KR101870012B1 - Composition comprising expression regulator for preventing or treating non-alcoholic fatty liver - Google Patents
Composition comprising expression regulator for preventing or treating non-alcoholic fatty liver Download PDFInfo
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- KR101870012B1 KR101870012B1 KR1020150097689A KR20150097689A KR101870012B1 KR 101870012 B1 KR101870012 B1 KR 101870012B1 KR 1020150097689 A KR1020150097689 A KR 1020150097689A KR 20150097689 A KR20150097689 A KR 20150097689A KR 101870012 B1 KR101870012 B1 KR 101870012B1
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Abstract
The present invention relates to a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver comprising an agonist of TIS21 gene as an active ingredient, a method for screening a therapeutic agent, or a health food. When the TIS21 gene is inhibited or deficient, Can be effectively used for the treatment of non-alcoholic fatty liver, and the candidate drug that promotes the expression of the TIS21 gene can be screened by examining the expression level of the TIS21 gene. Therefore, the pharmaceutical composition and the health food can be effectively used for the treatment of non- . ≪ / RTI >
Description
The present invention relates to a composition for preventing or treating non-alcoholic fatty liver.
Fatty liver disease is classified into alcoholic fatty liver disease caused by excessive alcohol consumption and nonalcoholic fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) is a disorder in which liver fat accumulates in the liver regardless of alcohol consumption, including nonalcoholic steatosis and nonalcoholic steatohepatitis.
Nonalcoholic lipidemia is classified into large bovine lipid and small bovine lipid according to the size of lipid droplets appearing in hepatocytes. Most nonalcoholic lipidemia is giant bacillary. In the early stages of development, several small fat droplets appear in hepatocytes, but as fat droplets grow larger, gigantic vesicles are formed. Giant vesicles push nuclei to one side of cell center and signet ring shape. Nonalcoholic fatty liver disease progresses slowly and chronically, so there is no general symptom, but liver function is gradually increasing, as the activity of alkaline phosphatase (ALP) or aminotransferase, which is a measure of liver function, is elevated. It gets worse.
In nonalcoholic fatty liver disease, fatty acids that migrate from adipose tissue to the liver are increased due to deficiencies in lipid metabolism caused by imbalance in energy expenditure. In addition, peroxisome proliferator-activated receptors-α (PPAR-α), a PPAR-γ and a sterol regulatory factor-coupled transcriptional regulator, which is a receptor molecule that regulates the action of enzymes necessary for the oxidation or synthesis of fatty acids A decrease in factor 1 (Sterol regulatory element-
Hepatocyte necrosis occurs in the liver accompanied by extensive hepatitis, and stellate cells become activated, leading to fibrosis. Especially, perisinusoidal fibrosis appears at the end of the hepatic vein. Therefore, hepatitis is improved by removing the cause of accumulation of fat, but if left untreated liver fibrosis or cirrhosis. Fibrosis or cirrhosis of the liver occurs in 15 to 50% of patients with nonalcoholic steatohepatitis, and 30% of patients with fibrosis show cirrhosis 10 years later.
Nonalcoholic lipidemia is highly associated with obesity or diabetes. Since both obesity and diabetic patients have been increasing rapidly in recent years, nonalcoholic fatty fat is becoming an increasingly important disease in modern society. The incidence of nonalcoholic lipidemia increases by 10-15% in people with normal weight, but by 80% in people with overweight. In particular, the prevalence of obesity over the age of 20 increased from 26.3% in 1998 to 31.5% in 2005 and increased from 29.1% to 34.8% over 30 years.
Nonalcoholic lipidemia is usually recovered by adequate intake of nutrients and control of ductal metabolic diseases. However, when left untreated, it progresses to hepatic fibrosis or cirrhosis following hepatitis, resulting in poor prognosis. In order to prevent the progression of nonalcoholic fatty liver to cirrhosis, it is necessary to prevent the accumulation of liver fat. However, in order to prevent fat accumulation in the liver, it is necessary to reduce calorie intake or increase calorie consumption by exercise. Is not presented.
Therefore, it is required to develop an effective treatment method for treating non-alcoholic fatty liver disease.
Accordingly, it is an object of the present invention to provide a composition for preventing or treating nonalcoholic fatty liver.
Another object of the present invention is to provide a method for screening a non-alcoholic fatty liver therapeutic agent.
Another object of the present invention is to provide a health food for preventing or ameliorating nonalcoholic fatty liver.
In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver comprising an expression regulator of TIS21 gene as an active ingredient.
In order to accomplish the above-mentioned further object, the present invention provides a method for screening a therapeutic agent for non-alcoholic fatty liver disease comprising the step of selecting a candidate drug that promotes the expression of TIS21 gene.
In order to achieve the above-mentioned further object, the present invention provides a health food for preventing or ameliorating non-alcoholic fatty liver comprising an expression regulator of TIS21 gene as an active ingredient.
The present invention relates to a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver comprising an agent for regulating the expression of TIS21 gene as an active ingredient, a method for screening a therapeutic agent or a health food. When the TIS21 gene is inhibited or deficient, Can be effectively used for the treatment of non-alcoholic fatty liver disease.
FIG. 1 shows the result of confirming fat deposition through oil-red O staining in the liver of a mouse in which TIS21 gene was lost,
FIG. 2 shows the results of confirming expression of transcription factors involved in fatty acid synthesis in normal mice fasted for 48 hours and mice lacking TIS21 gene.
FIG. 3 shows the results of confirming expression of an enzyme involved in fatty acid synthesis in a normal mouse fasted for 48 hours and a mouse in which the TIS21 gene was lost.
Hereinafter, the present invention will be described in detail.
The inventors of the present invention have completed the present invention upon confirming that liver fat deposition is significantly increased when the TIS21 gene is lost during research on the treatment of nonalcoholic fatty liver disease.
The TIS21 gene is a gene encoding the TIS21 protein represented by SEQ ID NO: 1.
The sequence of the TIS21 protein is shown in Table 1 below:
Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease comprising an expression-regulating agent for TIS21 gene as an active ingredient.
The expression regulator is an expression promoter.
The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.
Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.
In addition, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to conventional methods .
In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations 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 sucrose), lactose, gelatin, and the like.
In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .
The amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, body weight, and disease, but may be 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg, once or several times per day.
In addition, the dosage of the pharmaceutical composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.
The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. And can be administered orally or parenterally (for example, intravenously, subcutaneously, or intraperitoneally) depending on the intended method.
In addition, the present invention provides a screening method for a non-alcoholic fatty liver therapeutic agent comprising the step of selecting a candidate drug that promotes the expression of the TIS21 gene.
The screening method comprises the steps of: treating a candidate drug with a non-alcoholic fatty liver suspected sample; And analyzing the expression of the TIS21 gene in the sample treated with the candidate drug.
In addition, the present invention provides a health food for preventing or ameliorating a non-alcoholic fatty liver comprising the TIS21 gene expression regulator as an active ingredient.
The expression regulator is an expression promoter.
The food composition may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food may be used in combination with other food or food additives other than the expression control agent as an active ingredient, . The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
The effective dose of the expression control agent contained in the food composition may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes , It is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.
There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.
< Example 1> Frozen section (Frozen section) preparation
First, liver was extracted from normal C57BL6-TIS21WT and C57BL6-TIS21KO female mice deficient in TIS21 gene, and then OCT-Compound (Tissue-Tek) And the tissue was frozen.
The frozen tissue was cut into 8 μm thickness with frozen section micromachine and attached to a slide glass for oil-red o staining.
< Example 2> Confirmation of fat production in extracted liver
Oil-Red O staining was performed to confirm the fat production in the extracted liver.
First, the frozen sectioned tissue prepared in Example 1 was dried at room temperature for 5 minutes and fixed in 10% formalin for 10 minutes.
The fixed tissue was washed with 60% isopropyl alcohol and then stained with Sigma's Oil-red O reagent for 10 minutes.
Ten minutes later, the cells were rinsed three times with sterilized distilled water, stained with a hematoxylin reagent from Sigma, and then sealed.
Observation of the encapsulated tissue with an optical microscope revealed that fat deposition was significantly increased in the liver of C57BL6-TIS21KO mice deficient in TIS21 gene as shown in Fig.
< Example 3> Expression of transcription factors leading to fatty acid synthesis after abortion
Changes in the expression of transcription factors leading to the fatty acid synthesis after stopping feeding were examined from normal mouse and TIS21 deficient mice that were fasted for 48 hours.
For this, a quantitative real-time polymerase chain reaction was performed.
First, RNAiso plus (Takara, Japan) solution and chloroform were suspended in the hepatic tissue of the rapidly-quenched mouse, and the suspension was centrifuged, and the supernatant was extracted by insolubilization with an equal amount of isopropanol.
The extracted total RNA (1 μg) was converted into cDNA using HelixCript ™ 1 st -Strand cDNA Synthesis (Nano Helix, Korea) and the oligo-dT18 primer shown in Table 2 below.
The sequence of the converted cDNA is shown in Table 3 below and is represented by SEQ ID NO: 12.
Quantitative real-time polymerase chain reaction was performed using RealHelix ™ qPCR kit (Nanohelix, Korea) and SYBR Green (SYBR Green) using 40 ng of the transformed cDNA and CFX96 Touch ™ Real Time Detection System (Bio-Rad) Respectively. Actin was used for the expression level correction.
As a result, as shown in FIG. 2, PPAR-γ and peroxisome proliferator-activated receptor Gamma Coactivator-1alpha in C57BL6-TIS21KO mice deficient in TIS21 gene compared with normal mice after fasting , PGC1-α) was significantly increased.
In addition, the expression of PPAR-γ and PGC1-α in C57BL6-TIS21KO mice deficient in TIS21 gene after fasting was significantly increased.
Thus, unlike normal lipid metabolism, which promotes fatty acid oxidation when the feeding is interrupted, the expression of transcription factors such as PPAR-γ and PGC1-α, which rapidly store fatty acids to prevent lipolysis in the absence of TIS21, is increased Respectively.
< Example 4> Expression of enzymes leading to fatty acid synthesis after abortion
Changes in the enzymes responsible for the fatty acid synthesis by feeding were determined from normal and TIS21 - deficient mice that were fasted for 48 hours.
For this, quantitative real-time PCR was performed according to the method of Example 3 above.
Similar to the results of Example 3, unlike the normal lipid metabolism that promotes fatty acid oxidation when the feeding is stopped as shown in FIG. 3, when TIS21 is deficient, stearoyl CoA desaturase (SCD-2 ) And acetyl-Coenzyme A carboxylase alpha (ACACA) were increased, thus confirming that lipolysis was inhibited.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition of the expression regulator for preventing or 처리 non-alcoholic fatty liver <130> ADP-2015-0068 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 158 <212> PRT <213> Homo sapiens <400> 1 Met Ser His Gly Lys Arg Thr Asp Met Leu Pro Glu Ile Ala Ala Ala 1 5 10 15 Val Gly Phe Leu Ser Ser Leu Leu Arg Thr Arg Gly Cys Ala Ser Glu 20 25 30 Gln Arg Leu Lys Val Phe Ser Arg Ala Leu Gln Asp Ala Leu Thr Asp 35 40 45 His Tyr Lys His His Trp Phe Pro Glu Lys Pro Ser Lys Gly Ser Gly 50 55 60 Tyr Arg Cys Ile Arg Ile Asn His Lys Met Asp Pro Ile Ile Ser Lys 65 70 75 80 Val Ala Ser Gln Ile Gly Leu Ser Gln Pro Gln Leu His Arg Leu Leu 85 90 95 Pro Ser Glu Leu Thr Leu Trp Val Asp Pro Tyr Glu Val Ser Tyr Arg 100 105 110 Ile Gly Glu Asp Gly Ser Ile Cys Val Leu Tyr Glu Glu Ala Pro Val 115 120 125 Ala Ala Ser Tyr Gly Leu Leu Thr Cys Lys Asn Gln Met Met Leu Gly 130 135 140 Arg Ser Ser Pro Ser Lys Asn Tyr Val Met Ala Val Ser Ser 145 150 155 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mPPARa-Forward primer <400> 2 agggttgagc tcagtcagga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mPPARa-Reverse primer <400> 3 ggtcacctac gagtggcatt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mPPARg-Forward primer <400> 4 ttcagaagtg ccttgctgtg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mPPARg-Reverse primer <400> 5 gctggtcgat atcactggag a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mPgc1a-Forward primer <400> 6 atgtgtcgcc ttcttgctct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mPgc1a-Reverse primer <400> 7 cggtgtctgt agtggcttga 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mSCD2-Forward primer <400> 8 gtttgaaagc tttgggtagg g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mSCD2-Reverse primer <400> 9 aaggccctaa agcctctctc t 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mACACA-Forward primer <400> 10 gcctcttcct gacaaacgag 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mACACA-Reverse primer <400> 11 tgactgccga aacatctctg 20 <210> 12 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> TIS21 cDNA <400> 12 atgagccacg ggaagagaac cgacatgctc ccggagatcg ccgccgccgt gggtttcctc 60 tccagtctcc tgaggactcg gggctgcgcg agcgagcaga gactcaaggt tttcagtagg 120 gcgctccagg acgcactgac cgatcattac aaacaccact ggtttccaga aaaaccatcc 180 aaaggttctg gctatcgctg tatccgtatc aaccacaaga tggaccccat catcagcaag 240 gtggccagcc agattggact cagccagccc cagctgcacc ggctgctgcc cagtgagctg 300 accctgtggg ttgatcccta tgaagtgtct taccgcatcg gggaggatgg ctccatctgt 360 gtcctgtatg aggaggcccc ggtggctgcc tcctatgggc tcctcacctg caagaaccag 420 atgatgctgg gcaggagcag cccctcgaag aactatgtga tggccgtctc cagctag 477
Claims (5)
Analyzing the expression of the TIS21 gene in the sample treated with the candidate drug; And
Selecting a candidate drug that promotes the expression of the TIS21 gene.
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| Diabetes 2014;63:1870-1880 |
| Exp Mol Med. 2013 May; 45(5): e25. |
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