IMMUNOMODULATING PEPTIDE DERIVED FROM CONCAVE-EARED TORRENT FROG AND USE THEREOF

Abstract

The present invention relates to an immunomodulating peptide comprising an amino acid sequence as shown in SEQ ID NO:1. The present invention also discloses use of the immunomodulating peptide in the preparation of a drug or skin care product for promoting skin wound healing. The present invention provides a new immunomodulating peptide and use thereof, and discloses the mechanism of action of the immunomodulating peptide in promoting wound healing. The immunomodulating peptide is useful as a wounding healing polypeptide template.

Claims

1. An immunomodulating peptide, comprising an amino acid sequence as shown in SEQ ID NO:1.

2. The immunomodulating peptide according to claim 1, wherein the immunomodulating peptide is derived from a concave-eared torrent frog.

3. The immunomodulating peptide according to claim 1, wherein the immunomodulating peptide is derived from the skin of concave-eared torrent frog.

4. A drug or a skin care product for promoting skin wound healing comprising the immunomodulating peptide according to claim 1.

5. The drug or skin care product according to claim 4, wherein the drug or skin care product is used to promote the healing of human skin wounds.

6. The drug or skin care product according to claim 4, wherein in the presence of macrophages, the drug or skin care product causes the migration of neutrophils and macrophages.

7. The drug or skin care product according to claim 4, wherein the drug or skin care product induces the mouse macrophages and skin wounds to produce chemokines and cytokines.

8. The drug or skin care product according to claim 4, wherein the drug or skin care product activates the MAPKs, NF-κB and TGF-β/Smad signaling pathways in bone marrow-derived macrophages.

9. The drug or skin care product according to claim 4, wherein in the presence of macrophages, the drug or skin care product promotes the proliferation of keratinocytes and fibroblasts.

10. The drug or skin care product according to claim 4, wherein in the presence of macrophages, the concentration of immunomodulating peptide in the drug or skin care product is 20-100 μg/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 illustrates the active peak obtained at a wavelength of 280 nm in the process of separating and preparing Ot-WHP from the skin secretion of concave-eared torrent frog in Example 1;

[0023] FIG. 2 is a photograph showing the skin wound healing of mice in different experimental groups in Example 3;

[0024] FIG. 3 shows the test results of skin wound healing rate of mice in different experimental groups in Example 3;

[0025] FIG. 4 shows the immunohistochemical and quantitative statistical results of neutrophils and macrophages of mice in different experimental groups in Example 4;

[0026] FIG. 5 shows the influence of Ot-WHP at different concentrations on the migration of neutrophils and macrophages in Example 5;

[0027] FIG. 6 shows the effect of Ot-WHP at different concentrations on the production of chemokines and cytokines in mouse BMDMs cells and skin wounds in Example 6;

[0028] FIG. 7 shows the activation of different signal pathways in mouse BMDMs cells by Ot-WHP at different concentrations in Example 7;

[0029] FIG. 8 shows the effect of Ot-WHP at different concentrations on the proliferation of keratinocytes and fibroblasts in Example 8; and

[0030] FIG. 9 shows the test results of transformation of fibroblasts into fibrocytes and collagen deposition promoted by different concentrations of Ot-WHP in the presence of macrophages.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The specific embodiments of the present invention will be described in further detail with reference to embodiments. The following embodiments are intended to illustrate the present invention, instead of limiting the scope of the present invention.

EXAMPLE 1

Separation and Preparation of Ot-WHP From the Skin Secretion of Concave-Eared Torrent Frog

[0032] The concave-eared torrent frogs were collected from Huangshan Mountain, China. The concave-eared torrent frog was stimulated with a small amount of ether, and a large amount of secretion was produced on the back of the frog body. The secretion was collected, and centrifuged at 5000×g for 10 min. The supernatant was collected and lyophilized. The lyophilized powder was dissolved in 10 mL of PBS (0.1 M, pH 6.0) and centrifuged at 5000×g for 10 min. The supernatant was collected and loaded onto Sephadex G-50 (Superfine, Amersham Biosciences, 2.6 cm×100 cm) gel chromatographic column, and subjected to filtration chromatography with a PBS buffer (0.1 M, pH 6.0). The eluate was collected every 10 minutes in 3 mL each tube, and about 200 tubes were collected. After the collection, the absorbency of each tube at 280 nm was detected, and FIG. 1A was plotted according to the absorbency of each tube.

[0033] According to FIG. 1A, the skin secretion of concave-eared torrent frog was roughly divided into four absorption peaks after filtration chromatography by Sephadex G-50 gel. Each peak was combined, and whether each peak has the activity of promoting skin wound healing in mice was detected according to Example 3. One peak is found to have the function of promoting skin wound healing in mice (shown by the arrow in FIG. 1A). Then active peak in FIG. 1A was loaded onto a C18 (5 μm particle size, 110 Å pore size, 250 mm×4.6 mm, Gemini, CA, USA) reverse-phase high-performance liquid chromatographic column equilibrated with 0.1% trifluoroactic acid in water, and subjected to filtration chromatography eluting with acetonitrile over 0-60% gradient. The elution time was 80 min, and the absorbency at 280 nm was detected. The light absorption characteristics are shown in FIG. 1B. As described above, the activity to promote the repair of skin wounds was traced. The active peak in FIG. 1B is marked with an arrow. Next, under the same conditions as in FIG. 1B, the active peak in FIG. 1B was subjected to a second filtration chromatography by a reverse-phase high-performance liquid chromatographic column. The result is shown in FIG. 1C. The active peak is marked with an arrow and named Ot-WHP.

EXAMPLE 2

Amino Acid Sequencing of Ot-WHP

[0034] The Ot-WHP purified in Example 1 was loaded onto the Shimadzu protein pulse liquid sequencer (PPSQ-31A; Shimadzu, Kyoto, Japan), followed by Edman degradation sequencing following the instruction of instrument. After sequencing, the amino acid sequence is ATAWDLGPHGIRPLRPIRIRPLCG (SEQ ID NO:1).

[0035] In a practical application process, Ot-WHP can be directly chemically synthesized according to the known amino acid sequence of Ot-WHP.

EXAMPLE 3

Ot-WHP Can Significantly Promote Skin Wound Healing in Mice

[0036] Experimental animals BALB/c mice, female, 6-8 weeks old, weighed 18-20 g, were purchased from Shanghai Slac Animal Co., Ltd. The mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.1 mL/20 g), and the back was shaved with an electric razor and disinfected with iodophor. A hole was punched in the exposed back by a biopsy punch having a diameter of 8 mm. After the operation, the mice were kept in separate cages until the end of the experiment. The mice were randomly divided into 6 groups having 5 animals in each group, including a negative control group (20 μL PBS), a natural Ot-WHP group (20 μL, 200 μg/mL), a synthetic Ot-WHP group (20 μL, 200 μg/mL), a scrambled Ot-WHP group (SEQ ID NO:2, 20 μL, 200 μg/mL), an AH90 positive polypeptide control group (20 μL, 200 μg/mL), and an EGF growth factor control group (20 μL, 100 μg/mL). The drugs were dripped into corresponding wounds in mice once a day for 8 days.

[0037] The results are shown in FIGS. 2-3. FIG. 2 shows the wound healing of mice in different experimental groups taken at 0, 2, 4, 6, and 8 days after operation. FIGS. 3A1-A4 show the wound healing rate of mice in different experimental groups at 2, 4, 6, and 8 days after surgery. In the figures, Vehicle represents the negative control group; synthetic Ot-WHP represents the synthetic Ot-WHP group; Natural Ot-WHP represents the natural Ot-WHP group; and Scrambled Ot-WHP represents the scrambled Ot-WHP group. The results show that both natural Ot-WHP and synthetic Ot-WHP can significantly promote the healing of mouse skin wounds with an effect comparable to AH90 and EGF, while the scrambled Ot-WHP has no activity to promote wound repair.

EXAMPLE 4

Ot-WHP Recruits Neutrophils and Macrophages to Migrate to the Wound, to Accelerate the Initial Stage of Inflammation in Wound Healing

[0038] The mice were treated according to the method in Example 3. The drugs in the negative control group and the natural Ot-WHP group were dripped to wounds of corresponding mice. At specified time point (0.5, 1, 2, 3 days) after the operation, the mice were sacrificed. A biopsy tissue containing the wound center was taken, immobilized with 10% formalin, then dehydrated by ethanol, washed with xylene, and finally embedded in paraffin. The paraffin block was cut into 5 μm slices, deparaffinized, and hydrated, and antigen retrieval was performed for immunohistochemical analysis. The slice was blocked with 5% BSA for non-specific sites, incubated with the primary antibody (anti-Ly6G for neutrophils, and anti-F4/80 for macrophages) at 4° C., and immunostained with horseradish peroxidase and DBA. The infiltration by neutrophils and macrophages was measured by statistically counting the immunostained anti-Ly6G and anti-F4/80 cells.

[0039] The results are shown in FIG. 4. FIGS. 4A1-A4 show the immunohistochemical results of neutrophils recruited on days 0.5 and 1 after operation of mice in different groups. FIGS. 4C1-C4 show the immunohistochemical results of macrophages recruited on days 2 and 3 after operation of mice in different groups. FIGS. 4B1-B2 show the statistical results of neutrophils recruited on days 0.5 and 1 after operation of mice in different groups. FIGS. 4D1-D2 show the statistical results of macrophages recruited on days 2 and 3 after operation of mice in different groups. The results show that Ot-WHP can promote the migration of neutrophils and macrophages to the wound.

EXAMPLE 5

Ot-WHP Does Not Directly Cause the Migration of Neutrophils and Macrophages, But Requires the Coexistence of Macrophages to Cause the Migration of Neutrophils and Macrophages

[0040] To clarify how Ot-WHP chemoattracts neutrophils and macrophages to the wound area, the prepared mouse neutrophils or bone marrow-derived macrophages (BMDMs) were resuspended in RPMI 1640 medium containing 2% FBS at a cell density of 7×10.sup.6 cells/mL. 100 μL was pipetted to a Transwell chamber with a pore size of 3.0 μm (upper chamber, 24-well plate). Then 500 μL of Ot-WHP (25, 50, 100 μg/mL, dissolved in RPMI 1640 medium containing 2% FBS) or the culture medium (control) was pipetted to the lower chamber, and then cultured in a cell incubator at 37° C. for 8 h. The cells in the lower chamber were collected and counted with a hemocytometer.

[0041] In a co-incubation system, mouse bone marrow-derived macrophages (BMDMs, 4×10.sup.6 cells/mL, 500 μL) resuspended in in RPMI 1640 medium containing 2% FBS were plated in the lower chamber (24-well plate) of the co-incubation chamber. After adherence, the prepared mouse neutrophils (7×10.sup.6 cells/mL, 100 μL) or bone marrow-derived macrophages (BMDMs, 7×10.sup.6 cells/mL, 100 μL) were added to the Transwell chamber with a pore size of 3.0 μm (upper chamber, 24-well plate). Then 500 μL of Ot-WHP (25, 50, 100 μg/mL, dissolved in RPMI 1640 medium containing 2% FBS) or the culture medium (control) was added to the lower chamber, and then cultured in a cell incubator at 37° C. for 8 h. The cells in the upper chamber were collected and counted with a hemocytometer. The reduced cells in the upper chamber are the migrated cells

[0042] The results are shown in FIG. 5. Ot-WHP does not directly cause the migration of neutrophils (FIG. 5A) and macrophages (FIG. 5C) at a concentration in the range of 25-100 μg/mL. However, when macrophages coexist, Ot-WHP at a concentration in the range of 25-100 μg/mL significantly causes the migration of neutrophils (FIG. 5B) and macrophages (FIG. 5D) in a dose-dependent manner.

EXAMPLE 6

Ot-WHP Can Induce Mouse Macrophages and Skin Wounds to Produce Chemokines and Cytokines

[0043] To clarify why Ot-WHP can cause the migration of neutrophils and macrophages in the coexistence of macrophages, mouse bone marrow-derived macrophages (BMDMs, 5×10.sup.5 cells/well, DMEM medium, 2% FBS) were plated in a 24-well plate, Ot-WHP (25, 50, 100 μg/mL) or an equal volume of PBS (Vehicle) were added, and cultured for 24 h. The supernatant was collected, and centrifuged at 10000×g for 10 min. The chemokines and cytokines were determined.

[0044] The results are shown in FIGS. 6A1-A8. Compared with the Vehicle (PBS, control)-treated group, Ot-WHP induces mouse BMDMs to produce chemokines (CXCL1, CXCL2, CXCL3 and CCL2) and cytokines (TNF-α, IL-1β, IL-6 and TGF-β1) in a dose-dependent manner.

[0045] Then whether Ot-WHP can induce the production of chemokines and cytokines in the skin wounds of mice was tested. At specified time points (days 0, 0.5, 1, 2, 4, and 6), the wound tissue of the mice was removed and homogenized with 0.1 M PBS (containing 1 mM PMSF, 1 mL/g tissue) in a glass homogenizer. After the homogenization, the homogenate was centrifuged at 12000×g and 4° C. for 30 min. The supernatant was collected, and the chemokines (CXCL1 and CCL2) and cytokines (including TNF-α and TGF-β1) were determined.

[0046] The results are shown in FIGS. 6B1-B4. Compared with the Vehicle (PBS, control)-treated group, Ot-WHP can effectively induce the production of chemokines (CXCL1 and CCL2) and cytokines (TNF-α and TGF-β1) in skin wounds of mice.

EXAMPLE 7

Ot-WHP Can Activate MAPKs, NF-κB, TGF-β/Smad Signaling Pathways in Mouse Bone Marrow-Derived Macrophages

[0047] Mouse bone marrow-derived macrophages (BMDMs) were inoculated into a 6-well plate and cultured without serum for 16 h. For the detection of MAPKs and NF-κB, BMDMs were incubated with different concentrations of Ot-WHP (0, 25, 50, 100 μg/mL) for 30 min. For the detection of TGF-β/Smad signaling pathway, BMDMs were incubated with different concentrations of Ot-WHP (0, 25, 50, 100 μg/mL) for 24 h, or BMDMs were incubated with 100 μg/mL Ot-WHP, with or without TGF-β1 antibody.

[0048] The results are shown in FIG. 7. Compared with the Vehicle (PBS, control)-treated group, Ot-WHP activates the MAPKs (ERK, JNK subunit, FIGS. 7A and 6C), NF-κB (IκBα, p65 subunit, FIGS. 7B and 6C) and Smad (Smad2, Smad3, FIG. 7D) signaling pathways in mouse MAPKs in a dose-dependent manner, where the activation of Smad signaling pathway by Ot-WHP is dependent on the production of TGF-β induced by Ot-WHP (FIGS. 7E -G).

EXAMPLE 8

Ot-WHP Does Not Directly Promote the Proliferation of Keratinocytes and Fibroblasts, But Requires the Coexistence of Macrophages to Promote the Proliferation of Keratinocytes and Fibroblasts

[0049] Keratinocytes HaCat and mouse fibroblasts (5×10.sup.3 cells/well, 100 μL/well) were inoculated into a 96-well plate, and incubated with different concentrations of Ot-WHP (25, 50, and 100 μg/mL). PBS was used as a control. After HaCat was incubated with Ot-WHP for 24 h, and the fibroblasts were incubated with Ot-WHP for 72 h, 10 μl of Cell Counting Kit-8 (CCK-8) solution was added to each well, and incubated for another 2-4 h at 37° C. The absorbency at 450 nm was detected on a microplate reader.

[0050] HaCat was co-incubated with THP-1 derived macrophages, and mouse fibroblasts were co-incubated with mouse macrophages BMDMs. HaCat (1×10.sup.5 cells/well) or mouse fibroblasts (1×10.sup.5 cells/well) were inoculated into a 24-well plate (lower chamber) one day in advance. After adherence, corresponding macrophages (1×10.sup.5 cells/well, 100 μL) and different concentrations of Ot-WHP (25, 50, 100 μg/mL) or Vehicle (PBS, control) were added to the co-incubation chamber (upper chamber). After HaCat was co-incubated with THP-1-derived macrophages for 24 h, and the fibroblasts were co-incubated with mouse macrophages BMDMs for 72 h, the co-incubated upper chamber was discarded, and the cell activity in the lower chamber was detected by CCK-8.

[0051] The results are shown in FIG. 8. Ot-WHP cannot directly promote the proliferation of keratinocytes (FIG. 8A) and fibroblasts (FIG. 8B). Only when macrophages coexist, Ot-WHP can promote the proliferation of keratinocytes (FIG. 8C) and fibroblasts (FIG. 8D). It shows that Ot-WHP can promote the interaction between macrophages and keratinocytes/fibroblasts.

EXAMPLE 9

Ot-WHP Does Not Directly Promote the Transformation of Fibroblasts into Fibrocytes and Does Not Directly Promote the Collagen Production by Fibroblasts, But Requires the Coexistence of Macrophages to Promote the Transformation of Fibroblasts into Fibrocytes and Promote the Collagen Production by Fibroblasts

[0052] The method was the same as that in Example 8. The fibroblasts treated in Example 8 were lysed with RIPA cell lysis solution. The protein was extracted, and the expression of α-SMA was detected by Western blot. A higher expression level of α-SMA means a higher transformation of fibroblasts into fibrocytes. The supernatant of the fibroblasts treated in Example 8 was collected, and the production of collagen in the supernatant was quantitatively detected with an ELISA kit.

[0053] The results are shown in FIG. 9. Ot-WHP has no direct effect on the expression of α-SMA in fibroblasts. Ot-WHP does not directly promote the transformation of fibroblasts into fibrocytes (FIG. 9A), and Ot-WHP has no direct effect on the collagen production by fibroblasts and does not directly promote the collagen production by fibroblasts (FIG. 9B). Ot-WHP requires the coexistence of macrophages to promote the expression of α-SMA by fibroblast, that is, promote the transformation of fibroblasts to fibrocytes (FIG. 9C), and promote the collagen production by fibroblasts (FIG. 9D).

[0054] While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that some improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention, which are also contemplated to be within the scope of the present invention.