Composition for wound healing or inhibiting skin wrinkle formation or moisturizing skin

Abstract

The present invention provides: a pharmaceutical composition for wound healing, comprising a specific peptide; and a cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin. The peptide acts as an agonist for a GLP -2 receptor, thereby effectively inducing GLP-2 signaling and inducing cell proliferation, and promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier. Therefore, the peptide can be effectively used for: wound healing; and inhibiting skin wrinkle formation or moisturizing skin.

Description

Composition for wound healing or skin wrinkle prevention or skin moisturizing

[0001] The present invention relates to a pharmaceutical composition for wound treatment comprising a specific peptide; and a composition for inhibiting skin wrinkle formation or moisturizing the skin. The peptide has the activity of activating the glucagon-like peptide 2 (GLP-2) receptor, thereby promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier.

[0002] GLP-2 is a 33-amino acid peptide hormone produced by L cells in the small intestine, and plays a role in energy absorption and protection, as well as in activating enterocyte function (https: / www.proteinatlas.org / ENSG00000065325-GLP2R / tissue; Curr Protein Pept Sci.(2004) 5(1):51-65). In addition, GLP-2 is known to help skin regeneration by inducing skin cell proliferation (Daniel J Drucker, et al, GMol Endocrinol. 2003 Feb;17(2):161-171).

[0003] The skin barrier is a protective barrier formed in the stratum corneum, the outermost layer of the skin. It prevents moisture and electrolytes from escaping and bacteria and pathogens from penetrating the skin. Therefore, the skin barrier is known to play a very important role in skin hydration and aging. The skin barrier is composed of keratinocytes, which contain natural moisturizing factors, and intercellular lipids, that is, a wall structure of lipid components that connect keratinocytes and the lipid components that connect them. The intercellular lipid layer has a lamellar structure with multiple layers of membranes arranged in a parallel manner and is composed of lipid components such as ceramides and fatty acids. In addition, a structural support protein layer composed of proteins such as filaggrin, involucrin, and loricrin forms the cell shell of keratinocytes and plays a role in maintaining the skin barrier (Yeonjoon Kim, et al., Arch Pharm Res. 2021 Jan;44(1):36-48; Byung Eui Kim, et al., Allergy Asthma Immunol Res. 2018 May;10(3):207-215, etc.). Therefore, a substance that can activate the GLP-2 receptor can not only act as an active substance for wound healing or promoting wound healing, but also act as a functional cosmetic substance for inhibiting skin aging (e.g., skin wrinkle formation) or moisturizing the skin.

[0004] The present inventors have disclosed that a peptide derived from fibroblast growth factor (FGF) activates the fibroblast growth factor receptor to induce collagen synthesis and also has the activity of promoting skin keratinocyte proliferation and angiogenesis (Korean Patent Registration No. 10-1772048).

[0005] The present inventors have conducted various studies to develop an active substance based on small peptides as a substance capable of activating the GLP-2 receptor. As a result, we discovered that a specific peptide, namely a 4-mer peptide composed of Leu-Ser-Ser-Phe, acts as a substance capable of activating the GLP-2 receptor (i.e., an agonist for the GLP-2 receptor), thereby effectively inducing GLP-2 signaling, and not only inducing cell proliferation but also promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier, and thus can be usefully applied to wound healing, inhibition of skin wrinkle formation, or skin moisturizing.

[0006] Accordingly, the present invention aims to provide a pharmaceutical composition for wound treatment or wound healing promotion comprising the specific peptide as an active ingredient.

[0007] In addition, the present invention aims to provide a cosmetic composition for inhibiting skin aging or moisturizing skin, which comprises the specific peptide.

[0008] According to one aspect of the present invention, a pharmaceutical composition for wound treatment or wound healing promotion is provided, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] <Chemical Formula 1>

[0010]

[0011] According to another aspect of the present invention, a cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin is provided, comprising the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0012] The present invention has revealed that the peptide according to the present invention (i.e., a peptide composed of Leu-Ser-Ser-Phe) binds to an intracellular GLP-2 receptor, induces GLP-2 signaling in a concentration-dependent manner, induces cell proliferation in a concentration-dependent manner, and promotes the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier. The present invention has also revealed that the peptide according to the present invention effectively induces filaggrin synthesis in a 3D human skin model. Therefore, the peptide according to the present invention can be usefully applied to a pharmaceutical composition for wound healing or promoting wound healing; and a cosmetic composition for inhibiting skin aging or moisturizing the skin, including inhibiting skin wrinkle formation.

[0013] Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Glytin)-AMC of the present invention composed of Leu-Ser-Ser-Phe by GLP-2 siRNA treatment.

[0014] Figure 2 shows the results of analyzing changes in β-catenin / TCF interaction by peptide treatment of the present invention.

[0015] Figure 3 shows the results of analyzing changes in β-catenin / TCF interaction by treatment with the peptide of the present invention and GLP2 siRNA.

[0016] Figure 4 shows the results of analyzing the cell proliferation rate by peptide treatment of the present invention.

[0017] Figure 5 shows the results of analyzing changes in the expression level of skin barrier-related proteins by peptide treatment of the present invention.

[0018] Figure 6 shows the results of analyzing changes in the expression level of skin barrier-related proteins by peptide treatment of the present invention in a 3D human skin model.

[0019] The present invention provides a pharmaceutical composition for wound treatment or wound healing promotion, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] <Chemical Formula 1>

[0021]

[0022] In addition, the present invention provides a cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin, comprising the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0023] In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Chemical Formula 1 may also be expressed as "Leu-Ser-Ser-Phe". The amino acids constituting the peptide of Chemical Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Chemical Formula 1 include, but are not limited to, acid addition salts, for example.

[0024] The pharmaceutical composition of the present invention may include excipients such as lactose and corn starch, lubricants such as magnesium stearate, and known and usable emulsifiers, suspending agents, buffers, isotonic agents, etc., and may be formulated as a parenteral dosage form, preferably a parenteral dosage form including a topical skin preparation. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous administration forms, a sterile solution of the active ingredient is usually prepared, and a buffer capable of suitably adjusting the pH of the solution may be included, and in the case of intravenous administration, a isotonic agent may be included to impart isotonicity to the preparation. In addition, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4, and may be locally introduced into the intramuscular bloodstream of a patient in the form of a solution. In addition, it may be formulated as a transdermal dosage form such as a topical solution, emulsion, ointment, or patch according to a conventional pharmaceutical method. The pharmaceutical composition of the present invention can be administered to patients with various wounds at a daily dose of about 1 to 50 mg / kg. The appropriate dosage may generally vary depending on the patient's age, weight, and symptoms.

[0025] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an active ingredient. The cosmetic composition may be manufactured in various forms according to a conventional cosmetic manufacturing method. For example, the cosmetic composition may be manufactured in the form of a cosmetic product, toner, cream, lotion, etc. containing the peptide, which may be diluted with a conventional cleansing solution, astringent, or moisturizing solution and used. In addition, the cosmetic composition may include conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide is an amount effective for inhibiting skin aging, particularly for inhibiting skin wrinkle formation, and / or for moisturizing the skin, for example, 1 x 10 based on the total weight of the composition. -5 ~ 1 x 10 -2 It may be contained in a content of weight %, preferably about 1 x 10 -4 ~ 1 x 10 -3 It can be contained in a content of weight%.

[0026] Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are intended to illustrate the present invention, and the present invention is not limited to these examples and test examples.

[0027] Example 1. Synthesis of peptides

[0028] A peptide consisting of Leu-Ser-Ser-Phe was synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea). The synthesized peptide was purified and analyzed by reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan) using a C18 analytical RP column (Shiseido capcell pak), and identified using mass spectrometry (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

[0029] Example 2. Preparation of a composition containing a peptide

[0030] The peptide (peptide composed of Leu-Ser-Ser-Phe) prepared in Example 1 was dissolved in triple-distilled water to prepare a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

[0031] Test Example 1: GLP-2 Receptor Binding Evaluation

[0032] In order to confirm whether the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) binds to the target protein, GLP-2 receptor, the peptide-AMC of the present invention labeled with the fluorescent substance AMC (7-amino-4-methyl coumarine) was treated to cells, and whether it binds to the GLP-2 receptor was confirmed through immunofluorescence.

[0033] (1) Test materials

[0034] - Preparation of test substances

[0035] The peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0036] - Test system

[0037] 1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0038] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2-3 days.

[0039] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0040] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0041] - Test materials

[0042] 1) Peptide-AMC (7-amino-4-methyl coumarine) of the present invention

[0043] Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

[0044] 2) Phalloidin-Rhodamin

[0045] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, R415

[0046] 3) siRNA transfection

[0047] 3-1) GLP2R siRNA(h)

[0048] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-60695

[0049] 3-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0050] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0051] 3-3) Opti-MEM ® Medium

[0052] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0053] (2) Test method

[0054] - Composition of the test group

[0055]

[0056] - Exam process

[0057] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0058] 2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

[0059] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The mixture was mixed in a 1:1 ratio and slowly mixed, then left to react at room temperature for 5 minutes.

[0060] 4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

[0061] 5) The peptide-AMC (1:100) of the present invention was reacted at 4°C for 16 hours and then washed three times with a washing solution (PBS).

[0062] 6) To stain the cytoskeleton, phalloidin-rhodamine (1:2000) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

[0063] 7) Mounting was performed using a mounting solution.

[0064] 8) The fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0065] - Observe and judge results

[0066] The luminescence level of the peptide-AMC of the present invention in the GLP-2R siRNA treatment group was compared and analyzed based on the control siRNA negative control group.

[0067] (3) Test results

[0068] As a result of evaluating the degree of binding of the test substance to the GLP-2 receptor, in the control siRNA treatment group, the test substance bound to the GLP-2 receptor and an AMC (blue fluorescence) signal was observed, but in the GLP-2R siRNA treatment group in which the expression of the GLP-2 receptor was suppressed, the AMC signal was reduced (Fig. 1).

[0069] (4) Conclusion

[0070] Compared to the negative control group, a decrease in the peptide-AMC signal of the present invention was observed in the GLP-2R siRNA treatment group. Therefore, the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) is believed to specifically bind to the GLP-2 receptor.

[0071] Test Example 2: Evaluation of GLP-2 receptor signaling induction efficacy

[0072] In order to verify the possibility of the peptide of the present invention as a GLP-2 receptor agonist, changes in the interaction of β-catenin / TCF, a downstream signaling pathway of the GLP-2 receptor, were observed through in situ PLA, and whether the peptide of the present invention transmits a signal through GLP-2R was confirmed by treating with GLP-2R siRNA.

[0073] (1) Test materials

[0074] - Preparation of test substances

[0075] The peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0076] - Test system

[0077] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0078] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2 to 3 days.

[0079] 3) Medium: CEFOgro Human MSC Growth medium

[0080] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0081] - Test materials

[0082] 1) Anti-β-catenin (S33 / S37 / T41) antibody

[0083] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

[0084] 2) Anti-TCF-4 (D-4) antibody

[0085] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166699

[0086] 3) siRNA transfection

[0087] 3-1) GLP2R siRNA(h)

[0088] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-60695

[0089] 3-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0090] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0091] 3-3) Opti-MEM ® Medium

[0092] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0093] 4) NaveniFlex 100RM

[0094] Storage conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

[0095] 5) Prolong™ diamond antifade mountant with DAPI

[0096] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0097] 6) Digital Fluorescence Imaging System

[0098] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0099] (2) Test method

[0100] (2-1) in situ PLA

[0101] - Composition of the test group

[0102]

[0103]

[0104] - Exam process

[0105] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0106] 2) For the siRNA treatment group, the medium was replaced with a dedicated medium (DMEM + 10% FBS).

[0107] 3) siRNA + Opti-MEM ® The medium and transfection reagent + Opti-MEM® medium were mixed in a 1:1 ratio, slowly mixed, and reacted at room temperature for 5 minutes.

[0108] 4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

[0109] 5) The negative control and test substances were treated for 1 hour at the appropriate concentration for each treatment group.

[0110] 6) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0111] 7) Subsequent tests were conducted using the In Situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer's instructions.

[0112] 8) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

[0113] 9) Two antibodies for confirmation were diluted to 10 μg / mL in antibody diluent, reacted at 4°C for 16 hours, and washed three times with TTBS (0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4).

[0114] 10) After adding the PLA probe and reacting at 37°C for 1 hour, it was washed 3 times with TTBS.

[0115] 11) Reactions A, B, and C were sequentially processed and reacted at 37°C for 1 hour, 30 minutes, and 90 minutes, respectively. Finally, the cells were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).

[0116] 12) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0117] - Observe and judge results

[0118] To compare and analyze the luminescence signal due to the interaction of β-catenin / TCF in the test substance treatment group, the PLA fluorescence signal was quantitatively analyzed using NIS-Elements BR3.1 with the negative control group as the standard.

[0119] (3) Test results

[0120] Compared to the negative control group, it was observed that the interaction of β-catenin / TCF increased in a concentration-dependent manner in the test substance treatment group (Fig. 2), and when GLP-2R siRNA was treated to suppress the expression of GLP-2R, the signal was reduced (Fig. 3).

[0121] (4) Conclusion

[0122] The test substance was confirmed to activate a downstream signaling pathway through the GLP-2 receptor. Therefore, the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) is believed to be capable of acting as a GLP-2 receptor agonist.

[0123] Test Example 3: Evaluation of cell proliferation induction efficacy

[0124] GLP-2 is known to aid skin regeneration by inducing skin cell proliferation. Using a proliferation assay kit (CCK-8), we confirmed whether the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) induces the proliferation of keratinocytes, similar to GLP-2.

[0125] (1) Test materials

[0126] - Preparation of test substances

[0127] The peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0128] - Test system

[0129] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0130] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2 to 3 days.

[0131] 3) Medium: CEFOgro Human MSC Growth medium

[0132] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0133] - Test materials

[0134] 1) Cell viability assay kit

[0135] Storage conditions: Refrigerated at 4℃ / Manufacturer: BYLABS, BYVA0500

[0136] 2) Microplate reader

[0137] Manufacturer: BIO-TEK, EL808

[0138] (2) Test method

[0139] - Composition of the test group

[0140]

[0141] - Exam process

[0142] 1) 5X10 in a 96-well culture plate 3 0.1 mL of cells were dispensed into each well.

[0143] 2) The negative control and test substances were treated for 24, 48, and 72 hours according to the concentration of each treatment group.

[0144] 3) Each well was treated with 10 μl of cell viability assay solution and incubated at 37°C for 2 hours.

[0145] 4) The absorbance (450 nm) of each test group was measured using a microplate reader.

[0146] - Observe and judge results

[0147] The degree of cell proliferation was confirmed by measuring absorbance (450 nm) using a microplate reader, and the degree of cell proliferation in the test substance treatment group was compared and analyzed based on the negative control group.

[0148] (3) Test results

[0149] Compared to the negative control group, it was observed that cell proliferation increased in a concentration-dependent manner in the test substance treatment group (Fig. 4).

[0150] (4) Conclusion

[0151] As a result of evaluating the skin cell proliferation-inducing efficacy of the test substance, it was confirmed that cell proliferation was promoted in a concentration-dependent manner by treatment with the test substance. Therefore, it is determined that the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) has skin cell proliferation-inducing efficacy.

[0152] Test Example 4: Evaluation of the efficacy of inducing skin barrier-related protein synthesis

[0153] It was confirmed through Western blot analysis whether the peptide of the present invention has the effect of maintaining and strengthening the skin barrier by inducing the synthesis of lipid components and proteins related to the skin barrier.

[0154] (1) Test materials

[0155] - Preparation of test substances

[0156] The peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0157] - Test system

[0158] 1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0159] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2 to 3 days.

[0160] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0161] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0162] - Test materials

[0163] 1) Anti-Fatty acid synthase antibodies

[0164] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-20140

[0165] 2) Anti-LASS3 (CerS3) antibody

[0166] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABCAM, ab272552

[0167] 3) Anti-Filaggrin (AKH1) antibody

[0168] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-66192

[0169] 4) Anti-Involucrin (SY5) antibody

[0170] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-21748

[0171] 5) Anti-Loricrin (W-22) antibody

[0172] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-133757

[0173] 6) Goat anti-mouse IgG Fc-HRP

[0174] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0175] 7) NP40 cell lysis buffer

[0176] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0177] 8) Bovine serum albumin (BSA)

[0178] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0179] 9) Protein assay dye reagent concentrate

[0180] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0181] 10) Immuno-bolt for protein blotting® PVDF membrane

[0182] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0183] 11) WEST SAVE GOLD,

[0184] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0185] 12) DaVinci Western Imaging System

[0186] Manufacturer: DAVINCH-K, CAS-400SM

[0187] (2) Test method

[0188] (2-1) Immunofluorescence

[0189] - Composition of the test group

[0190]

[0191] - Exam process

[0192] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 50% or higher.

[0193] 2) The negative control and test substances were treated for 24 hours at concentrations appropriate to the concentrations of each treatment group.

[0194] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0195] 4) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0196] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0197] 6) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0198] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0199] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0200] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0201] - Observe and judge results

[0202] The expression levels of each protein treated with the test substance were evaluated by quantitative analysis using ImageJ based on the expression level of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. The changes in the expression levels of fatty acid synthase, CERS3 (ceramide synthase 3), filaggrin, involucrin, and loricrin in the test substance treatment group were compared and analyzed based on the negative control group.

[0203] (3) Test results

[0204] Compared to the negative control group, it was confirmed that the expression levels of lipid component synthesis enzyme (Fatty acid synthase, CERS3) and structural support proteins (Filaggrin, Involucrin, Loricrin) of the skin barrier increased in a concentration-dependent manner in the test substance treatment group (Fig. 5).

[0205] (4) Conclusion

[0206] It was confirmed that the test substance promoted the expression of lipid component synthesis enzyme (Fatty acid synthase, CERS3) and structural support proteins (Filaggrin, Involucrin, Loricrin) of the skin barrier in a concentration-dependent manner. Therefore, the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) is judged to be effective in maintaining and strengthening the skin barrier.

[0207] Test Example 5: Evaluation of the efficacy of inducing filaggrin synthesis in a 3D human skin model.

[0208] The above-described tests confirmed that the test substance strengthened the skin barrier by activating the GLP-2 receptor at the cellular level. Immunofluorescence was also used to confirm that the test substance also activated the GLP-2 receptor in a 3D human skin model (Neoderm-ED) similar to human skin, thereby promoting the synthesis of filaggrin, a representative skin barrier protein.

[0209] (1) Test materials

[0210] - Preparation of test substances

[0211] The peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0212] - Test system

[0213] 1) 3D human skin model: Neoderm-ED

[0214] 2) Management: Cultured in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0215] 3) Badge: Maintenance medium

[0216] Composition: 10% fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

[0217] - Test materials

[0218] 1) Anti-Filaggrin (AKH1) antibody

[0219] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-66192

[0220] 2) Prolong™ diamond antifade mountant with DAPI

[0221] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0222] 3) Digital Fluorescence Imaging System

[0223] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0224] (2) Test method

[0225] - Composition of the test group

[0226]

[0227] - Exam process

[0228] 1) After receiving Neoderm-ED, a dedicated medium was added and cultured for 24 hours.

[0229] 2) The negative control and test substances were treated for 48 hours at the concentrations appropriate for each treatment group.

[0230] 3) Neoderm-ED was separated from the insert well using a blade to create a paraffin block.

[0231] 4) Slides were prepared by cutting sections at 4 ㎛ thickness.

[0232] 5) Paraffin washing and water dehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

[0233] 6) To expose the antigen, citrate buffer was added, heated for 14 minutes, and then washed with PBS.

[0234] 7) Pretreatment was performed to increase antibody permeability by treating with 0.1% Triton X-100 for 10 minutes.

[0235] 8) The primary antibody was reacted at 4°C for 16 hours and washed three times with washing solution (PBS).

[0236] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution (PBS).

[0237] 10) Mounting was performed using a mounting solution containing DAPI (nuclear stain).

[0238] 11) The fluorescent signals detected in the tissue were observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0239] - Observe and judge results

[0240] The level of filaggrin expression in the test substance treatment group was compared and analyzed by comparing the amount of fluorescence signal based on the negative control group.

[0241] (3) Test results

[0242] Compared to the negative control group, an increase in the expression of Filaggrin (green fluorescence) was observed in the test substance treatment group (Fig. 6).

[0243] 6-5. Conclusion

[0244] In a 3D human skin model, the test substance was confirmed to promote the expression of filaggrin, a representative structural support protein of the skin barrier. Therefore, the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) was confirmed to maintain and strengthen the skin barrier not only at the cellular level but also in the skin model.

Claims

1. A pharmaceutical composition for wound healing or promoting wound healing, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Chemical formula 1> 2. A cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <Chemical formula 1>

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