Preparation of shell nacre protein hydrolysis anti-inflammatory peptide and application of shell nacre protein hydrolysis anti-inflammatory peptide in skin repair

Abstract

The invention discloses preparation of a shell nacre protein hydrolysis anti-inflammatory peptide and application of the shell nacre protein hydrolysis anti-inflammatory peptide in skin repair. The peptide PDFDNGF provided by the invention can effectively promote proliferation of macrophage RAW264.7 cells, can inhibit excessive production of nitric oxide and cytokines in the RAW264.7 cells induced by lipopolysaccharide, can increase the level of anti-inflammatory cytokines, and shows remarkable anti-inflammatory activity. In addition, the bioactive peptide has a remarkable proliferation effect on L929 cells, and it is indicated that the bioactive peptide has the potential of promoting skin wound healing. The method provides a theoretical basis for high-value utilization of the shell nacre layer.

Description

Technical Field

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

[0002] Shells can be divided into three layers according to the formation method and organizational structure. The outermost layer is the cuticle (periostracum); the middle prismatic layer (prismatic layer), also known as the "shell layer" (ostracum); the inner layer is the pearl layer (pearl layer), or "shell bottom" (hypostracum). The pearl layer is usually composed of leaf-shaped aragonite, which is formed by the secretion of the entire surface of the mantle. It increases in thickness and is shiny as the animal grows. Under an electron microscope, aragonite crystals appear in a brick-like structure, with extremely thin amino acids filling between each layer. The pearl layer is homologous to pearls and contains the same ingredients as pearls, mainly calcium, multiple amino acids and a small amount of trace elements. The horny shell protein contained in it contains unit amino acids that cannot be synthesized by the human body.

[0003] While people are getting pearls and pearl oyster meat, a large amount of shell resources are left as waste and are not effectively utilized. How to reasonably 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 nacreous 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 application 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: A shell nacre proteolytic anti-inflammatory peptide having the following amino acid sequence: Pro-Asp-Phe-Asp-Asn-Gly-Phe (PDFDNGF).

[0008] Furthermore, the shell nacre protein hydrolyzed anti-inflammatory peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0009] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre protein hydrolyzed anti-inflammatory peptide.

[0010] The invention relates to a solid phase synthesis product comprising the above-mentioned shell nacre protein hydrolyzed anti-inflammatory peptide.

[0011] A nucleic acid encoding the above-mentioned shell nacre proteolytic anti-inflammatory peptide.

[0012] A biological material, comprising the above-mentioned nucleic acid, is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0013] 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.

[0014] 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.

[0015] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0016] Furthermore, the active peptide is any one of an oral preparation and an external preparation.

[0017] 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 PDFDNGF (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.

[0018] The invention provides a theoretical basis for high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the study results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0020] 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)).

[0021] 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)).

[0022] Figure 4 This is a graph showing the study results on the effect of active peptides on the proliferation rate of L929 cells.

[0023] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] Example 1 1. Synthesis of anti-inflammatory peptides from nacreous layer proteolytic enzymes The anti-inflammatory peptide PDFDNGF obtained by hydrolysis of nacreous protein from shells through molecular docking was synthesized by solid phase synthesis method, and the purity of the synthesized peptide was ≥98% as determined by HPLC.

[0027] 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.

[0028] 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. 4After 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.

[0029] 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 5 After 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 shell nacre protein hydrolyzed anti-inflammatory peptides, the blank control group and the model group were added with an equal amount of complete culture medium, and 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.

[0030] 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.

[0031] result: (1) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from nacreous shells on the viability of RAW264.7 macrophages The MTT method was used to determine the effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from the nacre layer on the viability of RAW264.7 macrophages at a concentration of 50-800 μg / mL.

[0032] Depend on Figure 1It can be seen that after PDFDNGF treated RAW264.7 macrophages for 24 h, the cell viability did not weaken, and compared with the blank control group, shell nacre anti-inflammatory peptides promoted the proliferation of RAW264.7 macrophages. Therefore, subsequent studies chose to evaluate the anti-inflammatory activity in the concentration range of 10-200 μg / mL.

[0033] (2) Effect of anti-inflammatory peptide PDFDNGF hydrolyzed from nacreous shell protein on NO release in RAW264.7 macrophages The present invention constructs a cellular inflammation model by inducing the release of NO from macrophages RAW264.7 by LPS, and explores the anti-inflammatory activity of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre protein.

[0034] Depend on Figure 2 It 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 sample group of the anti-inflammatory peptide PDFDNGF from shell nacre protein hydrolyzed at different concentrations could significantly inhibit the release of NO by RAW264.7 macrophages. It is worth noting that its inhibition rate on NO release was concentration-dependent. At a concentration of 200 μg / mL, the NO release of PDFDNGF decreased by 74.16±2.39% (P<0.001), and was higher than that of the positive control group dexamethasone. The experimental results show that PDFDNGF can effectively inhibit the release of NO from RAW264.7 macrophages induced by LPS, and can regulate the inflammatory response to a certain extent.

[0035] (3) Effect of anti-inflammatory peptide PDFDNGF from shell nacre protein on the release of inflammatory factors from RAW264.7 macrophages Depend on Figure 3It 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 PDFDNGF 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 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%.

[0036] The above results indicate that the anti-inflammatory peptide PDFDNGF hydrolyzed from the shell nacre protein can effectively regulate the secretion of inflammatory factors in RAW264.7 macrophages and has good anti-inflammatory activity.

[0037] Example 2 1. Synthesis of anti-inflammatory peptides from nacreous layer proteolytic enzymes The anti-inflammatory peptide PDFDNGF obtained by hydrolysis of nacreous protein from shells through molecular docking was synthesized by solid phase synthesis method, and the purity of the synthesized peptide was ≥98% as determined by HPLC.

[0038] 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.

[0039] 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.

[0040] 4. L929 cell scratch assay Mouse fibroblast L929 cells were seeded into 6-well plates in a 5% CO 2 , incubated in a 37°C incubator overnight, 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, and 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 another 24 h with a medium containing 2% fetal bovine serum. After the incubation, the cell status was observed under a microscope and photographed and compared. The effect of active peptides on cell migration ability was analyzed.

[0041] result: (1) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre 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, PDFDNGF had no cytotoxicity to L929 cells. Compared with the blank control group, PDFDNGF had a significant proliferation effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0042] (2) Effect of the anti-inflammatory peptide PDFDNGF hydrolyzed from shell nacre on the migration of skin fibroblasts L929 In vitro scratch test was used to explore the effect of PDFDNGF on the migration ability of L929 cells. Image J software was used to process and compare the changes in scratch area at 0 h and 24 h.

[0043] like Figure 5 As shown in the figure, compared with the blank control group, 5-20 μg / mL PDFDNGF can significantly increase the migration of L929 cells, and the scratch distance is significantly shortened. In particular, after treatment with 5 μg / mL PDFDNGF, the wound was almost completely healed. It can be seen that PDFDNGF can significantly promote the migration and fusion of L929 cells and has a good skin repair effect.

[0044] 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 peptide hydrolyzed from pearly layer of shells, characterized in that: It has the following amino acid sequence: 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 shell nacre protein hydrolyzate comprising the shell nacre protein hydrolyzed anti-inflammatory peptide as claimed in claim 1.

4. A solid phase synthesis product comprising the shell nacre protein hydrolyzed anti-inflammatory peptide as claimed in claim 1.

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

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

7. Use of the shell nacre protein hydrolyzate anti-inflammatory peptide described in claim 1, the shell nacre protein hydrolyzate described in claim 3, the solid phase synthesis product described in claim 4, the nucleic acid described in claim 5 or the biomaterial described 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 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.

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