A shell nacre active peptide with anti-inflammatory and skin repair effects

Abstract

The present invention discloses an active peptide from shell nacre with anti-inflammatory and skin-repairing effects. The active peptide, NYRPVLGF, can promote macrophage proliferation and effectively inhibit LPS-induced NO release in RAW264.7 macrophages, effectively regulating the secretion of inflammatory factors from macrophages and, to a certain extent, modulating inflammatory responses. Furthermore, the active peptide has a significant proliferative effect on skin fibroblasts, promoting skin wound healing and possessing promising application value.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, in particular to a shell nacre active peptide with anti-inflammatory and skin repairing effects. Background Art

[0002] When tissues or cells are damaged or infected by external sources of infection (such as bacteria, fungi, viruses and various allergy-inducing substances), the inflammatory response is related to various inflammatory mediators and immune cells in local blood vessels and body fluids, resulting in complex physiological reactions such as enzyme activation, secretion of inflammatory mediators, body fluid infiltration, cell migration, tissue destruction, and external symptoms such as erythema, edema, fever and pain.

[0003] Specifically, if external bacteria invade specific tissues and proliferate, the white blood cells in the body recognize them and actively attack the proliferated external bacteria. The dead white blood cells produced in the above process accumulate in the tissues invaded by the bacteria. At the same time, the cells of the invading bacteria caused by apoptosis of the white blood cells destroy the stomach and dissolve into the invaded tissues, thereby forming an abscess.

[0004] Under normal circumstances, the inflammatory response serves to remove external sources of infection and restores the functions of the body by regenerating damaged tissues. However, if the antigens are not removed or internal substances become the cause, the inflammatory response will be excessive or persistent, and diseases that threaten the human life will appear, such as acute inflammation, diseases in the joints such as rheumatoid arthritis, skin diseases in the form of psoriasis, and allergic inflammatory diseases such as bronchial asthma, etc., and it will also become an obstacle in the treatment process such as blood transfusion, drug administration, and organ transplantation.

[0005] Currently, anti-inflammatory agents include dexamethasone and cortisone, which utilize adrenocortical hormones. While these agents are effective, they are highly toxic and can cause side effects such as edema. Therefore, there is an urgent need to develop non-steroidal agents without side effects. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a shell nacre active peptide with anti-inflammatory and skin repair effects.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0008] Another object of the present invention is to provide the application of the above-mentioned shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A shell nacre active peptide with anti-inflammatory and skin repair effects, having the following amino acid sequence: Asn-Tyr-Arg-Pro-Val-Leu-Gly-Phe (NYRPVLGF).

[0011] Furthermore, the shell nacre active peptide with anti-inflammatory and skin repairing effects is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0012] The invention relates to a shell nacre protein hydrolyzate containing the shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0013] The invention relates to a solid-phase synthesis product comprising the shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0014] A nucleic acid encoding the shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0015] A biological material comprising the above nucleic acid, which is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0016] The use of the above-mentioned shell nacre active peptides, shell nacre protein hydrolysates, solid-phase synthesis products, nucleic acids or biomaterials with anti-inflammatory and skin repair effects in the preparation of anti-inflammatory agents and / or skin wound repair products.

[0017] 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.

[0018] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0019] Furthermore, the anti-inflammatory agent and / or skin wound repair product is any one of an oral preparation and an external preparation.

[0020] The present invention has the following advantages and effects compared to the prior art:

[0021] Molecular docking simulations of the active peptide NYRPVLGF (965.497 Da) provided by this invention revealed that it binds to the receptor proteins cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) through hydrogen bonding and hydrophobic interactions, thereby exerting anti-inflammatory activity. This active peptide significantly promoted the proliferation of RAW264.7 macrophage cells and inhibited lipopolysaccharide (LPS)-induced overproduction of nitric oxide (NO) and cytokines (TNF-α, IL-6) in RAW264.7 cells, while increasing the level of the anti-inflammatory cytokine (IL-10), demonstrating significant anti-inflammatory activity. Furthermore, this active peptide exhibited a significant proliferative effect on L929 cells, suggesting its potential to promote skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the research results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0023] Figure 2 This is a study result of the effect of active peptides on NO release in RAW264.7 macrophages (Note: different lowercase letters indicate significant differences (P<0.05)).

[0024] Figure 3 The graph shows the results of a study on the effects of active peptides on the cytokine release of RAW264.7 macrophages induced by LPS; A represents the effect on the release of cytokine TNF-α, B represents the effect on the release of cytokine IL-6, and C represents the effect on the release of cytokine IL-10 (Note: different lowercase letters indicate significant differences (P<0.05)).

[0025] Figure 4 This is a graph showing the research results on the effect of active peptides on the proliferation rate of L929 cells.

[0026] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0027] 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 intended to limit the present invention.

[0028] Example 1

[0029] 1. Synthesis of active peptides from shell nacre with anti-inflammatory and skin-repairing effects

[0030] The shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects screened by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0031] 2. Cell Culture

[0032] 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 passaged when the cells reached the logarithmic growth phase.

[0033] 3. RAW264.7 Cytotoxicity Assay

[0034] RAW264.7 macrophages in the logarithmic growth phase were seeded into 96-well plates at a volume of 100 μL per well, with 5 × 10 cells per well. 4 After 24 hours of incubation, the original culture medium was discarded. A blank control group and sample groups were set up. The sample group received 100 μL of complete culture medium containing various concentrations of nacre anti-inflammatory peptides, while the blank control group received an equal amount of complete culture medium. After a further 24 hours of incubation, the original culture medium was discarded, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. The cells were then incubated in a cell culture incubator for 4 hours. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance at 490 nm was measured using a microplate reader.

[0035] 4. Determination of Inflammatory Factors in RAW264.7 Cells

[0036] RAW264.7 macrophages in the logarithmic growth phase were seeded into 24-well plates at a volume of 500 μL per well, with a cell count of 1 × 10 5 After culturing for 24 hours, 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 shell nacre active peptides with anti-inflammatory and skin repair effects. The blank control group and the model group were added with the same amount of complete culture medium. The positive control group selected dexamethasone as a control. After a further 24 hours of culture, the original culture medium was discarded. The blank group was added with 500 μL of complete culture medium. The other groups were added with 500 μL of μg / mL LPS solution. The culture was continued for 24 hours. The supernatant was collected and the levels of NO, IL-10, IL-6, and TNF-α in the cell culture supernatant were detected using NO kit and ELISA kit.

[0037] 5. Data Analysis

[0038] All experiments were performed in triplicate, and data are presented as mean ± standard deviation. Statistical graphs were generated using Origin 2019 software, and one-way analysis of variance was performed on the group means using IBM SPSS Statistics 26. P < 0.05 was considered statistically significant.

[0039] result:

[0040] (1) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the viability of RAW264.7 macrophages

[0041] The effect of the anti-inflammatory peptide NYRPVLGF hydrolyzed from nacreous shell protein on the viability of RAW264.7 macrophages at concentrations of 50-800 μg / mL was determined based on the MTT assay.

[0042] Depend on Figure 1 The results indicate that NYRPVLGF treatment of RAW264.7 macrophages for 24 hours did not diminish cell viability, and compared with the blank control group, the anti-inflammatory peptide from nacre promoted the proliferation of RAW264.7 macrophages. Therefore, subsequent studies evaluated the anti-inflammatory activity of the peptide within a concentration range of 10–200 μg / mL.

[0043] (2) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on NO release in RAW264.7 macrophages

[0044] In the present invention, a cellular inflammation model was constructed by inducing NO release from macrophages RAW264.7 cells using LPS, and the anti-inflammatory activity of NYRPVLGF, an active peptide from the shell nacre layer, which has anti-inflammatory and skin repair effects, was explored.

[0045] Depend on Figure 2 As shown, LPS-stimulated RAW264.7 macrophages in the model group significantly increased NO release (P < 0.001). Compared with the model group, NYRPVLGF, a nacre-derived peptide with anti-inflammatory and skin-repairing properties, significantly inhibited NO release from RAW264.7 macrophages at various concentrations. Notably, the inhibition of NO release was concentration-dependent. At a concentration of 200 μg / mL, NYRPVLGF decreased NO release by 68.73 ± 1.67% (P < 0.001), significantly exceeding that of the positive control group (dexamethasone). These results demonstrate that NYRPVLGF effectively inhibits LPS-induced NO release in RAW264.7 macrophages, modulating the inflammatory response to a certain extent.

[0046] (3) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the release of inflammatory factors from RAW264.7 macrophages

[0047] Depend on Figure 3 As shown, compared with the blank control group, the release of inflammatory cytokines TNF-α (a), IL-6 (b), and IL-10 (c) by RAW264.7 macrophages in the model group under LPS stimulation significantly increased. Dexamethasone, a positive control drug with proven anti-inflammatory properties, significantly decreased the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by RAW264.7 macrophages after dexamethasone treatment (P < 0.001), demonstrating that the cellular inflammation model used in this invention is suitable for evaluating the activity of anti-inflammatory peptides from shell nacre. The NYRPVLGF-treated 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 the effects were dose-dependent at concentrations of 10-200 μg / mL. At the highest concentration, the effect on IL-6 secretion was 65.31±1.85%; the effect on TNF-α secretion was 56.51±3.14%; and the effect on IL-10 secretion was 68.29±3.56%.

[0048] The above results indicate that the shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects can effectively regulate the secretion of inflammatory factors in RAW264.7 macrophages and has good anti-inflammatory activity.

[0049] Example 2

[0050] 1. Synthesis of active peptides from shell nacre with anti-inflammatory and skin-repairing effects

[0051] The shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects screened by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0052] 2. Cell Culture

[0053] Mouse fibroblast L929 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator and passaged when the cells reached the logarithmic growth phase.

[0054] 3. L929 Cytotoxicity Assay

[0055] Mouse fibroblast L929 cells in logarithmic growth phase were seeded into 96-well plates at 100 μL per well, with 5 × 10 cells per well. 4After 24 hours of incubation, the original culture medium was discarded. A blank control group and sample groups were set up. The sample group received 100 μL of complete culture medium containing various concentrations of nacre anti-inflammatory peptides, while the blank control group received an equal amount of complete culture medium. After a further 24 hours of incubation, the original culture medium was discarded, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. The cells were then incubated in a cell culture incubator for 4 hours. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance at 490 nm was measured using a microplate reader.

[0056] 4. L929 cell scratch assay

[0057] Mouse fibroblast L929 cells were seeded into 6-well plates and incubated overnight in a 5% CO2, 37°C incubator. A midline was drawn across the bottom of the 6-well plate. Two scratches perpendicular to the midline were made across the confluent cell monolayer at three equal points using a 200 μL pipette tip. Each well was rinsed with PBS to remove cell debris. After removal, the experimental groups were incubated for 24 hours with varying concentrations of shell nacre active peptides. To minimize the effects of cell proliferation, the blank control group was incubated for an additional 24 hours in culture medium supplemented with 2% fetal bovine serum. After incubation, the cells were observed microscopically and photographed for comparison. The effects of the active peptides on cell migration were analyzed.

[0058] result:

[0059] (1) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the activity of skin fibroblasts L929

[0060] like Figure 4 As shown in the data, within the mass concentration range of 5-40 μg / mL, NYRPVLGF had no cytotoxicity to L929 cells. Compared with the blank control group, NYRPVLGF had a significant proliferative effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0061] (2) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the migration of skin fibroblasts L929

[0062] The effect of NYRPVLGF on the migration ability of L929 cells was investigated using an in vitro wound wound test. Image J software was used to compare the changes in wound area at 0 h and 24 h.

[0063] like Figure 5As shown in the results, compared with the blank control group, NYRPVLGF at concentrations of 5 to 20 μg / mL significantly increased L929 cell migration and significantly shortened the wound distance. In particular, treatment with 5 μg / mL of NYRPVLGF resulted in almost complete wound healing. This suggests that NYRPVLGF significantly promotes L929 cell migration and fusion, demonstrating a promising skin repair effect.

[0064] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0065] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A shell nacre active peptide with anti-inflammatory and skin repairing effects, characterized by: The amino acid sequence is as follows: Asn-Tyr-Arg-Pro-Val-Leu-Gly-Phe.

2. The shell nacre active peptide with anti-inflammatory and skin repairing effects according to claim 1, characterized in that: The shell nacre active peptide with anti-inflammatory and skin repairing effects is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

3. A shell nacre protein hydrolyzate comprising the shell nacre active peptide having anti-inflammatory and skin repairing effects as claimed in claim 1.

4. A solid phase synthesis product comprising the shell nacre active peptide having anti-inflammatory and skin repairing effects as claimed in claim 1.

5. A nucleic acid, characterized in that: The nucleic acid encodes the shell nacre active peptide with anti-inflammatory and skin repairing effects as claimed in claim 1.

6. A biomaterial, characterized in that: The biological material comprises the nucleic acid according to claim 5 and is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

7. Use of the shell nacre active peptide with anti-inflammatory and skin repairing effects as claimed in claim 1, the shell nacre protein hydrolyzate as claimed in claim 3, the solid phase synthesis product as claimed in claim 4, the nucleic acid as claimed in claim 5 or the biomaterial as claimed in claim 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 effect 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 an external preparation.

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