KR20160057529A - A New Scolopendrasin-4 peptide for promoting growth of large intestine epithelial cell and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel scolopendrasin-4 peptide which promotes the growth of large intestine epithelial cells and a composition thereof. More specifically, the purpose of the present invention is to provide a peptide which promotes the growth of large intestine epithelial cells, wherein the peptide is synthesized from an amino acid which is represented by sequence number 1 and derived from a gene of a peptide isolated from Scolopendra subspinipes. According to the present invention, by providing the novel scolopendrasin-4 peptide which promotes the growth of large intestine epithelial cells, it is possible to provide a composition for preventing and treating inflammatory large bowel disease as a novel substance which improves the inflammatory large bowel without causing cytotoxicity.

Description

[0001] The present invention relates to novel scolopendrazine-4 peptides which promote the growth of large intestinal epithelial cells,

The present invention relates to a novel scolopendrazine-4 peptide and its composition for promoting the growth of colon epithelial cells, and more particularly to a novel scolopendrazine-4 peptide and a composition thereof which promote the growth of colon epithelial cells without cytotoxicity, 4 < / RTI > peptides and compositions thereof.

Diseases such as Crohn's disease and ulcerative colitis are inflammatory bowel disease and the exact pathogenesis has not yet been elucidated.

However, it is clear that one of the causes of the enteritis diseases is the destruction process of the intestinal cell layer. The reason for this is due to the barrier function, which is the main role of the epithelial layer that constitutes the digestive mucosa.

Because of the presence of various pathogenic microorganisms and immunologically active substances in the lumen of the intestine, the epithelial cells create strong intercellular junctions such as adhesion, blocking external pathogenic substances from entering the body.

However, like the skin, the intestinal mucosal epithelium, in which dead cells are severely dislodged, must be replaced continuously and rapidly by the use of another epithelial cell at the location of the fallen cell through rapid division of stem cells.

In other words, the process of promoting the division of stem cells located in the mucous membrane can provide a rapid supply of the absolute number of epithelial cells necessary for the construction of the mucosal layer, thereby rapidly blocking the vacancy of the detached cells.

In addition, the rapid division of epithelial cells can effectively cope with the process of epithelial cell detachment in a wide area found in the mucosa of an inflammatory disease patient.

In a variety of studies, it is well known that intestinal epithelial cell promoting factors are highly effective in treating enteritis.

Epidermal growth factor (EGF) was found to significantly lower Crohn's disease activity when applied to patients with moderate to severe Crohn's disease.

The keratinocyte growth factor (KGF) -2 secreted from fibroblasts also showed significant therapeutic efficacy when treated in patients with ulcerative colitis.

It has also been shown that linoleic acid, one of the fatty acids in human colon epithelial cells (HT-29), strongly stimulates cell proliferation. This means that the ingested food ingredient promotes epithelial cell growth and strengthens the barrier.

Furthermore, "probiotics", which are known to dramatically improve the symptoms of colonic inflammatory disease, have also proven to have a function that strongly stimulates the production and growth of epithelial-stem cells.

Therefore, in the case of a substance capable of enhancing the barrier function by promoting the growth of intestinal epithelial cells, it can be said that it is possible to develop it as a candidate substance for treatment of colonic inflammatory disease, and related prior arts include Korea Patent Publication No. 10-2003 -0021093 (a method for producing a human epithelial cell growth factor and an angiangiogenin fusion protein and derivatives thereof using insect cells) and Korean Patent No. 10-0824396 (a peptide having activity of epithelial growth factor and its Purpose) is known.

It is an object of the present invention to provide novel scolopendrazine-4 peptides and compositions thereof that promote the growth of colon epithelial cells.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

In order to achieve the above object, the present invention provides a cell growth peptide comprising an amino acid sequence isolated from Scolopendra subspinipes and represented by SEQ ID NO: 1.

The peptide is characterized in promoting the growth of colon epithelial cells.

The growth of the colon epithelial cells is characterized by the denaturation of the I &kgr; B-alpha protein.

The growth of the colon epithelial cells is characterized by the activation of NFkB.

The peptide is characterized by being non-toxic to human colon epithelial cells.

The peptide is characterized by inhibiting apoptosis caused by malnutrition.

The inhibition of apoptosis is characterized by the decrease of active caspase-3.

The present invention is to provide a composition for controlling growth of colon epithelial cells comprising the peptide as an active ingredient.

The present invention also provides a composition for preventing and treating inflammatory bowel disease comprising the peptide as an active ingredient, wherein the inflammatory bowel disease is any one of Crohn's disease and ulcerative colitis.

According to the present invention, there is provided a novel scolopendrazine-4 peptide which promotes the growth of colon epithelial cells, thereby providing a composition for preventing and treating inflammatory bowel disease as a novel colonic inflammation-improving substance without cytotoxicity.

Figure 1 is a graph showing the proliferation of human colon epithelial cells (HT29 cells) according to the treatment concentration of the novel scolopendrazine-4 peptide of the present invention.
FIG. 2 is a graph showing the proliferation of human colonic epithelial cells (HT29 cells) according to the treatment time of the novel scolopendrazine-4 peptide of the present invention.
FIG. 3 is a graph showing the degree of degradation of the amount of IκB-α protein according to the treatment of the novel scolopendrazine-4 peptide of the present invention. FIG.
FIG. 4 is a graph showing the degree of reduction of the amount of IκB-α protein when MG-132 and scolopendrazin-4, which are proteasome inhibitors, are treated. FIG.
FIG. 5 is a graph showing the degree of reduction of cell proliferation phenomenon during simultaneous treatment of MG132 and scallopendrathin-4. FIG.
6 is a graph showing the non-toxicity of the novel scolopendrazine-4 peptide of the present invention on human colon epithelial cells.
FIG. 7 is a graph showing the growth rate of mouse-colon tissue epithelial cells in the treatment of the novel scolopendrazine-4 peptide of the present invention. FIG.
FIG. 8 is a diagram showing a mouse-colon tissue epithelial cell suicide process caused by malnutrition in the treatment of the novel scolopendrazine-4 peptide of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention is represented by SEQ ID NO: 1 and provides a peptide synthesized from an amino acid deduced from a gene of a peptide isolated from Scolopendra subspinipes .

DISCLOSURE OF THE INVENTION The present invention has the disadvantage that the price of epithelial cell growth promoting factor (EGF) clinically applicable to treat the colon inflammation disease is high and that the EGF exhibits a strong growth signal to induce the tumor The inventors of the present invention intend to discover a new substance that enhances barrier function through promotion of growth of colon epithelial cells and thereby improves healing of mucosal injury.

In order to identify the gene of the peptide showing the activity of promoting the growth of the colonic epithelial cells from Scolopendra subspinipes , Wangzine was rapidly frozen in liquid nitrogen to isolate the total RNA, and then, using RNA seq analysis, Based on the sequence of translated amino acids from each transcript, we confirm the genetic information about the dead bodies and accelerate the growth of new colon epithelial cells through the secondary structure analysis and comparison of amino acid sequence using SVM algorithm Unigen is presumed to be a peptide showing activity.

Then, the amino acid sequence of the peptide thus produced was composed of 21 amino acids with SRSEKAGLQFPVGRIGRMLKK-NH ₂ , which was named Scolopendrasin-4 (SP-4) .

Such a peptide exhibits an activity of promoting the growth of colon epithelial cells, and in particular, it is non-toxic to human colon epithelial cells, and thus provides a composition for the prevention and treatment of inflammatory bowel disease as an effective ingredient.

Specifically, the growth promoting effect was confirmed by treating the human colon epithelial cell line HT29 cell with Scolopendrasin-4, a peptide extracted from Scolopendra subspinipes .

For this, HT29 cells were seeded in a 96-well plate at 1 × 10 ⁴ cells, cultured in an incubator for 48 hours, cultured at 37 ° C. in 5% CO 2, 4 (Scolopendrasin-4) were treated at 0.5, 1, and 5 μg / ml for 48 hours, and the growth rate was measured using a 450 nm absorbance meter.

In addition, proliferation confirmation using the BrdU Kit was performed to closely examine whether the growth rate was affected.

In order to confirm the change of intracellular protein, HT29 cells were treated with scolopendrasin-4 by time and concentration, and the expression pattern of IκB-α protein was confirmed by Western blotting Respectively.

To determine whether the cell growth effect of scolopendrasin-4 was related to the degradation of IκB-α protein, MG-132, a proteasome inhibitor, was added to the scolopendrasin-4, And then the amount of IκB-α protein was determined by Western blotting.

To confirm whether the scolopendrasin-4 showed toxic effects on human colon epithelial cells, the same cells were seeded in a 96-well plate, and the peptide was added at a concentration of 5 μg / ml And maintained for up to 48 hours for MTT assays to assess toxicity.

To determine whether the cell growth effect of scolopendrasin-4 was related to the degradation of IκB-α protein, MG-132, a proteasome inhibitor, was added to the scolopendrasin-4, ≪ / RTI > HT29 cells were seeded in a 96 well plate at 1 × 10 ⁴ cells and cultured in an incubator for 24 hours. MG-132 (10 μM) and Scolopendrasin-4 -4) and then cultured for 36 hours. BrdU uptake was then performed.

In order to re-verify in mouse tissues other than cell experiments, the colonic tissues (3 cm) of CD-1 mice were treated with Scolopendrasin-4 and cultured for 24 hours before performing BrdU uptake.

In addition, to determine whether the cell growth was promoted by scolopendrasin-4 treatment after forming an artificial condition for inhibiting cell growth by removing serum at the time of mouse tissue culture. To this end, the mouse colon tissue (3 cm) treated 1 hr before the scolopendrasin-4 was cultured in the serum-free culture medium for 48 hours and 72 hours, Were identified by Western blotting.

As a result, the novel scolopendrazine-4 peptide of the present invention promotes the growth of colon epithelial cells as shown in FIGS. 1 to 8, particularly promoting the growth of colon epithelial cells by causing denaturation of IκB-α protein It is possible to provide a composition for the prevention and treatment of inflammatory bowel disease as a novel colonic inflammation-improving substance.

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 only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1; Peptide selection promoting the growth of Wang Ji-yin-derived colon epithelial cells

In order to identify the peptide gene promoting the growth of colon epithelial cells from Wang Ji, the Wang Ji Nee was rapidly frozen with liquid nitrogen to isolate total RNA,

Peptides that promote the growth of colon epithelial cells were selected based on the amino acid sequence translated from Wangzine transcripts using RNA seq analysis (Table 1).

This peptide was highly homologous to Buforin2. The selected peptides were biosynthesized and used for experiments. The amino acid sequence of the biosynthetic peptide was 21 amino acids in the form of SRSEKAGLQFPVGRIGRMLKK-NH ₂ and was named 'Scolopendra-4 (SP-4)'.

Scolopendrazine-4
Peptide sequence
(SEQ ID NO: 1) SRSEKAGLQFPVGRIGRMLKK-NH ₂

Example 2 Synthesis and Isolation of Scolopendrazine-4 Peptide

In Example 1, the scolopendrazine-4 peptide derived from Wang Zyene was synthesized and isolated and purified.

First, in order to synthesize a scolopendrazine-4 peptide, the present inventors prepared peptide using Merrifield's liquid phase method using a Fmoc amino group protecting container.

Method of the peptide synthesis the Fmoc (9-fluorenylmethoxycarbonyl) amino acids of the N _α were synthesized and used as a protecting group (protecting group) amino Groom (amino group) to the conventional method (solid phase method).

Concretely, the peptide having carboxyl terminal in the form of -NH ₂ used Rink Amide MBHA-Resin as the starting material, and the peptide having carboxyl terminal in the form of -OH used Fmoc-amino acid-Wang Resin as a starting material.

Elongation of the peptide chain by coupling of Fmoc-amino acid was performed by DCC ( N -hydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide) method.

After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM) It was dried with gas.

A solution of TFA (trifluoroacetic acid) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added and the reaction was carried out for 3 hours. The peptides were separated and the peptide was precipitated with diethyl ether. The crude peptide thus obtained was subjected to a reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15 μ, 19.0 mM ㅧ) using an acetonitrile gradient containing 0.1% TFA 30, Waters).

After the synthetic peptide was hydrolyzed with 6 N HCl at 110 ° C for 24 hours, the resulting residue was concentrated under reduced pressure, dissolved in 0.02 N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A).

The molecular weight was calculated based on the sequence of the synthesized peptides, and the exact molecular weight was measured using a MALDI mass spectrometer (MALDI).

As a result, it was confirmed that a peptide that promotes the growth of colon epithelial cells having the correct amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight were identical.

Experimental Example 1: Confirmation of the growth promoting ability of human colonic epithelial cells by the treatment concentration of scolopendrazine-4 peptide

1. Experimental Method

To investigate the growth promoting activity of human colonic epithelial cells on scolopendrazine-4 peptide (SP-4) synthesized in Example 2, HT29 cells, a human colon epithelial cell line, were cultured in 96 wells at 1 × 10 ⁴ After seeding and culturing for 24 hours, SP-4 was treated at 0.5, 1, and 5 μg / ml, respectively, for 48 hours.

BrdU uptake was then performed and then measured at 450 nm. At this time, the average value of 4 wells of each treatment was obtained and BrdU Cell Proliferation Assay Kit of Exalpha Biological Co. was used.

2. Experimental results

As shown in FIG. 1, it was confirmed that the cell proliferation of HT29 cells treated with the scolopendrazine-4 peptide (SP-4) was significantly increased.

In addition, it was confirmed that the increase of cell growth rate by SP-4 was concentration-dependent.

Experimental Example 2: Confirmation of growth promoting ability of human colonic epithelial cells according to treatment time of scolopendrazine-4 peptide

1. Experimental Method

HT29 cells were seeded in 96 wells at a concentration of 1 × 10 ⁴ cells. The scolopendrazine-4 peptide (SP-4) synthesized in Example 2 was treated at a concentration of 5 μg / ml for 24 hours and 48 hours Lt; / RTI >

BrdU uptake was performed and then measured at 450 nm.

2. Experimental results

As shown in FIG. 2, it was confirmed that the growth of human colonic epithelial cells treated with scolopendrazine-4 peptide (SP-4) synthesized in Example 2 increased in a time-dependent manner.

Experimental Example 3: Measurement of IκB-α Protein Level by Treatment of Scolopendrazine-4 Peptide

1. Experimental Method

NFκB, known as a transcription initiation factor involved in cell growth, is generally bound to the IκB-α protein and its activity is inhibited. However, when the IκB-α protein is degraded and reduced, it is known that the free NFκB enters the nucleus and begins to be transcribed, thereby initiating cell growth.

Therefore, western blotting was performed to measure the activation of various intracellular proteins, which may influence changes in cell growth rate including IκB-α.

To this end, HT29 cells were treated with the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 and pulverized. The extracted protein was injected into 10% SDS-PAGE gel, Respectively.

They were then transferred to membranes and diluted at a ratio of 1000: 1 to skim milk using various antibodies, including IκB-α antibodies, and reacted overnight.

After that, anti-rabbit-HRP, which is a secondary antibody, was added to skim milk at a ratio of 2000: 1, followed by color development after reaction at room temperature for 2 hours.

2. Experimental results

As shown in FIG. 3, the amount of IκB-α protein was significantly decreased in human colon epithelial cells treated with scolopendrazine-4 peptide (SP-4) synthesized in Example 2, Indicating that NFκB activation is underway.

However, other proteins known to promote cell growth, such as RXR-α and c-Src proteins, were found to be unchanged.

EXPERIMENTAL EXAMPLE 4 Modification of IκB-α Protein by MG132 (Proteasome Inhibitor) Measure

1. Experimental Method

 To investigate whether the promoting effect of scolopendrazine-4 peptide (SP-4) synthesized in Example 2 on growth of colorectal epithelial cells is related to the degradation of IκB-α protein, To block the proteasome.

MG132, known as a proteasome inhibitor, was treated with scolopendrazine-4 peptide (SP-4) for 24 hours, and the amount of IκB-α was confirmed by western blotting in HT29 cells.

2. Experimental results

As shown in FIG. 4, inhibition of proteasome by MG132 blocked the reduction of IκB-α protein by the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 above.

This suggests that SP-4 promotes growth of colonic epithelial cells due to denaturation of IκB-α protein and its subsequent reduction.

EXPERIMENTAL EXAMPLE 5 Simultaneous Treatment of MG132 and Scolopendrazine-4 Peptide (SP-4) to Promote Growth Enhancement of Colonic Epithelial Cells

1. Experimental Method

 To further confirm whether the promoting effect of scolopendrazine-4 peptide (SP-4) synthesized in Example 2 on the colonic epithelial cell growth was related to the denaturation of IκB-α protein, MG132 was added to SP-4 And then the growth rate of the colon epithelial cells was measured.

HT29 cells (1 × 10 ⁴ ) were treated with 10 μM of MG132 and 5 μg / ml of scopolpradacin-4 peptide (SP-4) and cultured for 18 hours and 36 hours. BrdU uptake was then performed.

2. Experimental results

As shown in FIG. 5, in the group treated with the scolopendrazine-4 peptide (SP-4) synthesized in Example 2, the growth of the colon epithelial cells was increased but the group treated with SP-4 and MG-132 And the cell growth rate was significantly decreased.

This shows that the process of IκB-α protein modification by SP-4 is an important mechanism for promoting the growth rate of colon epithelial cells.

Experimental Example 6: Measurement of non-toxicity of scolopendrazine-4 peptide (SP-4) on human colon epithelial cells

1. Experimental Method

SP-4 (5 μg / ml) was incubated for 24 hours and 48 hours in order to confirm whether the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 exhibited cytotoxicity against human colon epithelial cells After each treatment, MTT (10 mM) was further treated for 2 hours.

After removing the treated culture media, 100 μl of DMSO, an organic solvent, was added to dissolve the MTT, and the absorbance was measured at a wavelength of 570 nm.

2. Experimental results

As shown in FIG. 6, it was confirmed that there was no change in cell health in the control group and the experimental group in which the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 was not treated. This shows that there is no cytotoxicity of SP-4 against human colon epithelial cells.

Experimental Example 7: Confirmation of growth promoting ability of scolopendrazine-4 peptide (SP-4) in mouse-colon epithelial tissue cells

1. Experimental Method

The same size of the large intestine tissue from the CD1 mice was added to the cell culture medium, and the experimental group treated with 5 μg / ml of the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 and untreated And cultured in an incubator (Incubator) for 24 hours as a control group.

Twenty-four hours later, BrdU was added to each well (2 μl / ml) and incubated for an additional 3 hours.

After fixation with 4% PFA for 1 hour, the membrane was punctured with 0.1% triton X-100 and reacted for 2 hours with BrdU antibody for 1 hour.

Subsequently, the cells were further reacted with secondary-mouse IgG-HRP antibody for 1 hour. The tissue was disrupted by ultrasonication and centrifuged at 12,000 rpm to isolate the supernatant.

The color development reagent was added to the supernatant and the degree of luminescence was measured at 450 nm.

Statistical analysis was performed using the mean values measured in four tissues and two repeated experiments were performed. Proteins of each tissue were quantified and standardized for color development.

2. Experimental results

As shown in FIG. 7, it was confirmed that the cell growth rate was increased by about 25% in the mouse colon tissue treated with the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 as compared with the control.

This is consistent with the results of cell line experiments and shows that the scolopendrazine-4 peptide (SP-4) is also effective in promoting normal colon epithelial cell growth in live mice.

Experimental Example 8: Suicidal activity of mouse-colon epithelial tissue cells induced by nutrient deficiency in the treatment of scolopendrazine-4 peptide (SP-4)

1. Experimental Method

The same size of colon tissue excised from CD1 mice was cultured in serum-free cell culture medium. This process was used as a model to promote suicide of colon epithelial cells through nutritional deficiency.

In order to confirm the inhibitory effect of the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 in the suicide test of mouse colonic epithelial cells caused by malnutrition.

For this purpose, mouse colon tissue was placed in a cell culture medium free from serum, and then treated with 5 μg / ml of scopolpradacin-4 peptide (SP-4) and incubated for 48 hours and 72 hours in an incubator .

To confirm the changes of epithelial cells only, mucosa-epithelial cells were selectively removed by scraping only the inside with a thin glass slide.

Cellular apoptosis was assessed by western blotting of activation of caspase-3 protein as a marker of programmed cell death.

2. Experimental results

As shown in Fig. 8, malnutrition significantly increased apoptotic cell apoptosis in mouse colon tissues (increased amount of active caspase-3 protein).

However, in the cells treated with the scolopendrazine-4 peptide (SP-4) synthesized in Example 2, the amount of active caspase-3 caused by malnutrition was significantly decreased.

This demonstrates that the scolopendrazine-4 peptide (SP-4) synthesized in Example 2 promotes the growth of epithelial cells in mouse colon tissues and also strongly inhibits the process of apoptosis due to malnutrition Lt; / RTI >

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 thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Republic of korea <120> A New Scolopendrasin-4 peptide for promoting growth of large          intestine epithelial cell and its synthetic composition <130> p2014-0037 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Ser Arg Ser Glu Lys Ala Gly Leu Gln Phe Pro Val Gly Arg Ile Gly   1 5 10 15 Arg Met Leu Lys Lys              20

Claims (10)

1. A cell growth peptide comprising the amino acid sequence shown in SEQ ID NO: 1, isolated from Scolopendra subspinipes .
SEQ ID NO: 1:
Ser Arg Ser Glu Lys Ala Gly Leu Gln Phe Pro Val Gly Arg Ile
Gly Arg Met Leu Lys Lys
The method according to claim 1,
Wherein said peptide promotes growth of colon epithelial cells.
3. The method of claim 2,
Wherein the growth of the colon epithelial cells is caused by denaturation of I &lt; [kappa] &gt; B-alpha protein.
3. The method of claim 2,
Wherein the growth of the colon epithelial cells occurs by progressive activation of NFkB.
The method according to claim 1,
Wherein said peptide is non-toxic to human colon epithelial cells.
The method according to claim 1,
Wherein said peptide inhibits apoptosis caused by malnutrition.
The method according to claim 6,
Wherein the inhibition of cell apoptosis is caused by decreasing active caspase-3.
A pharmaceutical composition comprising a peptide of any one of claims 1 to 7 as an active ingredient
A composition for regulating growth of colon epithelial cells.
A pharmaceutical composition comprising a peptide of any one of claims 1 to 7 as an active ingredient
A composition for the prevention and treatment of inflammatory bowel disease.
10. The method of claim 9,
Wherein the inflammatory bowel disease is any one of Crohn's disease and ulcerative colitis.
A composition for the prevention and treatment of inflammatory bowel disease.

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