Shell nacre active peptide with anti-inflammatory and skin repairing effects

Abstract

The invention discloses a shell nacre active peptide with anti-inflammatory and skin repairing effects. The shell nacre active peptide NYRPVLGF with the anti-inflammatory and skin repairing effects, provided by the invention, can contribute to macrophage proliferation, can effectively inhibit release of RAW264.7 macrophage NO induced by LPS, effectively regulates and controls secretion of macrophage inflammatory factors, and can regulate inflammatory response to a certain extent. Besides, the bioactive peptide has a remarkable proliferation effect on skin fibroblasts, can promote healing of skin wounds, and has good application value.

Description

Technical Field

[0001] The invention relates to the technical field of active peptides, and 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, molds, 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 movement, 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 will 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 restore 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 continuous, and diseases that threaten the human life may 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 may also become an obstacle in the treatment process such as blood transfusion, drug administration, and organ transplantation.

[0005] Currently, there are dexamethasone and cortisone that use adrenal cortical hormones as inflammatory therapeutic agents. Although they work as inflammatory therapeutic agents, they are highly toxic and cause symptoms such as edema as side effects. Therefore, it is urgent to develop non-steroidal drugs 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 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 with anti-inflammatory and skin repair effects.

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

[0010] Furthermore, the shell nacre active peptides with anti-inflammatory and skin repairing effects are prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0011] The invention relates to a shell nacre protein hydrolyzate comprising the shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0012] The invention relates to a solid phase synthesis product comprising the above-mentioned shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0013] A nucleic acid encoding the shell nacre active peptide having anti-inflammatory and skin repairing effects.

[0014] A biological material, comprising the above-mentioned nucleic acid, is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0015] 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.

[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 anti-inflammatory agent and / or skin wound repair product 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 molecular docking simulation of the active peptide NYRPVLGF (965.497 Da) provided by the present invention shows that it 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 significantly 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the study results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0021] 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)).

[0022] 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)).

[0023] Figure 4 This is a graph showing the study results on the effect of active peptides on the proliferation rate of L929 cells.

[0024] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1 1. Synthesis of active peptides from shell nacre with anti-inflammatory and skin repair effects The shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects screened out by molecular docking was synthesized by solid phase synthesis, and the purity of the synthetic 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 passaged when the cells reached 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 a cell number of 5 × 10 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.

[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 pearl active peptides with anti-inflammatory and skin repair effects. 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.

[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) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the viability of RAW264.7 macrophages The MTT method was used to determine the effect of the anti-inflammatory peptide NYRPVLGF hydrolyzed from the nacre layer on the viability of RAW264.7 macrophages at a concentration of 50-800 μg / mL.

[0032] Depend on Figure 1 It can be seen that after NYRPVLGF treated RAW264.7 macrophages for 24 hours, the cell viability did not weaken, 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.

[0033] (2) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, 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 using LPS, and explores the anti-inflammatory activity of the shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects.

[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 NYRPVLGF sample group, which has anti-inflammatory and skin repair effects, 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 NYRPVLGF decreased by 68.73±1.67% (P<0.001), and was higher than that of the positive control group dexamethasone. The experimental results show that NYRPVLGF 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) 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 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 NYRPVLGF 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 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%.

[0036] 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.

[0037] Example 2 1. Synthesis of active peptides from shell nacre with anti-inflammatory and skin repair effects The shell nacre active peptide NYRPVLGF with anti-inflammatory and skin repair effects screened out by molecular docking was synthesized by solid phase synthesis, and the purity of the synthetic 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. 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.

[0040] 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.

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

[0042] (2) Effects of NYRPVLGF, a shell nacre active peptide with anti-inflammatory and skin repair effects, on the migration of skin fibroblasts L929 The effect of NYRPVLGF 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.

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

[0044] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0045] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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 in that: It has the following amino acid sequence: 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 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 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 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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