Preparation of an anti-inflammatory peptide hydrolyzed from shell nacre protein and its application in skin repair

Abstract

The present invention discloses the preparation of an anti-inflammatory peptide derived from the hydrolysis of shell nacre protein and its application in skin repair. The peptide PDFDNGF provided by the invention can effectively promote the proliferation of RAW264.7 macrophage cells and inhibit the excessive production of nitric oxide and cytokines in RAW264.7 cells induced by lipopolysaccharide, while increasing the levels of anti-inflammatory cytokines, demonstrating significant anti-inflammatory activity. Furthermore, the active peptide has a significant proliferative effect on L929 cells, indicating its potential to promote skin wound healing. This invention provides a theoretical basis for the high-value utilization of shell nacre.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, in particular to the preparation of an anti-inflammatory peptide obtained by hydrolysis of shell nacre protein and its application in skin repair. Background Art

[0002] Shells are divided into three layers, depending on their formation method and structure. The outermost layer is the cuticle (periostracum); the middle prismatic layer, also called the "ostracum"; and the innermost layer is the pearl layer, or "hypostracum." The nacre is typically composed of leaf-shaped aragonite, secreted from the entire surface of the mantle. It thickens and develops a lustrous luster as the animal grows. Under an electron microscope, aragonite crystals appear like brickwork, with thin layers of amino acids filling the gaps between each layer. Nacre is homologous to pearls and contains the same ingredients: primarily calcium, various amino acids, and trace elements. The keratin protein contained in it contains amino acids that cannot be synthesized by the human body.

[0003] While people are obtaining pearls and pearl oyster meat, a large amount of shell resources are left as waste and are not effectively utilized. How to rationally utilize these shell resources has become an urgent problem to be solved. 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 peptide obtained by hydrolyzing shell nacre protein.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned shell nacre protein hydrolyzed anti-inflammatory peptide.

[0006] Another object of the present invention is to provide the use of the above-mentioned shell nacre protein hydrolyzed anti-inflammatory peptide.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A shell nacre protein hydrolyzed anti-inflammatory peptide having the following amino acid sequence: Pro-Asp-Phe-Asp-Asn-Gly-Phe (PDFDNGF).

[0009] Furthermore, the shell nacre protein hydrolysis anti-inflammatory peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0010] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre protein hydrolyzed anti-inflammatory peptide.

[0011] The invention relates to a solid phase synthesis product comprising the above-mentioned shell nacre protein hydrolysis anti-inflammatory peptide.

[0012] A nucleic acid encoding the above-mentioned shell nacre protein hydrolysis anti-inflammatory peptide.

[0013] A biological material comprising the above nucleic acid, which is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0014] The use of the above-mentioned shell nacre protein hydrolysis anti-inflammatory peptides, shell nacre protein hydrolysis products, solid-phase synthesis products, nucleic acids or biomaterials in the preparation of anti-inflammatory agents and / or skin wound repair products.

[0015] 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.

[0016] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0017] Furthermore, the active peptide is any one of an oral preparation and an external preparation.

[0018] The present invention has the following advantages and effects compared to the prior art:

[0019] This study isolated, purified, and identified multiple peptides from a protein hydrolyzate extracted from the nacreous layer of shells. A novel anti-inflammatory peptide, PDFDNGF (811.3024 Da), was subsequently identified through molecular docking. Molecular docking simulations revealed that the active peptide binds to the receptor proteins cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) through hydrogen bonds and hydrophobic interactions, thereby exerting anti-inflammatory activity. The active peptide effectively promoted the proliferation of RAW264.7 macrophages and inhibited lipopolysaccharide (LPS)-induced overproduction of nitric oxide (NO) and cytokines (TNF-α and IL-6) in RAW264.7 cells, while increasing the level of the anti-inflammatory cytokine (IL-10), demonstrating significant anti-inflammatory activity. Furthermore, the active peptide exhibited a significant proliferative effect on L929 cells, suggesting its 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 research results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0022] Figure 2This 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)).

[0023] 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)).

[0024] Figure 4 This is a graph showing the research 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 intended 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", "an", "the" 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 otherwise.

[0028] Example 1

[0029] 1. Synthesis of anti-inflammatory peptides from shell nacre protein hydrolysis

[0030] The anti-inflammatory peptide PDFDNGF obtained by hydrolysis of nacreous layer protein through molecular docking was synthesized by solid phase synthesis method, and the purity of the synthesized 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, model group, sample group, and positive control group were set up. The sample group was added with 500 μL of complete culture medium containing different concentrations of shell nacre protein hydrolyzed anti-inflammatory peptides. 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 further culturing for 24 hours, 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. After further culturing 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) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre on the viability of RAW264.7 macrophages

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

[0042] Depend on Figure 1The results indicate that PDFDNGF 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) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre on NO release in RAW264.7 macrophages

[0044] 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 peptide PDFDNGF hydrolyzed from shell nacre protein.

[0045] Depend on Figure 2 As shown, NO release from RAW264.7 macrophages in the model group was significantly increased after LPS stimulation (P < 0.001). Compared with the model group, PDFDNGF, an anti-inflammatory peptide hydrolyzed from nacreous shell protein, significantly inhibited NO release from RAW264.7 macrophages at various concentrations. Notably, the inhibition rate on NO release was concentration-dependent. At a concentration of 200 μg / mL, PDFDNGF decreased NO release by 74.16±2.39% (P < 0.001), significantly exceeding that of the positive control group (dexamethasone). These results demonstrate that PDFDNGF effectively inhibits LPS-induced NO release from RAW264.7 macrophages, modulating the inflammatory response to a certain extent.

[0046] (3) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre on the release of inflammatory factors from RAW264.7 macrophages

[0047] Depend on Figure 3As 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 PDFDNGF-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 the 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 63.52±1.31%; the effect on TNF-α secretion was 48.41±3.91%; and the effect on IL-10 secretion was 58.69±2.92%.

[0048] The above results indicate that the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre protein 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 anti-inflammatory peptides from shell nacre protein hydrolysis

[0051] The anti-inflammatory peptide PDFDNGF obtained by hydrolysis of nacreous layer protein through molecular docking was synthesized by solid phase synthesis method, and the purity of the synthesized 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 placed in a 5% CO 2 The cells were incubated overnight in a 37°C incubator. A midline was drawn on the bottom of the six-well plate. Two scratches perpendicular to the midline were made on 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 with varying concentrations of nacreous active peptides for 24 hours. To minimize the effects of cell proliferation, the blank control group was incubated with medium containing 2% fetal bovine serum for an additional 24 hours. After the incubation period, the cells were observed under a microscope and photographed for comparison. The effects of the active peptides on cell migration were analyzed.

[0058] result:

[0059] (1) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre 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, PDFDNGF had no cytotoxicity to L929 cells. Compared with the blank control group, PDFDNGF had a significant proliferative effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0061] (2) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre on the migration of skin fibroblasts L929

[0062] The effect of PDFDNGF 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, 5-20 μg / mL of PDFDNGF significantly increased L929 cell migration and significantly shortened the scratch distance. In particular, treatment with 5 μg / mL of PDFDNGF resulted in almost complete wound healing. This suggests that PDFDNGF significantly promotes L929 cell migration and fusion, demonstrating a beneficial effect on skin repair.

[0064] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An anti-inflammatory peptide obtained by hydrolysis of shell nacre protein, characterized in that: The amino acid sequence is as follows: Pro-Asp-Phe-Asp-Asn-Gly-Phe.

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

3. A nucleic acid, characterized in that: The nucleic acid encodes the shell nacre proteolytic anti-inflammatory peptide described in claim 1.

4. A biomaterial, characterized in that: The biological material comprises the nucleic acid according to claim 3 and is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

5. Use of the anti-inflammatory peptide obtained by hydrolysis of shell nacreous layer protein as claimed in claim 1, the nucleic acid as claimed in claim 3 or the biomaterial as claimed in claim 4 in the preparation of an anti-inflammatory agent and / or a skin wound repair product.

6. The use according to claim 5, 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.

7. The use according to claim 6, characterized in that: The skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

8. The use according to any one of claims 5 to 7, characterized in that: The anti-inflammatory agent and / or skin wound repair product is an external preparation.

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