KR20170135544A - Peptide and composition containing the same for treating or preventing of liver disease - Google Patents

Abstract

The present invention relates to a composition including AIMP1 peptide as an effective component for preventing or treating liver diseases. As the AIMP1 peptide controls a smad2 position through the activation of extracellular signal-regulated kinase (ERK) and suppresses the expression of fibrosis factors such as collagen I, the peptide has an excellent effect for preventing and treating hepatocirrhosis as well as hepatic fibrosis, and thus, the composition is able to be used as a pharmaceutical composition and functional healthy food for preventing or treating liver diseases.

Description

[0001] The present invention relates to peptides for the prevention or treatment of liver diseases,

The present invention relates to a peptide for preventing or treating liver disease and a composition comprising the same as an active ingredient.

Liver fibrosis can be defined as excessive deposition of extracellular matrix due to chronic inflammation of the liver, and when such chronic liver inflammation persists, liver fibrosis develops into cirrhosis due to a decrease in the number of liver cells.

Representative cells involved in hepatic fibrosis include hepatic stellate cells, kuffer cells, and endothelial cells. Hepatic stellate cells are the major source of extracellular matrix production and are involved in the production of various extracellular matrix, including collagen. Cooper cells are present in the sinusoidal space in the liver and promote the hepatic fibrosis by affecting peripheral hepatocytes, endothelial cells, and hepatic stellate cells in the activated Cooper cells. Endothelial cells play an important role in the regulation of blood flow in the liver. In addition, endothelial cells are involved in the production of growth factors and extracellular matrix, which are involved in the proliferation of hepatic stellate cells by inflammation and hepatic fibrosis.

When hepatic fibrosis occurs, activated hepatic stellate cells grow exponentially, and these cells secrete a profibrogenic cytokine resulting in α-SMA, collagen, and metalloproteinase tissue inhibitor (TIMP) ≪ RTI ID = 0.0 > and / or < / RTI >

Representative intracellular signaling involved in hepatic fibrosis is mediated by TGF-beta, the most potent fibrotic cytokine of hepatic stellate cells. TGF-β produced from hepatic specimens binds to type I receptors and phosphorylates type I receptors, which phosphorylate them in contact with smad2 and smad3. The smad2 and smad3 conjugates bind to smad4 and then migrate into the nucleus and regulate transcription of the target gene. Smad7, on the other hand, acts on the type I receptor to interfere with the phosphorylation of smad2 and smad3 and to inhibit liver fibrosis by interfering with smad signaling by TGF-β.

Until now, liver fibrosis has been known to be irreversible, but recently it has been reported that the liver fibrosis process is reversible through clinical and experimental studies, and that the degree of liver fibrosis can be controlled. Therefore, studies on the inhibition of hepatic fibrosis progression through activated hepatic stellate cells have begun to attract attention.

On the other hand, AIMP1 peptides are produced when hypoxia and hypoglycemia occur, and exhibit agonist or antagonist effects in vascular endothelial cells and immune cells. On the other hand, the effects of AIMP1 peptides on liver disease and its mechanisms have not yet been studied or reported.

The present inventors have conducted studies to develop drugs having excellent effects for the prevention and treatment of liver diseases, and have found that AIMP1 peptides are effective for preventing and treating liver diseases, more specifically, for preventing and treating liver fibrosis and cirrhosis of AIMP1 peptides And the present invention was completed.

It is an object of the present invention to provide a composition for the prevention or treatment of liver disease comprising an AIMP1 peptide as an active ingredient.

It is an object of the present invention to provide a composition for improving liver function comprising an AIMP1 peptide as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating liver disease comprising an AIMP1 peptide as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating liver disease comprising AIMP1 peptide as an active ingredient.

Further, the present invention provides a health functional food composition for improving liver function comprising an AIMP1 peptide as an active ingredient.

The AIMP1 peptide according to the present invention has an excellent effect for the prevention and treatment of liver fibrosis and further cirrhosis by controlling the location of smad2 through activation of extracellular signal-regulated kinase (ERK) and inhibiting the expression of fibrosis factors such as collagen I Therefore, it can be usefully used as a pharmaceutical composition for preventing or treating liver disease and a health functional food.

FIG. 1 shows the results of confirming the inhibition of ERK activation and smad2 / 3 phosphorylation by AIMP1 peptide over time in LX2 cell line (human interstitial cell line) through Western blotting (left) and immunoprecipitation (right) Fig.
FIG. 2 is a graph showing the results of confirming the inhibition of ERK activation and smad2 / 3 phosphorylation by AIMP1 peptide and U0126 over time in LX2 cell line through Western blotting (left) and immunoprecipitation (right).
FIG. 3 is a graph showing the results of western blot analysis of changes in the concentrations of p-smad, smad2 / 3, smad4, a-tubulin and lamin A / C by AIMP1 peptide and TGF-β in the nucleus and cytoplasm of LX2 cell line .
FIG. 4 is a graph showing the results of immunofluorescence analysis of p-smad2 / 3, smad4 and their binding patterns by AIMP1 peptide and TGF-beta in the nucleus and cytoplasm of LX2 cell line, and the ratio of p-smad2 / 3 Fig.
FIG. 5 is a graph showing the results of Western blotting, RT-PCR (left) and luminescence enzyme activity analysis (right) of the activity of collagen I by AIMP1 peptide and TGF-β in LX2 cell line.
FIG. 6 is a graph showing the results of confirmation of collagen I activity by AIMP1 peptide, TGF-β and UO126 in LX2 cell line through Western blot (top), RT-PCR (left) and lysozyme activity assay (right).
7 is H. The liver tissue after intraperitoneal administration of the AIMP1 polypeptide for fibrosis mouse model liver induced a CCl ₄ hematoxylin and eosin (H & E) and masong-tree chromium dye (masson-trichrome staining) indicating the pathological analysis result of tissue .
8 is a diagram showing the effect of different concentrations following administration of the peptide to the AIMP1 fibrosis mouse model induced by CCl ₄ liver, performing biochemical analysis was performed to obtain blood samples.
9 is a diagram showing a result of following administration of the AIMP1 polypeptide and peptide AIMP1 scrambled fibrosis mouse model induced by CCl ₄ liver, perform the histopathological examination and immunohistochemistry.
10 is a diagram showing the after the administration of the AIMP1 polypeptide and peptide AIMP1 scrambled fibrosis mouse model induced by CCl ₄ liver perform immunohistochemical staining and evaluating the result.

The present invention provides a composition for preventing or treating liver disease comprising AIMP1 peptide as an active ingredient.

The composition includes a pharmaceutical composition or a health functional food composition.

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

The AIMP1 peptide, which is an active ingredient of the present invention, is composed of a total of 312 amino acids represented by SEQ ID NO: 1 and is secreted from various types of cells including prostate cancer, immunity and transformed cells. Secreted AIMP1 is monocyte / macrophage, Such as endothelial cells and fibroblasts.

The AIMP1 peptide according to the present invention may be a peptide having an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 2, among the amino acids represented by SEQ ID NO: 1, and functional equivalents thereof, but is not limited thereto.

The functional equivalent means a peptide having at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i.e., identity) with the amino acid sequence of SEQ ID NO: 2. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% Sequence homology to the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, Quot; peptide " refers to a peptide exhibiting substantially the same physiological activity as the AIMP1 peptide. Here, "substantially homogenous physiological activity" means that the AIMP1 peptide inhibits collagen I and inhibits hepatic fibrosis, further inhibits cirrhosis, and exhibits the original activity of AIMP1 peptide. The functional equivalents may result in some of the amino acid sequence of SEQ ID NO: 1 being the result of addition, substitution or deletion. The substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur containing amino acids (Cys, Met). Such functional equivalents also include variants in which a portion of the amino acids are deleted in the amino acid sequence of the AIMP1 peptide of the present invention.

The deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the AIMP1 peptide of the present invention. In addition, deletion of the amino acid is preferably located in a portion not directly involved in the physiological activity of the AIMP1 peptide. Also included are variants in which some amino acids are added at both ends or sequences of the amino acid sequence of the AIMP1 peptide. The scope of the functional equivalents of the present invention also includes derivatives in which some basic structures of the AIMP1 peptide according to the present invention and its chemical structure are modified while maintaining its physiological activity. This includes, for example, structural modifications to alter the stability, shelf stability, volatility, or solubility of the AIMP1 peptides of the present invention.

In this specification, sequence homology and homology are defined as the percentage of amino acid residues of the candidate sequence for each amino acid sequence after aligning the candidate sequence with the amino acid sequence of the AIMP1 peptide and introducing gaps. If necessary, conservative substitutions as part of sequence homology are not considered to obtain maximum percent sequence homology. Also, the N-terminal, C-terminal or internal stretch, deletion, or insertion of an amino acid sequence of an AIMP1 peptide is not interpreted as a sequence that affects sequence homology or homology. In addition, such homology can be determined by standard standard methods used to compare similar portions of amino acid sequences of two peptides.

The content of the AIMP1 peptide contained in the pharmaceutical composition of the present invention can be appropriately controlled depending on the method of using the therapeutic agent, the condition of the recipient, the kind of the disease, and the severity.

The AIMP1 peptide can be readily prepared by chemical synthesis known in the art (Creighton, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983).

The AIMP1 peptide regulates the location of smad2 by activating extracellular signal-regulated kinase (ERK) and inhibits the expression of fibrosis factors such as collagen I, thereby inhibiting liver fibrosis and further inhibiting cirrhosis have. Thus, the AIMP1 peptide can be used for the prevention or treatment of liver disease.

The liver disease includes liver fibrosis or liver cirrhosis, acute chronic hepatitis, fatty liver or liver cancer, preferably liver fibrosis or liver cirrhosis, more preferably, but not limited to, liver fibrosis.

The composition of the present invention, together with the AIMP1 peptide, may contain at least one known active ingredient having a preventive or therapeutic effect on liver diseases.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, calcium stearate, White sugar, etc. may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but not limited thereto.

The composition of the present invention can be administered orally or parenterally in various clinical formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, And suitable formulations known in the art are preferably those described in Remington ' s Pharmaceutical Science (Mack Publishing Company, Easton PA, recently). Examples of carriers, excipients and diluents that can be contained in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like May be included.

The preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. The parenteral administration can be carried out using an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection or intra-thoracic injection.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, And may be administered several times.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of liver diseases.

The term "prevent" in the present invention means any action that inhibits or delays liver disease by the administration of the pharmaceutical composition of the present invention. The term " treatment " Or any benefit that is beneficially altered.

The present invention also provides a health functional food composition for preventing or ameliorating liver disease comprising an AIMP1 peptide as an active ingredient.

Further, the present invention provides a health functional food composition for improving liver function comprising an AIMP1 peptide as an active ingredient.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

When the AIMP1 peptide of the present invention is used as a food additive, the AIMP1 peptide may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the AIMP1 peptide of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention, but is not limited thereto.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited thereto.

Example  One. AIMP1 Of peptide  Produce

6-46 amino acids of the AIMP1 protein sequence consisting of a total of 312 amino acids were synthesized and used in this study. The AIMP1 information can be found at http://www.ncbi.nlm.nih.gov/protein/Q12904.2 .

Experimental Example  One. Western Blot  And Immunoprecipitation method  through AIMP1 Of peptide  Confirming the inhibitory effect of p-smad2 on LX2 cell line (human interstitial cell line)

Western blot and immunoprecipitation were performed to confirm whether the AIMP1 peptide inhibited the migration of LX2 cells to ERK phosphorylation into p-smad2 nuclei. The LX2 cell line was infected with 2% fetal bovine serum (PBS) and 1% streptomycin (Sigma) supplemented with 1% fetal bovine serum / Dulbecco ' s modified Eagle ' s medium supplemented with penicillin.

LX2 cells were treated with AIMP1 and then subjected to Western blotting with time using an antibody against p-EPK, EPK and tubulin in a conventional manner. Next, LX2 cells (5 × 10 ⁴ cells) cultured as described above were cultured for 4 hrs and treated with AIMP1 peptide (5 μg / ml) for immunoprecipitation. The cells were then lysed with RIPA buffer and incubated in the freezer for 30 minutes. Cell lysates were prepared by centrifugation at 25,000 x g for 10 min at 4 ° C. Protein extracts (300 μg) were incubated with anti -smad2 / 3 antibody (1.5 μg; Cell Signaling Technology) at 4 ° C for 4 hours and then incubated with agarose at 4 ° C for 4 hours. The beads were washed three times with RIPA buffer without? -Mercaptoethanol. Samples were heated and loaded with 9% SDS-PAGE. Phosphorylation of smad2 / 3 was confirmed by pSer antibody (Abcam). The results are shown in Fig. At this time, tubulin was used as a control.

Next, LX2 cells (5 × 10 ⁴ cells) cultured as described above were cultured for 4 hrs and treated with AIMP1 peptide (5 μg / ml) for immunoprecipitation. The cells were then lysed with RIPA buffer and incubated in the freezer for 30 minutes. Cell lysates were prepared by centrifugation at 25,000 x g for 10 min at 4 ° C. Protein extracts (300 μg) were incubated with anti -smad2 / 3 antibody (1.5 μg; Cell Signaling Technology) at 4 ° C for 4 hours and then incubated with X2 agarose at 4 ° C for 4 hours. The beads were washed three times with RIPA buffer without? -Mercaptoethanol. Samples were heated and loaded with 9% SDS-PAGE. Phosphorylation of smad2 / 3 was confirmed by p-Ser antibody (Abcam). The results of the above experiment are shown in Fig.

As shown in Fig. 1, as the treatment time of AIMP1 peptide increased, it was confirmed that ERK was activated in LX2 cells and the phosphorylation of serine residue of smad2 / 3 was increased.

Next, the LX2 cell line was treated with the AIMP1 peptide and the ERK inhibitor U0126 (10 [mu] M), followed by Western blotting and immunoprecipitation by the same method as described above. The results are shown in Fig.

As shown in FIG. 2, it was confirmed that the phosphorylation of p-ERK and smad2 / 3 increased by the treatment of AIMP1 peptide was reduced by treatment with U0126, an ERK inhibitor.

Experimental Example  2. Western Blot  And Immunofluorescence  through AIMP1 Of peptide  Confirming the inhibition of migration from LX2 cell line to smad2 / 3 nucleus

Western blot analysis was performed on p-smad, smad2 / 3, smad4, a-tubulin and lamin A / C to confirm whether AIMP1 peptide induces ERK phosphorylation in LX2 cells. And p-smad2 / 3, smad4 were subjected to immunofluorescence.

More specifically, LX2 cell lines were treated with AIMP 1 peptide or TGF- [beta] and then centrifuged at 15,000 rpm for 20 minutes. The cells were fractionated into nuclear and cytoplasmic fractions using a nuclear / cytoplasmic fractionation kit (BioVision), and then primary antibodies against p-smad2, p-smad3, smad2 / 3, smad4, Western blotting was performed in a usual manner. At this time, α-tubulin was used as a marker for cytoplasm, and lamin A / C was used as a marker for nuclear, respectively. The results are shown in Fig.

As shown in Fig. 3, p-smad2 and p-smad3 were found at a high concentration in the nucleus in the LX2 cell line induced by fibrosis in TGF-β. When the AIMP1 peptide was treated together, activated p-smad2 and p-smad3 And the concentration in the cytoplasm was increased. This indicates that when fibrosis is induced by TGF-β, p-smad2 and p-smad3 are phosphorylated and migrated to the nucleus, indicating that the treatment of this activated smad2 / 3 to the nucleus is inhibited by AIMP1 peptide treatment.

Next, cells were cultured to carry out immunofluorescence on the LX2 cells (2 × 10 ⁴ cells) in 24-well plates round glass slip curve (LabShop VWR, Batavia, IL, USA) for 24 hours. The cells were then starvated with 0.5% FBS for 12 hours, treated with 5 μg / ml of AIMP1 peptide for 30 minutes, and then treated with 2 ng / ml of TGF-β for 1 hour. The cells were fixed with 4% formaldehyde for 10 minutes and washed with PBST buffer for 5 minutes. The fixed cells were incubated for an additional 30 minutes in a PBST solution containing 5% BSA to prevent nonspecific binding of the antibody and incubated for 1 hour at room temperature with Alexa 488 and 594-conjugated goat anti-mouse or anti-rabbit secondary antibody (Molecular Probes). Nuclear DNA was counterstained with DAPI (4 ', 6-diamidino-2-phenylindol) and observed for fluorescence on a Leica confocal microscope TCS SP5 (Leica Microsystems, Wetzlar, Germany). The results of the calculation of the ratio calculation for P-Smad2 / 3 in fluorescence and nuclei are shown in FIG.

As shown in FIG. 4, when the AIMP1 peptide was treated with TGF-β, the degree of expression of phosphorylated smad2 / 3 was decreased in the nucleus. This indicates that the AIMP1 peptide inhibits the migration of smad2 / 3 to the nucleus, thereby inhibiting the action of liver fibrosis, and furthermore, the action of cirrhosis.

Experimental Example  3. Western Blot , PT- PCR  And luminescence enzyme activity assay ( Luciferase  activity assay AIMP1  Confirmation of collagen I synthesis inhibition effect on peptide LX2 cell line

Western blotting, RT-PCR and luciferase activity assays were performed to determine whether the APMP1 peptide inhibited collagen I synthesis.

More specifically, the LX2 cell line was treated with TGF-beta to induce hepatic fibrosis, and then the AIMP1 peptide was treated at different concentrations. After separating whole proteins from the cell line, western blotting was performed by a conventional method using a primary antibody against collagen I and tubulin.

Next, total RNA was extracted from the LX2 cell line treated with the TGF-β and AIMP1 peptides at the concentration as described above for the RT-PCR using an RNA isolation kit (iNtRON Biotechnology, Inc., Seoul, Korea). CDNA was prepared by reverse transcription of the total RNA (0.5 μg), and the cDNA was amplified by using AccuPower GreenStar qPCR PreMix (SYBR-Green PreMix; Bioneer Corp., Daejeon, Korea) and StepOne real-time PCR system (Applied Biosystem) Real-time PCR was performed according to the manufacturer's method. The primers used were as follows; Collagen I mRNA (NM_000088.3) (forward, 5'-CCCCTGGAAAGAATGGAGATG-3 'and reverse, 5'-TCCAAACCACTGAAACCTCTG-3'); GAPDH (forward, 5'-CGAGATCCCTCCAAAATCAA-3 'and reverse, 5'-TGTGGTCATGAGTCCTTCCA-3').

In addition, LX2 cells (5 x 10 ⁴ cells) treated with the TGF-β and AIMP1 peptides in a concentration-dependent manner for the luminescent enzyme activity were seeded in a 12-well plate and incubated with 2% fetal bovine serum (FBS) And cultured with DMEM supplemented with streptomycin / penicillin. The LX2 cells were incubated with SBE4-Luc vector containing SMAD binding element (SBE) and Renilla luminescent enzyme using Lipofectamine 2000 (Invitrogen) for 12 hours. The cells were incubated with AIMP1 peptide for 10 minutes after incubation for 4 hours with 0.5% FBS, and then TGF-β was added, followed by another 20 hours. The luminescence enzyme activity was determined by Dual-Luciferase Reporter Assay System (Promega) and quantified using GloMax (Promega). The luminescence enzyme was normalized to Renilla luciferase activity. The results of the above experiment are shown in Fig.

As shown in FIG. 5, it was confirmed that the expression of collagen I increased by treatment with TGF-β was decreased as the treatment concentration of AIMP1 peptide increased in LX2 cell line.

Further, U0126 was further treated with LX2 cell line, and Western blotting, RT-PCR and luminescence enzyme activity assay were performed in the same manner as described above. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the expression of collagen I reduced by AIMP1 peptide was reduced by the treatment with U0126, an ERK inhibitor.

These results suggest that AIMP1 peptide inhibits hepatic fibrosis and cirrhosis by inhibiting collagen I expression, which is known as a cell proliferation and fibrosis marker, in hepatic stellate cells.

Experimental Example  4. Histopathological examination ( histopathology ) In the mouse model AIMP1  Of peptide Liver fibrosis  Verify mitigation effectiveness

It was performed for histopathological examination to the AIMP1 peptides to determine the impact on liver fibrosis in CCl ₄ induced mouse model. Male BALB / c mice (weighing 20-22 g) required for the animal experiment were obtained from Orient Bio. Animal Inc. (Seoul, Republic of Korea). Animal management and all experimental procedures were carried out in accordance with the approval and guidelines of Inha Institutional Animal Care and Use Committee (Inha IACUC). The animals were fed standard food and water ad libitum and incubated at 21 ° C for 12 hours in an arm / light cycle. Severe liver damage was induced by intraperitoneal injection of cone oil containing one dose (150 μl) 40% CCl ₄ twice a week for one month. Control mice were treated with only the same volume of cone oil, and mice in the experimental group received intraperitoneal injections twice a week for two weeks of scrambled AIMP1 peptide and AIMP1 peptide administration of CCl ₄ (scrambled AIMP1 peptide-treated mouse group was negative Lt; / RTI > All mice were anesthetized with ether after 4 weeks of treatment, and the liver was sacrificed and weighed and immediately fixed with 10% neutral buffered formalin for histopathology.

More specifically, the paraffin slice technique was used for liver samples fixed with 10% formaldehyde buffer for histopathological examination. The liver tissue was stained with hematocylin and eosin (H & E). More specifically, the tissue was stained with hematocrit for 3 minutes and washed, then stained with 0.5% eosin for an additional 3 minutes. To confirm the degree of liver fibrosis, the liver tissue samples were stained with Masson's trichrome method according to standard protocols. After dyeing, further washing with water was followed by dehydration of the slides continuously with 70, 95 and 100% ethanol and washing with xylene. The extent of liver injury was observed with an optical microscope (Olympus) and the results are shown in FIG.

As shown in FIG. 7, it was confirmed that the degree of liver damage and hepatic fibrosis was reduced by the treatment of AIMP1 peptide through the size of the stained region, and it was confirmed that AIMP1 peptide had CCl ₄ induced liver damage, .

Experimental Example  5. In a mouse model with biochemical parameters AIMP1 Of peptide Liver fibrosis  Verify mitigation effectiveness

For the AIMP1 peptides to determine the impact on liver fibrosis in CCl ₄ induced mouse model were carried out biochemical tests. Blood was collected from a mouse treated under the same conditions as Experimental Example 4 through a cardiac puncture. After the serum was separated from the collected blood samples, the liver damage markers including aspartate transaminase (ALT), alanine transaminase (ALT) and total bilirubin (ALT) Serum total bilirubin, D-bilirubin, albumin and blood urea nitrogen (BUN) were measured by the Green Cross Reference Lab (Seoul, Republic of Korea) The results are shown in Table 1 and FIG. 8

CCl ₄ Control group sAIMP1 AIMP1 AST 155.50 ± 23.46 232.17 ± 21.60 ^a 192.20 ± 17.68 169.33 ± 26.91 ^c ALT 57.83 + - 5.81 135.00 ± 12.07 ^b 123.60 ± 19.17 102.00 ± 9.16 ^c albumin 2.34 0.33 1.66 + 0.03 ^a 2.06 ± 0.26 2.60 ± 0.26 ^d BUN 38.00 + - 4.82 56.53 +/- 10.95 ^a 56.90 + - 4.47 28.82 ± 1.5 ^c

(Control vs. CCl ₄ , P <0.05; P <0.01; CCl ₄ vs. AIMP 1, P <0.05; P <0.01)

As shown in Table 1 and FIG. 8, it was confirmed that the concentration of serum total bilirubin, D-bilirubin, AST, ALT and BUN increased in the negative control group when the AIMP1 peptide was treated. Thus, it was confirmed that AIMP1 peptide has no hepatotoxicity in vivo and shows therapeutic effect of hepatic fibrosis.

Experimental Example  6. Immunohistochemistry ( immunohistochemistry ) In the mouse model AIMP1 Of peptide Liver fibrosis  Verify mitigation effectiveness

It was performed immunohistochemistry for the AIMP1 peptides to determine the impact on liver fibrosis in CCl ₄ induced mouse model. A sample of liver tissue was obtained from the mice treated under the same conditions as in Experimental Example 4 and placed in normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 hour. TGF-beta, collagen I, And cultured with SMA (Sigma-Aldrich) specific primary antibody. After removal of unbound primary antibody, the liver tissue was incubated with secondary antibody contained in 1.5% horse serum / PBS at room temperature for 1 hour. The liver tissue was visualized as an avidin-biotin peroxidase complex solution using ABC kit (Vector Laboratories). This was stained with a diaminobenzidine tetrahydrochloride substrate for 15 minutes and then the nuclei were contrasted stained with hematocylin separately. A photograph of the result is shown in Fig. 9, and the result is shown in a graph in Fig.

As shown in FIG. 9 and FIG. 10, it was confirmed that the AIMP1 peptide decreased the expression level of TGF-beta, collagen I and alpha-SMA, which are the collagen fibrous markers in mouse liver tissue. This confirms that the AIMP1 peptide inhibits liver fibrosis and further inhibits cirrhosis.

The results were expressed as means ± SD and analyzed by ANOVA and unpaired Student's t-test. AP-value ≤0.05 represents statistically significant results and statistical calculations were performed using SPSS software from a system operating in Windows (version 10.0; SPSS, Inc., Chicago, IL, USA).

Formulation example  1. Preparation of pharmaceutical preparations

One. Sanje  Produce

AIMP1 peptide 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

AIMP1 peptide 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

AIMP1 peptide 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

AIMP1 peptide 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Liquid  Produce

AIMP1 peptide 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Manufacture of food preparation

1. Manufacture of Health Functional Foods

AIMP1 peptide 100 mg

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Health functional drinks  Produce

AIMP1 peptide 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered, sterilized in a sterilized 2 L container, sealed, &Lt; / RTI &gt; Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Peptide and composition containing the same for treating or          preventing of liver disease <130> 195p <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 312 <212> PRT <213> AIMP1 <400> 1 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala   1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu              20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu          35 40 45 Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln      50 55 60 Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Pro Phe  65 70 75 80 Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val                  85 90 95 Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln             100 105 110 Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu         115 120 125 Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser     130 135 140 Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly 145 150 155 160 Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val                 165 170 175 Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly             180 185 190 Leu Val Asn His Val Leu Glu Gln Met Gln Asn Arg Met Val Ile         195 200 205 Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln     210 215 220 Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala 225 230 235 240 Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe                 245 250 255 Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu             260 265 270 Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr         275 280 285 Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln     290 295 300 Thr Met Ser Asn Ser Gly Ile Lys 305 310 <210> 2 <211> 41 <212> PRT <213> AIMP1 <400> 2 Val Leu Lys Arg Leu Glu Gln Lys Gly Ala Glu Ala Asp Gln Ile Ile   1 5 10 15 Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu Lys Glu Lys Ala Ile Leu              20 25 30 Gln Ala Thr Leu Arg Glu Glu Lys Lys          35 40

Claims (7)

A pharmaceutical composition for preventing or treating liver disease comprising an AIMP1 (ARS-interacting multi-functional protein 1) peptide as an active ingredient.
The method according to claim 1,
Wherein the AIMP1 peptide comprises the amino acid sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the AIMP1 peptide is an amino acid sequence represented by SEQ ID NO: 2. 2. A pharmaceutical composition for preventing or treating liver disease,
The method according to claim 1,
Wherein the liver disease is at least one selected from the group consisting of liver fibrosis or liver cirrhosis, acute chronic hepatitis, fatty liver and liver cancer.
The method according to claim 1,
Wherein said AIMP1 peptide inhibits the expression of collagen I.
A health functional food composition for preventing or ameliorating liver disease comprising AIMP1 peptide as an active ingredient.
A health functional food composition for improving liver function comprising AIMP1 peptide as an active ingredient.

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