KR102430640B1 - Composition for suppressing adipocyte differentiation and method for screening material for suppressing adipocyte differentiation - Google Patents

Abstract

Disclosed herein are a composition comprising a substance that inhibits SRBC expression or activity as an active ingredient, and a method for screening a substance that inhibits adipocyte differentiation. According to an aspect of the present invention, the composition comprising a substance inhibiting the expression or activity of SRBC as an active ingredient inhibits the differentiation of adipocytes, and thus can be used for preventing or improving obesity. In addition, the screening method for a substance that inhibits adipocyte differentiation, which is an aspect of the present invention, simply, quickly and efficiently screens a substance that inhibits adipocyte differentiation by treating skin adipocytes with a test substance and checking the relative expression level of the SRBC gene. can do.

Description

Composition for suppressing adipocyte differentiation and method for screening material for suppressing adipocyte differentiation

Disclosed herein are a composition comprising a substance that inhibits SRBC expression or activity as an active ingredient, and a method for screening a substance that inhibits adipocyte differentiation.

Obesity generally refers to a state of being overweight, but more precisely, it refers to a state in which excess fat is accumulated in the body. Being overweight, including obesity, is known to cause many health problems, including high blood pressure, type 2 diabetes, cardiovascular disease, fatty liver, and various chronic diseases such as hyperlipidemia, and various drugs and natural substances for the treatment of obesity worldwide. Research on etc. is ongoing. On the other hand, obesity is induced by the active differentiation of adipocytes, and it has been reported that the differentiation of adipocytes occurs through a complex process of regulating intracellular gene expression by external stimuli such as hormones. In order to find the substance more accurately and efficiently, research on new markers related to adipocyte differentiation is also being conducted.

US 2010-0311609 A1

In one aspect, it is an object of the present invention to provide a novel adipocyte differentiation marker.

In another aspect, it is an object of the present invention to inhibit the differentiation of adipocytes.

In another aspect, an object of the present invention is to suppress obesity by inhibiting the differentiation of adipocytes.

In another aspect, it is an object of the present invention to conveniently screen for substances that inhibit adipocyte differentiation.

In one aspect, the present invention provides a composition for inhibiting adipocyte differentiation comprising a substance that inhibits the expression or activity of serum deprivation response factor-related gene product that binds to c-kinase (SRBC) as an active ingredient.

In another aspect, the present invention provides a composition for preventing or improving obesity comprising a substance that inhibits the expression or activity of SRBC as an active ingredient.

In another aspect, the present invention comprises the steps of: treating adipocytes with a test substance; And in the test substance-treated skin cells, before and after the test substance treatment SRBC (serum deprivation response factor-related gene product that binds to c-kinase) comprising the step of confirming the relative expression level, adipocyte differentiation inhibitory substance screening provide a way

According to an aspect of the present invention, the composition comprising a substance that inhibits the expression or activity of SRBC as an active ingredient suppresses the differentiation of adipocytes and can be used for anti-obesity purposes. In addition, the screening method for a substance that inhibits adipocyte differentiation, which is an aspect of the present invention, simply, quickly and efficiently screens a substance that inhibits adipocyte differentiation by treating skin adipocytes with a test substance and checking the relative expression level of the SRBC gene. can do.

1 is a result confirming the expression change of factors related to adipogenesis when SRBC expression is suppressed.
2 is a result of visual confirmation after staining adipocytes in which the expression of SRBC is suppressed.
3 is a result of measuring the content of triglycerol (TG) during SRBC overexpression.

Hereinafter, the present specification will be described in detail.

In one aspect, the present invention provides a composition for inhibiting adipocyte differentiation comprising a substance that inhibits SRBC (serum deprivation response factor-related gene product that binds to c-kinase) expression or activity as an active ingredient.

In one aspect, the present invention, SRBC (Serum deprivation response factor-related gene product that binds to c-kinase) is a composition for preventing or improving obesity comprising an expression or activity inhibitory material as an active ingredient.

The relationship between the expression of SRBC and the differentiation of adipocytes is still unknown. The present inventors found that, while the expression of PPAR-γ (Peroxisome proliferator-activated receptor gamma) and fatty acid synthase (FAS) and triglycerides, which are factors known to be related to adipocyte differentiation during adipocyte differentiation, increased, SRBC It was found that the expression of these factors and triglycerides decreased when the expression of SRBC was suppressed, and it was found for the first time that SRBC is a marker of adipocyte differentiation.

In the above aspect, the composition may increase the migration of pref-1 (predipocyte factor-1) into the nucleus. In addition, the composition may increase the expression of pref-1.

In the above aspect, the composition is peroxisome midsacchae activated receptor gamma (PPARγ), fatty acid synthase (fatty acid synthase, FAS), stearoyl-CoA desaturase 1 (stearoyl-CoA desaturase 1, SCD-1) , may decrease the expression of one or more selected from the group consisting of adipocyte fatty acid-binding protein 2 and adiponectin.

In the above aspect, the substance inhibiting the expression or activity of the SRBC may include an RNA nucleic acid molecule.

The RNA nucleic acid molecule may include one or more of siRNA, shRNA, crRNA, and miRNA that bind to mRNA or DNA of SRBC. For example, siRNA, shRNA and miRNA may bind to mRNA of SRBC, and crRNA may bind to DNA of SRBC.

siRNA, shRNA and miRNA are RNA fragments that cause RNA interference (RNAi). The RNA interference phenomenon means that target mRNA is degraded by double-stranded RNA (dsRNA) and the expression of a specific gene is down-regulated.

miRNA is a type of endogenous small RNA that exists in cells. It is derived from DNA that does not synthesize proteins and is generated from a hairpin-shaped transcript. miRNA binds to the complementary sequence of the 3'-UTR of the target mRNA and induces translational inhibition or destabilization of the mRNA, and ultimately serves as a repressor to inhibit protein synthesis of the target mRNA. . Such a role is also called RNAi. It is known that one miRNA can target multiple mRNAs, and mRNA can also be regulated by multiple miRNAs. Other RNAs that induce RNAi include short interfering RNA (siRNA), which is a short RNA of about 19 to 27 mer, and shRNA having a short hairpin structure. Generally speaking, when siRNA and shRNA are artificially introduced into cells to induce RNAi, miRNA refers to a substance naturally present in cells.

In the above aspect, the siRNA may include sequences capable of hybridizing with them, or sequences complementary thereto, respectively.

As used herein, “hybridizable” means that it can bind to a specific single-stranded RNA to form a double-stranded RNA.

crRNA may refer to RNA involved in the CRISPR system. crRNA complementarily binds to the target sequence, and then the DNA cleaving enzyme recognizes and binds to the crRNA binding site. Then, tracrRNA (trans-activating crRNA) binds to the crRNA complementary sequence, and the DNA cleaving enzyme cuts the target sequence. It works to cut. The CRISPR system may be CRISPR/Cas, such as CRISPR/Cas9, or CRISPR/Cpf1.

The RNA nucleic acid molecules may be expressed by being inserted into a vector.

In one embodiment, the siRNA may include one or more sequences selected from the group consisting of sequences of SEQ ID NOs: 1 to 4 and sequences capable of hybridizing to these sequences, respectively. Specifically, in one embodiment, the siRNA may include the sequences of SEQ ID NO: 1 and SEQ ID NO: 3, and may include the sequences of SEQ ID NO: 2 and SEQ ID NO: 4. The sequence of SEQ ID NO: 1 or 2 may be a sense strand, SEQ ID NO: 3 may be the antisense strand of the sequence of SEQ ID NO: 1, and the sequence of SEQ ID NO: 4 may be an antisense strand with respect to the sequence of SEQ ID NO: 2.

For example, the sequence of SEQ ID NO: 1 may include the sequence of 5'-ggagcuuuca gccuaauuut t-3', and the sequence of SEQ ID NO: 2 may include the sequence of 5'-cccgugcuuc aaauagagattt-3', and the sequence The sequence of No. 3 may include the sequence of 5'-aaauuaggcu gaaagcucctc-3', and the sequence of SEQ ID NO: 4 may include the sequence of 5'-ucucuauuugaagcacgggtt-3'.

In the above aspect, the composition, SRBC expression or activity inhibitory material, based on the total weight of the composition, may include 0.0001 to 70% by weight. For example, 0.0001 wt% or more, 0.01 wt% or more, 0.1 wt% or more, 10 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, 60 wt% or more, 70 Weight % or less, 60 wt% or less, 50 wt% or less, 40 wt% or less, 30 wt% or less, 20 wt% or less, 10 wt% or less, 1 wt% or less, 0.1 wt% or less, 0.001 wt% or less .

The cosmetic composition may be provided in any formulation suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol composition. Compositions of such formulations can be prepared according to conventional methods in the art.

The cosmetic composition may contain, in addition to the above substances, other ingredients capable of giving a synergistic effect to the main effect, preferably within a range that does not impair the main effect. The cosmetic composition according to the present specification may include a substance selected from the group consisting of vitamins, polymer peptides, polymer polysaccharides, and sphingolipids. In addition, the cosmetic composition according to the present specification may include a moisturizer, an emollient, a surfactant, a UV absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or a limiting agent. . The blending amount of the component can be easily selected by those skilled in the art within the range that does not impair the purpose and effect of the present specification, and the blending amount may be 0.01 to 5 wt%, specifically 0.01 to 3 wt%, based on the total weight of the composition have.

The pharmaceutical composition may be provided in any dosage form suitable for topical application. For example, it may be a skin external solution, suspension, emulsion, gel, patch or spray, but is not limited thereto. Formulations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions or suppositories. The formulation can be easily prepared according to conventional methods in the art, and surfactants, excipients, wettable powders, emulsification accelerators, suspending agents, salts or buffers for osmotic pressure control, coloring agents, spices, stabilizers, preservatives, preservatives, or Other commercially available adjuvants may be appropriately used.

The active ingredient of the pharmaceutical composition according to an embodiment of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject, the route of administration, or the judgment of the prescriber. Determination of the dosage based on these factors is within the level of those skilled in the art, and the daily dosage thereof used is, for example, from 0.0001 mg/kg/day to 1000 mg/kg/day, more specifically from 0.02 mg/kg/day to 100 mg/kg. / can be, but is not limited to.

The dosage of the formulation is different depending on the subject's age, sex, weight, symptoms, and administration method, but it is preferable to apply it 1 to 5 times a day at 1.0 to 3.0 ml per day and continue it for at least 1 month.

In addition, health food is manufactured using raw materials or ingredients (functional raw materials) that are easily deficient in daily meals or have useful functions for the human body, and maintains normal functions of the human body or maintains health by activating physiological functions. It may mean a food to improve, but is not limited thereto. The health food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., but is not limited thereto and may be manufactured and processed in any form according to the law.

In one aspect of the present invention, the health drink composition is not particularly limited in other ingredients except for containing the compound as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. have. Examples of natural carbohydrates are conventional sugars such as monosaccharide polysaccharides, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

In general, the amount of active ingredient administered by the health food composition may be in the range of 0.0001 mg/kg/day to about 1000 mg/kg/day. A more preferred dosage may be 0.02 mg/kg/day to 100 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses.

In another aspect, the present invention is a cosmetic method comprising the step of treating the skin with the composition. In one embodiment, the composition can be used to remove or reduce unnecessary fat by dissolving fat by treating local obese areas such as the abdomen.

In another aspect, the present invention comprises the steps of: treating adipocytes with a test substance; And in the test substance-treated skin cells, before and after the test substance treatment SRBC (serum deprivation response factor-related gene product that binds to c-kinase) comprising the step of confirming the relative expression level, adipocyte differentiation inhibitory substance screening way.

In the above aspect, the method may further include determining the adipocyte differentiation inhibitory substance when the expression level of SRBC is reduced after the test substance treatment, compared to before the test substance treatment. For example, if the expression level of SRBC in adipocytes treated with the test substance is lower than the expression level of SRBC in adipocytes not treated with the test substance, it can be determined that the treated test substance suppressed the expression level of SRBC. . As described above, if the treated test substance suppresses the expression level of SRBC, it can be determined that it is a substance that inhibits adipocyte differentiation.

As used herein, the "relative expression level" may be the expression level when comparing the expression level of SRBC in adipocytes not treated with the test substance with the expression level of SRBC in adipocytes not treated with the test material. In the above, the expression level may include an expression amount and expression quality.

In one embodiment, the step of determining the adipocyte differentiation inhibitory substance may be determining the adipocyte differentiation inhibitory substance when the expression level of SRBC after treatment with the test substance is 95% or less of the expression level of SRBC before treatment with the test substance. have. For example, when the expression level of the SRBC gene is 94% or less, 95% or less, or 96% or less after the test substance treatment compared to the expression level before the test substance treatment, it can be judged as an adipocyte differentiation inhibitory substance, but is limited thereto. not. The expression level is a result measured in a state in which statistical significance is secured. The concept of statistical significance is a case where a significant difference is shown through a biological statistical analysis method, and in a quantitative case, the p value differs by less than 0.05.

In one embodiment, the expression level of the SRBC gene can be confirmed using known techniques, such as reverse transcription polymerase chain reaction (RT-PCR), ELISA, Western blot or immune blot. , but is not limited thereto.

In another aspect, the present invention, adipocytes; and instructions, wherein the instructions describe the screening method.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. However, the following examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] SRBC expression inhibition cell line preparation

[Example 1-1] Cell culture

In order to proliferate mouse 3T3-L1 fibroblasts (ATCC, CL-173) under undifferentiated conditions, using a 6-well plate, in a 5% CO2 incubator, Dulbecco's modified Eagle's medium (DMEM) (Lonza, MD, USA) 10% Calf serum (Gibco BRL, NY, USA) and 5% penicillin streptomycin were added for 2 days.

[Example 1-2] Cell differentiation

After culturing the undifferentiated adipocytes in Example 1-1 to 95% of the 6-well plate, 10% fetal bovine serum (FBS) (PAA, Austria), 10 μg/ml insulin (Sigma- Aldrich, St. Louis, USA), 0.5 mM 3-isobutyl-1-methylxantine (IBMX) (Sigma-Aldrich, St. Louis, USA), and 1 μM dexamethasone (DEX) (Sigma-Aldrich, St. Louis, USA) ) were incubated for two days, and then incubated again for 3 days under conditions containing 10% FBS and 10 μg/ml insulin in DMEM. Thereafter, the differentiated adipocytes were obtained by culturing for two days in DMEM containing only 10% FBS.

[Example 1-3] RNA extraction and cDNA synthesis

RNA was extracted by applying the method using TRIzol (Gibco BRL, NY, USA), and cDNA was synthesized using the Reverse Transcription System (Promega Co, WI, USA).

[Example 1-4] Real-time quantitative RT-PCR (RT-qPCR)

Rotor-gene 3000 (Corbett Research, AUS) was added to the cDNA synthesized in Example 1-3 with TaqMan® probe (Life technologies, CA, USA) and master mix (Life technologies, CA, USA) for expression of each gene. was used for analysis.

[Example 1-5] SRBC expression inhibition cell line production

The siSRBC sequence (5'-GGAGCUUUCAGCCUAAUUUtt-3 that inhibits expression from the SRBC open reading frame (ORF) of mouse fibroblasts cultured in Example 1-1 using silencer pre-designed RNAi software (Ambion, Inc. USA) ' and 5'-AAAUUAGGCUGAAAGCUCCtc-3') were secured and then inserted into BamH I/Hind III of pSilencer2.1-U6puro plasmid (Ambion, Inc. Austin, USA). The synthesized plasmid was transfected into cells using FuGene® 6 Transfection Reagent (Roche Diagnostics, IN, USA). The transfected cells were obtained through the cells surviving in the medium containing puromycin (1.5 μg/ml).

[Example 2] Oil Red O staining and staining photography after inhibition of SRBC expression

To measure the amount of Triacylglycerol (TG) in fibroblasts of differentiated mice, Oil Red O (Sigma-Aldrich, St. Louis, USA) was added to 60% propylene glycol (PG) (Santa Cruz Biotechnology, Inc. CA, USA). A stock solution was obtained. In each of Examples 1-1 and 1-2, undifferentiated and differentiated cells were obtained and washed twice with cold phosphate-buffered saline (PBS) (Welgene, Daegu, Korea), followed by 3.7% formaldehyde (Sigma-Aldrich, St. Louis, USA) was used to fix the cells for 1 hour. Next, after washing twice with cold PBS, it was stained for 30 minutes using the ORO stock solution prepared previously. After washing with running water, cell staining pictures were taken using Olympus IX71 (Tokyo, Japan). After imaging, the stained cells were collected by dissolving ORO again using 100% PG, and absorbance was measured at 450 nm to quantify the amount of TG.

As a result, it was found that when the expression of SRBC was suppressed, the differentiation of adipocytes was suppressed ( FIGS. 1 and 2 ).

<110> Amorepacific corporation <120> Composition for suppressing adipocyte differentiation and method for screening material for suppressing adipocyte differentiation <130> 16P250IND <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sense strand_SRBC-1 siRNA <400> 1 ggagcuuuca gccuaauuut t 21 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> sense strand_SRBC-2 siRNA <400> 2 cccgugcuuc aaauagagat tt 22 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sense strand_SRBC-1 siRNA <400> 3 aaauuaggcu gaaagcucct c 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sense strand_SRBC-2 siRNA <400> 4 ucucuauuug aagcacgggt t 21

Claims (13)

As a composition for preventing or improving obesity,
The composition contains a substance that inhibits the expression or activity of SRBC (serum deprivation response factor-related gene product that binds to c-kinase) as an active ingredient,
The SRBC expression or activity inhibitory substance comprises siRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 and 3 and sequences capable of hybridizing to these sequences, respectively, the composition for preventing or improving obesity. According to claim 1,
The composition increases the migration into the nucleus of pref-1 (predipocyte factor-1), or increases the expression, composition. According to claim 1,
The composition comprises, 0.0001 to 70% by weight of the SRBC expression or activity inhibitory material based on the total weight of the composition. 4. The method according to any one of claims 1 to 3,
The composition is a pharmaceutical composition. 4. The method according to any one of claims 1 to 3,
The composition is a health food composition. 4. The method according to any one of claims 1 to 3,
The composition is a cosmetic composition, composition. delete delete delete treating adipocytes isolated from humans with a test substance; and
In adipocytes treated with a test substance, a screening method for an adipocyte differentiation inhibitory substance comprising the step of confirming the relative expression level of SRBC (serum deprivation response factor-related gene product that binds to c-kinase) before and after the test substance treatment . 11. The method of claim 10,
The screening method is
After the test substance treatment, when the expression level of SRBC is reduced compared to before the test substance treatment, further comprising the step of determining the adipocyte differentiation inhibitory substance screening method. 12. The method of claim 11,
In the step of determining the adipocyte differentiation inhibitory substance, when the expression level of SRBC after the test substance treatment is 95% or less of the SRBC expression level before the test substance treatment, the adipocyte differentiation inhibitor is judged as the adipocyte differentiation inhibitory substance. Substance screening methods. adipocytes; and instructions;
A kit for screening an adipocyte differentiation inhibitory substance, wherein the method of any one of claims 10 to 12 is described in the instructions.

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