KR20190070312A - Therapeutic and preventive effect of astaxanthin in ethanol-induced liver injury - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a food composition for treating or preventing alcoholic liver disease containing astaxanthin (ASX, 3,3′-dihydroxy-β, β′-carotene-4,4′-dione), which has an effect of treating, preventing, and alleviating alcoholic liver disease, as an active component. More specifically, the present invention relates to the pharmaceutical composition or the food composition for treating or preventing alcoholic liver disease, which exhibits the effect of preventing or treating alcoholic liver disease by inhibiting the activity of STAT3 by binding to STAT3, thereby inhibiting inflammatory cytokine production and inhibiting oxidative stress caused by ROS.

Description

TECHNICAL FIELD The present invention relates to a therapeutic and preventive effect of astaxanthin in ethanol-induced liver injury,

The present invention relates to a pharmaceutical composition containing astaxanthin (ASX, 3,3'-dihydroxy-β, β'-carotene-4,4'-dione) having an effect of treating, preventing and alleviating alcoholic liver disease The present invention relates to a composition for treating or preventing alcoholic liver disease, and more particularly, to a composition for preventing or treating alcoholic liver disease by inhibiting the production of inflammatory cytokines and inhibiting oxidative stress caused by ROS by binding STAT3 to inhibit STAT3 activity And to a pharmaceutical composition and a food composition for the treatment or prevention of an alcoholic liver disease.

The liver is the first organ to break down toxic substances in the body. Therefore, toxic alcohol is oxidized to acetaldehyde at the time of drinking and finally produces fatty acids.

Overeating has been shown to induce liver complications, including alcoholic hepatitis, often characterized by fatty liver and inflammation. When excessive alcohol is consumed, a large amount of fatty acid accumulates and fatty liver and hepatitis can be induced. Alcoholic hepatitis eventually leads to liver cirrhosis (scarring) and liver hardening, and can lead to liver cirrhosis or liver cancer .

Astaxanthin is known to have immune modulatory activity and cardioprotective effect (Jyonuchi et al. (1993) Nutr. Cancer, 19: 269-280). To date, astaxanthin No toxicity has been reported. Thus, ingestion of astaxanthin is associated with neurodegenerative disease, cancer (Nishino, H. (1998) Mutat. Res., 402: 159-163), immune disorder (Bendich, A. 1989) J. Nutr., 119: 112-115), cardiovascular disease and the like (Beal, HF (1991) The Neuroscientist, 3: 21-27; Chew , et al. (1999) Anticancer Res., 19: 1849-1853, Murillo, E. (1992) Arch. Latinoam. Nutr., 42: 409-413).

In addition, astaxanthin protects the eyesight and central nervous system from health promotion and UV damage (Mathews-Roth, MM. (1990) Am. J. Clin. Nutr., Sep 52: And prevention of arteriosclerosis, prevention of cataract, reduction of senile plaques, antiplasmic reaction, anti-stress effect are recognized.

However, it is not known that astaxanthin has the effect of treating or preventing alcoholic liver disease.

An object of the present invention is to provide a composition for treating or preventing alcoholic liver diseases, which contains astaxanthin as a main component and has therapeutic or preventive effects in alcoholic liver diseases.

The present invention provides a pharmaceutical composition for the treatment or prevention of alcoholic liver diseases comprising astaxanthin as an active ingredient in order to solve the above problems.

The present invention also provides a food composition for the treatment or prevention of alcoholic liver disease comprising astaxanthin as an active ingredient.

In the present invention, "astaxanthin" is a carotenoid pigment belonging to a class of phytochemicals known as terpenes and is represented by the following formula (3,3'-dihydroxy- beta -carotene- - polyion).

[Chemical Formula 1]

The pharmaceutical compositions and food compositions for the treatment and prevention of alcoholic liver diseases comprising astaxanthin according to the present invention as an active ingredient inhibit STAT3 activity by binding to signal and transcriptional activator-3 (STAT3) .

The term " STAT3 (signal transducers and activators of transcription 3) inhibition "in the present invention means that STAT3 is activated and acts as an electron regulatory protein to reduce or block the activity of STAT3 acting on the carcinogenesis process.

The STAT3 protein is a transcription factor (txn factor), and is a potent inhibitor of apoptosis such as Bcl-xL, Mcl-1 and survivin, cell-cycle regulators (eg cyclin D1 and c-Myc) Is known to function as an important mediator of oncogenic signaling through the transcriptional activity of genes encoding angiogenesis inducers (e. G., Vascular endothelial growth factor, etc.) .

One of the major reasons for liver disease is inflammation and fatty liver formation due to the activation of signal and transcription activator-3 (STAT3) and the accumulation of reactive oxygen species (ROS). STAT3 is an important factor in the regulation of hepatitis and fatty liver and is activated by interleukin-6 (IL-6). When alcohol is ingested, STAT3 is activated by IL-6 activated by alcohol metabolism. In the present invention, astaxanthin directly binds to STAT3 to block the activity of STAT3, thereby reducing the incidence of alcoholic liver disease have. It also provides a potential approach for alcoholic liver disease by reducing the activation of STAT3 by IL-6 produced by ROS.

The pharmaceutical composition and food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient is characterized by reducing fatty liver disease.

Fatty liver disease, also called fatty liver disease, is a disease caused by abnormal accumulation of fat (triglycerides, etc.) in hepatocytes, leading to liver damage. It is known that the initial condition of fatty liver disease is a simple fatty liver that recognizes only fat deposition in hepatocytes, and then the condition progresses to fatty liver (including liver fibrosis), and also cirrhosis or hepatocellular carcinoma.

In general, the causes of fatty deposits in the liver include alcohol consumption, obesity, diabetes, lipid metabolism disorders, drugs (steroids, tetracyclines, etc.), Cushing's syndrome, poisoning . The cause of fatty liver disease is largely divided into alcohol and non-alcoholic. Liver disease caused by the former is called alcoholic fatty liver disease (also called alcoholic liver disease), and alcoholic fatty liver disease is the progressive Liver transplantation, and liver cirrhosis.

The pharmaceutical composition and food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient is characterized by preventing or treating damage to liver tissue.

The pharmaceutical composition and the food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient are characterized by decreasing the expression amount of COX-2 and iNOS.

The pharmaceutical composition and the food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient are also characterized by decreasing the expression amount of CYP2E1.

CYP2E1 (cytochrome P450 2E1) plays a key role in the oxidation of alcohol in MEOS with NADPH-cytochrome P-450 reductase. Acute alcohol is oxidized by acetylcholinesterase (ADH) and converted to acetaldehyde. In the case of alcoholics or chronic alcoholics, CYP2E1 is involved and produces HER and other ROS simultaneously with the production of acetaldehyde. In particular, CYP2E1 induced by induction of alcohol by chronic intake of alcohol induces excessive radicals, which are not eliminated by GSH but cause lipid peroxidation and damage by reactive oxygen species.

The degree of induction of CYP2E1, increased malondialdehyde (MDA) production and lipid peroxidation due to increased iron content from ferritin are directly related. Alcohol-induced CYP2E1 also affects drug metabolism and increases normal microsomal detoxication activity, resulting in excessive drug administration, resulting in drug deprivation symptoms (Albert Y. Sun et al. Ethanol and Oxidative stress, Alcoholism; Clinical and Experimental Research, 25, No. 5, 237S-243 S, 2001). Therefore, it has been shown that the inhibition and inhibition of the biosynthesis and activity of CYP2E1, which directly acts as a causative factor in the formation of phytochemicals to prevent hepatic damage and hepatic injury caused by lipid peroxidation in alcoholic liver disease Search is essential.

The food composition for treating or preventing an alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient is characterized by reducing the amount of active oxygen.

The food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient is characterized by decreasing the expression amount of interleukin-6 (IL-6).

Interleukin-6 (IL-6), a cytokine, also called B-cell stimulating factor 2 (BSF2) or interferon-2 (INF-2), was found as a differentiator involved in the activation of B lymphocytes. It has since been found to be a multifunctional cytokine that affects various cell functions. IL-6 carries its biological activity on the cell membrane through two types of proteins. One is the IL-6 receptor, a protein that IL-6 binds. The IL-6 receptor is expressed through the membrane and is a membrane-bound protein with a molecular weight of about 80 kDa. The other is the membrane protein gp130, which has a molecular weight of about 130 kDa, which belongs to signaling of non-ligand binding. IL-6 and IL-6 receptors form an IL-6 / IL-6 receptor complex, which in turn binds to gp130. After binding of these ligands and receptors, JAK2 (Janus Kinase 2) is activated in the cell by transphosphorylation. Activated JAK2 phosphorylates several tyrosine residues of the cytoplasmic domains and is responsible for signal transducers and activators of STAT3, which have SH2 or other phosphotyrosine binding motifs transcription 3) as a docking site for proteins in the cytoplasm.

STAT3 bound to the cytoplasmic domain of the receptor is phosphorylated by JAK2 and then released from the receptor. These activated STAT3 bind to each other in the cytoplasm to form a homodimer or a heterodimer, Into the recognition sequence of the target gene to increase transcription.

The pharmaceutical composition and the food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient are characterized by decreasing the expression amount of TNF-α and IL-1β.

TNF-α is a cytokine that is produced by macrophages in animals, including humans, in response to a variety of immunostimulating substances including endotoxin, a toxic substance of bacteria, and is a substance that plays an important role in the immune system to be. When TNF-α is administered to animals or humans, inflammation, heat, cardiovascular effects, hemorrhage, flocculation, and acute phase similar to those in acute infection and shock conditions occur. Production of TNF-α in the body It is known that many diseases are caused if not overdosed or controlled.

IL-1β, which plays a key role in inflammatory cytokines, binds to specific receptors and activates inflammatory transcription factors such as NF-kB, thereby inducing the synthesis and secretion of inflammatory cytokines including IL-1β, Cytokines activate immune cells and induce and persist inflammatory responses. Thus, inflammatory cytokines such as IL-1 [beta] are increased in autoimmune diseases and inflammatory diseases, and the quantitative increase of these inflammatory cytokines leads to the problem of hypersensitivity to IL-1 [beta].

The pharmaceutical composition and food composition for the treatment or prevention of alcoholic liver diseases comprising astaxanthin according to the present invention as an active ingredient is characterized in that the expression level of chemokine (CC motif) ligand 2 (CCL2) Is reduced.

Chemokine (CC motif) ligand 2 (CCL2) is known as monocyte chemoattractant protein-1 (MCP-1) or a small inducible cytokine A2, And is the best known chemokine in CC chemokines. It is largely expressed in monocyte / macrophages and is an antiviral immunity such as endothelial cells, smooth muscle, fibroblasts, epithelial cells, mesangial cells, astrocytic and microglial cells It is widely expressed in reactive cells. Mature CCL2 is composed of 76 amino acids, and various glycosylations represent native proteins of different molecular weights. Although homodimers can be formed at high concentrations, they are predominantly monomers at physiological concentrations, and dimers can not bind or activate CCR2. Structural experiments revealed a secondary structure of CCL2 consisting of three anti-parallel β-strands and a carboxy-terminal α-helix. The four cysteine residues form two intramolecular disulfide bonds between Cys34 and Cys59 and between Cys35 and Cys75. The N-terminal region was found to be important for interaction with the receptor. Changes in the two residues have been reported to attract neutrophils instead of monocytes.

The pharmaceutical composition and food composition for the treatment or prevention of alcoholic liver diseases comprising astaxanthin according to the present invention as an active ingredient is characterized by decreasing the expression amount of Neutrophil.

Neutrophil is a polymorphonuclear leukocyte with a short life span in bone marrow, spleen, lymph node, liver, lung, and blood in a steady state. These populations play an important role in the initial immune response to infection. Neutrophils induce bactericidal effects through phagocytosis, ROS production, and neutrophil extracellular trap formation. And recruits and activates various innate or acquired immune cells through cytokine secretion and antigen presentation.

The pharmaceutical composition for the treatment and prevention of alcoholic liver diseases comprising astaxanthin according to the present invention as an active ingredient can be in the form of a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution, an emulsion, a syrup, Aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories, and may be oral or parenteral formulations.

Examples of the food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, , Dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in the conventional sense, including functional foods and health functional foods.

The present invention also provides a composition for inhibiting the activity of STAT3 (signal transducers and activators of transcription 3) in vitro comprising astaxanthin represented by the following formula (1) .

[Chemical Formula 1]

The present invention also provides a method for detecting a STAT3 protein comprising the steps of: 1) treating a test substance with activated STAT3 protein; And 2) selecting a test substance which inhibits the activity of the STAT3 protein as compared to the untreated control group. The present invention also provides a method for screening a therapeutic agent for an alcoholic liver disease.

The above-mentioned alcoholic liver disease may have the above-mentioned characteristics, and may be specifically fatty liver disease.

Further, the present invention relates to a method for producing a STAT3 protein comprising the steps of: 1) treating a test substance with a cell line expressing an activated STAT3 protein; And 2) screening a test substance that inhibits the activity of STAT3 protein in the cell line as compared to the untreated control group. The present invention also provides a method for screening a therapeutic agent for an alcoholic liver disease.

The pharmaceutical composition and food composition for treating or preventing alcoholic liver disease comprising astaxanthin according to the present invention as an active ingredient alleviate alcoholic liver disease by anti-inflammatory and antioxidative effects.

The pharmaceutical composition and food composition for the treatment or prevention of alcoholic liver diseases comprising astaxanthin according to the present invention as an active ingredient are useful for the treatment of astaxanthin by directly binding STAT3 to reduce the activity of STAT3, Thereby decreasing the expression amount of CYP2E1 and reducing the amount of active oxygen. Therefore, it is possible to reduce the expression level of interleukin-6 (IL-6), TNF-α and IL-1β and reduce the expression amount of CCL2, thereby decreasing the expression amount of neutrophils and treating liver diseases including alcoholic fatty liver, The composition containing astaxanthin according to the present invention as an active ingredient can be developed as a medicament and a functional food for prevention, treatment or improvement of alcoholic liver disease.

FIGS. 1A and 1B show the results of oral administration of astaxanthin after inducing an alcoholic liver disease in mice and comparing the weights of the liver and the liver.
Figure 1c shows that AST levels are decreased in astaxanthin concentration-dependent manner.
Figure 1d shows H & E staining to confirm the effect of astaxanthin on alcoholic liver disease.
2A shows docking modeling performed using AutoDock VINA.
FIG. 2b shows that the amount of expression in astaxanthin-bound beads is larger than that in pull-down assays.
FIG. 2c shows the western blot analysis of the expression level of STAT3 protein when astaxanthin was administered and when astaxanthin was not administered.
FIG. 3A shows western blot analysis of the degree of inflammatory protein expression in order to examine the difference in inflammation between when astaxanthin was administered and when it was not.
FIG. 3B shows that the concentration of NO is decreased depending on the concentration of astaxanthin.
FIG. 3C shows that the amount of CYP2E1 expression decreases in astaxanthin concentration-dependent manner.
FIG. 3D shows that total GSH increases in astaxanthin concentration-dependent manner.
Figure 4a shows that the expression level of IL-6 is decreased in astaxanthin concentration-dependent manner.
4B shows that the amount of TNF-a expression decreases in astaxanthin concentration-dependent manner.
FIG. 4c shows that the amount of IL-1? Expression is decreased in astaxanthin concentration-dependent manner.
Figure 4d shows that the expression level of CCL2 decreases in astaxanthin concentration-dependent manner.
FIG. 5A shows that the expression level of neutrophils decreases in astaxanthin concentration-dependent manner.
FIG. 5B shows that the expression amount of Ly6g decreases in astaxanthin concentration-dependent manner.
FIG. 5C shows that Ly6g was stained by cutting off liver tissue to confirm the amount of neutrophils.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example 1: Effect of astaxanthin on the reduction of fatty liver in liver-induced rats

To investigate whether astaxanthin inhibits fatty liver formation in alcoholic liver disease through antiinflammatory and antioxidant effects, astaxanthin at 0.2, 2 and 20 mg / kg was orally administered to 8-week-old mice for 11 days oral gavage). At the same time, ethanol (5% v / v) was added to liquid-diet to induce alcoholic liver disease.

In order to measure the degree of recovery of fatty liver, the first stage of alcoholic liver disease, mice were weighed during induction and administration, and the weight of the last day was measured to compare the weight of the liver. (Figs. 1A and 1B)

As shown in FIG. 1, the astaxanthin-treated group according to the present invention showed a decrease in body weight compared to the comparative example in which ethanol was administered.

<Example 2> The effect of astaxanthin on liver tissue damage treatment

In Example 1, biochemical liver function tests were performed using blood of mice induced with alcoholic liver disease and astaxanthin.

Aspartate aminotransferase (AST), which is a pyridoxal phosphorylase, and alanine aminotransferase (ALT), which is a transaminase, are characterized by being liberated into blood when a tissue is damaged. As shown in FIG. 1C, , Astaxanthin showed a higher AST level in the ethanol-treated group than the astaxanthin-treated group, indicating that inflammation-like damage occurred in liver tissue and AST level was decreased in dependence on drug concentration. Of the patients. (Fig. 1C)

H & E staining was performed to determine the effect of astaxanthin on alcoholic liver disease.

As shown in FIG. 1d, hepatic tissues were fixed and H & E staining was performed. As a result, according to the examples of the present invention, compared to the group administered with astaxanthin, the inflammatory cells and adipocytes increased Respectively. On the other hand, according to the embodiment of the present invention, in astaxanthin-treated group, astaxanthin is effective for alcoholic liver disease because the number of inflammatory cells is decreased and the size of adipocytes is decreased.

Example 3 Confirmation of Binding of astaxanthin to STAT3

To confirm whether astaxanthin directly binds to STAT3, docking modeling is performed using AutoDock VINA, and the results are shown in FIG. .

The degree of binding affinity was as high as -9.0 kcal / mol. STAT3 was transfected into HEK293 cells, and STAT3 protein, which was lysed and cell-free system, was directly bound to astaxanthin through a pull-down assay.

As shown in FIG. 2B, the amount of astaxanthin-bound beads in the beads is larger than that of astaxanthin, indicating that astaxanthin and STAT3 bind directly to each other.

<Example 4> Effect of astaxanthin on STAT3 activity

To determine the difference in STAT3 activity between astaxanthin-treated mice and non-astaxanthin-treated mice, western blot analysis was performed.

As shown in FIG. 2C, the amount of phospho-STAT3 (p-STAT3) expression was increased in the ethanol-treated group (EtOH-fed), which was a control group, compared with the astaxanthin-treated group according to the example of the present invention.

STAT3 was activated by ethanol and p-STAT3 expression decreased in a dose-dependent manner, indicating that astaxanthin binds to STAT3, reducing the activity of STAT3, reducing oxidative stress and inflammatory cytokines that induce liver damage can see.

Example 5 Effect of astaxanthin on inflammation

The expression of inflammatory proteins was determined by western blot analysis to determine the difference in inflammation when astaxanthin was administered or not in mice induced with alcoholic liver disease.

The expression level of COX-2 and iNOS was increased in the ethanol-treated group (EtOH-fed) as compared with the astaxanthin-administered group according to the example of the present invention. As a result, EtOH induced inflammation of liver tissues, The decrease in expression of COX-2 and iNOS suggests that astaxanthin alleviates the inflammatory response. (Fig. 3A)

<Example 6> Reduction effect of astaxanthin on active oxygen

Fig. 3 shows the results of western blot and qPCR in order to examine the expression level of CYP2E1 that produces ROS.

FIG. 3 shows that the expression level of CYP2E1 decreases in a drug concentration-dependent manner.

NO concentration was also measured to compare the degree of ROS production. The concentration of NO in the astaxanthin-treated group according to the example of the present invention was lower than that of the control group (EtOH-fed) (FIG. 3B).

Total GSH was also measured. As a result, when GSH was oxidized in response to ROS, it changed into GSSG form, and total GSH increased depending on the drug concentration. As a result, astaxanthin- It can be seen that the degree of oxidation is lower than that of mouse liver. (Fig. 3d)

Finally, TBARS assay was performed to measure the amount of lipid peroxidation. As a result, the concentration of astaxanthin in the group treated with astaxanthin was reduced according to the present invention, ).

<Example 7> Reduction effect of astaxanthin on cytokine expression

QPCR was performed to compare the expression levels of inflammatory cytokines.

The expression level of IL-6, a cytokine that activates STAT3, was higher in the ethanol-treated group (EtOH-fed) than in the pair-fed group and decreased in a dose dependent manner. (Fig. 4A)

In addition, the expression levels of TNF-α and IL-1β, which are important cytokines for inflammation, also decreased in a dose dependent manner. (Figs. 4B and 4C)

The expression level of CCL2, which is a chemokine that attracts immune cells when inflammation occurs, is confirmed by qPCR. As a result, the amount of CCL2 expression is decreased depending on the drug concentration.

<Example 8> Reduction effect of astaxanthin on neutrophil expression

To compare the expression levels of neutrophils, which break down the external toxic substances entering the body into pharmacological effects, we checked them by CBC test.

As shown in FIG. 5A, the expression level of neutrophils in blood was higher in the EtOH-fed group administered with ethanol compared to the pair-fed group administered with astaxanthin according to the example of the present invention, And decreased in a concentration-dependent manner in one group.

As shown in FIG. 5B, Ly6g, a marker of neutrophils, was confirmed by qPCR, and the amount of expression of Ly6g was decreased depending on the drug concentration.

In FIG. 5c, to determine the amount of neutrophils expressed in liver tissues, fixed liver tissue was cut and stained with Ly6g. As a result, the amount of neutrophils was reduced in the drug-treated group than in the non-treated group.

Claims (4)

A composition for inhibiting the activity of STAT3 (signal transducers and activators of transcription 3) in vitro comprising astaxanthin represented by the following formula (1).
[Chemical Formula 1]

1) treating the test substance with the activated STAT3 protein; And
2) screening a test substance which inhibits the activity of the STAT3 protein compared to the untreated control group.
3. The method according to claim 2, wherein the alcoholic liver disease is fatty liver disease.
1) treating the test substance with a cell line expressing the activated STAT3 protein; And
2) screening a test substance that inhibits the activity of STAT3 protein in the cell line as compared to the untreated control group.

Images