KR20030046119A - A composition for treating hepatic fibrosis and cirrhosis containing butein - Google Patents

Abstract

PURPOSE: A medicinal composition containing albumin as an active ingredient for hepatic cirrhosis and fibrosis is provided to normalize the damaged hepatic cell by reducing the collagens overly cumulated at the problematic hepatic tissue, and recovering blood albumin level. CONSTITUTION: A medicinal composition for the hepatic cirrhosis and the fibrosis induced by carbon tetrachlororide contains an active amount of butein as an active ingredient. The butein inhibits the expression of the collagen genes coding TIMP-1 and 1 (I) collagen and facilitates the expression of the collagenase, metalloproteinase-13 (MMP-13). The butein reduces the amount of collagens in the hepatic tissue, and prohibits the activation and growth of the hepatic stellate cell.

Description

A composition for treating hepatic fibrosis and cirrhosis containing butein}

The present invention relates to a hepatic fibrosis and liver cirrhosis therapeutic composition containing butane (Butein) as an active ingredient, and more particularly, the present invention, 1) to promote collagen expression inhibitory and collagen degradation in liver tissue By inhibiting the proliferation and activation of hepatic stellate cells that produce collagen, thereby inhibiting overaccumulation of collagen, a characteristic finding of liver fibrosis and cirrhosis; 2) Hepatic fibrosis and cirrhosis therapeutic composition which can restore the decrease of blood albumin level caused by chronic liver damage caused by chronic liver damage to the normal level by inhibiting hepatocellular function or damage of liver function in liver fibrosis and cirrhosis patients It is about.

Liver fibrosis refers to a disease in which liver tissue in chronic inflammatory state is repeatedly damaged and regenerated, resulting in excessive accumulation of connective tissue such as collagen in the tissue, thereby causing scars in the liver tissue.

In general, hepatic fibrosis, unlike cirrhosis, is reversible, consists of thin fibrils, and has no nodule formation. In addition, normal recovery may be possible if the cause of liver damage is lost. However, if the hepatic fibrosis mechanism continues repeatedly, crosslinking between connective tissues increases, thick fibrils accumulate, and the nodule is formed by losing the normal structure of hepatic lobule. It progresses to irreversible cirrhosis.

Liver diseases may have various causes, but if they become chronic, they eventually lead to liver fibrosis or cirrhosis regardless of the cause. Liver disease is difficult to diagnose early due to the lack of early self-consciousness, and is usually found only when it is chronic. Therefore, it is not easy to treat, and mortality is high, causing social problems. In addition, therapeutic agents with high efficacy have not yet been developed.

The pathogenesis of hepatic fibrosis is known in the art to be caused by complex interactions between cells, cytokines and extracellular matrix (ECM). Overproduction of ECM during hepatic fibrosis promotes the activation of hepatic stellate cells (HSCs) and binds to activated hepatic stellate cells by the activation of several cytokines secreted by the activation of intracellular macrophages, Kupffer cells. It is known to be caused by increased production of tissue.

Hepatic stellate cells were discovered by Kupffer in 1876, described by Ito, and established by Wake in 1971. Hepatic stellate cells are cells located in the space of Disse between homologous vascular endothelial cells and hepatocytes and are star-shaped. Lipids contain hepatic stellate cells, Ito cells, and vitamin A. It has been named as a vitamin A storing cell, a fat storing cell, or a perisinusoidal hepatic lipocyte because it has droplets. Hepatic stellate cells (HSCs) have been determined and used to date.

Cooper cells are activated by phagocytosis of hepatocytes damaged by various toxins or directly activated by toxins, thereby transforming growth factor-beta (TGF-beta) and platelet-induced growth factors. It secretes cytokines, such as platelet-derived growth factor (PDGF), and the cytokines themselves autocrine again by these cytokines. In addition, cytokines secreted by Cooper cells activate sinusoidal endothelial cells and hepatic stellate cells in liver tissue. The basement membrane-like matrix (ECM) is broken down by the breakdown of the extracellular matrix (ECM) by collagen type IV and increased collagen production secreted by activated hepatic stellate cells. For example, collagen 4 is degraded, and interstitial type matrices (e.g., type 1 and 3 collagen) are intertwined with each other to form fibril-forming collagen and then the space of Dissipates, and hepatic stellate cells are activated by degraded basal membrane ECM and accumulated intercellular ECM.

In addition, the activated hepatic stellate cells promote the production of macroglobulins and TIMPs, which are inhibitors of collagen proteinase (MMP) activity, and also inhibit the excessive production of intercellular ECM. By intercellular ECM accumulated in the Dice Seol inhibits the exchange of blood and hepatocytes, hepatocytes are difficult to supply nutrients and discharge of toxic substances, hepatic cells are also continuously damaged. As this series of reactions are repeated, connective tissue accumulates in the liver tissue, resulting in liver fibrosis or cirrhosis.

Currently, studies on hepatic fibrosis are being actively conducted in various fields such as research on ECM, the relationship between cells and various cells involved, cell activation mechanisms, and various cytokines and antifibrogenic agents. It aims to develop early diagnosis and treatment of liver fibrosis. In particular, the development of drugs that can inhibit the activity and activation of the stellate cells, as it was found that the biggest inducer of liver fibrosis and cirrhosis associated with the production of over-accumulated connective tissue is the activation of hepatic stellate cells The research on this is ongoing.

Conventionally, a substance that inhibits hepatic fibrosis or cirrhosis, penicillamine, 16, 16- dimethyl estigladdin E2, biphenyl dimethyl dicarboxylic acid, colchicine, glucocorticoid, malotilate, gamma-interferon, Pentoxifylline, pyridine-2,4-dicarboxylic-diethylamide, pyridine-2,4-dicarboxylic-di (2-methoxyethyl) amide, and the like have been developed, but have been applied clinically. Because of its weak visual effects and severe side effects, it can be said that there are currently no therapeutic agents for liver fibrosis and cirrhosis (Boker et al., J. Hepatol., 13, s35, 1991; Tamayo et al., Ibid, 3). , 112, 1983; Jenkins et al., Ibid, 1, 489, 1985; Kershenobich et al., Ibid, 7, 1104, 1987; Svegliati et al., 18 209A, 1993; Guzelian et al., Gastro., 86 , 897, 1984; Ala-Kokko et al., J. Lab. Clin. Med., 113, 177, 1989).

Thus, the present inventors have conducted a number of studies to find a substance that can be used more effectively in the treatment of liver fibrosis and cirrhosis, but as a result of butane (Butein) inhibits the production of collagen in the liver tissue, promotes the breakdown of collagen It is possible to restore the liver function lowered by liver damage, and found that a composition containing butane can be used for the treatment of liver fibrosis and cirrhosis, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a composition for treating liver fibrosis and cirrhosis.

FIG. 1 shows that immune tissues of α-Smooth muscle actin (α-Smooth muscle actin) inhibit the proliferation of butane-activated hepatic stellate cells in liver tissues derived from carbon tetrachloride-induced liver fibrosis and cirrhosis experimental animal models. It is shown by the chemical staining method.

Figure 2 shows that butane inhibits increased mRNA expression of α1 (I) collagen and TIMP-1 genes in liver tissues of hepatic fibrosis and cirrhosis induced animals induced by carbon tetrachloride.

FIG. 3 shows that butane inhibits mRNA expression of genes encoding α1 (I) collagen and TIMP-1 in primary cultured activated hepatic stellate cells, and mRNA expression of a gene encoding MMP-13, a collagenase Is a diagram showing the increase.

Figure 4 shows that butane inhibits the expression of collagen and α-SMA at the protein level in primary cultured activated hepatic stellate cells.

In order to achieve the above object, the present invention is characterized in that it contains butane (Butein) as an active ingredient as a substance that can be used in the treatment of patients diagnosed with liver fibrosis and cirrhosis.

Hereinafter, the present invention will be described in more detail.

According to the present invention, butane inhibits the expression of the alpha 1 (I) collagen gene known as a gene encoding the collagen protein, and is known as metalloproteinase-13 (MMP-13) as a collagen protease. The present invention contains butane as an active ingredient because it can increase the expression of the gene and inhibit the expression of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene known as the activity inhibitory protein of the MMP-13 protein. The composition according to the present invention can inhibit collagen expression in liver tissues, promote collagen degradation, and inhibit excessive accumulation of collagen in liver tissues.

In addition, the composition according to the present invention, by inhibiting the proliferation and activation of hepatic stellate cells, by inhibiting the production of excessively increased collagen and connective tissue (liver tissue) in the liver tissue, to liver cirrhosis expression and liver cirrhosis Can prevent the development of

In addition, the composition according to the present invention can restore to normal levels the decrease in blood albumin levels caused by chronic liver damage caused by liver damage, which is a composition according to the present invention containing butane as an active ingredient Impaired hepatocyte function or liver function.

Butane is a flavonoid component separated by Dalbergia odorifera T. Chen, and is a 2 ', 4', 3,4-tetrahydroxychalcone (2 ', 4). ', 3,4-tetrahydroxychalcone) has the chemical name and has the following structural formula.

At present, there is no exact report on the effect of chalcone-structured substances on human health, but until now it has been reported that some chalcone-structured substances have anti-inflammatory, anticancer and antifungal effects (Anto et al. , Cancer Lett., 97, 33, 1995; Chin et al., Cancer Lett., 82, 65, 1994). In addition, these substances have recently been reported to be involved in the inhibition of cAMP-phosphodiesterase and glutathione S-transferase (Kuppusamy et al., Biochem. Pharmacol., 44, 1307, 1992; Zhang et al., Biochem.Pharmacol., 47, 2063, 1994). Butane is also reported to inhibit lipid peroxidation in rat liver microsomes as an inhibitor of xanthine oxidase (Sogawa et al., Biol. Pharmacol. Bull). , 17, 251, 1994).

On the other hand, butane may be chemically synthesized using conventional methods in the art, for example, using a conventional method to replace the desired position of the phenyl group in the chalcone structure with a hydroxy group to convert to chalcone or its Synthesis from derivatives; Condensation, oxidation, and appropriate acetophenyl groups of the Winjet can also be prepared by reduction with a suitable benzaldehyde; It is also available as a commercially available product number, B-178, available from Sigma (see Sigma RBI Homepage).

The effective amount of butane as a therapeutic agent for liver fibrosis and cirrhosis of the present invention may be appropriately selected depending on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease, but is not specifically limited. In general, butane may be formulated to be administered at a concentration of 1 to 2,000 mg / kg body weight. Of course, the dose of the active ingredient may be an amount outside the above range.

Formulations for the administration of butane within the above range may have a conventional form, for example, may be used in the form of pills, capsules, drinks, medicines and the like. They may be administered orally or various parenteral routes of administration for the treatment of liver fibrosis and cirrhosis and are suitable for the dosage form and are already well known to those skilled in the art and can be easily selected by those skilled in the art and can be readily selected by those skilled in the art. And the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto, and modifications such as insertion, deletion, and modification, which can be easily implemented by those skilled in the art, are also included within the scope of the present invention. . In addition, although the present invention showed the therapeutic effect of these diseases of butane through carbon tetrachloride-induced liver fibrosis and cirrhosis experimental animal model and primary cultured hepatic stellate cell experiment, the present invention is induced by carbon tetrachloride It will be readily appreciated by those skilled in the art that the therapeutic effect of liver fibrosis and / or cirrhosis patients is not only in the liver fibrosis and cirrhosis experimental animals but also in the actual clinical field. Therefore, the scope of application of the therapeutic agent according to the present invention includes not only applied to carbon tetrachloride-induced liver fibrosis and liver cirrhosis experimental animal models, but also to other liver fibrosis and / or liver cirrhosis patients, such as clinical settings.

Reference Example 1 Treatment of Experimental Liver Cirrhosis Animal Model and Butane

Typically, laboratory cirrhosis and hepatic fibrosis animal models are obtained by exposing experimental animals to carbon tetrachloride, ethanol, methotrexate, dimethylnitrosamine or lacking vitamin B ₁₂ . In this example, carbon tetrachloride-induced hepatic fibrosis and liver cirrhosis rats were used to test the effects of butane on hepatic fibrosis and cirrhosis.

Preparation of experimental liver fibrosis and cirrhosis animal models and methods of treating carbon tetrachloride and / or butane to such animals are as follows.

First, male rats (obtained from Korea Experimental Animal Center; Chungbuk Negative) weighing 190-220 g were acclimated to the laboratory environment for one week, and were provided with sufficient feed and water. Then, butane and carbon tetrachloride were treated as shown in Table 1.

Military number Military name Number of doses Administration substance (oral administration) One Control 5 Corn Oil + Distilled Water 2 Butane-only group 5 10 mg / kg / day butane + corn oil 3 Butane-only group 5 Butane + corn oil at 25mg / kg / day 4 Carbon tetrachloride alone 12 1.0 ml / kg of carbon tetrachloride + distilled water 5 Carbon tetrachloride and butane simultaneous administration group 14 1.0 ml / kg of carbon tetrachloride + 10 mg / kg / day butane 6 Carbon tetrachloride and butane simultaneous administration group 14 1.0 ml / kg carbon tetrachloride + 25 mg / kg / day butane

Each of the administration substances listed in Table 1 was orally administered using sonde. The control group 1 was given corn oil twice a week (Monday, Thursday) while distilled water, which is a solvent of butane, was administered daily (Monday, Thursday). Twice a week, carbon tetrachloride was given twice a week (Monday, Thursday) while distilled water was administered daily to group 4, and carbon tetrachloride was given once a week with butane suspended in water every day. Administration was twice. Carbon tetrachloride was orally administered with carbon tetrachloride and the same amount of corn oil (orientated oil product). Liver fibrosis was induced by oral administration of carbon tetrachloride twice a week for 6 weeks. 72 hours after the last oral administration of carbon tetrachloride, the rats of each group were anesthetized, and then cardiopuncture to collect blood, and left at room temperature for at least 1 hour, followed by centrifugation to obtain serum. At the same time as the cardiac puncture, the liver was immediately extracted and a part of the left lobe of the liver was placed in 10% formalin. Serum of each group separated in the above and liver tissue placed in 10% formalin was stored at minus 20 ℃ to perform the following experiment.

Reference Example 2 Hepatic Stellate Cell Separation Culture and Butane Treatment

Hepatic fibrosis Hepatic stellate cells play a decisive role in the accumulation of excess connective tissue in the liver and in about 7% of normal livers.

Hepatic stellate cells were isolated from rats by modifying the method of Friedman et al. In brief, anesthetized rats were dissected, then intubated into the portal vein, and a balanced salt of Ca ²⁺ , Mg ²⁺ -free Hank at 37 ° C. was blown with a mixture of 5% CO ₂ and 95% O ₂ . A solution (Ca ²⁺ , Mg ²⁺ -free Hank's balaced salt solution) was flowed through the tube to remove hepatic blood. Then, 0.05% collagen type IV (collagenase type IV) and 1 mM CaCl ₂ solution was flowed.

After removing the liver tissue from the body, hepatocellular suspension was made, centrifuged at 50 x g for 2 minutes, and the precipitated hepatocytes were removed. The supernatant from which hepatocytes were removed was stacked on 11.5% and 17% metrizamide (Sigma) layers and centrifuged at 1,700 xg for 15 minutes to obtain a hepatic stellate cell layer. The hepatic stellate cell layer was washed with Hank's balanced salt solution and then, in an uncoated culture vessel, 10% (v / v) fetal bovine serum (FBS, GibcoBRL), 50 U / ml penicillin and 50 µg / Cell number 5 × 10 using Williams culture medium E (Williams' Medium E; WME, GibcoBRL, USA) to which ml of streptomycin (Sigma) was added as a culture medium.⁵ 5% CO after cell count / ml₂And cultured at 37 ℃. After incubation for 5 days, the culture medium was changed to a culture medium in which 0.5% FBS was added instead of 10%, and then cultured for 1 day, followed by butane treatment. Butane was treated for 1 day or 2 days at 0.5 μg / ml and 1.0 μg / ml, which were not toxic to hepatic stellate cells.

Example 1 Inhibition Effect of Butane on Collagen Production in Liver Tissue

Collagen is an important component of liver connective tissue. However, as described above, when liver fibrosis is induced or liver fibrosis progresses to cirrhosis, excessive amounts of collagen accumulate in liver tissue. Therefore, conventionally, by measuring the amount of collagen in liver tissue, the possibility of inducing liver fibrosis or the occurrence of cirrhosis has been diagnosed.

Therefore, in this Example, the liver fibrosis inhibitory effect of butane was examined by measuring the amount of collagen in liver tissue treated with carbon tetrachloride and / or butane.

The amount of collagen in this example was measured by the following method. That is, the amount of collagen expressed in liver tissue was measured by quantifying the amount of hydroxyproline as an amino acid component that is specific to collagen and occupies about 10% of collagen (Schuppan, J. Hepatol., 13, s17, 1991). Hydroxyproline was quantified according to the method of Jamal et al. (Jamall et al., Anal. Biochem., 112, 70, 1981).

That is, as in Reference Example 1, 0.2 g of liver tissue of each group treated with butane and carbon tetrachloride was homogenized with 6 ml of 6N hydrochloric acid, and the 5% liver homogenate was hydrolyzed at 110 ° C. and filtered. 50 μl of the filtrate was taken, evaporated to dryness at 80 ° C. under reduced pressure, dissolved in 1.2 ml of 50% isopropanol solution, and oxidized to 200 μl of chloramine-T (manufactured by Sigma). Then, color development was carried out with 0.1 ml of Erlich reaction solution, and the absorbance at 558 nm was measured. The results are shown in Table 2 below.

As shown in Table 2, it was confirmed that butane can reduce the amount of hydroxyproline, that is, the amount of collagen in the liver tissue increased by carbon tetrachloride. In other words, these results show that butane can be used to treat liver fibrosis or cirrhosis.

Example 2 Recovery Effect of Butane on Blood Albumin Reduction

Albumin is a blood protein synthesized in hepatocytes and is characterized by a decrease in hepatocyte synthesis when hepatic function is reduced.

The amount of albumin was measured from the serum of animals treated with carbon tetrachloride and butane by the same method as in Reference Example 1 above. The amount of albumin in the blood was measured using a kit provided by ARKRAY FACTORY (Japan) and Spotchem automatic analyzer II (Spotchem-SP4410, Japan), and the results are shown in Table 2 below.

As shown in Table 2, it was confirmed that in the animal model of hepatic fibrosis and cirrhosis induced by carbon tetrachloride, butane recovers to normal levels the decrease in blood albumin caused by hepatic impairment caused by chronic liver damage. These results indicate that butane can inhibit hepatocellular function or hepatic function from being impaired by hepatic fibrosis or cirrhosis.

Military number Hydroxyproline (μg / g) Albumin (mg / dl) One 247 ± 24 4.7 ± 0.1 2 238 ± 32 4.7 ± 0.1 3 249 ± 58 4.7 ± 0.1 4 878 ± 78 ^a 3.7 ± 0.4 ^a 5 611 ± 67 ^{a, b} 4.5 ± 0.1 ^{a, b} 6 474 ± 39 ^{a, b} 4.5 ± 0.2 ^{b a} P <0.001, showing statistically significant difference compared to group 1 ^b P <0.001, showing statistically significant difference compared to group 4

<Result>

As shown in Table 2, it was confirmed that the albumin level of the experimental animal model (group 4) induced by carbon tetrachloride was restored to almost normal level by butane. In addition, the amount of collagen, that is, hydroxyproline, increased during the treatment of carbon tetrachloride is reduced by 30% (group 5) and 46% (group 6) in a dose-dependent manner by butane, butane is a substance capable of inhibiting liver fibrosis. Confirmed. On the other hand, in the level of hydroxyproline, the results of group 2 and 3 did not show a statistically significant difference compared to group 1, which means that butane itself does not cause any adverse effects on liver function. do.

Example 3 Inhibitory Effect of Butane on Activation and Proliferation of Hepatic Stellate Cells

It is known in the art that during the progression of liver fibrosis and cirrhosis, hepatic stellite cells are activated. Therefore, the progress of liver fibrosis and cirrhosis can be determined by confirming the activation of hepatic stellate cells in liver tissue using an immunohistochemical staining method. Therefore, in the present invention, by confirming the activation of these hepatic stellate cells in the carbon tetrachloride and / or butane-treated liver tissue, it was intended to measure the inhibitory effect of butane liver hepatic fibrosis and cirrhosis. It is known in the art that the degree of activation of hepatic stellate cells in liver tissue can be determined by the degree of formation of α-smooth muscle actin (α-SMA) in liver tissue. Therefore, in the present embodiment, it is possible to know the state of activated hepatic stellate cells by immunohistostaining α-SMA in liver tissue and determining the distribution and degree of formation thereof. Therefore, in this example, it was intended to determine whether butane can inhibit the proliferation of hepatic stellate cells activated by carbon tetrachloride.

First, carbon tetrachloride and / or butane were treated as in Reference Example 1, and livers were extracted from experimental animals in each group and fixed in 24% of 10% neutral formalin. The liver tissue was then paraffin-blocked, 3 μm sections were attached to the slide glass, deparaffinized with xylene I and II according to the usual method, and 100%, 95% ethanol. Were sequentially treated to dehydration and then washed with distilled water. Then use, ^ⓡ LSAB 2 kit (Dako), following the manual attached to the kit, streptavidin-biotin-peroxidase complex formation (biotin- streptavidin-peroxidase complex) method (Guesdon et al, J. Histochem. Cytochem., 1979; 27: 1131, Warnke and Levy, J. Histochem. Cytochem., 1980; 28: 771). That is, liver tissue was treated with 3% hydrogen peroxide to remove endogenous peroxidase and blocked with 1% bovine serum albumin (BSA) for 10 minutes to inhibit nonspecific binding of the primary antibody and to prevent primary antibody. And reacted with α-SMA antibody diluted 200-fold at room temperature for 2 hours to bind α-SMA in liver tissue. The biotinylated anti-rabbit / anti-mouse immunoglobulin is then treated according to the user instructions attached to the kit and then streptavidin-linked horseradish peroxidase ( streptavidin conjugated horse radish peroxidase) and stained with 3% 3-amino-9-ethylcarbazole. Then, nuclear staining with Mayer's hematoxylin, added with 0.25% ammonia water, washed with distilled water, and then observed under a microscope. The results are shown in FIG.

As shown in FIG. 1, group 1 liver tissue (FIG. 1A) is stained with α-SMA only in a portion of vascular smooth muscle cells and the surrounding cells, but only carbon tetrachloride was administered (group 4). In liver tissue (FIG. 1B), α-SMA was detected in significant portions along the fibrous septum, indicating that activated hepatic stellate cells increased significantly. On the other hand, α-SMA is significantly decreased in liver tissues (FIG. 1C and FIG. 1D) of the group of 10, 25 mg / kg / day butane administration (groups 5 and 6, respectively) with carbon tetrachloride. Could confirm. That is, it can be seen from the results of FIG. 1 that butane can inhibit the proliferation of activated hepatic stellate cells, which play an important role in the development of liver fibrosis and cirrhosis.

From the results of these experiments, it was confirmed that butane did not induce hepatotoxicity in the rats of the normal group, but it was possible to protect liver fibrosis and cirrhosis induced by carbon tetrachloride and to inhibit liver fibrosis at the same time.

Example 4 Effect of Butane on Expression of Collagen, TIMP-1, and MMP-13

As confirmed above, butane can reduce the amount of collagen in liver tissue caused by carbon tetrachloride. In this regard, in the present embodiment, it was intended to confirm the action of butane in the expression of genes involved in the synthesis and degradation of collagen.

Expression of genes involved in collagen synthesis and degradation was confirmed using Reverse Transcription-Polymerase Chain Polymerization (RT-PCR). The genes involved in collagen synthesis and degradation were alpha-1 (I) collagen gene, MMP-13 gene known as collagen degrading enzyme in rats, and TIMP-1 gene known to inhibit the activity of collagen degrading enzyme MMP-13. , RT-PCR was used to test the expression of these genes into mRNA.

RT-PCR analysis was performed on each liver tissue extracted from each of 5 rats and 6 rats treated with carbon tetrachloride and butane simultaneously in Reference Example 1, and extracted from each liver tissue, and was negative control (C). As a positive control (N), three groups of liver tissues treated with carbon tetrachloride and distilled water were used.

In addition, as a cell experiment, RT-PCR was also performed on hepatic stellate cells treated with butane at 0, 0.5, and 1.0 µg / ml for one day, as in Reference Example 2, to determine the genes involved in collagen production and degradation. Expression was confirmed.

First, total RNA in liver tissue and hepatic stellate cells of each group was extracted using RNeasy Mini Kit (Qiagen). Single-strand cDNA was synthesized under the following conditions using oligo-dT primer and moloney murine leukaemia virus-reverse transcriptase (Superscript ™ II RT, GibcoBRL). ; 1 μg of oligo (dT) _12-18 primer (GibcoBRL) was added per 1 μg of extracted RNA, followed by reaction at 70 ° C. for 10 minutes and 37 ° C. for 5 minutes, followed by RNase inhibitors (RNasin ^® , Promega), 0.1 mM. Dithiothreitol (GibcoBRL), 2.5 μM dNTP (Prime-a-gene ^® , Promega) and reverse transcriptase (Superscript ™ II RT, GibcoBRL) were further added, followed by reaction at 42 ° C. for 60 minutes to synthesize cDNA. It was. The synthesized cDNA was diluted 10-fold, and PCR was performed using the primers of Table 3 below to amplify specific portions of alpha 1 (I) collagen, TIMP-1, and MMP-13.

Alpha 1 (I) Collagen Sense primer 5'-TAC TAC CGG GCC GAT GAT G-3 ' Antisense primer 5'-CTT GGG GTT TGG GCT GAT GT-3 ' TIMP-1 Sense primer 5'-CCA CAG ATA TCC GGT TCG CCT ACA-3 ' Antisense primer 5'-GCA CAC CCC ACA GCC AGC ACT AT-3 ' MMP-13 Sense primer 5'-AAA GAA CAT GGT GAC TTC TAC C-3 ' Antisense primer 5'-ACT GGA TTC CTT GAA CGT C-3 '

PCR reaction conditions were repeated 35 cycles of 30 seconds of denaturation at 94 ° C, 30 seconds of annealing at 55-57 ° C, 30 seconds of extension at 72 ° C in the presence of 1.5 mM MgCl ₂ , and finally The expansion reaction was carried out at 72 ° C. for 5 minutes.

The GAPDH gene was used as a control for the PCR product, and the primers for PCR amplification of the GAPDH gene were 5'-CCC CTT CAT TGA CCT CAA CTA C-3 'and a 5'-CAT GGT GGT. Antisense primers with the sequence of GAA GAC GCC AG-3 'were used.

After performing the PCR reaction under the above conditions, PCR amplification products were agarose gel electrophoresis, visualized by EtBr (ethidium bromide staining) staining, and confirmed by using a photometer analysis. The results for liver tissue are shown in FIG. 2, and the lack of hepatic stellate cells is shown in FIG. 3.

As shown in Figure 2, butane significantly reduced the mRNA expression of the alpha 1 (I) collagen gene in liver tissue, and also the mRNA expression of TIMP-1 gene as an inhibitor of collagenase, a collagen degrading enzyme. Reduced.

In addition, as shown in Figure 3, butane in primary cultured hepatic stellate cells in the same manner as the results of Figure 2, inhibited the mRNA expression of alpha 1 (I) collagen gene and TIMP-1 gene, collagen degradation Expression of the enzyme MMP-13 was increased. These results indicate that butane inhibits collagen biosynthesis at the gene level in the progression of liver fibrosis and / or cirrhosis and also increases collagen degradation at the gene level. Therefore, it was confirmed that butane may be very useful in treating liver fibrosis and cirrhosis.

Example 5 Inhibitory Effect of Butane on Collagen and Hepatic Stellate Cell Activation at Protein Level

As confirmed above, butane acts on the expression of genes involved in collagen expression and degradation in liver tissues induced by carbon tetrachloride. In this regard, in this example, it was intended to confirm whether butane can inhibit the expression of collagen and α-SMA gene at the protein level using an immunoblotting test.

To determine the amount of α-SMA protein, hepatic stromal cells treated as in Reference Example 2 were treated with RIPA buffer (50 mmol / L Tris-HCl pH 7.4; 1% (v / v) Triton X-. Suspension with 100, 1 mmol / L EDTA, 1 mmol / L leupeptin, 1 mmol / L phenylmethylsulfonyl fluoride, followed by centrifugation at 14,000 rpm and 4 ° C. for 30 minutes to separate the supernatant. It was. Since collagen is a secreted protein, the amount of protein in the culture was measured directly. Protein concentration in culture and cell lysate was measured using the Bio-Rad DC Protein Test Kit (Bio-Rad Laboratories). 20 ㎍ of protein isolated from the culture and cell lysate were electrophoresed at 200V for 1 hour on 10% (w / v) SDS gel (sodium dodecyl sulfate polyacrylamide gel). The protein in the gel was separated by electrophoresis according to the molecular weight, and the protein was transferred to a nitrocellulose membrane (Millipore) at 30V for 16 hours. The transferred nitrocellulose membrane was blocked with 5% skim milk and then 200-fold diluted rabbit anti-collagen type I antibody (Calbiochem) or 250-fold diluted mouse anti-alpha SMA antibody. (mouse monoclonal anti-alpha SMA antibody; Sigma) was used as the primary antibody and reacted at room temperature for 2 hours. As secondary antibodies, horseradish peroxidase-linked anti-rabbit immunoglobulin G (horseradish peroxidase (HRP) -conjugated anti-rabbit IgG; Santacruz) for collagen and horseradish peroxidase- for α-SMA Bound anti-mouse immunoglobulin G (horseradish peroxidase (HRP) -conjugated anti-mouse IgG (Santacruz)) was used for 1 hour using a 2,000-fold dilution. Color development was confirmed by exposure to X-ray films (Agfa, EC) using an enhanced chemiluminescence (ECL) western blotting detection system kit (Amersham) in accordance with the user instructions. Collagen and α-SMA in the identified film was measured by using a photo densitometer analysis, the results are shown in FIG.

As shown in FIG. 4, butane significantly reduced the amount of collagen synthesized in hepatic stellate cells at the protein level, and significantly decreased the expression of α-SMA at the protein level, which is an indicator of activation of hepatic stellate cells. . These results show that butane inhibits collagen biosynthesis in hepatic stellate cells during hepatic fibrosis and / or cirrhosis, and also inhibits the activation of hepatic stellate cells. Therefore, it was confirmed at the level of cell experiment that butane can be used to treat liver fibrosis and cirrhosis.

As described above, butein can reduce collagen accumulated excessively in liver fibrosis and cirrhosis liver tissue through proper regulation of enzymes involved in collagen synthesis and degradation; Inhibits proliferation and activation of hepatic stellate cells by induction of hepatic fibrosis; By restoring blood albumin levels, which are reduced as a result of liver damage due to the progression of hepatic fibrosis and cirrhosis, to normal levels can lead to normalization of hepatocytes. Thus, a composition containing butane can be effectively used for the prevention and treatment of liver fibrosis and cirrhosis.

Claims (8)

A therapeutic composition for liver fibrosis and cirrhosis, characterized in that it contains an amount of butein effective to treat hepatic fibrosis and hepatic cirrhosis. [Claim 2] The composition of claim 1, wherein the liver fibrosis and cirrhosis are caused by carbon tetrachloride. [Claim 2] The composition for treating liver fibrosis and cirrhosis of claim 1, wherein the butane reduces the amount of collagen in liver tissue. 4. The method of claim 3, wherein the butanes cause inhibition of expression of genes encoding TIMP-1 and α1 (I) collagen in hepatic tissues and hepatic stellate cells and increase expression of genes encoding MMP-13, a collagen degrading enzyme. A composition for treating liver fibrosis and cirrhosis, characterized in that [Claim 2] The composition for treating liver fibrosis and cirrhosis of claim 1, wherein the butane inhibits the activation and proliferation of hepatic stellate cells. [Claim 2] The composition for treating liver fibrosis and cirrhosis of claim 1, wherein the butane recovers damage of liver tissue and liver cells infected by chronic inflammation. [Claim 7] The composition for treating liver fibrosis and cirrhosis according to claim 6, wherein the butane recovers blood albumin levels reduced in liver tissue to normal levels during chronic liver injury. The therapeutic agent for liver fibrosis and cirrhosis according to any one of claims 1 to 6, wherein the therapeutic agent contains butane at a concentration of 1 to 2,000 mg / kg body weight.