Anti-inflammatory active peptide with effect of promoting skin repair and application of anti-inflammatory active peptide

Abstract

The invention discloses an anti-inflammatory active peptide with an effect of promoting skin repair and application of the anti-inflammatory active peptide. The anti-inflammatory active peptide GGVGFYG with the effect of promoting the skin repair, provided by the invention, can promote the proliferation of skin fibroblasts and has the potential of promoting the healing of skin wounds. Moreover, experiments show that the GGVGFYG can effectively promote proliferation of macrophages RAW264.7 cells, can inhibit excessive production of nitric oxide and cytokines in the RAW264.7 cells induced by lipopolysaccharide, increases the level of anti-inflammatory cytokines, and shows remarkable anti-inflammatory activity.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, and in particular to an anti-inflammatory active peptide capable of promoting skin repair and application thereof. Background Art

[0002] The skin is one of the largest organs in the human body, and its integrity is essential for maintaining physiological functions and resisting the invasion of the external environment. However, skin trauma caused by various reasons is a common and serious health problem, which has an unignorable impact on the patient's quality of life and physical health. Skin trauma refers to damage or injury to the skin or subcutaneous tissue caused by physical injury, irritation or external factors. This injury can include different types such as cuts, tears, abrasions, burns, bruises, punctures, ulcers, etc. Different types of skin trauma can cause different degrees of damage, from minor abrasions to severe burns or deep wounds. After trauma, the skin usually recovers as quickly as possible through its own repair mechanism.

[0003] Therefore, finding effective methods to promote the repair and healing of skin wounds has always been one of the important topics in clinical medicine and biomedical research. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an anti-inflammatory active peptide with the effect of promoting skin repair.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned anti-inflammatory active peptide having the effect of promoting skin repair.

[0006] Another object of the present invention is to provide the application of the above-mentioned anti-inflammatory active peptide having the effect of promoting skin repair.

[0007] The purpose of the present invention is achieved through the following technical solutions: An anti-inflammatory active peptide with the effect of promoting skin repair, which has the following amino acid sequence: Gly-Gly-Val-Gly-Phe-Tyr-Gly (GGVGFYG).

[0008] Furthermore, the anti-inflammatory active peptides having the effect of promoting skin repair are prepared by hydrolysis of shell nacre protein, solid phase synthesis or genetic engineering technology.

[0009] The invention relates to a shell nacre protein hydrolyzate containing the anti-inflammatory active peptide having the effect of promoting skin repair.

[0010] The solid phase synthesis product comprises the anti-inflammatory active peptide having the effect of promoting skin repair.

[0011] The above anti-inflammatory active peptide having the effect of promoting skin repair is a cosmetically acceptable salt or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt comprises a metal salt of a peptide, wherein the metal comprises lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

[0013] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an organic base, and the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.

[0014] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an inorganic acid or an organic acid, wherein the organic acid includes acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; and the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0015] Application of the above-mentioned anti-inflammatory active peptides, shell nacre protein hydrolysates, solid phase synthesis products, cosmetically acceptable salts or pharmaceutically acceptable salts with skin repair promoting effects in the preparation of anti-inflammatory agents and / or skin wound repair products.

[0016] Furthermore, the anti-inflammatory agent has the effects of promoting macrophage proliferation, promoting macrophage NO release, promoting macrophage anti-inflammatory factor secretion and / or reducing pro-inflammatory factor secretion.

[0017] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0018] Furthermore, the active peptide is any one of an oral preparation and an external preparation.

[0019] Compared with the prior art, the present invention has the following advantages and effects: The present invention separates, purifies and identifies multiple peptides from the protein hydrolysate extracted from the pearl layer of shells, and then screens out a new anti-inflammatory peptide GGVGFYG (811.3024 Da) through molecular docking. Molecular docking simulation shows that the active peptide can bind to the receptor protein cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) through hydrogen bonds and hydrophobic interactions, thereby exerting anti-inflammatory activity. The active peptide can effectively promote the proliferation of macrophage RAW264.7 cells, and can inhibit the excessive production of nitric oxide (NO) and cytokines (TNF-α, IL-6) in RAW264.7 cells induced by lipopolysaccharide (LPS), while increasing the level of anti-inflammatory cytokines (IL-10), showing significant anti-inflammatory activity. In addition, the active peptide has a significant proliferation effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0020] The invention provides a theoretical basis for high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the study results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0022] Figure 2 This is a study result of the effect of active peptides on NO release from RAW264.7 macrophages (Note: different lowercase letters indicate significant differences (P<0.05)).

[0023] Figure 3 This is a study result of the effect of active peptides on the cytokine release of RAW264.7 macrophages induced by LPS; A is the effect on the release of cytokine TNF-α, B is the effect on the release of cytokine IL-6, and C is the effect on the release of cytokine IL-10 (Note: different lowercase letters indicate significant differences (P<0.05)).

[0024] Figure 4 This is a graph showing the study results on the effect of active peptides on the proliferation rate of L929 cells.

[0025] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0028] Example 1 1. Synthesis of anti-inflammatory active peptides that promote skin repair The anti-inflammatory active peptide GGVGFYG with skin repair promoting effect screened out by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0029] 2. Cell Culture Mouse macrophage RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator, and the cells were passaged when they grew to the logarithmic growth phase.

[0030] 3. RAW264.7 Cell Cytotoxicity Assay 100 μL of RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, with 5×10 cells per well. 4 After culturing for 24 h, the original culture medium was discarded. A blank control group and a sample group were set up. The sample group was added with 100 μL of complete culture medium containing different concentrations of shell nacre anti-inflammatory peptides, and the blank control group was added with an equal amount of complete culture medium. After continuing to culture for 24 h, the original culture medium was discarded, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and then placed in a cell culture incubator for 4 h. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and after shaking for 10 min, the absorbance value at a wavelength of 490 nm was measured using an enzyme marker.

[0031] 4. Determination of inflammatory factors in RAW264.7 cells 500 μL of RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 24-well plate, with a cell number of 1×10 5After culturing for 24 h, the original culture medium was discarded. A blank control group, a model group, a sample group, and a positive control group were set up. The sample group was added with 500 μL of complete culture medium containing different concentrations of anti-inflammatory active peptides that promote skin repair. The blank control group and the model group were added with an equal amount of complete culture medium. The positive control group selected dexamethasone as a control. After continuing to culture for 24 h, the original culture medium was discarded, 500 μL of complete culture medium was added to the blank group, and 500 μL of μg / mL LPS solution was added to the other groups. After continuing to culture for 24 h, the supernatant was taken, and the levels of NO, IL-10, IL-6, and TNF-α in the cell culture supernatant were detected using NO kits and ELISA kits.

[0032] 5. Data Analysis All experiments were set up in three parallel groups, and the final data were expressed as mean ± standard deviation. Statistical graphs were completed in Origin 2019 software, and the experimental results were analyzed by one-way analysis of variance for the means of each group using IBM SPSS Statistics 26 software. P < 0.05 was considered statistically significant.

[0033] result: (1) Effect of the anti-inflammatory peptide GGVGFYG, which promotes skin repair, on the activity of RAW264.7 macrophages The MTT method was used to determine the effect of the anti-inflammatory peptide GGVGFYG, which has the effect of promoting skin repair, on the viability of RAW264.7 macrophages at a concentration of 50-800 μg / mL.

[0034] Depend on Figure 1 It can be seen that after 24 h of treatment of RAW264.7 macrophages with GGVGFYG, the cell viability was not weakened, and compared with the blank control group, the shell nacre anti-inflammatory peptide promoted the proliferation of RAW264.7 macrophages. Therefore, the subsequent study chose to evaluate the anti-inflammatory activity in the concentration range of 10-200 μg / mL.

[0035] (2) Effect of the anti-inflammatory peptide GGVGFYG, which promotes skin repair, on NO release in RAW264.7 macrophages In the present invention, LPS was used to induce the release of NO from macrophages RAW264.7 to construct a cellular inflammation model and explore the anti-inflammatory activity of the anti-inflammatory active peptide GGVGFYG that has the effect of promoting skin repair.

[0036] Depend on Figure 2It can be seen that the NO release of RAW264.7 macrophages in the model group increased significantly after LPS stimulation (P<0.001). Compared with the model group, the anti-inflammatory active peptide GGVGFYG sample group, which has the effect of promoting skin repair, can significantly inhibit the release of NO by RAW264.7 macrophages at different concentrations. It is worth noting that its inhibition rate on NO release is concentration-dependent. At a concentration of 200 μg / mL, the NO release of GGVGFYG decreased by 57.86±3.04% (P<0.001), and was higher than that of the positive control group dexamethasone. The experimental results show that GGVGFYG can effectively inhibit the release of NO from RAW264.7 macrophages induced by LPS, and can regulate the inflammatory response to a certain extent.

[0037] (3) Effect of the anti-inflammatory peptide GGVGFYG, which promotes skin repair, on the release of inflammatory factors from RAW264.7 macrophages Depend on Figure 3 It can be seen that compared with the blank control group, the release of inflammatory factors TNF-α (a), IL-6 (b) and IL-10 (c) in the model group RAW264.7 macrophages under LPS stimulation increased significantly. The positive control dexamethasone is a drug that has been proven to have anti-inflammatory effects. After dexamethasone treatment, the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by RAW264.7 macrophages was significantly reduced (P<0.001), indicating that the cell inflammation model used in the present invention can be applied to the activity evaluation of anti-inflammatory peptides from shell nacre. The GGVGFYG treatment group not only significantly reduced the secretion of pro-inflammatory cytokines TNF-α and IL-6 in RAW264.7 macrophages, but also significantly increased the secretion of anti-inflammatory cytokine IL-10, and showed a dose-dependent effect at concentrations of 10-200 μg / mL. At the highest concentration, the effect on IL-6 secretion was 48.55±1.52%; the effect on TNF-α secretion was 42.86±3.58%; and the effect on IL-10 secretion was 38.39±2.27%.

[0038] The above results indicate that the anti-inflammatory active peptide GGVGFYG, which promotes skin repair, can effectively regulate the secretion of inflammatory factors in RAW264.7 macrophages and has good anti-inflammatory activity.

[0039] Example 2 1. Synthesis of anti-inflammatory active peptides that promote skin repair The anti-inflammatory active peptide GGVGFYG with skin repair promoting effect screened out by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0040] 2. Cell Culture Mouse fibroblast L929 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator, and the cells were passaged when they grew to the logarithmic growth phase.

[0041] 3. L929 cytotoxicity assay Mouse fibroblast L929 cells in logarithmic growth phase were inoculated into 96-well plates at 100 μL per well, with 5×10 cells per well. 4 After culturing for 24 h, the original culture medium was discarded. A blank control group and a sample group were set up. The sample group was added with 100 μL of complete culture medium containing different concentrations of shell nacre anti-inflammatory peptides, and the blank control group was added with an equal amount of complete culture medium. After continuing to culture for 24 h, the original culture medium was discarded, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and then placed in a cell culture incubator for 4 h. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and after shaking for 10 min, the absorbance value at a wavelength of 490 nm was measured using an enzyme marker.

[0042] 4. L929 cell scratch assay Mouse fibroblast L929 cells were inoculated into 6-well plates and incubated overnight in a 5% CO2, 37°C incubator. A midline was drawn on the bottom of the six-well plate, and two scratches perpendicular to the midline were made on the monolayer of fused cells at the three equal points with a 200 μL pipette tip. Each well was rinsed with PBS solution to remove cell debris. After removal, the experimental group was incubated for 24 h with different concentrations of shell nacre active peptides. In order to avoid the influence of cell proliferation, the blank control group was incubated for 24 h with a medium containing 2% fetal bovine serum. After the incubation, the cell status was observed under a microscope and photographed and recorded for comparison. The effect of active peptides on cell migration ability was analyzed.

[0043] result: (1) Effect of the anti-inflammatory peptide GGVGFYG, which promotes skin repair, on the activity of skin fibroblasts L929 like Figure 4 As shown in the data, within the mass concentration range of 5-40 μg / mL, GGVGFYG had no cytotoxicity to L929 cells. Compared with the blank control group, GGVGFYG had a significant proliferation effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0044] (2) Effect of the anti-inflammatory peptide GGVGFYG, which promotes skin repair, on the migration of skin fibroblasts L929 The effect of GGVGFYG on the migration ability of L929 cells was investigated by in vitro scratch test. Image J software was used to process and compare the changes of scratch area at 0 h and 24 h.

[0045] like Figure 5 As shown in the figure, compared with the blank control group, 5-20 μg / mL of GGVGFYG can significantly increase the migration of L929 cells, and the scratch distance is significantly shortened. In particular, after treatment with 5 μg / mL of GGVGFYG, the wound was almost completely healed. It can be seen that GGVGFYG can significantly promote the migration and fusion of L929 cells and has a good skin repair effect.

[0046] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An anti-inflammatory active peptide having the effect of promoting skin repair, characterized in that: It has the following amino acid sequence: Gly-Gly-Val-Gly-Phe-Tyr-Gly.

2. The anti-inflammatory active peptide having the effect of promoting skin repair according to claim 1, characterized in that: The anti-inflammatory active peptide with skin repair promoting effect is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

3. A shell nacre protein hydrolyzate comprising the anti-inflammatory active peptide having skin repair promoting effect as claimed in claim 1.

4. A solid phase synthesis product comprising the anti-inflammatory active peptide having skin repair promoting effect as claimed in claim 1.

5. The cosmetically acceptable salt or pharmaceutically acceptable salt thereof of the anti-inflammatory active peptide having skin repair promoting effect as claimed in claim 1.

6. The cosmetically acceptable salt or pharmaceutically acceptable salt thereof of the anti-inflammatory active peptide having the effect of promoting skin repair according to claim 5, characterized in that: The cosmetically acceptable salt or pharmaceutically acceptable salt includes a metal salt of a peptide, wherein the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an organic base, wherein the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an inorganic acid or an organic acid, wherein the organic acid includes acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

7. Use of the anti-inflammatory active peptide with skin repair promoting effect as claimed in claim 1, or the shell nacre protein hydrolyzate as claimed in claim 3, or the solid phase synthesis product as claimed in claim 4, or the cosmetically acceptable salt or pharmaceutically acceptable salt thereof of the anti-inflammatory active peptide with skin repair promoting effect as claimed in any one of claims 5 to 6 in the preparation of anti-inflammatory agents and / or skin wound repair products.

8. The use according to claim 7, characterized in that: The anti-inflammatory agent has the effects of promoting macrophage proliferation, promoting macrophage NO release, promoting macrophage anti-inflammatory factor secretion and / or reducing pro-inflammatory factor secretion.

9. The use according to claim 7, characterized in that: The skin wound repair product has the function of promoting the proliferation and / or migration of fibroblasts.

10. The use according to any one of claims 7 to 9, characterized in that: The anti-inflammatory agent and / or skin wound repair product is any one of an oral preparation and an external preparation.

Images