A hexapeptide LR6 with anti-inflammatory activity and a preparation method and application thereof

Abstract

This invention discloses a hexapeptide LR6 with anti-inflammatory activity, its preparation method, and its applications, belonging to the field of small molecule peptide technology. The amino acid sequence of the hexapeptide LR6 is LDAVDR, and it can be prepared by solid-phase synthesis and enzymatic hydrolysis. The hexapeptide LR6 was identified from the protein hydrolysate of *Spirulina platensis* and has a potential interaction with Keap1. LPS-induced RAW264.7 cell assays showed that both low-dose (200 mM) and high-dose (400 mM) treatment with hexapeptide LR6 significantly inhibited TNF-α, IL-1β, and IL-10. The high-dose hexapeptide LR6 showed a better inhibitory effect than the positive control dexamethasone. Hexapeptide LR6 has better anti-inflammatory activity than dexamethasone and can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a hexapeptide LR6 with anti-inflammatory activity, its preparation method, and its application. Background Technology

[0002] The pathogenesis of inflammatory diseases is closely related to oxidative stress. In the cellular defense system, the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-associated factor 2 (Nrf2) signaling pathway is a core hub regulating oxidative stress and inflammatory responses. Keap1, as the substrate recognition subunit of E3 ubiquitin ligases, maintains cellular redox homeostasis by mediating the ubiquitination and degradation of Nrf2. Therefore, targeting Keap1 and activating the Nrf2 signaling pathway has become a novel strategy for treating inflammatory diseases. Currently, the widely used glucocorticoid anti-inflammatory drug dexamethasone effectively controls inflammatory responses, with a biological duration of action lasting 36-54 hours. However, its potent anti-inflammatory effect can lead to hypothalamic-pituitary-adrenal axis suppression and metabolic disorders. This situation has prompted researchers to turn their attention to the development of food-derived anti-inflammatory peptides. These bioactive peptides not only possess better biosafety but also have a lower risk of toxic side effects due to their origin in natural proteins.

[0003] Anti-inflammatory peptides derived from proteins of different origins after enzymatic hydrolysis exhibit significant functional differences. Han Jiaojiao et al., in "The novel peptides ICRD and LCGEC screened from tuna roe show antioxidative activity via Keap1 / Nrf2-ARE pathway regulation and gut microbiotamodulation," reported that the tetrapeptide ICRD and pentapeptide LCGEC, screened using Keap1 as a target, possessed significant antioxidant and anti-inflammatory activities. Liu Tong et al., in "Preparation of Anti-inflammatory Peptides from White Kidney Bean by Ultrasound-Assisted Enzymatic Hydrolysis and Its Activity Study," reported that the tetrapeptide FFFR, screened using iNOS as a target, possessed anti-inflammatory activity. Jiang Yu et al., in "Screening of Anti-inflammatory Peptides from Manila Clams Using Molecular Docking Technology," reported that the tetrapeptides DQTF and GYTR exhibited significant anti-inflammatory activity at the cellular level. In their paper "Research Progress on the Types and Mechanisms of Action of Fish-Derived Anti-inflammatory Peptides", Wang Aodong et al. compared and analyzed the research progress on fish-derived anti-inflammatory peptides at home and abroad over the past 10 years. They found that fish have been shown to contain more than 50 kinds of fish-derived anti-inflammatory peptides, such as anti-colitis peptides, anti-arthritis peptides, anti-nephritis peptides, and anti-dermatitis peptides. These anti-inflammatory peptides have significant differences in sequence from the anti-inflammatory peptides involved in this invention.

[0004] Marine organisms, due to their unique living environment, often possess protein amino acid sequences with special structures not found in terrestrial organisms, making them an excellent source for discovering novel bioactive peptides. Spirulina platensis, belonging to the phylum Cyanobacteria, class Cyanophyta, family Oscillatiaceae, and genus Spirulina, is a high-protein microalga with a protein content as high as 60-70%, making it a high-quality raw material for preparing bioactive peptides. The anti-inflammatory potential of Spirulina platensis, particularly its mechanism of action through the Keap1-Nrf2 pathway, has not yet been fully explored. Systematic exploration of Spirulina platensis proteins and the discovery of peptides with specific anti-inflammatory activities can realize the high-value utilization of this marine resource. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule peptide with a novel sequence structure, identified from spirulina protein hydrolysate, and possessing strong anti-inflammatory activity, as well as a method for preparing the small molecule peptide and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A hexapeptide LR6 with anti-inflammatory activity, wherein the amino acid sequence of the hexapeptide LR6 is LDAVDR.

[0008] The aforementioned method for preparing the anti-inflammatory hexapeptide LR6 employs a solid-phase synthesis method, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize the hexapeptide LR6 in a solid phase.

[0009] The aforementioned application of the hexapeptide LR6 with anti-inflammatory activity in the preparation of anti-inflammatory drugs.

[0010] The advantages of this invention are as follows: The hexapeptide LR6 provided by this invention was identified from the protein hydrolysate of Spirulina platensis. Molecular docking revealed that hexapeptide LR6 has a potential interaction with Kelch-like ECH-associated protein 1 (Keap1). In vitro cell experiments showed that in a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model, both low-dose (200 mM) and high-dose (400 mM) treatment with hexapeptide LR6 significantly inhibited TNF-α, IL-1β, and IL-10. Among them, the inhibitory effect of high-dose hexapeptide LR6 was better than that of the positive control dexamethasone (1 μM). Hexapeptide LR6 has a better anti-inflammatory effect than dexamethasone and can be used to prepare anti-inflammatory drugs. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the binding mode between hexapeptide LR6 and Keap1;

[0012] Figure 2This is a graph showing the effect of hexapeptide LR6 treatment on TNF-α, IL-1β, and IL-10. In the graph, A is the effect of hexapeptide LR6 treatment on TNF-α, B is the effect of hexapeptide LR6 treatment on IL-1β, C is the effect of hexapeptide LR6 treatment on IL-10, LPS is the model group, PC is the dexamethasone group, LD is the low-dose (200 mM) hexapeptide LR6 group, and HD is the high-dose (400 mM) hexapeptide LR6 group. ** represents p < 0.01, and *** represents p < 0.001. Detailed Implementation

[0013] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0014] I. Preparation of Spirulina platensis protein peptides

[0015] The method for preparing Spirulina platensis protein peptides specifically includes the following steps:

[0016] (1) Put 100g of Spirulina platensis into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze at this temperature for 2h;

[0017] (2) Continue heating until the temperature reaches 48°C. Then add 3g of alkaline protease and 2g of neutral protease and hydrolyze at this temperature for 2 hours.

[0018] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.

[0019] (4) Continue to heat up to 85℃ and hold for 30 minutes;

[0020] (5) The enzymatic hydrolysis product is precipitated, centrifuged and filtered, and the resulting solution is spray-dried to obtain a powdered product, which is Spirulina platensis protein peptide.

[0021] II. Obtaining the polypeptide sequence from Spirulina platensis protein peptides

[0022] The obtained Spirulina platensis protein peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry software to obtain several peptide sequences.

[0023] The LC-MS / MS determination conditions are as follows:

[0024] (1) Liquid chromatography method: The chromatographic column is C18, 3μm, 250mm×75μm (Eksigent). The mobile phase A is water, 0.1% formic acid, and the mobile phase B is acetonitrile, 0.1% formic acid. The flow rate is 300nL / min, the injection volume is 1μL, and the chromatographic gradient is 70min. The specific elution gradient is as follows: 0-55min, phase A decreases uniformly from 95% to 65%; 55-63min, phase A decreases uniformly from 65% to 50%; 63-64min, phase A decreases uniformly from 50% to 0%; 64-70min, phase A is maintained at 0%.

[0025] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120,000, AGC set to 310, scan range of 110-2000 m / z. MIPS mode is peptide, valence state 1-6 is selected, secondary resolution is 17,500, separation window is 1.6 m / z.

[0026] III. Screening for bioactive peptides with peak area ≥ 1.00 × 10⁸ and amino acid count ≤ 6.

[0027] From the several polypeptide sequences obtained above, 22 peak areas ≥ 1.00 × 10⁻⁶ were finally selected. 8 The screening results for active peptides with ≤6 amino acid counts are shown in Tables 1-1 and 1-2.

[0028] Table 1-1 High-abundance bioactive peptides in Spirulina platensis protein peptides (Part 1)

[0029]

[0030] Table 1-2 High-abundance bioactive peptides in Spirulina platensis protein peptides (II)

[0031]

[0032] IV. Screening for bioactive peptides with strong binding affinity to Keap1

[0033] Using Discovery Studio software, the active peptide sequences in Tables 1-1 and 1-2 were molecularly docked with Keap1. Before docking, the 2D structure of the active peptides was converted into a 3D structure by minimizing energy, and active peptide sequences with strong binding ability to Keap1 were screened.

[0034] The 3D structure of Keap1 can be downloaded from the RCSB protein database (PDB ID: 6QMK). Docking results are expressed as docking scores; the higher the docking score, the stronger the binding affinity between the active peptide and Keap1.

[0035] The molecular docking results of the above 22 active peptides with Keap1 are shown in Tables 2-1 and 2-2.

[0036] Table 2-1 Predicted results of the interaction between bioactive peptides and Keap1 (Part 1)

[0037]

[0038] Table 2-2 Predicted results of the interaction between bioactive peptides and Keap1 (Part 2)

[0039]

[0040] V. Molecular docking analysis

[0041] Among the 22 bioactive peptides listed in Tables 2-1 and 2-2, LDAVDR (denoted as hexapeptide LR6, SEQ ID NO: 3) had the highest docking score of 104.1570 kcal / mol. Therefore, LDAVDR (hexapeptide LR6) was selected for further molecular docking analysis.

[0042] Analysis revealed that the binding mode between hexapeptide LR6 and Keap1 is as follows: Figure 1 As shown, the molecular docking is as follows:

[0043] The hexapeptide LR6 forms one salt bridge interaction, 10 HH bond interactions, 4 CH bond interactions and 3 electrostatic interactions with Keap1. Eleven amino acid residues are involved in the interaction between the hexapeptide LR6 and Keap1.

[0044] VI. Evaluation of the anti-inflammatory activity of hexapeptide LR6

[0045] A solid-phase synthesis method was adopted, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize hexapeptide LR6 (purity >90%).

[0046] The anti-inflammatory activity of solid-phase synthesized hexapeptide LR6 was evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. Specifically:

[0047] (1) Prepare DMEM high-glucose complete culture medium;

[0048] (2) Place RAW264.7 cells in a T25 cell culture flask, add 6 mL of DMEM high glucose complete culture medium, shake gently to mix, and incubate in a constant temperature incubator at 37℃ and 5% CO2.

[0049] (3) Take RAW264.7 cells that are growing well in the logarithmic growth phase and prepare a cell suspension with DMEM high-glucose complete culture medium, with a cell density of 1×10⁻⁶ cells / cells. 5cells / mL;

[0050] (4) Seed RAW264.7 cells into 96-well plates, 100 μL per well, and place them in a constant temperature incubator at 37°C and 5% CO2 for 12 h. Then discard the culture medium.

[0051] (5) RAW264.7 cells were subjected to different treatments, specifically:

[0052] (i) Control group: 100 μL of DMEM high glucose complete culture medium was added to RAW264.7 cells, 6 replicates;

[0053] (ii) Model group: 99 μL of DMEM high glucose complete culture medium and 1 μL of lipopolysaccharide (LPS) solution with a concentration of 100 μg / mL were added to RAW264.7 cells, with 6 replicates;

[0054] (iii) Dexamethasone group: 89 μL of DMEM high glucose complete culture medium, 1 μL of LPS solution with a concentration of 100 μg / mL and 10 μL of dexamethasone (PC) solution with a concentration of 10 μM were added to RAW264.7 cells, with 6 replicates;

[0055] (iv) Low-dose hexapeptide LR6 group: 89 μL of DMEM high-glucose complete culture medium, 1 μL of LPS solution with a concentration of 100 μg / mL and 10 μL of hexapeptide LR6 solution with a concentration of 2M were added to RAW264.7 cells, with 6 replicates.

[0056] (v) High-dose hexapeptide LR6 group: 79 μL of DMEM high-glucose complete culture medium, 1 μL of LPS solution with a concentration of 100 μg / mL and 20 μL of hexapeptide LR6 solution with a concentration of 2M were added to RAW264.7 cells, with 6 replicates;

[0057] (6) Place each group of cells in a constant temperature incubator at 37℃ and 5% CO2 for 12 hours. Collect the cell culture medium and use an ELISA kit to detect the content of inflammatory factors (TNF-α, IL-1β, IL-10).

[0058] The results of the detection of inflammatory factors (TNF-α, IL-1β, IL-10) in each group are shown in the figure. Figure 2 .

[0059] Depend on Figure 2As shown in A, the TNF-a content in the control group was 164.32±12.4 pg / mL, which significantly increased to 1209.16±220.0 pg / mL after LPS induction (p<0.001). The positive control dexamethasone (1 μM) significantly reduced the TNF-a content to 604.26±29.7 pg / mL (p<0.001) on the basis of the model, while the low-dose (200 mM) and high-dose (400 mM) hexapeptide LR6 significantly reduced the TNF-a content to 404.58±134.5 pg / mL (p<0.001) and 380.63±74.7 pg / mL (p<0.001), respectively, on the basis of the model.

[0060] Depend on Figure 2 As shown in B, the IL-1β level in the control group was 417.70±68.9 pg / mL, which significantly increased to 868.44±16.7 pg / mL after LPS induction (p<0.001). The positive control dexamethasone (1 μM) significantly reduced the IL-1β level to 597.75±23.0 pg / mL (p<0.001) on the basis of the model, while the low-dose (200 mM) and high-dose (400 mM) hexapeptide LR6 significantly reduced the IL-1β level to 629.89±117.1 pg / mL (p<0.01) and extremely significantly reduced it to 557.67±91.8 pg / mL (p<0.001), respectively, on the basis of the model.

[0061] Depend on Figure 2 As shown in C, the IL-10 content in the control group was 618.5±82.7 pg / mL, which significantly increased to 944.76±61.7 pg / mL after LPS induction (p<0.001). The positive control dexamethasone (1 μM) significantly reduced the IL-10 content to 472.69±10.8 pg / mL (p<0.001) on the basis of the model, while the low-dose (200 mM) and high-dose (400 mM) hexapeptide LR6 significantly reduced the IL-10 content to 528.57±114.5 pg / mL (p<0.01) and 382.93±44.3 pg / mL (p<0.01), respectively, on the basis of the model.

[0062] In summary, in the LPS-induced RAW264.7 macrophage inflammation model, both low-dose (200 mM) and high-dose (400 mM) hexapeptide LR6 treatment significantly inhibited TNF-α, IL-1β, and IL-10. Among them, the high-dose hexapeptide LR6 showed better inhibitory effects than the positive control dexamethasone (1 μM). Hexapeptide LR6 has better anti-inflammatory effects than dexamethasone and can be used to prepare anti-inflammatory drugs.

[0063] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the protection scope of this invention.

Claims

1. A hexapeptide LR6 with anti-inflammatory activity, characterized in that, The amino acid sequence of the hexapeptide LR6 is LDAVDR.

2. The method for preparing the hexapeptide LR6 with anti-inflammatory activity as described in claim 1, characterized in that, A solid-phase synthesis method was adopted, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize hexapeptide LR6.

3. The use of the hexapeptide LR6 with anti-inflammatory activity as described in claim 1 in the preparation of anti-inflammatory drugs.

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