An anti-inflammatory peptide derived from pearl oyster and application thereof

Abstract

This invention discloses an anti-inflammatory peptide derived from pearl oysters and its applications, relating to the field of bioactive peptide technology. The amino acid sequence of this anti-inflammatory peptide is TAMY. The anti-inflammatory peptide derived from pearl oysters provided by this invention can regulate the release of cellular inflammatory mediators, reduce the levels of NO and pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-1β), and simultaneously promote the secretion of anti-inflammatory cytokines (such as IL-10), exhibiting excellent anti-inflammatory activity and suitable for use in functional foods, cosmetics, or pharmaceutical ingredients.

Description

Technical Field

[0001] This invention belongs to the field of active peptide technology, and more specifically, relates to an anti-inflammatory peptide derived from pearl oysters and its applications. Background Technology

[0002] Inflammation is a normal physiological defense response of the body's immune system to harmful stimuli and tissue damage. It is an adaptive immune response. Excessive inflammatory responses of the immune system can lead to the accumulation of inflammatory mediators (such as NO, cellular inflammatory factors, and chemokines), exacerbating the inflammatory response and damaging the body and tissues, resulting in local inflammatory diseases. Although inflammatory diseases have various causes, they share common pathological mechanisms in the inflammatory response, including cellular pathways through which activated immune cells regulate inflammatory mediators. Therefore, studying important cellular pathways in the inflammatory response process can lead to the development of anti-inflammatory active substances for inflammation-related diseases. Due to the increasing incidence of inflammation-related diseases and concerns about the side effects of anti-inflammatory drugs, public attention has increased significantly for safer natural compounds and their derivatives that can prevent, control, and treat inflammation-related diseases. Food-derived derivatives can regulate chronic inflammation, and bioactive peptides extracted from food can regulate the secretion of pro-inflammatory and anti-inflammatory cytokines by immune cells, demonstrating good anti-inflammatory effects in in vitro cell studies and in vivo animal experiments.

[0003] The prior art discloses the application of small molecule peptides from pearl oysters in skin wound repair. It obtains enzymatic hydrolysates of peptides with different molecular weights through enzymatic hydrolysis, ultrafiltration separation and other steps, and points out that the main therapeutic effects of these enzymatic hydrolysates include hemostasis, antibacterial, anti-inflammatory, regulation of cytokines, promotion of wound healing and inhibition of scar formation. However, it does not study which specific active ingredients play the corresponding therapeutic role. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing research on the anti-inflammatory active components of pearl oyster meat and to provide an anti-inflammatory peptide derived from pearl oysters. This anti-inflammatory peptide can inhibit the activity of cyclooxygenase-2, regulate the release of inflammatory mediators, including reducing the levels of NO and pro-inflammatory cytokines and promoting the secretion of anti-inflammatory factors, and can be used in the preparation of functional foods, cosmetics or pharmaceuticals.

[0005] Another object of the present invention is to provide an application of the above-mentioned anti-inflammatory peptide in the preparation of a drug.

[0006] Another object of the present invention is to provide an application of the above-mentioned anti-inflammatory peptide in the preparation of functional foods.

[0007] Another object of the present invention is to provide an application of the above-mentioned anti-inflammatory peptide in the preparation of cosmetics.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] An anti-inflammatory peptide derived from pearl oysters, the anti-inflammatory peptide having the sequence shown in SEQ ID NO: 1.

[0010] The inventors of this invention extracted an anti-inflammatory peptide from pearl oysters, with the sequence TAMY (Thr-Ala-Met-Tyr). This anti-inflammatory peptide can inhibit the activity of cyclooxygenase-2 by occupying a key region of the cyclooxygenase-2 active site, thereby suppressing the inflammatory response. Furthermore, cell experiments have confirmed that the anti-inflammatory peptide TAMY derived from pearl oysters can regulate the release of cellular inflammatory mediators, including reducing the levels of NO and pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokines, exhibiting excellent anti-inflammatory activity. Therefore, this anti-inflammatory peptide can be widely used in the preparation of functional foods, cosmetics, or pharmaceuticals.

[0011] This invention specifically protects the application of an anti-inflammatory peptide derived from pearl oysters in the preparation of pharmaceuticals.

[0012] Furthermore, the use of the anti-inflammatory peptide in the preparation of a drug that inhibits cyclooxygenase-2 activity is protected.

[0013] Prostaglandin intraperoxide synthase, commonly known as cyclooxygenase (COX), is mainly composed of cyclooxygenase-1 and cyclooxygenase-2 isoforms. It is a key enzyme in the biosynthetic pathway that induces prostaglandin (PG) production and regulates inflammatory responses, and prostaglandins play a crucial role in inflammation. Inhibiting cyclooxygenase activity, especially cyclooxygenase-2, can reduce the expression of pro-inflammatory cytokines downstream of the inflammatory pathway, interfere with the pathogenesis of inflammatory diseases, and effectively alleviate inflammatory symptoms. The anti-inflammatory peptide described in this invention can occupy a key region of the cyclooxygenase-2 active site, inhibiting cyclooxygenase-2 activity and thus suppressing the inflammatory response.

[0014] More specifically, the use of the anti-inflammatory peptide in the preparation of a drug in which it is bound by hydrogen bonds to residues Thr 94, Leu 352, Ile 517, Phe 518, and Glu 524 of the cyclooxygenase-2 protein.

[0015] More specifically, the use of the anti-inflammatory peptide in the preparation of a drug in which it binds to residues Pro 514, Ala 516, and Ala 527 of the cyclooxygenase-2 protein via hydrophobic bonds is protected.

[0016] Hydrophobicity of peptides is one of the main factors inducing anti-inflammatory responses. Anti-inflammatory active peptides are generally rich in hydrophobic and polar amino acids, with hydrophobic amino acids mainly concentrated at the N-terminus, while the C-terminus mainly contains polar groups. The presence of hydrophobic amino acids can enhance the interaction between the peptide and the cell membrane, and can exert a stronger anti-inflammatory effect by inhibiting the release of LPS-induced inflammatory cytokines and inhibiting the phosphorylation of signaling proteins to regulate downstream signaling pathways. The anti-inflammatory peptide (TAMY) provided by this invention can form hydrogen bonds with residues Thr94, Leu 352, Ile 517, Phe 518, and Glu 524 of cyclooxygenase-2 protein, and form hydrophobic bonds with Pro514, Ala 516, and Ala527. Furthermore, the anti-inflammatory peptide can form hydrophobic bonds with amino acid residues Ala 516 and Ala 527 involved in protein ligand complementarity activity, and form hydrogen bonds with Leu 352 and Phe 518 in the hydrophobic pocket.

[0017] Furthermore, the use of the anti-inflammatory peptide in the preparation of drugs that reduce cellular NO release is protected.

[0018] NO is an important intracellular and intercellular signaling molecule that participates in regulating various physiological and pathological processes. Under normal conditions, NO can regulate physiological functions such as neurotransmission, vasodilation, and immune response. However, excessive intracellular NO is not conducive to alleviating inflammatory responses and can damage cells, leading to apoptosis. The anti-inflammatory peptides described in this invention can reduce NO levels and inhibit inflammatory responses by controlling cell secretion of NO.

[0019] Furthermore, the use of the anti-inflammatory peptide in the preparation of drugs that reduce the secretion of pro-inflammatory factors is protected.

[0020] More specifically, the pro-inflammatory factor is at least one of TNF-α, IL-6, and IL-1β.

[0021] Furthermore, the use of the anti-inflammatory peptide in the preparation of a drug that promotes the secretion of the IL-10 anti-inflammatory factor is protected.

[0022] The anti-inflammatory peptides described in this invention can significantly reduce the secretion of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β, while increasing the secretion of the anti-inflammatory cytokine IL-10.

[0023] Furthermore, the amount of the anti-inflammatory peptide added to the drug is 0.0625–2.0 mg / mL.

[0024] This invention also protects the use of an anti-inflammatory peptide derived from pearl oysters in the preparation of functional foods.

[0025] This invention also protects the use of an anti-inflammatory peptide derived from pearl oysters in the preparation of cosmetic ingredients.

[0026] The anti-inflammatory peptides described in this invention have good anti-inflammatory activity and can be used to prepare cosmetics, playing an anti-inflammatory and soothing role and relieving skin inflammation problems.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The anti-inflammatory peptides derived from pearl oysters provided by this invention have good cyclooxygenase-2 inhibitory activity and can also regulate the release of inflammatory mediators, including reducing the levels of NO and pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokines. They have excellent anti-inflammatory activity and can be widely used in the preparation of functional foods, cosmetics or drugs. Attached Figure Description

[0029] Figure 1 The effect of pearl oyster meat hydrolysate, <3kDa fraction and ≥3kDa fraction on NO release from mouse macrophages RAW264.7.

[0030] Figure 2 The absorption spectrum at 280 nm for the <3kDa fraction separated and purified by RP-HPLC.

[0031] Figure 3 The figure shows the effect of components F1, F2, and F3 on the survival rate of LPS-induced RAW264.7 macrophages.

[0032] Figure 4 The figure shows the effect of components F1, F2, and F3 on LPS-induced NO release from RAW264.7 macrophages.

[0033] Figure 5 The absorption spectrum of component F1 at 280 nm is shown.

[0034] Figure 6 This is the total electron flow graph for component F1.

[0035] Figure 7 The molecular docking model of the interaction between cyclooxygenase-2 and anti-inflammatory peptides is shown in 3D (A) and 2D (B).

[0036] Figure 8 The figure shows the effect of anti-inflammatory peptides on the survival rate of RAW264.7 cells.

[0037] Figure 9 The figure shows the effect of different concentrations of anti-inflammatory peptides on NO release from RAW264.7 macrophages.

[0038] Figure 10 The figure shows the effect of anti-inflammatory peptides on the release of TNF-α factor from RAW264.7 macrophages.

[0039] Figure 11The figure shows the effect of anti-inflammatory peptides on the release of IL-6 from RAW264.7 macrophages.

[0040] Figure 12 Figure showing the effect of anti-inflammatory peptides on the release of IL-1β from RAW264.7 macrophages.

[0041] Figure 13 The figure shows the effect of anti-inflammatory peptides on the release of IL-10 factor from RAW264.7 macrophages. Detailed Implementation

[0042] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from the conventional market. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0043] The main materials and reagents used in each embodiment are described below:

[0044] The meat of Pinctada martensii (also known as pearl oyster) is provided by Beihai Black Pearl Marine Biotechnology Co., Ltd.

[0045] Neutral protease (10 U / g) was purchased from Guangxi Nanning Pangbo Biotechnology Co., Ltd. (Guangxi, China);

[0046] DMEM medium and fetal bovine serum (FBS) were purchased from Gibco (New York, USA).

[0047] Dexamethasone (DEX), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0048] Mouse macrophages RAW264.7 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China);

[0049] The NO detection kit was purchased from Beyotime Biotechnology (Shanghai, China).

[0050] The TNF-α, IL-6, IL-1β, and IL-10 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Xinbosheng Biotechnology Co., Ltd. (Shenzhen, China).

[0051] All other chemicals and reagents were of analytical grade.

[0052] Example 1: Enzymatic hydrolysis, ultrafiltration, and separation and purification of pearl oyster meat

[0053] (1) Enzymatic hydrolysis: The pearl oyster meat was enzymatically hydrolyzed using neutral protease. The specific process is as follows: The pearl oyster meat was washed and drained, then crushed into a pearl oyster meat homogenate. Deionized water was added at a material-to-liquid ratio (mass ratio) of 1:1 to adjust the pH to 7.0. Neutral protease was added at an enzyme-to-liquid ratio of 0.31%, and the mixture was enzymatically hydrolyzed in a water bath at 46.3℃ for 84 min. After inactivating the enzyme in a water bath at 90℃ for 10 min, the mixture was cooled to room temperature, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The mixture was freeze-dried to obtain the pearl oyster meat hydrolysate and stored at -20℃ for subsequent analysis.

[0054] (2) Ultrafiltration: The enzymatic hydrolysate of pearl oyster meat was separated by using an ultrafiltration tube with a molecular weight cutoff of 3kDa. The mixture was centrifuged at 4000r / min for 30min, and the components with molecular weight <3kDa and ≥3kDa were collected. After vacuum freeze-drying, the mixture was stored at -20℃.

[0055] Pearl oyster meat enzymatic hydrolysate, <3kDa fraction, and ≥3kDa fraction were used as samples for cellular anti-inflammatory activity evaluation. The specific process for evaluating cellular anti-inflammatory activity was as follows:

[0056] Cell Culture: Mouse RAW264.7 macrophages were cultured in DMEM medium containing 10% FBS (inactivated at 56°C for 30 min) in a 5% CO2, 37°C cell culture incubator. Cells were passaged after reaching the logarithmic growth phase. When the cell deposition rate exceeded 80% of the culture flask area, the original culture medium was aspirated, and 2 mL of culture medium was pipetted to completely detach the cells. Cells were counted, and the cell suspension concentration was adjusted based on the count results. The cells were then seeded into wells and incubated in a constant temperature incubator.

[0057] Cytotoxicity assay: Cytotoxicity of the samples was evaluated using mouse RAW264.7 macrophages. The concentration of RAW264.7 macrophages was adjusted to 1×10⁻⁶. 4 Cells were seeded at a rate of 100 μL / mL into 96-well plates (6 replicates per group) and incubated overnight. The old culture medium was discarded. A control group and a sample group were set up. 100 μL of complete culture medium containing the sample was added to the sample group, and an equal volume of complete culture medium was added to the blank group. After culturing for 24 h, the old culture medium was discarded, and 100 μL of MTT solution (0.5 mg / mL) was added to each well and incubated in an incubator for 4 h. Finally, the MTT solution was discarded, and 150 μL of DMSO was added to each well to dissolve the solids. The mixture was shaken for 10 min to fully dissolve the crystals. The absorbance of each well at 490 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control group. A sample concentration with a reduction in cell viability of more than 10% compared to the control group was considered cytotoxic.

[0058] NO release was measured by adjusting the concentration of RAW264.7 macrophages to 5 × 10⁻⁶. 4 Cells were seeded at a rate of 500 μL per well in 24-well plates (three replicates per group). Incubation was performed overnight at 37°C with 5% CO2. Dexamethasone (DEX) was used as the positive control. Different concentrations of sample were added to the sample groups. The blank and model groups were added with an equal volume of complete culture medium (the medium used for cell culture). After culturing for 24 hours, the supernatant was aspirated. The model, sample, and positive control groups were stimulated with 500 μL of LPS (1 μg / mL) for 24 hours. The blank group was added with an equal volume of complete culture medium. The cell culture supernatant was collected, and the NO content in the supernatant was measured using the Griess method, which represents the NO release.

[0059] The results of the evaluation of the anti-inflammatory activity of pearl oyster meat enzymatic hydrolysate, the <3kDa fraction, and the ≥3kDa fraction are as follows: Figure 1 As shown, Figure 1 In the diagram, ### indicates P < 0.001 compared to the control group; *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001 compared to the model group, respectively. From Figure 1 It was found that, compared with the model group, the NO release in the positive control group (DEX, 1 μg / mL) decreased by 44.84%. At a concentration of 2.0 mg / mL, the pearl oyster meat hydrolysate, the ≥3 kDa fraction, and the <3 kDa fraction all significantly inhibited NO release (P<0.001), with NO release decreasing by 70.00±0.69%, 62.60±1.98%, and 74.08±1.83%, respectively (P<0.001). This indicates that the <3 kDa fraction exhibited the strongest anti-inflammatory activity, and its further separation and purification are planned.

[0060] (3) Separation and purification: High performance liquid chromatography (RP-HPLC) was used to separate and purify the <3kDa components. The specific process is as follows:

[0061] First, the fractions <3kDa were filtered through a 0.45μm filter membrane and then separated using preparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a C18 self-assembled column (10μm, 10mm × 250mm). The chromatographic conditions were as follows: mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid methanol solution; injection volume: 3mL; elution flow rate: 10mL / min; detection wavelength: 280nm; the elution program is shown in Table 1 below.

[0062] Table 1. Elution Procedure

[0063] time mobile phase 0-45min 5-10% mobile phase B 45-65min 10-20% mobile phase B 65-75min 20-50% mobile phase B 75-80min 50-95% mobile phase B 80-85min 95%–5% mobile phase B

[0064] Collect the elution peaks, concentrate them using rotary evaporation, and separate the fractions F1, F2, and F3. The high-performance liquid chromatogram is shown below. Figure 2 As shown, the anti-inflammatory activity of cells was evaluated after concentration and freeze-drying. Components F1, F2, and F3 were used as samples, and their anti-inflammatory activity was evaluated according to the aforementioned method. The results are as follows. Figure 3 and Figure 4 As shown. Figure 3 The effect of components F1, F2, and F3 on LPS-induced RAW264.7 macrophage cell survival is shown in the figure. Figure 4 The figure shows the effect of components F1, F2, and F3 on LPS-induced NO release from RAW264.7 macrophages; among them, ### ** indicates P < 0.001 compared to the control group; ** and *** indicate P < 0.01 and P < 0.001 compared to the model group, respectively; from Figure 3 It was found that components F1, F2, and F3 had no toxic side effects on RAW264.7 macrophages within the concentration range of 0–2.0 mg / mL. From Figure 4 It was found that LPS-induced NO release from RAW264.7 macrophages in the model group was significantly increased (P<0.001), and the inhibitory effect of components F1, F2, and F3 on NO release from RAW264.7 macrophages gradually increased with increasing concentrations. Compared with the NO release from RAW264.7 cells in the model group, at a concentration of 2.0 mg / mL, the NO release from RAW264.7 macrophages treated with components F1, F2, and F3 decreased by 58.98%, 50.76%, and 53.45%, respectively (P<0.001). Therefore, component F1 had the strongest inhibitory effect on NO secretion from RAW264.7 cells, and component F1 was selected for further isolation and purification.

[0065] Component F1 was subjected to secondary separation. The chromatographic conditions were as follows: C18 column (5 μm, 4.6 mm × 200 mm); mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid methanol solution; injection volume: 1 mL; elution flow rate: 10 mL / min; detection wavelength: 280 nm; the elution program is shown in Table 2 below.

[0066] Table 2. Elution Procedure

[0067] time mobile phase 0-45min 5-10% mobile phase B 45-50min 10-20% mobile phase B 50-55min 20-95% mobile phase B 55-70min 95%–5% mobile phase B

[0068] Further separation and purification of component F1 revealed only one absorption peak, such as Figure 5 As shown, component F1 was therefore used for subsequent peptide composition and amino acid sequence analysis.

[0069] Example 2: Structural identification, molecular docking screening, and synthesis of anti-inflammatory peptides

[0070] 2.1 Structural Identification: Component F1 was dissolved in deionized water containing 0.1% formic acid. After desalting on a C18 desalting column (Acclaim PepMap 100, 75 μm × 2 cm), the peptide composition and amino acid sequence of the sample were analyzed by LC-MS / MS using an online nanospray ionization source. Chromatographic conditions: C18 column (Acclaim PepMap, 75 μm × 25 cm); column flow rate controlled at 300 nL / min; column temperature at 40℃; injection volume: 3 μL; electrospray voltage 2 kV. Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; elution program: 0–3 min, 2–6% mobile phase B; 3–42 min, 6–20% mobile phase B; 42–47 min, 20–35% mobile phase B; 47–48 min, 35–100% mobile phase B; 48–60 min, 100% mobile phase B. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: ① Primary mass spectrometry scan range (m / z): 350–1800; resolution: 70000; automatic gain control target: 3e6; maximum injection time: 50 ms; scan charge: 2-6; ② HCD-MS / MS, resolution: 17500; isolation window: 2 m / z; automatic gain control target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s. Tandem mass spectra were analyzed using De Novo software (PEAKS Studio X+). The uniprot-Mytilus coruscus database was searched using PEAKS DB, and enzymatic digestion was set to None. Search parameters: Fragment ion mass tolerance: 0.02 Da; Parent ion mass tolerance: 7 ppm; Maximum missed cuts: 2; Variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98; Protein card value: -10lgP ≥ 0, containing at least 1 unique peptide; Peptide card value: -10lgP ≥ 15. Identification results are as follows: Figure 6 As shown, more than 260 peptides of different molecular weights were identified from component F1. All identified peptides were short peptides with 3 to 10 amino acid residues and molecular weights ranging from 349.167 to 1102.58.

[0071] 2.2 Molecular docking screening: First, the 3D structure of cyclooxygenase-2 (COX-2, PDB ID: 1CX2) was preprocessed and optimized, and output as a mol2 format file for subsequent molecular docking. The 3D structure of cyclooxygenase-2 was downloaded from the Protein Data Bank (PDB) (https: / / www1.rcsb.org / ). PyMol software was used to remove repeating chains, water molecules, and the original ligand from the receptor protein molecule, and the result was saved as a PDB format file. Then, Autodock Tools 1.5.6 software was used to add polar hydrogen atoms to the receptor protein, calculate the gasteiger charge, and set rotation bonds, and the result was saved as a PDBQT format file. The 2D structure of the ligand was drawn using Marvin Sketch software and optimized for energy minimization to construct the 3D structure, which was then saved as a PDBQT format file. AutoDock Vina 1.1.2 was used to perform molecular simulation flexible docking of the above receptor and ligand. The docking results are expressed as binding energy values. The docking parameters are as follows: The following amino acid residues in the receptor protein were set to be flexibly conformational: His... 90, Arg 120, Gln192, Val 349, Leu 352, Ser 353, Tyr 355, Tyr 385, Trp 387, Arg 513, Ala 516, Phe 518, Val 523, Gly 526, Ala 527, Leu351. The center positions of the grid boxes were set as follows: center_x = 28.684, center_y = 28.603, center_z = 9.285, and the box size was 40×40×40. All other parameters used default values. Finally, Discovery Studio 4.5 software was used to visualize and analyze the molecular docking results.

[0072] By setting a confidence level (-10lgP) ≥ 36.0, a total of 70 peptides were screened for molecular docking simulation experiments to study their interaction with cyclooxygenase-2. Molecular docking simulation analysis was performed on each peptide with the cyclooxygenase-2 receptor protein, and each peptide was repeated 10 times. The binding energy results were averaged. Peptides with lower binding energy to the cyclooxygenase-2 receptor protein in the molecular docking were selected for further analysis of their anti-inflammatory mechanism.

[0073] Among them, the anti-inflammatory peptide (TAMY, Thr-Ala-Met-Tyr) has a low binding energy, as shown in Table 3, indicating that the anti-inflammatory peptide can spontaneously and stably bind to the key active site in the cyclooxygenase-2 receptor protein.

[0074] Table 3. Results of simulated docking of anti-inflammatory peptides with cyclooxygenase-2 receptor protein

[0075]

[0076] Furthermore, the hydrophobicity of peptides is one of the main factors that cause anti-inflammatory responses. Anti-inflammatory peptides (TAMY) contain two hydrophobic amino acids (Ala and Met), and the two ends of the peptide chain are composed of polar amino acid groups or hydrophobic amino acid groups. Specifically, the C-terminus of the peptide chain is a polar amino acid group, and the N-terminus is a hydrophobic amino acid group. These structures indicate that anti-inflammatory peptides have anti-inflammatory potential.

[0077] Cyclooxygenase-2 has three important active regions. The first region is a hydrophobic pocket composed of amino acid residues Tyr 385, Trp 387, Phe 518, Ala 201, Tyr 248, and Leu 352. The second region is the entrance to the active site, composed of hydrophilic amino acid residues Arg 120, Glu 524, and Tyr 355. The third region is a lateral pocket composed of His 90, Arg 513, and Val 523. Amino acid residues His 90, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Ala 516, Phe 518, Val 523, Gly 526, Ala 527, and Leu 531 associated with the A chain of the cyclooxygenase-2 protein (the main region of the cyclooxygenase-2 active site) are involved in protein-ligand complementarity activity. Figure 7 This is a molecular docking model diagram of the interaction between cyclooxygenase-2 and anti-inflammatory peptides. From... Figure 7 As shown in Table 4, the anti-inflammatory peptide (TAMY) can form hydrogen bonds with residues Thr 94, Leu 352, Ile 517, Phe 518, and Glu 524 of the cyclooxygenase-2 protein, and hydrophobic bonds with Pro 514, Ala 516, and Ala 527. Specifically, the anti-inflammatory peptide forms polar interactions with amino acid residues Ala 516 and Ala 527, which are involved in the complementary activity of the cyclooxygenase-2 protein ligands, and hydrogen bonds with Leu 352 and Phe 518 in the hydrophobic pocket. This indicates that the anti-inflammatory peptide binds to different amino acid residues within the active site of cyclooxygenase-2, and the interactions within different active regions can affect the activity of the cyclooxygenase-2 protein, inhibiting its catalytic activity and inducing anti-inflammatory function.

[0078] Table 4. Binding energy and interaction sites between cyclooxygenase-2 and anti-inflammatory peptides based on molecular docking simulations

[0079]

[0080] 2.3 Synthesis: The anti-inflammatory peptide was synthesized by solid-phase synthesis. The anti-inflammatory peptide was synthesized by Jietai Biotechnology Co., Ltd. and had a purity of over 95%. It was used for further research on its anti-inflammatory activity.

[0081] Example 3

[0082] Anti-inflammatory peptides were used as samples to evaluate their cellular anti-inflammatory activity, and the NO release from RAW264.7 macrophages was measured. At the same time, the contents of cellular inflammatory factors (TNF-α, IL-6, IL-1β and IL-10) in the culture supernatant were measured by quantitative enzyme-linked immunosorbent assay (ELISA).

[0083] Cytotoxicity assay evaluation:

[0084] The cytotoxicity of the anti-inflammatory peptide (TAMY) on RAW264.7 macrophages at concentrations of 0.0625–2.0 mg / mL was assessed using the MTT assay. The results are as follows: Figure 8 As shown, the anti-inflammatory peptide did not produce significant cytotoxicity after 24 h of treatment with RAW264.7 macrophages, and it even promoted the proliferation of RAW264.7 macrophages. That is, the anti-inflammatory peptide did not produce cytotoxicity even at a concentration of 2.0 mg / mL. Therefore, the NO release was subsequently measured in a low concentration range of 1–200 μg / mL.

[0085] Determination of NO release from RAW264.7 macrophages:

[0086] Depend on Figure 9 Cellular experiments showed that LPS stimulation significantly increased NO release from RAW264.7 macrophages in the model group (P<0.001). Compared with the model group, the positive control group (DEX, 1 μg / mL) significantly inhibited NO release from RAW264.7 macrophages, with a decrease in NO release of 40.85±5.55% (P<0.001). The anti-inflammatory peptide treatment group significantly inhibited NO release from RAW264.7 macrophages at different concentrations, all in a concentration-dependent manner. At a concentration of 200 μg / mL, the NO release in the anti-inflammatory peptide treatment group decreased by 43.36±1.85% (P<0.001). These results indicate that anti-inflammatory peptides can effectively inhibit LPS-induced NO release from RAW264.7 macrophages.

[0087] Determination of RAW264.7 macrophage cytokines:

[0088] Figure 10 Figure showing the effect of anti-inflammatory peptides on the release of TNF-α factor from RAW264.7 macrophages; Figure 11 The effect of anti-inflammatory peptides on the release of IL-6 from RAW264.7 macrophages is shown in the figure. Figure 12Figure showing the effect of anti-inflammatory peptides on the release of IL-1β from RAW264.7 macrophages; Figure 13 This is a graph showing the effect of anti-inflammatory peptides on the release of IL-10 from RAW264.7 macrophages. From... Figures 10-13 As can be seen, compared with the blank control group, the release of various inflammatory factors TNF-α (A), IL-6 (B), IL-1β (C), and IL-10 (D) in RAW264.7 macrophages under LPS stimulation was significantly increased. Compared with the model group, the positive control group (DEX, 1 μg / mL) could significantly reduce the release of pro-inflammatory factors TNF-α, IL-6, and IL-1β. That is, DEX treatment can significantly inhibit the secretion of inflammatory mediators TNF-α, IL-6, and IL-1β by LPS-stimulated RAW264.7 macrophages (P<0.001), indicating that the cellular inflammation model used in this invention can serve as an effective means of evaluating anti-inflammatory substances. The anti-inflammatory peptide treatment group not only significantly reduced the secretion of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in RAW264.7 macrophages, but also showed a dose-dependent effect at concentrations of 1–200 μg / mL. When the concentration reached 200 μg / mL, it also significantly increased the secretion of anti-inflammatory factor IL-10. The effect of the highest concentration on the secretion of cellular inflammatory factors is shown in Table 5.

[0089] Table 5. Effects of each sample group on inflammatory factors in RAW264.7 macrophages (P<0.001)

[0090]

[0091] Examples 2 and 3 demonstrate that the anti-inflammatory peptide of the present invention can bind to the active site of cyclooxygenase-2, inhibit the activity of cyclooxygenase-2, and at the same time reduce the levels of NO and pro-inflammatory factors, promote the secretion of anti-inflammatory factors, thereby effectively regulating the secretion of inflammatory factors in RAW264.7 macrophages and exhibiting excellent anti-inflammatory activity.

[0092] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content of this invention should be considered within the scope of protection of the claims of this invention.

Claims

1. The application of anti-inflammatory peptides derived from pearl oysters in the preparation of anti-inflammatory drugs, characterized in that, The sequence of the anti-inflammatory peptide is shown in SEQ ID NO: 1.

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