KR101869308B1 - SREBP-1 decoy oligodeoxynucleotide and pharmaceutical composition for preventing or treating fatty liver disease containing the same as active ingredient - Google Patents

Abstract

The present invention relates to an SREBP-1 decoy oligodeoxynucleotide and a composition containing the same as an active ingredient for preventing or treating fatty liver disease. More specifically, the SREBP-1 decoy oligodeoxynucleotide of the present invention inhibits a transcription factor action of SREBP-1 which is a transcription factor to control expression of genes associated with biosynthesis of lipid and to inhibit accumulation of fat in the liver when the SREBP-1 decoy oligodeoxynucleotide is processed on an animal model with an induced fatty liver, reduce alanine aminotransferase in blood, aspartic acid aminotransferase, total cholesterol, and triglyceride indices, and reduce liver and blood infection indices. Therefore, the SREBP-1 decoy oligodeoxynucleotide can be used as a pharmaceutical composition to prevent or treat fatty liver disease.

Description

TECHNICAL FIELD The present invention relates to a SREBP-1 decoy oligodeoxynucleotide and a pharmaceutical composition for preventing or treating fatty liver disease containing the same as an effective ingredient. The present invention relates to a SREBP-1 decoy oligodeoxynucleotide,

The present invention relates to a SREBP-1 decoy oligodeoxynucleotide and a pharmaceutical composition for preventing or treating fatty liver disease which comprises the SREBP-1 decoy oligodeoxynucleotide as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for inhibiting transcription factor SREBP- SREBP-1 decoy oligodeoxynucleotide, which exhibits an effect of suppressing fat accumulation in liver tissue and an effect of reducing inflammation reaction in a liver animal model by controlling expression of genes related to biosynthesis, and prevention of fatty liver disease containing it as an active ingredient Or pharmaceutical compositions for therapeutic use.

Gene therapy is a very effective method for the treatment and prevention of various diseases by specifically controlling the expression of a specific gene by regulating the gene expression system and delivering it to the disease site. In particular, the method of administering decoy oligodeoxynucleotide (decoy ODN) to the transcription factor in gene therapy has recently been attempted as a tool to suppress the expression of a specific gene by inhibiting the action of the transcription factor. Theoretically, the strategy to inhibit the activity of these transcription factors implies injecting the Decoy ODN into the cell, which binds to the transcription factor of interest in the cell. That is, the DNA binding site of the transcription factor is bound by the decoy, so that the transcription factor can not bind to the promoter of the target gene, and the expression of the specific gene is reduced by interrupting the activity of the transcription factor.

Fatty liver is a state of accumulation of fat in hepatocytes. The proportion of fat in normal liver is about 5%. The state in which more fat is accumulated is called fatty liver. When the fatty liver becomes worse and fat mass in hepatocyte becomes larger, important components of the cells including nucleus are pushed to one side, the function of the hepatocyte is lowered, and the expanded hepatocytes due to the accumulated fat in the cells pressurize the microvascular and lymphatic vessels between the hepatocytes Resulting in obstacles to the circulation of blood and lymph in the liver. In this case, hepatocytes can not receive oxygen and nutrient supply properly, and liver function is deteriorated.

Fatty liver can be divided into alcoholic fatty liver and nonalcoholic fatty liver. Alcoholic fatty liver is caused by alcohol. The more you drink, the more likely it is to develop, and if you consume it on a continuous basis, the liver will not be able to metabolize alcohol, leading to more fatty liver disease. It also occurs when the nutritional status is bad. Some alcoholic fatty liver can progress to alcoholic hepatitis and cirrhosis and die.

Nonalcoholic fatty liver disease occurs when a large amount of fat is consumed irrespective of alcohol, and fat is not synthesized or released in the liver. Nonalcoholic fatty liver is caused by obesity, hyperlipidemia, diabetes and the like. It may also be caused by medication such as steroids. Nonalcoholic fatty liver may progress to nonalcoholic fatty liver disease.

Alcoholic fatty liver disease should be treated this week. In nonalcoholic fatty liver, maintaining healthy lifestyle, such as diet control and weight loss by proper exercise, is helpful in the treatment.

One of the proteins that stimulate the synthesis of fatty acids in the liver is the Sterol regulatory element-binding protein (SREBP). This gene is a lipid homeostasis regulatory factor that plays a role in increasing the gene transcription activity of enzymes important in fatty acid biosynthetic pathways and has three forms of SREBP-1a, SREBP-1c and SREBP-2. While SREBP-1c mainly induces fatty acid synthesis and glucose metabolism of insulin, SREBP-2 is relatively involved in cholesterol synthesis. In addition, SREBP-1a is associated with both paths. SREBP-1, involved in the synthesis of fatty acids, contains SRE-1, a DNA binding site that binds for transcription in the nucleus, and an E-box motif.

 Fatty liver has been found to cause and serious illness, but fatty liver patients who need to improve their symptoms through dietary control and exercise often fail to practice it. Therefore, there is a need for the development of an effective fatty liver therapeutic drug.

Korean Patent Laid-Open Publication No. 2016-0089000 discloses a novel synthetic oligodeoxynucleotide that simultaneously inhibits DNA and RNA, and a pharmaceutical composition for preventing and treating fibrosis disease containing the same as an effective ingredient. Korean Patent No. 0874798 Discloses a circular dumbbell decoy oligonucleotide comprising a DNA binding site. In Korean Patent No. 1161118, a novel Chi decoy oligodeoxynucleotide and a pharmaceutical composition for preventing and treating renal failure containing the same as an active ingredient are disclosed. It has not been disclosed about the SREBP-1 decoy oligodeoxynucleotide of the present invention and a pharmaceutical composition for preventing or treating fatty liver disease containing it as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating fatty liver disease comprising SREBP-1 decenoyldeoxynucleotide and an effective ingredient thereof. The present invention provides a pharmaceutical composition for preventing or treating SREBP-1 The present inventors have completed the present invention by confirming that decoy oligodeoxynucleotide reduces hepatic fat content in liver model animals induced by fatty liver and decreases liver function indicator, blood total cholesterol, triglyceride and inflammation index.

In order to solve the above-mentioned problems, the present invention is characterized in that SRE-1 oligodeoxynucleotide of SEQ ID NO: 1 and E-box motif oligodeoxynucleotide of SEQ ID NO: 2 are covalently linked to form DNA of two loop structures and transcription factors 1 < / RTI > decoy oligodeoxynucleotide having a stem structure capable of binding to the binding site.

In addition, the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease comprising dumbbell-shaped SREBP-1 decoy oligodeoxynucleotide as an active ingredient.

The present invention relates to a SREBP-1 decoy oligodeoxynucleotide and a pharmaceutical composition for preventing or treating fatty liver disease comprising the same as an active ingredient, wherein the SREBP-1 decoy oligodeoxynucleotide of the present invention is used in an animal model in which fatty liver is induced It has the effect of suppressing liver fat content, and has the effect of reducing liver function indicator substance, total cholesterol, triglyceride and inflammation index in blood. Therefore, the composition of the present invention comprising SREBP-1 decoy oligodeoxynucleotide as an active ingredient may be used as a functional material for prevention and treatment of fatty liver disease.

Figure 1 shows the structure of SREBP-1 decoy oligodeoxynucleotides. 1 shows the structure of SREBP-1 decoy oligodeoxynucleotides containing SRE-1 and E-box motif moieties which are sites for binding to DNA.
FIG. 2 shows the results of confirming the damage recovery and fat accumulation inhibition by hepatic tissue following SREBP-1 decoy oligodeoxynucleotide administration using an animal model. NC was a negative control of the normal diet, SREBP-1 decoy ODN was treated with SREBP-1 decoy oligodeoxynucleotide in the general dietary group, HFD + ODN was treated with the high-fat diet group and oligodeoxynucleotide negative control group, HFD + SREBP-1 Decoy ODN is a group treated with SREBP-1 decoy oligodeoxynucleotide in a high-fat diet.
FIG. 3 shows the results of immunohistochemical staining of the expression level of cholesterol synthase (HMG-CoA reductase, HMGCR) in liver tissues upon administration of SREBP-1 decoy oligodeoxynucleotide in an animal model. NC was a negative control of the normal diet, SREBP-1 decoy ODN was treated with SREBP-1 decoy oligodeoxynucleotide in the general dietary group, HFD + ODN was treated with the high-fat diet group and oligodeoxynucleotide negative control group, HFD + SREBP-1 Decoy ODN is a group treated with SREBP-1 decoy oligodeoxynucleotide in a high-fat diet.
FIG. 4 shows the result of confirming the expression level of the gene related to lipid biosynthesis in liver tissue by administration of SREBP-1 decoy oligodeoxynucleotide using an animal model. NC is a negative control with no treatment, and ODN is an oligodeoxynucleotide negative control.
FIG. 5 shows the results of the serum level of IL-6 expression, which is an inflammatory factor upon administration of SREBP-1 decoy oligodeoxynucleotide in an animal model. NC was a negative control of the normal diet, SREBP-1 decoy ODN was treated with SREBP-1 decoy oligodeoxynucleotide in the general dietary group, HFD + ODN was treated with the high-fat diet group and oligodeoxynucleotide negative control group, HFD + SREBP-1 Decoy ODN is a group treated with SREBP-1 decoy oligodeoxynucleotide in a high-fat diet. * Indicates that the level of IL-6 expression was significantly increased in the HFD group treated with the high-fat diet compared to the normal diet group, which means that p <0.005. † indicates that when SLEBP-1 decoy ODN was administered compared to ODN, the expression level of IL-6 decreased statistically when the high-fat diet method was applied, and † indicates that p <0.005.
FIG. 6 shows the results of the mRNA expression of TNF-.alpha., IL-1beta, and IL-6, which are inflammatory factors in the animal model, according to administration of SREBP-1 decoy oligodeoxynucleotide. NC was a negative control of the normal diet, SREBP-1 decoy ODN was treated with SREBP-1 decoy oligodeoxynucleotide in the general dietary group, HFD + Con ODN was treated with high-fat diet and oligodeoxynucleotide negative control And HFD + SREBP-1 decoy ODN were treated with SREBP-1 decoy oligodeoxynucleotide in a high-fat diet.

In order to accomplish the object of the present invention, the present invention relates to an oligonucleotide comprising an SRE-1 oligodeoxynucleotide of SEQ ID NO: 1 and an E-box motif oligodeoxynucleotide of SEQ ID NO: 2 linked by a covalent bond, The present invention provides a circular dumbbell-shaped SREBP-1 decoy oligodeoxynucleotide consisting of a stem structure capable of binding to a DNA binding site of a dendritic cell.

The dumbbell-type SREBP-1 decoy oligodeoxynucleotide comprises an SRE-1 oligodeoxynucleotide (SRE-1 ODN) comprising the nucleotide sequence of SEQ ID NO: 1 and an E-box motif oligodeoxynucleotide comprising the nucleotide sequence of SEQ ID NO: (E-box motif ODN) are connected by a covalent bond and consist of two loop structures and a stem structure. The stem structure is the site that can bind to the DNA binding site of the transcription factor.

SRE-1 ODN: 5'-GAATTCGTGTGGGGTGATTGAAAACAATCACCCCACAC-3 '(SEQ ID NO: 1)

E-box motif ODN: 5'-GAATTCGTATCACGTGATGAAAACATCACGTGATAC-3 '(SEQ ID NO: 2)

The present invention also provides a pharmaceutical composition for the prevention or treatment of fatty liver disease comprising the above-mentioned dumbbell-shaped SREBP-1 decenoyldeoxynucleotide as an active ingredient.

The fatty liver disease includes alcoholic fatty liver disease and non-alcoholic fatty liver disease, preferably non-alcoholic fatty liver disease. The non-alcoholic fatty liver disease is characterized in that it is any one selected from the group consisting of simple fatty liver, nutritive fatty liver, starvation fatty liver, obese liver fat, diabetic fatty liver, fatty liver, liver fibrosis and cirrhosis.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned dumbbell-shaped SREBP-1 decoy oligodeoxynucleotide. The composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method, but is not limited thereto . Examples of carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 ) Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use 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 have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Witepsol, macrogol, Tween 61, cacao paper, laurin, glycerogelatin and the like may be used as a base for suppositories. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The appropriate dosage of the pharmaceutical composition according to the present invention may vary depending on such factors as the formulation method, the administration method, the patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .

The concentration of the active ingredient contained in the composition of the present invention may be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

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

Example  1. Decoy Oligodeoxynucleotides  making

The base sequence of decoy oligodeoxynucleotide (hereinafter, Decoy ODN) used in Example 1 is as follows.

SREBP-1 Decoy ODN:

SRE-1 ODN: 5'-GAATTCGTGTGGGGTGATTGAAAACAATCACCCCACAC-3 '(SEQ ID NO: 1)

E-box motif ODN: 5'-GAATTCGTATCACGTGATGAAAACATCACGTGATAC-3 '(SEQ ID NO: 2)

Con ODN:

5'-GAATTCCAGGTACGGCAAAAAATTGCCGTACCTG-3 '(SEQ ID NO: 3)

The prepared oligodeoxynucleotide (ODN) was denatured at 95 ° C for 3 minutes and then annealed while lowering the temperature to 80 to 25 ° C. The annealed ODN was ligated with T4 ligase for 16-18 hours to make spherical Con ODN and SREBP-1 decoy ODN. The structure of the ligated spherical dumbbell-shaped SREBP-1 decoy ODN is shown in Fig. A ligated decoy ODN was identified in a 15% non-denaturating gel.

Example  2. Production of fat-derived animal model

High fat diet (HFD: 21% fat and 1% cholesterol) was consumed for 12 weeks in order to produce an animal model that induced fatty liver. During the high fat diet, 10 μg of decoy ODN was injected through the tail vein of the mouse. The general diet group (NC) was fed with commercial rodent feed, and decoy ODN was injected under the same conditions as above. After 12 weeks, liver tissue was removed or blood was collected and used in the experiment.

Example  3. Identification of liver tissue damage and fat accumulation in animal models

The liver obtained from the animal model of Example 2 was prepared with paraffin sections and H & E staining was performed to confirm liver damage and fat accumulation. As shown in FIG. 2, the liver of the liver treated with the high-fat diet and Con ODN showed a higher level of tissue damage and fat accumulation than the normal diet group. However, the SREBP-1 decoy ODN- The degree of liver damage and fat accumulation were significantly decreased in the liver tissues compared with the Con ODN - injected group. Therefore, it was confirmed that SREBP-1 decoy ODN inhibits liver damage and fat accumulation induced by high fat diet.

Example  4. Blood analysis of animal models

ALT, alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglyceride were measured in an animal model of Example 2 using a commercial assay kit. As a result, as shown in Table 1, the activities of plasma alanine aminotransferase and aspartic acid aminotransferase, which are liver function indicators, were significantly increased in the high-fat diet group compared with the normal diets, and the high fat diet group had SREBP-1 decoy When ODN was injected, the activities of alanine aminotransferase and aspartic acid aminotransferase were significantly lower than those of Con ODN injected group. In addition, the total cholesterol and triglyceride levels were significantly increased in the high-fat diet group compared to the normal diet group, and the SREBP-1 decoy ODN-infused group had higher total cholesterol and triglyceride Respectively. Therefore, SREBP-1 decoy ODN can prevent liver function deterioration induced by high fat diet.

Blood analysis of animal models NC SREBP-1
Decoy ODN HFD +
Con ODN HFD +
SREBP-1 Decoy ODN AST
(U / L) 109.33 + - 4.2 111.5 ± 6.7 261.25 ± 35.2 245.13 ± 27.2 ALT
(U / L) 81.77 ± 2.9 97.31 ± 5.2 245.73 + - 23.8 238.16 ± 19.6 Total cholesterol
(Mg / dl) 108.5 ± 5.2 125.41 ± 3.5 382.32 ± 45.1 267.34 ± 33.4 Triglyceride
(Mg / dl) 112.75 + - 4.2 111.2 ± 2.9 419.2 ± 24.3 287.11 + - 22.5

Example  5. Expression of cholesterol biosynthesis related genes in animal models

Expression of cholesterol synthase (HMG-CoA reductase, HMGCR), a gene involved in biosynthesis of cholesterol, was observed in the animal model of Example 2. [ Immunochemical staining was performed to confirm the expression of HMGCR protein in liver tissue, and the results are shown in Fig. The expression of HMGCR protein was significantly increased when Con ODN was injected into the high-fat diet group compared with the normal diet group. When the SREBP-1 decoy ODN was injected into the high fat diet, HMGCR protein expression was decreased compared to the Con ODN- The expression of HMGCR protein in the group injected with SREBP-1 decoy ODN was similar to that of the normal diet group. Thus, treatment with SREBP-1 decoy ODN can inhibit cholesterol biosynthesis by reducing HMGCR protein expression.

Example  6. Expression of lipid biosynthesis-related factors in animal models

In the animal model of Example 2, liver tissue was extracted and protein expression of the gene related to lipid biosynthesis was confirmed by Western blotting. SREBP-1C, FAS (Fatty acid syntase), ACC (Acetyl-CoA carboxylase) and SCD-1 (Stearoyl-CoA desaturase) were selected for lipid biosynthesis-related proteins. As a result, it was confirmed that the expression of lipid biosynthetic proteins was induced by the high-fat diet as shown in FIG. 4, and the expression was significantly reduced upon treatment with SREBP-1 decoy ODN. Therefore, lipid biosynthesis induced by high-fat diet can be reduced by inhibiting the expression of related genes by SREBP-1 decoy ODN treatment, which in turn can improve fat accumulation in the liver by SREBP-1 decoy ODN There will be.

Example  7. Analysis of inflammatory factors in animal models

In the animal model of Example 2, it was confirmed that the expression of blood IL-6 increased by high-fat diet decreased in the group injected with SREBP-1 decoy ODN as shown in Fig. As shown in FIG. 6, mRNA expression of TNF-.alpha., IL-1.beta. And IL-6, which are inflammatory factors, in the liver tissue was decreased in the group in which SREBP-1 decoy ODN was injected with high fat diet. Thus, SREBP-1 decoy ODN may help to prevent hepatitis by lowering the inflammatory response of the liver induced by high-fat diet.

<110> CATHOLIC UNIVERSITY OF DAEGU INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> SREBP-1 decoy oligodeoxynucleotide and pharmaceutical composition          for preventing or treating fatty liver disease containing the          same as active ingredient <130> PN17010 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> SRE-1 oligodeoxynucleotide <400> 1 gaattcgtgt ggggtgattg aaaacaatca ccccacac 38 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> E-box motif oligodeoxynucleotide <400> 2 gaattcgtat cacgtgatga aaacatcacg tgatac 36 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Con oligodeoxynucleotide <400> 3 gaattccagg tacggcaaaa aattgccgta cctg 34

Claims (5)

The SRE-1 oligodeoxynucleotide of SEQ ID NO: 1 and the E-box motif oligodeoxynucleotide of SEQ ID NO: 2 are covalently linked to form a stem structure capable of binding to the DNA binding sites of two loop structures and transcription factors RTI ID = 0.0 &gt; SREBP-1 &lt; / RTI &gt; decoy oligodeoxynucleotides. A pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease comprising the dumbbell-type SREBP-1 decoy oligodeoxynucleotide of claim 1 as an active ingredient. delete The non-alcoholic fatty liver disease according to claim 2, wherein the non-alcoholic fatty liver disease is any one selected from the group consisting of a simple fatty liver, a nutritive fatty liver, a starvation fatty liver, an obese liver liver, a diabetic liver liver, a fatty liver, liver fibrosis, A pharmaceutical composition for preventing or treating alcoholic fatty liver disease. 3. The pharmaceutical composition according to claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the dumbbell-type SREBP-1 decoy oligodeoxynucleotide. .

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