KR101502221B1 - Hepatoprotective Composition Comprising Thiacremonone As Active Ingredient - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a composition for protecting the liver, which comprises tiocrimmonium as an active ingredient. The present invention provides a method for effectively protecting hepatocyte damage induced by a hepatotoxic substance. Chiacormonone reduces ALT levels, AST levels, and lipid peroxidation levels and inhibits NO (nitric oxide) production in liver damage induced by excessive hepatotoxic drug administration. In addition, it inhibits the penetration of Kupffer cells, T cells, and NK cells induced by hepatotoxic substances in hepatocytes and the expression of hepatocyte cytochrome P450 2E1, and proinflammatory cytokines I-309, M-CSF, MIG, , MIP-1 ?, IL-10, IL-13, IL-7 and IL-17. Therefore, dia-crmmonone can be developed as an active substance for drugs and functional food that protects liver damage by hepatotoxic substances.

Description

(Hepatoprotective Composition Comprising Thiacremonone As Active Ingredient)

TECHNICAL FIELD The present invention relates to a composition for protecting the liver, which comprises tiocrimmonium as an active ingredient.

N-acetyl-p-aminophenol (APAP) is known to be safe when used in therapeutic doses and is used as an analgesic and antipyretic. However, overdose can lead to severe hepatocellular toxicity, called centrilobular hepatic necrosis (Agarwal et al., 2012). APAP is metabolically activated by cytochrome P450 2E1 to form the active metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and NAPQI binds to the protein (Slitt et al., 2005). At therapeutic doses, NAPQI forms an APAP-GSH conjugate by glutathione (GSH) and is effectively detoxified by excretion in the kidney (Prescott, 2005). However, overdosage of APAP saturates the sulfate and glucuronide conjugation pathways, greatly increasing the amount and rate at which NAPQI is formed (Hu et al., 1996). NAPQI covalently associates with hepatocyte proteins to form 3- (cysteine-S-yl) -acetaminophen (APAP-Cys) adducts (James et al., 2003; Hinson et al., 2010; Agarwal et al ., 2012). Excess quantities of NAPQI also peroxynitrite proteins and oxidize macromolecules such as lipids, proteins, and DNA, leading to hepatocyte injury and necrosis (Rubbo et al., 1994; Sies et al., 1997). Acetaminophen is widely used as a standard hepatotoxic agent to test the efficacy of new hepatoprotectants or therapeutic agents (DJ Jollow et al., J. Pharmacol. Exp. Ther. 187, pp 195-202, 1973; JR Mitchell et al., J. Pharmacol. Exp. Ther. 187, pp 185-194, 1973).

Kupffer cells, natural killer (NK) cells, neutrophils and macrophages have been implicated in tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN- Induction of hepatotoxicity through the release of proinflammatory cytokines and mediators such as IL-1α, IL-1β and nitric oxide (NO) (Michael et al., 2001; Martin-Murphy et al., 2010) . In APAP treated mice, excessive production of IFN-y induces a significant degree of inflammatory cytokines, chemokines, adhesion molecules, Fas, and inducible nitric oxide synthase (iNOS) (Ishida et al. 2002; Liu et al., 2004). In addition, excessive production of IFN-y induces necrotic hepatotoxicity, as can be seen through the histopathological evaluation of serum ALT (alanine aminotransferase) and AST (aspartate transaminase) levels and necrosis (Blazka et al. , 1995b; Liu et al., 2004). In mice, inhibition of TNF-α or IL-1α using antibodies can protect APAP-induced liver damage (Gandhi et al., 2010). IL-1ra is structurally similar to IL-1 and can bind IL-1 receptors strongly to block the IL-1 signaling pathway (Hu et al., 2010). The level of IL-1 in hepatocytes correlates with the severity of APAP-induced liver damage (Blazka et al., 1995b). Activated Kupffer cells are important for NO and superoxide production to form peroxynitrite (Rubbo et al., 1994; Sies et al., 1997). Kupffer cells can respond to inflammation, or can play a role in liver regeneration through changes in IL-10 secretion (Ju et al., 2002). Penetrating NK cells and NK-T cells activated by Kupffer cells are the major cell types that produce IFN-y in acetaminophen toxicity (Ishida et al., 2002; Liu et al., 2004). IL-1 and IL-lra are produced in large quantities by various types of cells such as neutrophils, macrophages and fibroblasts in patients with inflammatory conditions (Hu et al., 2010).

If the defense mechanism is not sufficient to withstand the attack, the cells begin to synthesize the following chemokines, which are believed to attract inflammatory cells such as granular leukocytes and mononuclear cells or to activate resident macrophages (Ren), 2002), cytokine-induced neutrophil chemoattractant (KC), and MIP-1 (Gene-dependent interferon-inducible protein , Macrophage inflammatory proteins (Li et al., 2004) such as MIP-2 and MIP-3. The above chemokines are secreted by damaged hepatocytes, T cells, NK cells and Kupffer cells in acute inflammatory conditions (Ishida On the other hand, IP-10 (CXCL10) and MIP-2 (CXCL-2) are major signal transporters in hepatocyte regeneration, STAT3 signal transducer and the activator of transcription 3) nuclear localization of transcription factor (loc IP-10 can induce HGF (hepatocyte growth factor) (Hinson et al., 1999; Hogaboam et al., 2000b; Ren et al., 2003) (Bone-Larson et al., 2001). Thus, an increase in the production of MIP-2 and IP-10 is associated with an increased risk of APAP toxicity (Bone-Larson et al., 2001; Hinson et al., 2010).

Nuclear transcription factor-kB (NF-kB) is a transcription factor that regulates various genes involved in the production of inflammatory cytokines, chemokines, cell adhesion molecules and growth factors (Richmond, 2002). NF-kB regulates the expression of multiple anti-apoptotic proteins as well as proliferative genes (Wang et al., 1998).

After overdose of APAP, the DNA binding activity of NF-kB in the liver is increased, and the protective effect against APAP-induced toxicity is associated with reduced NF-kB (Meraz et al., 1996). STAT-1 is involved in liver inflammation and damage as well as inhibition of liver regeneration. STAT-1 is activated in response to IFN-y in concavalin-A-induced hepatitis and lipopolysaccharide / d-galactosamine-induced hepatitis models and plays a deleterious role in these hepatotoxicity models (Hong et al., 2002 Ghosh and Sil, 2009). On the other hand, STAT3 and its related cytokines, which are mainly activated by IL-6, play a very important role in acute-state responses, including protection against liver damage and promoting liver regeneration (Hecht et al., 2001; Hong et al., 2004; Gao, 2005). Inhibition of STAT3 gene aggravates liver regeneration and activated STAT3 mitigates fatty liver disease (Hecht et al., 2001; Hong et al., 2004; Gao, 2005). These results suggest that STAT3 plays an important role in liver protection and liver regeneration by inducing a variety of anti-atocytosis and mitogen-promoting proteins, while STAT1 mediates liver damage.

Garlic acts by altering risk factors such as hypertension, hypercholesterolemia, and thrombosis, as well as by acting on other acute and chronic diseases associated with oxidative stress and aging, leading to cancer, obesity, diabetes, hepatotoxicity, renal toxicity and cardiovascular disease (Rahman, 2001; Yeh and Liu, 2001; Tanaka et al., 2006). The pharmacological effect of such garlic has been shown to be due to the presence of active sulfur components such as diallyl sulfide, diallyl disulfide, allicin and dipropyl sulfide (Ban et al., 2009a; Kim et al., 2012). These compounds, along with their antioxidant activity (Pari et al., 2007), increase the activity of enzymes involved in the metabolism of carcinogens (Fisher et al., 2007) and increase the anti-inflammatory effect in in vitro and in vivo (Narayanaswami and Sies, 1990; Son et al., 2006; Sabayan et al., 2007). Thiacremonone is a compound isolated from a fraction having antioxidative activity from heat-treated garlic juice (Kwon OC, Woo KS, Kim TM, Kim DJ, Hong JT, Jeong HS. 2006; 38: 331-336). The compound is a fungus Acremonium sp. It is also known that strain HA33-95 is able to produce and induce differentiation of animal cells (Gehrt, A. Erkel, G. Anke, T. and Sterner, O. Nat. Prod. Lett. 2000; 14: 281-284.). (Korean Patent Laid-Open No. 2009-0035946), therapeutic efficacy against metabolic diseases such as obesity, diabetes and hyperlipidemia (Korea Patent Publication No. 2011-0011187), anti-inflammatory activity 2011-0030756) has been disclosed, but the hepatocyte protective action from hepatotoxic substances has not been reported yet.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 2009-0035946 Korea Patent Publication No. 2011-0011187 Korea Patent Publication No. 2011-0030756

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The present inventors have sought to find a substance having an effect capable of protecting the liver from hepatotoxic substances from natural products. As a result, it was experimentally confirmed that chitosanmonone, which is a sulfur-containing compound separated from garlic, can very effectively protect hepatocytes from hepatotoxicity induced by acetaminophen administration in animal experiments in mice, Respectively.

Accordingly, it is an object of the present invention to provide a composition for protecting the liver, which comprises dicryptomone as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an antimicrobial composition for protecting the liver comprising, as an active ingredient, dicamphoronone represented by the following formula (1).

[Chemical Formula 1]

"Chiacormonone" as an active ingredient of the composition of the present invention is a compound isolated from a fraction having antioxidative activity, which is fractionated from the juice of heat-treated garlic. The compound is a fungus Acremonium sp. It is also known that the strain HA33-95 is capable of producing and is capable of inducing the differentiation of animal cells and also has an anticancer activity, an inflammatory disease and a metabolic disease treatment effect such as diabetes, hyperlipemia and cardiovascular disease .

The present invention relates to a new use for the protection of the teeth crest.

According to a preferred embodiment of the present invention, in the present invention, the liver protection is liver protection from hepatotoxic substances.

According to a more preferred embodiment of the present invention, in the present invention, the hepatotoxic substance is acetaminophen.

As demonstrated in the following specific example of the present invention, the dicamcinuron of the present invention highly effectively protects and restores damage of hepatocytes by administration of acetaminophen, which is known as a hepatotoxic inducer in animal experiments in mice .

According to a preferred embodiment of the present invention, the dacrimemicone reduces ALT level, AST level, and lipid peroxidation level induced by hepatotoxic substances in hepatocytes and inhibits nitric oxide (NO) production.

According to another preferred embodiment of the present invention, the dental crmmonon reduces penetration of Kupffer cells, T cells, and NK cells induced by hepatotoxic substances in hepatocytes and expression of hepatocyte cytochrome P450 2E1.

According to another preferred embodiment of the present invention, the diphtheria-crmmonon is a pro-inflammatory cytokine I-309, M-CSF, MIG, MIP-1α, MIP-1β, -13, IL-7 and IL-17 and increases the levels of cytokines IL-lra, IP-10 and MIP-2.

According to another preferred embodiment of the present invention, the tooth crushmonon is extracted and separated from garlic.

The composition of the present invention may be provided in the form of a pharmaceutical composition, and the pharmaceutical composition includes a pharmaceutically acceptable carrier other than the dicamphonone as an active ingredient.

The pharmaceutical composition of the present invention can be used for the treatment of liver diseases by hepatic toxic substances.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day. The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like. In the pharmaceutical composition of the present invention, the route of administration is preferably determined depending on the type of disease to which it is applied. The concentration of the active ingredient in the pharmaceutical composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention may be provided in the form of a functional food composition. When the composition of the present invention is provided in the form of a food composition, the composition of the present invention may contain, in addition to the above-mentioned active ingredient, chiacemphonone, a component ordinarily added during the manufacture of a food. Examples thereof include protein, carbohydrate, Fat, nutrients, flavoring agents and sweetening agents. Examples of such carbohydrates include, but are not limited to, monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose, oligosaccharides and the like, and polysaccharides such as dextrins, cyclodextrins, And sugar alcohols such as xylitol, sorbitol and erythritol. Natural sweeteners (tau Martin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic sweetening agents (saccharin, aspartame, etc.) can be used as sweeteners. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, etc., .

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a novel liver protection application for dicrecromone.

(Ii) dacrimemonon effectively protects hepatocyte damage induced by hepatotoxic agents.

(Iii) Tia-crmmonone reduces ALT levels, AST levels, and lipid peroxidation levels and inhibits NO (nitric oxide) production in liver damaged rat models treated with excess acetaminophen.

(Iv) It also inhibits the infiltration of Kupffer cells, T cells, and NK cells induced by excessive acetaminophen administration in hepatocytes and the expression of hepatocyte cytochrome P450 2E1 and proinflammatory cytokines I-309, M-CSF , MIP, MIP-1 alpha, MIP-1 beta, IL-10, IL-13, IL-7 and IL-17.

TECHNICAL FIELD The present invention relates to a composition for protecting the liver, which comprises tiocrimmonium as an active ingredient. The present invention provides a method for effectively protecting hepatocyte damage induced by a hepatotoxic substance. Chiacormonone reduces ALT levels, AST levels, and lipid peroxidation levels and inhibits NO (nitric oxide) production in liver damage induced by excessive hepatotoxic drug administration. In addition, it inhibits the penetration of Kupffer cells, T cells, and NK cells induced by hepatotoxic substances in hepatocytes and the expression of hepatocyte cytochrome P450 2E1, and proinflammatory cytokines I-309, M-CSF, MIG, , MIP-1 ?, IL-10, IL-13, IL-7 and IL-17. Therefore, dia-crmmonone can be developed as an active substance for drugs and functional food that protects liver damage by hepatotoxic substances.

FIGS. 1A and 1B show the effect of inhibiting hepatocellular toxicity in mice pretreated with dicamycorone.
FIG. 1A shows the results of measurement of the survival rate, ALT and AST levels of APAP-treated and dicammenone pretreated mice. The mice survival was monitored for 40 h after intraperitoneal (ip) infusion of 500 mg / kg APAP (n = 12) in the control, APAP-treated, and dicamperone pretreated mice. Log-rank test of mice pretreated with dicamcuronone showed a very high survival rate compared to APAP-treated mice (panel A). Levels of ALT (Panel B) and AST (Panel C) were measured for 40 hours after injection in untreated control mice and 500 mg / kg APAP in ip injected mice. Mice were pre-dosed with 10 mg / kg, 10, 20, 50 mg / kg for 7 days before APAP treatment. Mice pre-dosed with tooth crmmonon significantly reduced ALT and AST levels compared to APAP-treated mice.
Figure 1B shows the results of analysis of liver sections with H & E staining. Data are expressed as mean ± SD, * indicates a significant difference from control mice (p <0.05), and # indicates significant difference from APAP-treated mice (p <0.05).
FIGS. 2A to 2E show the results of measuring cytochrome P450 2E1 expression, GSH / GSSG ratio, NO (nitric oxide) and lipid peroxidation in liver tissues of mice previously administered with dicamycoronone.
In the results of FIG. 2A, expression of cytochrome P450 2E1, GSH / GSSG ratio, NO and lipid peroxidation levels were determined by Western blotting in total protein extracts of mouse liver tissue. FIG. 2B shows the results of immunohistochemistry analysis of cytochrome P450 2E1, showing the staining intensity of cytochrome P450 2E1 in the liver tissues of control, APAP-treated group and mice pretreated with dicamphorone.
Figure 2c shows that the hepatocyte GSH / GSSG ratio was significantly increased in mice pretreated with dicamcuronone compared with APAP-treated mice.
FIG. 2d shows that NO in the liver tissues of mice pre-administered with dicamcrimonone was remarkably inhibited compared with APAP-administered mice. The expression and the intensity of cytochrome P450 2E1 decreased in mice pretreated with dicamphorone.
Figure 2e shows that hepatocyte lipid peroxidation was significantly reduced in mice pre-dosed with dicamarcone compared with APAP-treated mice. Data are expressed as mean ± SD, * indicates a significant difference from control mice (p <0.05), and # indicates significant difference from APAP-treated mice (p <0.05).
FIGS. 3A through 3E show that the expression of proinflammatory cytokines is down-regulated in the liver of mice pre-administered with dicamcorone.
Figure 3a shows mouse cytokine array panel coordinates. In the nitrocellulose membrane, 40 different anti-cytokine antibodies are redundantly printed.
In the mouse cytokine array panel shown in Fig. 3b, there is a difference in cytokine expression in the liver tissues of control mice, APAP-treated mice (APAP) and mice treated with dicaprimonone (APAP + Thiacremonone 200 mg / kg). Figures 3c and 3d show representative values in three independent experiments. The positive control represents the manufacturer's internal positive control sample on the membrane. Expression levels of IL-1α, IL-7, IL-17, I-309, MIG, M-CSF, MIP-1a and MIP-1b in mice pre- Lt; / RTI &gt; FIG. 3E shows the results of measurement of expression of IL-1ra (IL-1 alpha receptor antagonist), IP-10 and MIP-2 cytokine. Expression of the cytokines was significantly increased in mice liver tissues pre-dosed with dicryptomone compared to the control and APAP-injected mice.
FIGS. 4A and 4B show the results of decreasing NF-kB and STAT1 in the liver of mice pretreated with dicamcuronone. 4A, the expression of phosphorylation of p50 and p65 in the nuclear extract (NE) was measured by Western blotting and the expression of NF-κB in nuclear extract (NE) of hepatic tissue of mice pre-administered with APAP- The DNA binding activity of kB was measured by EMSA.
In FIG. 4b, expression of p-STAT1 and STAT1 was measured by Western blotting in the nuclear extract (NE) of hepatic tissue of mice pre-administered with APAP-injected mice and dental crmmonon, and DNA binding activity of STAT1 was measured by EMSA .

FIGS. 5A to 5D show the effect of decreasing the infiltration of immune cells and Kupffer cells into blood and liver tissues of mice previously administered with dicamycoronone. Blood (Figs. 5A, 5B and 5C) and liver tissue (Fig. 5D) were isolated from the mouse model after study termination. Flow cytometry was performed using a FACSCalibur flow cytometer. Representative data were presented and data were expressed as the mean ± SD of four laboratory animals. Figure 5d is a photograph of liver tissue sections for Kupffer cells and NK cells after treatment of APAP in combination with vehicle or diafculonone. Immunoreactivity with Kupffer cells and NK cells was expressed in hepatocytes around the portal vein (central lobule). In the APAP-treated group, strong expression of Kupffer cells, NK cells and Tc cells was observed in the central nervous system sinusoid of liver. However, when pretreated with dichromocarnonine, the staining intensity of Kupffer cells and NK cells was weak in the sinusoid of the middle leaflet.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Methods and Materials

1. Experimental animals and treatment methods

C58BL6 / J mice were purchased from Orient Bio (Sungnam, Kyonggi-do, Korea). Mice were housed and maintained under sterilization conditions in facilities compliant with current regulations and standards approved by the American Laboratory Animal Care and Accreditation Association and Korea Food and Drug Administration and Chungbuk National University. The mice (n = 4 / cage) were maintained in a room at a constant temperature of 22 ± 3 ° C, a relative humidity of 50 ± 10% and a 12 hour light / dark cycle, Were randomly supplied. Mice matched with age (9 weeks old) and body weight (18-24 g) (n = 12 / group) were used. Before injecting APAP, the mice were intraperitoneally injected ip with a dose of 10, 20 or 50 mg / kg in the form of diphtheria (97% purity, dissolved in PBS (phosphate buffered saline) Day once a day. Cho et al., 2005; Cho et al., 2006; Cho et al., 2009). APAP was heated at 60 占 폚 and dissolved in saline. Mice were fasted for 16 h and then administered intraperitoneally with APAP (500 mg / kg; Sigma-Aldrich, St. Louis, Mo.) or saline. Mice were sacrificed at 40 hours after APAP administration. The mice were observed for 40 hours and then the overall survival rate was calculated.

2. Measurement of serum AST and ALT

Mice were anesthetized with an excess of pentobarbital (100 mg / kg) and the heart was perforated to collect blood. Levels of ALT and AST in serum were determined using an assay kit (Sigma Diagnostics, St. Louis, Mo.).

3. Measurement of glutathione ion / oxidized glutathione ion ratio and NO (NO) in hepatocytes

Levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) in hepatocytes were measured using a commercially available assay kit (Cayman Chemical, Ann Arbor, Mich.). Briefly, the frozen tissue is placed in a buffer at a 10% ratio (tissue weight / buffer volume), 0.01 M N-ethylmaleimide is added for GSSG measurements, and for GSH measurements Tissue homogenates were prepared in 0.1 M phosphate buffer (pH = 7.4) with EDTA, except N-ethylmaleimide. The content of GSH and GSSG in tissues was measured by the enzyme recycling method (Adams et al., 1983) which has been described previously. Liver tissues were homogenized in ice-cold lysis buffer (pH 7.5, 50 mM Tris, 1% Nonidet P-40, 150 mM NaCl and protease inhibitor cocktail) and centrifuged at 2000 g for 20 min. The supernatant was stored at -80 &lt; 0 &gt; C until use. The concentration of nitric oxide (NO) was determined by indirect measurement of nitrite (nitrite), which is a byproduct of NO conversion in biological tissues. The concentration of NO was analyzed using a NO detection kit (iNtRON Biotechnology, Seoul, Korea) and analysis was performed according to the manufacturer's protocol.

4. Western blotting

Liver tissue was homogenized with protein extraction solution (PRO-PREP ™, iNtRON Biotechnology), incubated at -20 ° C for 60 minutes and then lysed. The tissue homogenate was centrifuged at 15,000 rpm for 15 minutes at 4 ° C. Protein equivalent (30 μg) was separated using 10% SDS-PAGE and transferred to a PVDF membrane (GE Water and Process Technologies, Trevose, Pa.). Blots were incubated with TBST [Tris-Buffered Saline Tween-20; Was blocked with 5% (w / v) skim milk in a 150 mM NaCl solution containing 10 mM Tris (pH 8.0) and 0.05% Tween-20. After washing with TBST, the membranes were immunoblotted using the following primary antibodies: cytochrome P450 2E1 (1: 2000 dilution; Abcam PLC, Cambridge, MA), p65 and p50 (1: 2000 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif.), STAT1 or p-STAT1 (1: 2500 dilutions; Santa Cruz Biotechnology). Each blot was incubated with each horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1: 4000 dilution; Santa Cruz Biotechnology). Immunoreactive proteins were detected using an ECL detection system.

5. Cytokine Immune Array

Liver tissue was excised and homogenized in PBS with protease inhibitor cocktail (Sigma-Aldrich) and Triton X-100 (final concentration 1%). Samples were frozen at -70 ° C, thawed, and centrifuged at 10,000 × g for 5 minutes to remove cell debris. The tissue lysates were used to perform protein arrays in the same manner as in Western blotting. Protein 4.5 mg was collected from three samples per group and a Proteome Profiler (TM) mouse cytokine array was performed according to the manufacturer's protocol (# ARY006; R & D Systems, Minneapolis, MN). Briefly, antibody-printed membranes were blocked for 1 hour at 23 ° C. Hepatocyte lysis solution (0.5 mL) was mixed with biotinylated antibody cocktail and incubated at 23 ° C for 1 hour. The mixture was then added to the membrane and incubated overnight at &lt; RTI ID = 0.0 &gt; 4 C. &lt; / RTI &gt; The membrane was washed and then incubated in a streptavidin-horseradish peroxidase solution at room temperature for 30 minutes at 23 ° C. Immunoconjugates were detected using ECL.

6. Flow cytometry

Immune-related cells were recovered from the liver for phenotypic analysis. Liver tissues were isolated from fresh liver biopsy specimens obtained from 3 mice per group. Liver biopsies were mechanically homogenized in PBS, filtered using a 100 μm cell filter (BD Biosciences, Franklin Lakes, NJ), and placed in ACK lysis buffer (Lonza, Basel, Switzerland) to remove erythrocytes. One million hepatocytes were washed once in PBS containing 0.5% BSA and resuspended in 100 μL of PBS / BSA buffer. Meanwhile, 1 x ¹⁰⁶ separated cells were incubated with various conjugated monoclonal antibodies for 20 minutes on ice: CD4-APC (1: 400 dilution; BD Biosciences), CD3-FITC (1: 100 (1: 100 dilution; BD Biosciences), CD45R-PE (1: 400 dilution; BD Biosciences), CD49b-APC (1: 200 dilution; eBioscience, San Diego, Calif.), CD11c -PE (1: 200 dilution; BD Biosciences) and F4 / 80-APC (1: 100 dilution; eBioscience). It was then washed twice in PBS / BSA and resuspended in 500 [mu] L of PBS / BSA buffer. Flow cytometry was performed using a FACSCalibur instrument (BD Biosciences) and data were analyzed using WinMDI statistical software (Scripps, La Jolla, Calif.). Front and side scatter parameters were used to gate stained cells.

7. Gel electromobility shift assay

Gel electromobility shift assay (EMSA) was performed according to the manufacturer's instructions (Promega, Madison, Wis.). Liver tissues were homogenized in 200 μL of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM dithiothreitol and 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 6 min Incubation and centrifugation at 6,000 rpm for 6 minutes. The pelleted nuclei were resuspended in solution C (solution with 420 mM NaCl and 20% glycerol added to solution A) and incubated for 20 minutes with vortexing vigorously every 5 minutes on ice. The resuspended pellet was centrifuged at 15,000 rpm for 15 minutes and the resulting nuclear extract supernatant was collected in cold Eppendorf tubes. The consensus oligonucleotides were end-labeled with T4 polynucleotide kinase and [P ³² ] -ATP at 37 ° C for 10 minutes. The gel transfer reaction was combined and incubated at room temperature. Subsequently, 1 μL of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturing gel. The gel was electrophoresed until 4/5 of the gel was dyed, then the gel was dried at 80 ° C for 2 hours and exposed to film at -70 ° C overnight.

8. Histopathology and immunohistochemistry

Liver tissue was fixed in 4% paraformaldehyde and cut into 30μm sections using a freezing microtome (Thermo Scientific, Germany). The sections were stained with H & E (hematoxylin and eosin) for pathological examination. For immunohistochemical staining, liver sections were incubated with the following primary antibodies: CD49 (1: 5000 dilution; Santa Cruz Biotechnology), CD3 (1: 1000 dilution; Abcam, Cambridge, UK), cytochrome P450 2E1 Primary antibody to 1: 2000 dilution; Abcam) and F4 / 80 (1: 2000 dilution; Abcam). After washing in PBS, the sections were incubated in biotinylated secondary antibodies. Tissues were then incubated in an avidin-peroxidase complex (ABC Elite Kit; Vector Laboratories, Burlingame, Calif.) For 1 hour. After washing in PBS, the immune complexes were visualized using a 3,3-diaminobenzidine solution containing 0.08% hydrogen peroxide in PBS. The sections were dehydrated by successive treatment of concentrated gradient alcohol, removed with xylene, and then placed on cover slips using Permount (Sigma-Aldrich).

9. Statistical analysis

Data were analyzed using GraphPad Prism 6 software (Version 6.00; GraphPad Software, San Diego, Calif.). Data were expressed as mean ± SD. Group differences in APAP hepatotoxicity were analyzed using two-way ANOVA, the factor to be treated and genotype, and Bonferroni's post-hoc test was performed. The rest were analyzed using a two-tailed student t-test. p <0.05 was considered statistically significant. Survival data were calculated by Kaplan-Meier survival estimation and compared by Log-rank (Mantel-Cox) test within GraphPad Prism. All values were expressed as mean ± SEM. Significance was set at p <0.05 for all tests.

Experiment result

1.Tiamcurenonone reduced hepatocellular toxicity.

Acetaminophen (APAP) is known to cause central hepatotoxicity when overdosed. In order to confirm the protective effect of the tooth crmomonas, the survival rate of the mice was measured by administering an excess amount of acetaminophen after the dental crmmonium was previously administered to the mice. To determine the survival rate, fasted mice were administered 500 mg / kg of APAP alone and monitored for 40 hours (Fig. 1A Panel A). The survival rate of APAP-treated mice was significantly lower than that of control mice and dacrimemonone-pretreated mice. Survival rates of APAP-treated mice were reduced to 50%, whereas survival rates of mice pretreated with 10, 20, and 50 mg / kg of dacrimemone were respectively restored to 58.3%, 83.3%, and 100%, respectively. Serum levels of ALT and AST were measured at the end of the experiment. The levels of ALT and AST were 8.6 and 3.0 IU / L in the control group and 11754.8 and 18631.8 IU / L (p <0.001) in the APAP- , Showing significant differences in serum levels of ALT and AST. However, ALT and AST measurements were 9235.7 and 11063.8 IU / L for 10 mg / kg of dacrimemone, respectively, and 5797.8 and 6526.8 IU / L for dacrimemone (20 mg / <0.05), and the values of 4264.8 and 6185.0 IU / L (p <0.01) were obtained when 50 mg / kg of dacrimemonone was administered, respectively. That is, dia-clemmonon decreased ALT and AST levels in a concentration-dependent manner (FIG. 1A panels B and C).

Histopathological examination of mouse hepatic tissue from the control group revealed normal hepatic cells with central vein and sinusoidal dilatation. However, in the APAP-treated group, severe hepatocellular toxicity . Histopathological analysis showed that pathological damage by APAP administration was minimal in the group pre - administered with dicryptone. Namely, normal hepatocytes with regenerating hepatocytes were observed in the dacrimemonone-treated group and only weak inflammation was observed in the portal vein region (Fig. 1B).

2. Tachylemonium inhibited APAP-induced GSH depletion, NO production and lipid peroxidation by inhibiting cytochrome P450 2E1 expression.

Overexpression of cytochrome P450 2E1 plays an important role in APAP-induced hepatotoxicity. To evaluate whether dicamconeone is involved in the expression of cytochrome P450 2E1 in liver tissue, the expression of cytochrome P450 2E1 protein was examined by Western blotting and immunohistochemistry in liver tissue. Upregulation of the constant expression of cytochrome P450 2E1 by APAP administration was significantly reduced in mouse liver tissue pretreated with dicamcuronone (Fig. 2a). Immunohistochemistry showed that strong staining for cytochrome P450 2E1 was reduced in mice pretreated with dicamcone (Fig. 2B). Metabolites of APAP can deplete GSH and cause lipid peroxidation and increase nitric oxide (NO) production. Thus, we examined the differences in GSH / GSSG ratio, NO production and lipid peroxidation in APAP-treated mice and mice pretreated with dicamphorone. The GSH / GSSG ratio was significantly reduced in APAP-treated mice by about 67.4% (p <0.05) compared to the control group, but the GSH / GSSG ratio in APC-treated mice was pre-administered with 50 mg / Was significantly increased by about 123.6% (p < 0.05) compared with that of one mouse (Fig. 2C). On the other hand, NO levels and lipid peroxidation increased to about 156.5% and 661.5% (p < 0.05) in APAP-treated mice, respectively, as compared to control mice. NO and lipid peroxidation levels were reduced to about 56.1% and 194.2% (p < 0.05), respectively, in APAP-treated mice in mice pretreated with 50 mg / kg of dicamcrimonone (Figures 2d and 2e ).

3. Tachylemonon reduced the overproduction of APAP-induced proinflammatory cytokines.

To investigate the difference in cytokine levels in hepatic tissues of APAP-treated mouse liver tissue and mice pretreated with dicryptone, a cytokine array assay was performed using a Mouse Proteome Array (FIGS. 3A and 3B) . Among the 40 tested cytokines, IL-1α, IL-7, IL-17, I-309 (chemokine CC motif ligand 1, CCL1), macrophage colony-stimulating factor (M-CSF), MIG , CXCL9), MIP-1a (CCL3) and MIP-1b (CCL4) levels were increased about 10-fold in APAP-treated mice compared to control mice (FIGS. However, the cytokines significantly decreased in mice pre-dosed with dicamcorone. In contrast, IL-1ra (IL-1 alpha receptor antagonist), IP-10 and MIP-2 were significantly increased compared to control and APAP-administered mice in mice liver tissues pre-dosed with dicryptomone ).

4. NF-kB and STAT-1 activity decreased in liver tissues of mice pretreated with dicamcone.

DNA binding activity of NF-kB in hepatic tissues was measured by EMSA in order to evaluate whether hepatotoxicity in liver of APAP-treated mice was associated with inactivation of NF-kB. DNA-binding activity was higher in APAP-treated liver tissue compared to liver in mice pretreated with dicamarone. In addition, it was also observed that the migration of p50 and p65 into the nucleus was inhibited in the liver tissue pretreated with dicamcrimonone (Fig. 4A).

The reduction of hepatic toxicity in the liver of mice pretreated with dicamcuronone was also examined to determine whether it was associated with inactivation of STAT1 in the APAP-treated liver. DNA binding activity of STAT1 in liver tissue was measured by EMSA. As a result, DNA-binding activity was higher in hepatic tissues of APAP-treated mice than in mice pretreated with dicryptone. Western blotting experiments showed that STAT1 and STAT1 phosphorylation was low in the liver of mice pretreated with dicryptomone than in APAP-administered mice (Fig. 4B).

5. Tachylemonon reduced the penetration of APAP-induced cytotoxic immune cells.

In order to investigate whether the inhibition of APAP-induced hepatotoxicity was associated with the penetration of cytotoxic immune cells in mice pretreated with dicryptomone, the pattern of distribution of cytotoxic immune cells in liver tissues was analyzed. Lymphocytes were isolated from liver tissues and the phenotype of the cells was analyzed by fluorescence activated cell sorting. The proportion of T cells, B cells, cytotoxic T (Tc) cells and helper T (Th) cells in APAP-treated mice was 3.1%, 10.1%, 4.1% and 6.0%, respectively, , 6.4%, and 12.2%, respectively. The proportion of each cell group was 6.7%, 59.4%, 6.3%, and 10.9%, respectively. The proportion of each cell group in mice previously administered with dicryptone was 7.6%, 6.4% And Figure 5b). In the liver tissues of APAP-treated mice, the ratio of the cell population of the NK cells and the T cells to the dichotomous cells, the NK cells and the B cells were 2.1% and 0.9%, respectively. In contrast, And 0.6% and 0.2%, respectively, and the ratios of the above cell groups to mice pre-administered with dicryptone were 0.4% and 0.1%, respectively (Fig. 5C). The experimental data above show that Th cells and NK cells; Double positive cells of NK cells and T cells; And that the enhancement of the penetration of the double positive cells of NK cells and B cells could be reduced by dicamcone.

Observation of staining of liver tissue sections of APAP-treated mice revealed a significant inflow of Kupffer cells in the liver of APAP-treated mice as compared to control mice. However, it was observed that the expression of Kupffer cells was significantly reduced in the dendritic cell treated liver tissue.

NK cells and cytotoxic T cells were found to be higher in the liver of APAP-treated mice compared to the control mice. However, the immunohistochemical staining intensity and number of cells of NK cells and cytotoxic T cells were decreased in the liver tissues of mice treated with dia-crmmonon (Fig. 5d). These data show that mice can be pre-administered with dicryptomone to inhibit penetration of Kupffer cells, NK cells, and cytotoxic T cells, resulting in protection of APAP-induced liver damage.

Review

Chiacormonone inhibited APAP-induced ALT, AST and NO production, lipid peroxidation, and increased the GSH / GSSG ratio. In addition, dia-crmmonon reduced apoptosis-induced central nodule hepatocyte necrosis, Kupffer cells, T cells and NK cell infiltration and cytochrome P450 2E1 expression in the liver. Ticlcarmonone also inhibited the proinflammatory cytokine expression of I-309, IL-7, IL-10, IL-13, IL-17, M-CSF, MIG, MIP- Respectively. However, dicamconeone increased levels of IL-lra, IP-10 and MIP-2. These results suggest that dacremonone is highly likely to be used as a treatment for acute hepatotoxicity by hepatotoxic agents.

Excess APAP in hepatocytes is metabolized to NAPQI primarily by cytochrome P450 2E1. Accumulated NAPQI kills hepatocytes through glutathione ion depletion and consequent oxidative stress (Jaeschke et al., 2002). NAPQI reacts with GSH and depletes GSH to about 90%. This metabolite then covalently binds to the hepatocyte protein producing a 3- (cysteine-S-yl) -acetaminophen (APAP-Cys) adduct (James et al., 2003; Hinson et al., 2010 ; Agarwal et al., 2012). Peroxynitrite is formed by a rapid reaction between NO and superoxide, and NO synthesis is increased by APAP treatment (Hinson et al., 1998). Peroxynitrite induces nitration of tyrosine, which attacks various kinds of biological molecules such as lipids, proteins and DNA. Peroxynitrite is generally detoxified by GSH / GSSG peroxidase, and GSH is depleted in APAP toxicity. In addition, peroxynitrite is highly toxic under conditions of reduced cell oxidase clearing activity (Pryor and Squadrito, 1995; Beckman and Koppenol, 1996; Gardner et al., 1998). As a result, the normal detoxification mechanism for peroxynitrite has been impaired and hepatocytes are damaged by oxidative stress (Sies et al., 1997). As a result of the experiment of the present invention, the production of liver NO decreased by the treatment with dicammonon. By increasing the ratio of GSH / GSSG and decreasing lipid peroxidation, it was possible to confirm that dicryptone can protect hepatocytes from oxidative cell dysfunction.

Increased levels of ALT and AST are characteristic of acute overdosage of acetaminophen or other drugs or hepatocellular dysfunction caused by viruses (Ostapowicz et al., 2002). High serum levels of ALT and AST in APAP-treated mice are closely related to histopathological liver damage (Blazka et al., 1995a; Blazka et al., 1995b; Blazka et al., 1996; al., 2012). Histopathologic and serological analysis showed that pathologic sites developed by APAP in the liver of mice treated with dicryptomone were minimal. These results suggest that dicryptone is very useful as a hepatotoxic agent, for example, to protect liver damage by APAP overdose.

T cells, NK cells and Kupffer cells are activated by APAP treatment, and activated T cells, NK cells and Kupffer cells secrete various signal molecules such as hydrolytic enzymes, eicosanoids, nitric oxide and superoxide. Kupffer cells can secrete proinflammatory cytokines such as IL-1, IL-6, IL-7, IL-17 and TNF-α (Blazka et al., 1995b; Hogaboam et al., 1999; al., 2002), these cytokines are involved in the development of inflammatory reactions and the development of autoimmune diseases such as rheumatoid arthritis, experimental autoimmune encephalomyelitis, Crohn's disease (Iwakura et al., 2011). The other cytokines I-309, M-CSF, MIG, MIP-1α and MIP-1β belong to the family of inflammatory chemokines involved in acute inflammatory conditions that collect and activate some immune cells (Ishida et al. et al., 2004; Dragomir et al., 2012). These chemokines have been reported to be secreted by damaged hepatocytes, T cells, NK cells, and Kupffer cells, promoting the invasion of NK cells, mononuclear cells, and various other immune cells into the hepatic necrosis region (Lawson et al. 2000; James et al., 2003; Bourdi et al., 2007; Agarwal et al., 2012). Expression levels of I-309, M-CSF, MIG, MIP-1α, MIP-1β, IL-7, IL-13 and IL-17 were increased by APAP pretreatment, but these chemokine profiles were pretreated with , And the activation of APAP - induced Kupffer cells, cytotoxic T cells and NK cells was also decreased. Therefore, dicryptone can exert a liver protective effect by inactivating Kupffer cells, NK cells and Tc cells and decreasing the secretion of proinflammatory cytokines and chemokines. Some chemokines such as IP-10, IL-lra and MIP-2 can regenerate damaged liver by promoting hepatocyte proliferation (Gardner et al., 2012). Treatment with MIP-2 is more effective in protecting APAP-damaged liver damage. In addition, MIP-2 can maintain hepatocyte proliferation in APAP-exposed cells (Hogaboam et al., 1999; Hogaboam et al., 2000a). IP-10 may induce hepatocyte growth factors like mitogens. IP-10 has a protective effect on APAP-induced hepatotoxicity, and this protective effect is associated with the induction of MIP-2 receptors on hepatocytes (Bone-Larson et al., 2001; Ishida et al. al., 2004). IL-1ra can alleviate or completely treat APAP-induced liver necrosis. (Blazka et al., 1996; Ishibe et al., 2009; Hu et al., 2010), by administering IL-1ra and lowering serum levels of IL-1α. These chemokines can be secreted from damaged NK cells, T cells and Kupffer cells. Damage to Kupffer cells, T cells and NK cells is reduced by pretreatment with dia. The present inventors have also confirmed that dicryptomone increases the levels of MIP-2 and IP-10 that promote hepatocyte regeneration. The increased efficacy of these chemokines by dicamcurenon results in a direct recovery of hepatotoxicity or an indirect reduction of damaged Kupffer cells, T cells, and NK cells, and ultimately reduces hepatotoxicity.

Transfer of NF-kB to the nucleus is essential for induction of multiple cytokines related to APAP-induced hepatotoxicity (Arsura et al., 1996). Inflammatory stimuli such as TNF-α and IL-1β are activated by the NF-kB pathway. NF-kB is activated by the binding of the TNF receptor (TNFR) and the IL-1β receptor (IL-1βR) (O'Dea and Hoffmann, 2009). NF-kB regulates the expression of genes regulating inflammatory mediators such as NOS II (nitric oxide synthase II), TNF-α, IL-1β and cyclooxygenase-2, which are associated with hepatotoxicity (Dambach et al., 2006). Materials such as curcumin, anetholdithiolthione and silymarin protect the liver from damage by inhibiting NF-kB (Sen et al., 1996; Manna et al., 1999; Reyes-Gordillo et al., 2007). Kupffer cells activated through NF-kB inhibition inhibit TNF-α production (Wheeler et al., 2001). Thus, NF-kB inhibition reduces inflammatory mediator-dependent liver damage. Previous studies have shown that STAT-1 expression is increased in a time-dependent manner in APAP-treated hepatocytes (Ghosh and Sil, 2009). IFN-y and TNF-a activate STAT1 and induce hepatocyte apoptosis. Recent studies have shown that NK cells and NK T cells produce IFN-γ and play a key role in APAP-induced liver damage (Liu et al., 2004). IFN-y activates STAT1, which initiates inflammatory signals by inducing multiple cytokines, chemokines, adhesion molecules and Fas / FasL in APAP-induced hepatitis. TNF-α also activates STAT1, and STAT1-deficient cells are resistant to TNF-α-induced apoptosis (Meraz et al., 1996; Ishida et al., 2002; Jaruga et al., 2004). Thus, NF-kB and STAT1 activation in APAP-treated hepatocytes appears to be the main cause of liver injury (Numata et al., 2007). NF-KB and STAT1 activation were increased in the APAP-treated liver, but NF-KB and STAT1 were down-regulated in mice pretreated with dicryptomone. These experimental results suggest that inhibition of both STAT1 and NF-kB pathways is associated with liver protection from APAP-induced toxicity in dental crmomonas. Taken together with these experimental results, the experimental results of the present invention suggest that dicryptomone can be used very effectively for the treatment of hepatic toxicity by administration of hepatotoxic agents, for example, excessive APAP.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

1. A composition for protecting liver damage induced by acetaminophen comprising an effective ingredient of dicamphorone represented by the following formula (1).
[Chemical Formula 1]

delete delete The liver protecting composition according to claim 1, wherein said dicryptone is reduced in an increase in ALT level, AST level, and lipid peroxidation level induced by hepatotoxic substances in hepatocytes and inhibits NO (nitric oxide) production .
2. The composition of claim 1, wherein said dicryptone reduces the penetration of Kupffer cells, T cells, and NK cells induced by hepatotoxic agents in hepatocytes and the increase of hepatocyte cytochrome P450 2E1 expression.
2. The method of claim 1, wherein the diphtheria toxin is selected from the group consisting of proinflammatory cytokines I-309, M-CSF, MIG, MIP-1 ?, MIP-1 ?, IL- -7 and IL-17, and the levels of the cytokines IL-lra, IP-10 and MIP-2 are increased.
2. The composition of claim 1, wherein the dental crumomone is isolated from garlic.
The composition of claim 1, wherein the composition is in the form of a pharmaceutical composition.
The composition of claim 1, wherein the composition is in the form of a functional food composition.

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