KCs. (d) Pretreatment with control exosomes (Exo-native) or Exo-srIκB (KC:exosomes = 1:10,000) for 3 h, followed by LPS (100 ng ml -1 ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of isolated mouse KCs pretreated for 6 h (n=3 per group). Data are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001). Data were analyzed by one-way ANOVA with Tukey's multiple comparison test.

[0026] Fig.16 The results confirm that a high dose of Exo-srIκB according to one embodiment of the present invention exhibits the effect of reducing alcoholic fatty liver (AFL) and inflammatory macrophage infiltration. (ah) Wild-type (WT) male mice were fed EtOH for 10 days and randomly divided into Exo-native or Exo-srIκB groups (n=6 / group). A single high dose (5.0 x 10 per mouse) was intravenously injected 10 Particles) of exosomes from liver tissue. One hour later, mice were given acute EtOH (4 g / kg of 40% EtOH by gavage) and sacrificed 6 hours later. (a) Schematic diagram of the experiment. (b) Representative gross findings and H&E staining of liver tissue. CV denotes central vein, and PT denotes portal triad. Scale bar, 50 μm. (c) Measurement of hepatic triglyceride (TC) levels. (d) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic triglycerides (TG), and total cholesterol (TC). (e) Representative Western blot analysis and densitometry analysis of hepatocyte nuclear fractions. (f) qRT-PCR analysis of whole liver tissue. (g) Number of mononuclear cells (MNCs) per gram of liver tissue. (h) Flow cytometric analysis of liver MNCs, with representative panels and bar graphs indicating the frequency or absolute number of the indicated populations. (i) Results of body weight measurements according to administration of Exo-srIκB. Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01). Data were analyzed by one-way ANOVA with Tukey's multiple comparison test.

[0027] Fig.17 The results confirm the alleviation effect of ALI, AFL and ALF in the experimental group injected with Exo-srIκB for 3 consecutive days according to one embodiment of the present invention. (af) WT male mice were fed with EtOH for 9 days and randomly divided into Exo-natural (10 9 particles / day / mouse), low-dose Exo-srIκB (10 8 particles / day / mouse) or high dose Exo-srIκB (10 9 Particles / day / mouse) groups (n=6 per group). The indicated doses of exosomes were injected intravenously once every 24 hours for 3 days. Six hours after the last injection, mice were given acute EtOH (4 g / kg of 40% EtOH by gavage) and sacrificed 6 hours later. (a) Schematic diagram of the experiment. (b) Serum levels of ALT and AST were determined. (c) Representative gross findings, H&E, Oil Red O, and TUNEL staining of liver tissues. Scale bar, 50 μm. In TUNEL staining, apoptotic hepatocytes (HEPs) (yellow triangles) and non-parenchymal cells (blue triangles) are shown. (d) Measurement of liver TC levels. (e) TUNEL+ apoptotic HEPs were counted in 15 random images at x100 magnification. (f) qRT-PCR analysis of isolated hepatic stellate cells (HSCs). Body weight was measured on day 9 (g) or after sacrifice (h). (i) Serum levels of TG and TC were measured. Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). Data were analyzed by one-way ANOVA with Tukey's multiple comparison test.

[0028] Fig.18 It was confirmed that Exo-srIκB according to one embodiment of the present invention effectively alleviated alcoholic liver inflammation by inhibiting the activation of KC in mice. (ah) Male mice were fed EtOH for 9 days and randomly divided into Exo-native, low-dose Exo-srIκB or high-dose Exo-srIκB groups. The indicated dose of exosomes was injected intravenously once every 24 hours for 3 days. 6h after the last injection, mice were given acute EtOH (4g / kg of 40% EtOH gavage) and killed 6h later. (a, b) F4 / 80 高Flow cytometric analysis of CD11b+ KCs with C-type lectin-like receptor 2 (CLEC2) and T-cell immunoglobulin and mucin domain-containing 4 (TTIM4) with insets (a) and bar graphs (b). (c) Representative C-type lectin domain family 4 member F (CLEC4F) immunostaining of liver tissue. Yellow triangles indicate morphological changes in CLEC4F+ KCs. (d) Representative Western blot analysis and densitometry analysis of nuclear fractions of hepatocytes. (e) qRT-PCR analysis of isolated KCs. (f) Determination of serum IL-6 levels. (g) Representative myeloperoxidase (MPO) immunostaining of liver tissue (upper panel). MPO+ neutrophils are indicated by blue triangles and counted in 15 random images at x200 magnification (lower panel). (h) Flow cytometric analysis of liver MNCs with insets and bar graphs. Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). Data were analyzed by one-way ANOVA with Tukey's multiple comparison test. Scale bar, 50 μm.

[0029] Fig.19 It was confirmed that Exo-srIκB according to one embodiment of the present invention showed an effect of reducing the infiltration of immune cells into the liver. (a) qRT-PCR analysis of isolated HEPs. (b) Analysis of the number of MNC cells per gram of liver tissue. (ce) Flow cytometry analysis. Data are expressed as mean ± SEM (*P<0.05, **P<0.01, ***P<0.001). Data were analyzed by one-way ANOVA with Tukey's multiple comparison test. Scale bar, 50 μm. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below. Meanwhile, each description and embodiment disclosed herein can be applied to other descriptions and embodiments respectively. That is to say, all combinations of various elements disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the following specific description.

[0031] Furthermore, those skilled in the art will be able to identify or confirm, using no more than routine experimentation, numerous equivalents to the specific aspects of the invention described herein. Additionally, such equivalents are also intended to be encompassed by the present invention.

[0032] One aspect of the present invention provides a pharmaceutical composition for preventing or treating liver disease, the pharmaceutical composition comprising exosomes. Specifically, the exosome refers to an exosome containing super repressor-IκB (srIκB). Super repressor-IκB (srIκB) is an active ingredient that provides a preventive or therapeutic effect on liver disease, which is contained in the exosomes of the present invention and can be used interchangeably with cargo proteins.

[0033] Exosomes can contain super-repressor-IκB (srIκB).

[0034] As used herein, the term "super repressor-IκB (srIκB)" is a protein that is not phosphorylated by IκB kinase (IKK) and is not degraded by the proteasome, and is an S32A, S36A mutant form of IκB protein (IκBα). In the present invention, srIκB includes IκB protein or a fragment thereof. Super repressor-IκB (srIκB) can be an amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95% sequence homology thereto, or a fragment thereof, but is not limited thereto.

[0035] In the following, srIκB may be expressed as a cargo protein.

[0036] As used herein, the term "exosome" refers to a carrier capable of loading cargo proteins, and cargo proteins can be loaded by various methods known in the art. Examples of known methods for loading cargo proteins include: a method of overexpressing cargo proteins in exosome-producing cells to load them into exosomes; a method of overexpressing fusion proteins in exosome-producing cells using a carrier, wherein exosome-specific markers and cargo proteins are fused to increase loading efficiency, thereby loading them into exosomes; or a method of loading cargo proteins into exosomes by photodynamic reversible protein-protein interactions, etc., but are not limited thereto.

[0037] Specifically, as a method for producing exosomes containing super repressor-IκB (srIκB) protein of the present invention, the present invention can be combined with the entire disclosure of U.S. Patent No. 10702581 and Korean Patent No. 10-1733971 by reference to provide a composition and method for preparing the exosomes disclosed herein. In the present invention, "exosomes containing super repressor" can be used interchangeably with "Exo-srIκB".

[0038] The exosome may have a diameter of about 50 nm to about 200 nm, specifically about 50 nm to about 150 nm, but is not limited thereto.

[0039] As used herein, "liver disease" refers to a disease occurring in the liver, specifically, it may include liver fibrosis, cirrhosis, fatty liver, alcoholic liver disease, cholestasis or a combination thereof, but is not limited thereto, as long as liver tissue and function are abnormal.

[0040] As used herein, "liver fibrosis" refers to the result of the wound healing process of repeated liver damage. It is well known that, unlike cirrhosis, liver fibrosis is reversible, consisting of fine fibers and without nodule formation, and the liver can return to a normal state when the cause of liver damage is eliminated. However, when this liver fibrosis process continues repeatedly, liver fibrosis develops into cirrhosis.

[0041] As used herein, "liver cirrhosis" is a chronic disease that occurs with repeated increase in liver cell and fibrous tissue regeneration, which is pathologically accompanied by necrosis and fibrosis, and eventually develops into complications of liver cirrhosis such as liver failure and diseases such as hepatocellular carcinoma, leading to death. In particular, since there is no awareness of its symptoms in the early stages of the disease, liver cirrhosis is discovered only after a considerable period of development, and is therefore difficult to treat.

[0042] As used herein, "fatty liver" refers to a disease in which fat accumulates in liver cells, and specifically, it may include alcoholic fatty liver caused by excessive drinking and non-alcoholic fatty liver caused by obesity, diabetes, hyperlipidemia or metabolic syndrome.

[0043] As used herein, "alcoholic liver disease" refers to liver disease occurring due to excessive alcohol consumption, and specifically, it may be various diseases such as alcoholic fatty liver, alcoholic steatohepatitis, alcoholic cirrhosis, and alcoholic liver fibrosis, but is not limited thereto.

[0044] Specifically, "alcoholic liver disease" refers to several groups of liver diseases caused by chronic drinking. As the first stage of alcoholic liver disease, alcoholic fatty liver, in which fat accumulates in liver cells due to continued drinking, occurs. As the disease progresses, severe fat accumulation, liver necrosis, and acute inflammatory reactions occur, and these stages are called alcoholic hepatitis. Thereafter, as the last step, collagen accumulates in the liver tissue and may develop into alcoholic cirrhosis accompanied by liver fibrosis. Therefore, this type of alcoholic liver disease rarely develops sequentially or appears as a single disease, and in most cases, the development stages overlap and appear in the form of a complex disease group.

[0045] As used herein, "cholestasis" refers to a disease in which bile is retained in the liver due to an autoimmune disease or bile duct obstruction. Cholestatic diseases can be largely divided into extrahepatic cholestasis (external compression of the bile duct, internal obstruction of the bile duct, biliary stenosis, Caroli's disease, etc.) and intrahepatic cholestasis. Cholestasis can be used interchangeably with cholestatic liver disease. Cholestatic liver disease includes primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC) and Alagille syndrome (AS) in decreasing order of frequency.

[0046] The composition of the present invention can show (a) a decrease in relative liver weight; (b) a decrease in jaundice; (c) a decrease in liver abscess levels; (d) a decrease in liver fibrosis; (e) a decrease in T-BIL levels; (f) an increase in HDL levels; (g) a decrease in the number of α-SMA changes in hepatic stellate cells; (h) a decrease in serum ALT / AST levels; and / or (i) a decrease in alcoholic fatty liver, thereby preventing or treating liver disease, but is not limited thereto.

[0047] The exosomes or pharmaceutical compositions comprising exosomes of the present invention may further include a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients may include binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injection, which may be used in combination; and bases, excipients, lubricants, preservatives, etc. for topical administration, although not particularly limited thereto.

[0048] The formulation type of the composition of the present invention can be prepared in various ways by combining with the above-mentioned pharmaceutically acceptable excipients. For example, the composition of the present invention can have any one formulation type selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid drugs for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations and suppositories. The composition of the present invention can be a physiologically acceptable aqueous solution or suspension of exosomes. In the case of injection, the composition can be formulated into a unit dose ampoule or a multi-dose form.

[0049] In addition, the composition can be formulated into a unit dosage form suitable for administration to a patient according to conventional methods in the pharmaceutical field, so that it can be administered orally or parenterally (including skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal routes) using conventional administration methods in the art, but the administration route is not limited thereto.

[0050] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug as an active ingredient together with various factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, the severity of the disease, etc. Specifically, the composition of the present invention may contain a pharmaceutically effective amount of exosomes, but is not limited thereto.

[0051] Exosomes containing a pharmaceutically effective amount refer to a level at which the desired pharmacological activity (e.g., prevention, improvement, or treatment of liver disease) can be obtained by the exosomes of the present invention, and may refer to a pharmaceutically acceptable level, which is a level at which toxicity or side effects do not occur or occur at an insignificant level in the subject to be administered, but the level is not limited thereto. The pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, patients, preparations, etc.

[0052] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the above-mentioned ingredient (active ingredient) in an amount of 0.01% (W / V) to 99% (W / V).

[0053] The total effective dose of the composition of the present invention can be administered to the patient in a single dose. In the pharmaceutical composition of the present invention, the content of one or more active ingredients can vary depending on the severity of the disease. Specifically, the total daily dose of the exosomes of the present invention can be about 1x10 per 1 kg of patient body weight. 6 Up to 1x10 15 pn. However, the determination of the effective dose of exosomes is subject to various factors, including the patient's age, weight, health status, gender, disease severity, diet and excretion rate, as well as the administration route and treatment frequency of the pharmaceutical composition. In this regard, those skilled in the art can easily determine the effective dose suitable for a specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to the type of preparation, route of administration and mode, as long as it shows the effect of the present invention.

[0054] Another aspect for achieving the present invention provides a method for preventing or treating liver disease, comprising exosomes containing super repressor-IκB (srIκB) protein.

[0055] Example

[0056] Hereinafter, Preparation Examples are only used to describe the present invention in more detail, and the scope of the present invention is not limited to these Preparation Examples.

[0057] Preparation Example 1. Preparation of exosomes containing target protein (Exo-srIκB)

[0058] Exosomes containing super repressor-IκB (srIκB) as a target protein were prepared by the method described in Korean Patent No. 10-1877010.

[0059] Preparation Example 2. Preparation of control exosomes (Exo-native)

[0060] Expi293F cells (Thermofisher) were incubated under light irradiation conditions for 4 days, and then the culture medium was harvested and purified in the same manner as in Preparation Example 1 to obtain Exo-native.

[0061] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only used to describe the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0062] Example 1. srIκB-loaded exosomes (Exo-srIκB) in a miniature pig model of liver cirrhosis induced by bile duct stricture Efficacy evaluation in

[0063] 1-1. Construction of animal model of biliary stricture

[0064] In order to confirm whether the exosomes containing super repressor (srIκB) of the present invention can prevent or treat liver disease, animal models of liver disease induced by various reasons were constructed for confirmation.

[0065] First, an animal model of induced biliary stricture was constructed to confirm the effectiveness of exosomes containing super repressor (srIκB) (Exo-srIκB).

[0066] Specifically, endoscopic retrograde cholangiography (ERC) guided by endobiliary radiofrequency ablation (EB-RFA) was performed on miniature pigs using an RFA catheter (ELRA RF catheter; STARmed) together with a temperature sensing system to construct a miniature pig model of biliary stricture induction. The area where the inserted RFA electrode and the bile duct wall meet is called EB-RFA, and the device is used to perform RF energy at 80°C, 7W for 90 seconds (Table 1 below).

[0067] [Table 1]

[0068]

[0069] Three weeks after RFA induction, a stent (Nexent biliary stent, total length: 4 cm) was inserted into the EB-RFA to open the obstructed bile duct, and the animals were necropsied 5 weeks after RFA induction.

[0070] The test substance (vehicle-buffer, Exo-SriκB) was administered intravenously (iv) using an infusion pump 1 hour after RFA induction for a total of 4 times at weeks 1, 2, 3 and 4, and the dose for each individual was converted based on the immediate body weight before dosing, which was converted to a corresponding concentration of 1.6e+10 pn / kg / 1 mL.

[0071] Based on RFA, blood was collected 6 times before dosing (cargo-buffer, Exo-srIκB), before induction (0 hours), and at 1, 2, 3, 4, and 5 weeks.

[0072] The detailed method of making animal models is described in Figure 1 as shown in .

[0073] 1-2. Confirmation of relative weights of liver and liver tissue after autopsy and histopathological examination

[0074] After the test is completed, the animals are anesthetized, and blood is taken from the vein and bled, and then the liver tissue of each individual is removed. The weight of the extracted liver tissue is measured and photographed (Appendix 9.8). Then, the excised liver tissue is fixed in 10% neutral buffered formalin. The fixed tissue is cut into a certain thickness and embedded in paraffin through a conventional tissue processing process to prepare 4-5 μm tissue sections, followed by H&E (hematoxylin & eosin staining), which is a staining method.

[0075] Next, the relative weight (%) of the liver was measured and the average value was calculated, and the results were as follows Figure 2 shown.

[0076] As a result, from Figure 2 As can be seen from the graph, the relative weight of G1 (carrier control group) is 3.5, G2 (Exo-srIκB administration group) is 2.9, and G3 (normal control group) is 1.8, which confirms that the relative weight of G1 (carrier control group) is 2 times that of G3 (normal control group), and the relative weight of G2 (Exo-srIκB administration group) is reduced by 17% compared with G1 (carrier control group). Figure 2 b).

[0077] In addition, as a result of confirming liver abscess as a clinical symptom, Figure 3 As can be seen from the figure, liver abscess was observed in the vehicle control group, but no liver abscess was found in the Exo-srIκB administration group of the present invention.

[0078] In addition, as a result of confirming that jaundice symptoms are clinical symptoms, Figure 4 As can be seen in the figure, jaundice symptoms were observed in the gums and sclera of the vehicle control group, but no jaundice symptoms were found in the Exo-srIκB administration group of the present invention.

[0079] As a result of observing subcutaneous sections after laparotomy on the day of necropsy, jaundice that was not observed during the observation period was additionally confirmed in the vehicle control group and Exo-srIκB administration group. In addition, it was confirmed that jaundice was slightly observed in the Exo-srIκB administration group compared with the vehicle control group.

[0080] Next, as findings of observation of liver tissue, liver tissue was evaluated by scoring the following four items: fragmentary necrosis, lobular necrosis, histological activity score, and fibrosis, and they were scored, and the results are shown in Figure 5 middle.

[0081] from Figure 5As can be seen, as a result of calculating the average values ​​of the scores observed in the liver tissues, in the case of G1 (load control group), the scrappy necrosis score was 2.8, the lobular necrosis score was 2.0, the histological activity score was 3.0, and the fibrosis score was 3.8, adding up to a total score of 11.5.

[0082] In contrast, in the case of G2 (Exo-srIκB-administered group), the scrappy necrosis score was 1.5, the lobular necrosis score was 1.5, the histological activity score was 2.0, and the fibrosis score was 2.5, adding up to a total score of 7.5.

[0083] Based on the results, it was confirmed that G2 of the present invention showed a 46% reduction in the debris necrosis score, a 25% reduction in the lobular necrosis score, a 33% reduction in the histological activity score, a 34% reduction in the fibrosis score, and a 35% reduction in the total score compared to G1 (vehicle control group).

[0084] 1-3. Confirmation of collagen fiber distribution

[0085] To confirm the collagen fiber distribution, MT (Masson's Trichrome) staining, which stains only collagen fibers, was performed. The entire tissue slide of each individual was photographed, and the staining area relative to the total area was measured, and the average value was calculated.

[0086] The fibrosis sections in the liver tissue were stained and measured (%), the mean and standard deviation were calculated, and the results were presented as Figure 6 shown.

[0087] As a result, from Figure 6 It can be seen that the collagen area of ​​G1 (carrier control group) is 8.1, the collagen area of ​​G2 (Exo-srIκB administration group) is 5.0, and the collagen area of ​​G3 (normal control group-sham operation (Sham)) is 3.5, which confirms that the collagen area of ​​G2 (Exo-srIκB administration group) of the present invention is reduced by 38.3% compared with that of G1 (carrier control group).

[0088] 1-4. Confirmation of blood biochemical tests

[0089] The blood obtained in Example 1-1 was centrifuged at 3000 rpm, 4°C for 10 minutes to obtain serum in the supernatant, and then a total of 11 items of T-total bilirubin (BIL) and high-density lipoprotein cholesterol (HDL-C) were measured using an automatic blood biochemical analyzer.

[0090] Blood biochemistry tests were performed before the RFA procedure, before test substance administration, and on the day of necropsy. Blood was analyzed on the day of necropsy, and the mean and standard deviation were calculated, and the results are presented as Figure 7 shown.

[0091] from Figure 7 As can be seen, in G1 (carrier control group), T-BIL was found to be 2.09 mg / dL and HDL-C was 27.7 mg / dL, and in G2 (Exo-srIκB administration group), T-BIL was found to be 0.87 mg / dL and HDL-C was 37.4 mg / dL.

[0092] In most of the measurement items, there was no difference between G1 (carrier control group) and G2 (Exo-srIκB administration group) of the present invention, but in G2 (Exo-srIκB administration group), compared with G1 (carrier control group), the decrease in T-BIL concentration of G2 was 1 / 2.4 of that of G1, and the increase in HDL level of G2 was 1.4 times that of G1.

[0093] In summary, in the animal model of cirrhosis induced by bile duct stricture, it was confirmed that the composition comprising the exosomes (Exo-srIκB) of the present invention containing the super repressor (srIκB) had the effect of reducing the relative weight (%) of the liver and reducing liver abscesses compared with the load control group, and the effective reduction of liver fibrosis was shown in the MT staining confirming liver tissue fibrosis. As a result of blood biochemical examination, Exo-srIκB significantly reduced T-BIL and showed the effect of increasing HDL. Based on the above results, it can be confirmed that the Exo-srIκB of the present invention effectively treats fatty liver, cholestasis, liver fibrosis and cirrhosis, and further shows excellent effects of protecting the liver and improving liver function.

[0094] Example 2. Evaluation of the effectiveness of Exo-srIκB using a TAA-induced liver fibrosis animal model

[0095] 2-1. Construction of animal model

[0096] Next, an animal model of liver fibrosis induced by TAA (thioacetamide (172502-500G, SIGMA)) was prepared, and the effectiveness of Exo-srIκB of the present invention was evaluated.

[0097] Specifically, TAA was intraperitoneally administered to mice (C57BL / 6, 9-week-old, male) at a dose of 200 mg / kg three times a week for 8 weeks to prepare an animal model of liver fibrosis.

[0098] Since preliminary experiments confirmed that blood biochemical biomarkers began to increase and histopathological fibrosis began to increase 2 weeks after TAA 200 mg / kg administration, the administration time of Exo-srIκB was determined to be 2 weeks after TAA administration. Exo-srIκB was administered by intravenous push injection 3 times a week for 6 weeks. Since the administration time of Exo-srIκB was the same as that of TAA, TAA was administered in the morning and Exo-srIκB was administered in the afternoon. Thereafter, the body weight of the animals was measured within 8 weeks, and the dose was administered according to the body weight, and Exo-srIκB was administered at a dose of 4E+11pn / kg.

[0099] When 200 mg / kg of TAA was administered, the mortality rate of animals reached 20% to 30% within 2 weeks, so about 1.5 weeks after TAA administration, before Exo-srIκB administration, groups were formed. These groups consisted of a blank control group (sham operation), a negative control group administered only 200 mg / kg of TAA, and a group administered Exo-srIκB. Nine animals were selected for each group. Among them, 4 animals in each group were used for the indocyanine green (ICG) test.

[0100] At the 8th week of experimental termination, autopsy was performed, whole blood was collected, and serum was separated. Then, the liver of mice was excised to isolate protein, and some were fixed for histopathological experiments. Four animals of each group for ICG test were tested the next day, and 5 mg / kg of ICG was intravenously administered, and blood was collected after 15 minutes to measure the concentration of ICG present in the blood.

[0101] The construction of animal models Figure 8 Shown in.

[0102] 2-2. Analysis results of ICG test

[0103] More than 90% of ICG is taken up by hepatocytes and excreted only by the liver, and the retention rate after ICG administration is used as an indicator for evaluating liver function.

[0104] Specifically, after 5 mg / kg of ICG was administered to the animals, blood was collected 15 minutes later to evaluate the ICG concentration, and the results are shown in Fig. 9 middle.

[0105] As a result, from Fig. 9 As can be seen in the figure, it was confirmed that the ICG concentration in the blood of the negative control group increased compared with the blank control group. In contrast, in the Exo-srIκB administration group of the present invention, it was confirmed that the ICG concentration was significantly reduced by about 52.9% compared with the negative control group.

[0106] 2-3. Analysis results of blood biochemical biomarkers

[0107] Blood concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), markers of liver damage, were measured, and the results were as follows: Fig. 9 shown.

[0108] For reference, AST and ALT are aminotransferases present in the liver, which are involved in gluconeogenesis and are released into the blood when the liver is damaged, and are therefore enzymes used to diagnose liver disease.

[0109] As a result, from Fig.10 As can be seen in FIG. 1 , it was confirmed that AST and ALT increased in the negative control group compared with the blank control group, whereas they were inhibited in the Exo-srIκB-administered group of the present invention.

[0110] 2-4. Analysis results of fibrosis markers

[0111] Protein was isolated from mouse liver and changes in a-SMA (a-smooth muscle actin), a marker of fibrosis, were measured by Western blotting, and the amount of protein loaded in the experiment was normalized and analyzed to the amount of a-tubulin. Fig.11 shown.

[0112] from Fig.11 As can be seen, it was confirmed that the amount of a-SMA in the liver tissue of the negative control group increased compared with the blank control group, while the amount of a-SMA in the Exo-srIκB administration group of the present invention decreased.

[0113] 2-5. Analysis results of liver necrosis

[0114] Hematoxylin-eosin staining (H&E staining) was performed on the fixed liver tissue to evaluate the extent of liver necrosis, which was scored by a histopathologist. Fig.12 shown.

[0115] from Fig.12 As can be seen from the data, it was confirmed that the liver necrosis in the negative control group increased significantly compared with the blank control group, but decreased in the Exo-srIκB administration group of the present invention.

[0116] In conclusion, in the animal model of liver fibrosis prepared by intraperitoneal administration of TAA causing liver damage to mice, the blood concentrations of AST and ALT associated with liver damage were significantly reduced in the group administered with Exo-srIκB of the present invention. In addition, as a result of intravenous administration of ICG excreted only by the liver and measurement of blood concentration, it was confirmed that the ICG concentration in the blood was significantly reduced in the Exo-srIκB administration group of the present invention compared with the negative control group.

[0117] In addition, as a result of analyzing the amount of a-SMA, it was confirmed that a-SMA increased in the negative control group compared with the blank control group, but decreased in the group administered with Exo-srIκB. As a result of scoring the quantitative changes in necrotic cells, it was confirmed that the Exo-srIκB-administered group showed a decreasing effect compared with the negative control group.

[0118] Based on the above results, Exo-srIκB can be effectively used to treat liver fibrosis, cirrhosis and fatty liver by inhibiting hepatocyte necrosis and liver fibrosis.

[0119] Example 3. Evaluation of the effectiveness of Exo-srIκB against alcoholic liver disease (ALD)

[0120] 3-1. Construction of animal model

[0121] In order to confirm the therapeutic efficacy of Exo-srIκB of the present invention in an acute / chronic alcoholic liver injury mouse model (ethanol-induced steatohepatitis, NIAAA model), an animal model was prepared as follows.

[0122] Specifically, 8- to 10-week-old C57BL / 6JWT male mice were used as approved by the Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST). The mice were fed with 4.5% EtOH and liquid diet for 10 days.

[0123] In the 3-day infusion experiment, mice were randomly divided into Exo-native group (10 9 particles / day / mouse), low-dose Exo-srIκB group (10 8 particles / day / mouse) or high-dose Exo-srIκB group (10 9 The indicated doses of exosomes were delivered intravenously at 24-hour intervals for 3 consecutive days. Six hours after the last exosome injection, mice were given acute EtOH (4 g / kg of 40% EtOH by gavage) and sacrificed six hours later.

[0124] Hepatocytes (HEPs), hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatic mononuclear cells (MNCs) were isolated from C57BL / 6J WT male mice.

[0125] 3-2. Confirmation of the main target cell types of Exo-srIκB

[0126] scRNA-seq analysis was used to examine the transcriptional profiles of different hepatocytes to investigate the major target cell types of Exo-srIκB.

[0127] Specifically, single-cell RNA sequencing (scRNA-seq) analysis of normal mouse liver tissue can be studied on the Tabula MurisSenis website (https: / / tabula-muris-senis.ds.czbiohub.org / ) or found in the NCBI Gene Expression Omnibus under accession number GSE132042.

[0128] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https: / / david.ncifcrf.gov / ). scRNA-seq analysis of human liver samples is publicly available in the NCBI Gene Expression Omnibus under accession number GSE136.

[0129] When analyzing scRNA-seq of normal mouse liver, a total of nine clusters were confirmed to be KCs, liver sinusoidal endothelial cells (LSECs), ductal epithelial cells, hepatic stellate cells (HSCs), hepatocytes (HEPs), myeloid cells, natural killer (NK) cells, B cells, and dendritic cells ( Fig.14 a).

[0130] In addition, it was confirmed that the expression levels of genes related to exosome uptake processes such as clathrin-dependent endocytosis (Ap2a2 and Picalm), caveolin-mediated endocytosis (Cav2 and Pascin2), and vesicle fusion (Stx3 and Snap23) were enriched in LSECs or KCs ( Fig.14 b) Due to the anatomical location of KCs on LSECs, KCs may primarily take up Exo-srIκB after intravenous injection.

[0131] 3-3. Confirmation of increased expression levels of genes related to exosome uptake process

[0132] Next, to confirm whether inflammatory activation of KCs affects the expression levels of genes associated with the exosome uptake process, bulk RNA-seq analysis of KCs was performed. Bulk RNA-seq of vehicle- or LPS-treated mouse primary KCs is publicly available in the NCBI Gene Expression Omnibus under accession number GSE86397.

[0133] As a result, the expression levels of genes related to clathrin-dependent or caveolin-mediated endocytosis pathways were increased. In addition, the expression levels of genes involved in inflammatory response and NF-kB signaling pathways were increased in lipopolysaccharide (LPS)-treated KCs compared with vehicle-treated KCs ( Fig.14 Furthermore, in scRNA-seq analysis of MARCO+ human KCs isolated from cirrhotic patients, the expression levels of genes associated with clathrin-dependent endocytosis, caveolin-mediated endocytosis, and positive regulation of NF-κB transcription factor activity were upregulated compared with controls ( Fig.14 f and Fig.14 g).

[0134] Based on these results, it can be found that Exo-srIκB can be effectively delivered to activated KCs and inhibit NF-κB-mediated inflammation in ALD.

[0135] 3-4. In vitro confirmation of inhibition of inflammatory gene expression in LPS-treated mice

[0136] The effective delivery of Exo-srIκB in KC and its inhibitory effect on LPS-induced inflammatory gene expression levels in vitro were confirmed. The delivery of Exo-srIκB in KC was confirmed by DiI labeling. Specifically, the isolated and purified Exo-srIκB (1.0× 10 12 The particles were incubated with 10 μL of DiI at 37°C for 30 min in the dark. Then, 1X filtered phosphate buffered saline was added to 4 mL and transferred to Ultra-4 centrifugal filter unit (Merck, Rahway, NJ, USA). The solution was eluted by centrifugation at 3200Xg, 4°C for 15 min until the residual volume reached approximately 200 μL. First, in order to study the delivery of Exo-srIκB by KC, mouse primary KCs were treated with DiI-labeled Exo-srI_B in a time (1 or 3 hours) and dose (KC:Exo-srIκB=1:1000 or 1:10,000) dependent manner. Although Exo-srIκB-loaded KCs could not be detected after 1 hour, it was confirmed that Exo-srIκB was delivered to about 8% of KCs at a low dose (KC:Exo-srIκB=1:1000) and to 70% of KCs at a high dose (KC:Exo-srIκB=1:10,000) after 3 hours of treatment. Fig.15 ac).

[0137] To explore the inhibitory effect of Exo-srIκB on LPS-induced KC inflammatory activation, mouse KC were pretreated with Exo-native or Exo-srIκB and subsequently pretreated with LPS, and qRT-PCR was performed.

[0138] qRT-PCR analysis showed that Exo-srIκB treatment normalized the increased expression levels of inflammatory genes (Il1b, Tnf, Il6, and Ccl2) induced by LPS ( Fig.15 d). In addition, it was confirmed that LPS treatment significantly enhanced the uptake of Exo-srIκB by KC ( Fig.15 ac).

[0139] Based on this result, it could be found that Exo-srIκB was efficiently taken up by KC cells activated by inflammation.

[0140] 3-5. Confirmation of the therapeutic effect of Exo-srIκB administration on alcoholic fatty liver (AFL) and alcoholic liver injury (ALI)

[0141] Based on the above findings from previous experiments, the therapeutic potential of Exo-srIκB in the acute-on-chronic ALI experimental model was confirmed. After 10 days of EtOH feeding, mice were randomly divided into 2 groups and injected intravenously with Exo-native or Exo-srIκB ( Fig.16 a). For histological analysis, similar regions of the left and middle lobes of mouse liver were used. Liver tissues were fixed with 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature overnight. After deparaffinization and rehydration, paraffin-embedded tissues were sectioned at 4 μm thickness and stained with hematoxylin & eosin (H&E).

[0142] As a result, a significant reduction in alcoholic fatty liver disease (AFL) was confirmed in Exo-srIκB-treated mice compared with Exo-naive-treated mice, as confirmed by liver TG measurements ( Fig.16 b and Fig.16 c).

[0143] In addition, for biochemical analysis, liver triglyceride (TG) was measured. Specifically, liver lipids were extracted from about 20 to 30 mg of frozen liver tissue using chloroform / methanol (2:1 ratio) solution. Then, freeze-dried liver lipids were resuspended in 5% bovine serum albumin (BSA) saline. Liver TG levels and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG and total cholesterol (TC) were measured using VetTest chemical analyzer (IDEXX Laboratories, Westbrook, ME, USA).

[0144] As a result, serum levels of ALT and AST were decreased by Exo-srIκB, and there were no differences in serum TG and TC levels and body weight between the two groups ( Fig.16 d and Fig.16 i).

[0145] Next, to confirm the liver delivery of Exo-srIκB, NF-kB levels in the nuclear fraction of whole liver tissue were measured by Western blotting. For each sample, nuclear protein levels were normalized to the expression of lamin B1. Densitometry analysis was performed using ImageJ (National Institute of Health, Bethesda, MD, USA).

[0146] As a result, a significant decrease in the nuclear translocation of NF-kB was detected in Exo-srIκB-treated mice ( Fig.16 e). In addition, the hepatic expression levels of CXCL1 and CCL2 were suppressed, resulting in a lower number of MNCs per gram of liver tissue in Exo-srIκB-treated mice compared to control mice ( Fig.16 f and Fig.16 g).

[0147] In addition, flow cytometry analysis was performed to confirm whether the frequency of liver-infiltrating macrophages was reduced. As a result, F4 / 80 in the liver was observed in the Exo-srIκB-treated group. + CD11b + The frequency of infiltrating macrophages decreased, especially F4 / 80 in the liver + Ly6C 高 The frequency of pro-inflammatory macrophages was significantly reduced. + Ly6C 低 The frequency of anti-inflammatory macrophages increased in the Exo-srIκB-treated group, but Ly6G + CD11b + Neutrophils or lymphocytes showed similar frequencies between the two groups ( Fig.16 h).

[0148] 3-6. Alleviation of alcoholic liver injury (ALI), alcoholic fatty liver (AFL) and alcoholic liver fibrosis (ALF) in mice by multiple injections of Exo-srIκB

[0149] Various therapeutic and preventive effects of Exo-srIκB on alcoholic liver injury (ALI) have been confirmed.

[0150] Specifically, WT mice were fed EtOH (4 g kg -1 The mice were randomly divided into three groups and received 40% EtOH gavage for 3 consecutive days before drinking. 9 particles / day / mouse), low dose Exo-srIκB (10 8 particles / day / mouse) or high doses of Exo-srIκB (10 9 particles / day / mouse)( Fig.17 a).

[0151] The results confirmed that the serum ALT and AST levels of mice injected with low and high doses of Exo-srIκB were significantly reduced compared with control mice with similar body weight and serum TG and TC levels ( Fig.17 b and Fig.17 gi).

[0152] In addition, as a result of H&E staining and Oil Red O staining to confirm the total lipid content, it was confirmed that lipid accumulation in mid-zone apoptotic hepatocytes (HEP) was significantly suppressed by Exo-srI_B treatment ( Fig.17 c and Fig.17 d).

[0153] Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Abcam, Cambridge, UK) was performed according to the manufacturer's instructions to confirm whether apoptosis of HEPs and non-parenchymal cells in liver tissues was reduced, and images were captured using an Olympus BX51 microscope equipped with a CCD camera (Olympus, Tokyo, Japan) and analyzed with DP2-BSW.

[0154] As a result, in the case of Exo-srIκB treatment, apoptosis of HEPs and non-parenchymal cells in the midzone region of Exo-naïve treated mice was reduced ( Fig.17 c and Fig.17 d).

[0155] Finally, in isolated hepatic stellate cells (HSCs), the expression levels of genes associated with fibrosis activation (Acta2, Col1a1, and Tagln) were significantly decreased in the low-dose Exo-srIκB group and also decreased in the high-dose Exo-srIκB group compared with the control group ( Fig.17 f).

[0156] 3-7. Alleviation of alcoholic liver inflammation by inhibiting KC activity

[0157] In order to explore the potential mechanism of the protective effect of Exo-srIκB in ALD, the main target cell type KC of Exo-srIκB was analyzed from the multiple Exo-srIκB injection animal models of Examples 3-6. KC was analyzed based on known studies, which have confirmed that in the progression of non-alcoholic fatty hepatitis, embryo-derived KC (emKC) is gradually lost and eventually replaced by bone marrow-derived macrophages (bmKC) (Remmerie, A. et al., Immunity 2020, 53, 641-657; Bonnardel, J.; T'Jonck, W. et al., Immunity 2019, 51, 638-654.). In particular, CLEC2 and TIM4 were used as markers of emKC (CLEC2+TIM4+), which can distinguish emKC from bmKC (CLEC2+TIM4-), and flow cytometry was performed.

[0158] As a result, there was no difference in the frequencies of F4 / 80 high CD11b + KC (F4 / 80hiCD11b + KC), emKC (CLEC2 + TIM4 +), and bmKC (CLEC2 + TIM4 -) among the three groups ( Fig.18 a and Fig.18 b).

[0159] Next, for histological analysis, similar areas of the left and middle lobes of the mouse liver were used. As a result, immunostaining of liver tissues showed similar numbers of CLEC4F (a marker for both emKC and bmKC) expressing cells ( Fig.18 c). However, in the liver tissues of Exo-naïve treated mice, morphological changes were found in the mid-zone CLEC4F+ KCs, which were abolished by Exo-srIκB treatment, suggesting that Exo-srIκB causes functional changes in KCs ( Fig.18 c).

[0160] Furthermore, we confirmed that nuclear translocation of NF-κB and expression levels of pro-inflammatory genes (Tnf, Il1b, and Il6) were reduced in whole liver tissues or isolated KCs from Exo-srIκB-treated mice compared with control mice ( Fig.18 d and Fig.18 e). It was also confirmed that serum IL-6 levels were reduced in Exo-srIκB-treated mice ( Fig.18 f). In addition, the expression levels of CXCL1 and CCL2 were decreased in isolated KCs and HEPs from Exo-srIκB-treated mice compared with those in control mice ( Fig.18 e and 19a).

[0161] Indeed, in intact liver tissue, especially in the midzone, the number of MPO+ neutrophils was significantly reduced in Exo-srIκB-treated mice ( Fig.18 g) In addition, although FSC 高 Granulocytes such as Ly6G + CD11b + Neutrophils showed similar frequencies in all groups, but in Exo-srIκB-treated mice, liver F4 / 80 + CD11b + Macrophage frequency, especially F4 / 80 + Ly6C 高 The frequency of pro-inflammatory macrophages was reduced, as well as F4 / 80 + Ly6C 低 Increased frequencies of anti-inflammatory macrophages were demonstrated ( Fig.18 h and 19d).

[0162] These results suggest that multiple Exo-srIκB injections have a hepatoprotective effect in ALD mediated by the inhibition of inflammatory KCs.

[0163] In conclusion, it was confirmed that Exo-srIκB of the present invention can effectively inhibit lipid accumulation, apoptosis, acute inflammation and liver fibrosis of liver tissue after administration, and based on this, it is provided as an effective therapeutic agent for alcoholic liver disease in which the above symptoms appear in combination.

[0164] According to the foregoing, those skilled in the art to which the present invention belongs will be able to understand that, without modifying the technical concept or essential features of the present invention, the present invention can be implemented in other specific forms. In this regard, the exemplary embodiments disclosed herein are merely for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments, but also various replacements, modifications, equivalents and other embodiments, which may be included in the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A pharmaceutical composition for preventing or treating liver disease, comprising exosomes containing super repressor-IκB (srIκB) as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The liver disease is liver fibrosis, liver cirrhosis, fatty liver, alcoholic liver disease cholestasis or a combination thereof.

3. The pharmaceutical composition according to claim 2, wherein The alcoholic liver disease is alcoholic fatty liver, alcoholic steatohepatitis, alcoholic cirrhosis, alcoholic liver fibrosis or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein The composition is administered orally, transdermally, intraperitoneally, intravenously (IV), intramuscularly, subcutaneously, or a combination thereof.

5. The pharmaceutical composition according to claim 1, wherein The dose of exosomes is 1x10 6 pn / kg to 1x10 15 pn / kg.

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