A hexapeptide LE6 with anti-inflammatory activity and a preparation method and application thereof

Abstract

The application discloses a kind of hexapeptide LE6 with anti-inflammatory activity and its preparation method and application, belong to small molecule peptide technical field.The amino acid sequence of the hexapeptide LE6 is LEPGFE, and can be prepared by solid-phase synthesis method and enzymatic hydrolysis method.This hexapeptide LE6 is identified from polysiphondryalis protease hydrolysate, has potential interaction with Keap1, and is found that low-dose (200mM) and high-dose (400mM) hexapeptide LR6 treatment can significantly reduce cell TNF-alpha, IL-1beta and IL-10 level in LPS-induced RAW264.7 cell test, wherein, the reducing effect of hexapeptide LR6 on TNF-alpha is superior to positive control dexamethasone, and hexapeptide LR6 can be used for preparing anti-inflammatory drug.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a hexapeptide LE6 with anti-inflammatory activity, its preparation method, and its application. Background Technology

[0002] Chronic inflammation is a common pathological basis for many major diseases. In the body's innate immune response, pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) released after activation of immune cells like macrophages are key mediators driving the inflammatory response. Interleukin-10 (IL-10), as an important anti-inflammatory factor, is crucial for controlling the appropriate development of inflammation. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-associated factor 2 (Nrf2) signaling pathway is a central regulator of cellular oxidative stress responses. Regulating the balance of the inflammatory factor network by intervening in this pathway has become an important target for developing novel anti-inflammatory strategies.

[0003] While dexamethasone, widely used in clinical practice, possesses potent non-specific anti-inflammatory effects, long-term or high-dose use can lead to adverse reactions such as immunosuppression and metabolic disorders. Studies have shown that dexamethasone exhibits broad inhibitory effects on multiple cytokines, including TNF-α, IL-1β, and IL-10. This indiscriminate immunosuppression may weaken the body's normal immune defense function. Against this backdrop, food-derived anti-inflammatory peptides have attracted widespread attention due to their mild effects, good targeting, and high safety profile.

[0004] Proteins from different sources can produce bioactive peptides with varying structures and functions after enzymatic hydrolysis. Zhang Laidi et al., in their study "Isolation, Identification, and Activity Analysis of Anti-inflammatory Peptides from Duck Liver Enzymatic Hydrolysates," identified and screened 10 novel peptides with molecular weights below 1300 Da. LVYPFPGPI and VIESPPEI dose-dependently inhibited NO release from inflammatory cells, with inhibition rates of 48.24% and 56.32%, respectively. Xiang Huan et al., in their study "Screening of Anti-inflammatory Peptides from Snakehead Fish Based on Virtual Screening, Molecular Docking, and Cell Models," reported the isolation of QWWR and DEECWF from snakehead enzymatic hydrolysates, which significantly inhibited lipopolysaccharide-induced excessive secretion of NO and inflammatory factors in cells. Miao Jianyin et al., in their invention patent "Preparation of an anti-inflammatory peptide derived from hydrolyzed nacre protein in seashells and its application in skin repair" (CN202411635295.X), reported a polypeptide PDFDNGF with anti-inflammatory activity. This polypeptide inhibited the excessive production of NO and cytokines in RAW264.7 cells induced by lipopolysaccharide, while simultaneously increasing the level of anti-inflammatory cytokines, demonstrating significant anti-inflammatory activity. Wang Aodong et al., in their research "Research Progress on the Types and Mechanisms of Action of Fish-Derived Anti-inflammatory Peptides," compared and analyzed the research progress on fish-derived anti-inflammatory peptides both domestically and internationally over the past 10 years. They found that fish have been shown to contain more than 50 fish-derived anti-inflammatory peptides, including anti-colitis peptides, anti-arthritis peptides, anti-nephritis peptides, and anti-dermatitis peptides, which have significant sequence differences from the anti-inflammatory peptides involved in this invention.

[0005] Polysiphonia urceolata (mainly distributed along the coast of the Yellow and Bohai Seas in China), a type of marine red algae, has not yet had its protein resources fully explored. In particular, research on its anti-inflammatory effects through the Keap1-Nrf2 pathway is scarce, making it a valuable resource for discovering novel anti-inflammatory peptides. Summary of the Invention

[0006] The purpose of this invention is to provide a small molecule peptide with a novel sequence structure and strong anti-inflammatory activity, which is identified from the protein hydrolysate of Polypodiatomaceous earth, as well as the preparation method and application of the small molecule peptide.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A hexapeptide LE6 with anti-inflammatory activity, wherein the amino acid sequence of the hexapeptide LE6 is LEPGFE.

[0009] The aforementioned method for preparing the anti-inflammatory hexapeptide LE6 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 LE6 in a solid phase.

[0010] The aforementioned application of the hexapeptide LE6 with anti-inflammatory activity in the preparation of anti-inflammatory drugs.

[0011] The advantages of this invention are as follows: The hexapeptide LE6 provided by this invention was identified from the protein hydrolysate of *Polygonum multiflorum*. Molecular docking revealed that hexapeptide LE6 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 LE6 significantly inhibited TNF-α, IL-1β, and IL-10. Among these, the inhibitory effect of hexapeptide LE6 on TNF-α was superior to that of the positive control dexamethasone (1 μM), the inhibitory effect on IL-1β was comparable to that of the positive control dexamethasone, and the inhibitory effect on IL-10 was inferior to that of the positive control dexamethasone. Since TNF-α and IL-1β are pro-inflammatory factors, while IL-10 is an anti-inflammatory factor, this differential regulation is more beneficial for maintaining immune balance while effectively inhibiting excessive inflammation. Hexapeptide LE6 has a better anti-inflammatory effect than dexamethasone. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the binding mode between hexapeptide LE6 and Keap1;

[0013] Figure 2 This is a graph showing the effect of hexapeptide LE6 treatment on TNF-α, IL-1β, and IL-10. In the graph, A is the effect of hexapeptide LE6 treatment on TNF-α, B is the effect of hexapeptide LE6 treatment on IL-1β, C is the effect of hexapeptide LE6 treatment on IL-10, LPS is the model group, PC is the dexamethasone group, LD is the low-dose (200 mM) hexapeptide LE6 group, and HD is the high-dose (400 mM) hexapeptide LE6 group. ** represents p < 0.01, and *** represents p < 0.001. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0015] I. Preparation of Polytubularia protein peptides

[0016] The method for preparing polytubular algal protein peptides specifically includes the following steps:

[0017] (1) Put 100g of Polytubularia into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze at this temperature for 2h;

[0018] (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.

[0019] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.

[0020] (4) Continue to heat up to 85℃ and hold for 30 minutes;

[0021] (5) The enzymatic hydrolysis product is precipitated, centrifuged and filtered, and the resulting solution is spray-dried to obtain a powdered product, which is the multitube algae protein peptide.

[0022] II. Obtaining the polypeptide sequence from the polytubular algae protein peptide

[0023] The obtained polychaete algae protein peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry analysis software to obtain several polypeptide sequences.

[0024] The LC-MS / MS determination conditions are as follows:

[0025] (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%.

[0026] (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.

[0027] III. Screening peak area > 5.00 × 10 7 Active peptides with ≤6 amino acids

[0028] From the several polypeptide sequences obtained above, 38 peak areas > 5.00 × 10⁻⁶ were finally selected. 7 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1, 1-2, and 1-3.

[0029] Table 1-1 High-abundance bioactive peptides in Polychaete algae protein peptides (Part 1)

[0030]

[0031] Table 1-2 High-Abundance Bioactive Peptides in Polychaete Algae Protein Peptides (Part 2)

[0032]

[0033] Table 1-3 High-Abundance Bioactive Peptides in Polychaete Algae Protein Peptides (Part 3)

[0034]

[0035] IV. Screening for bioactive peptides with strong binding affinity to Keap1

[0036] Using Discovery Studio software, the active peptide sequences in Tables 1-1, 1-2, and 1-3 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.

[0037] 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.

[0038] The molecular docking results of the above 38 active peptides with Keap1 are shown in Tables 2-1, 2-2 and 2-3.

[0039] Table 2-1 Predicted results of the interaction between bioactive peptides and Keap1 (Part 1)

[0040]

[0041] Table 2-2 Predicted results of the interaction between bioactive peptides and Keap1 (Part 2)

[0042]

[0043] Table 2-3 Predicted results of interactions between bioactive peptides and Keap1 (Part 3)

[0044]

[0045] V. Molecular docking analysis

[0046] Among the 38 bioactive peptides listed in Tables 2-1, 2-2, and 2-3, AREVY (denoted as pentapeptide AY5, SEQ ID NO: 24) and LEPGFE (denoted as hexapeptide LE6, SEQ ID NO: 6) had the highest docking scores, at 100.0140 kcal / mol and 98.8038 kcal / mol, respectively. Considering that the peak area of ​​pentapeptide AY5 is 9.83 × 10⁻⁶... 7 The peak area of ​​hexapeptide LE6 is 13.2 × 10⁻⁶. 7The latter has a larger peak area, so LEPGFE (hexapeptide LE6) was selected for further molecular docking analysis.

[0047] Analysis revealed that the binding mode of hexapeptide LE6 to Keap1 is as follows: Figure 1 As shown, the molecular docking is as follows:

[0048] The hexapeptide LE6 forms one salt bridge interaction, seven HH bond interactions, four CH bond interactions, and two electrostatic interactions with Keap1. Eight amino acid residues are involved in the interaction between the hexapeptide LE6 and Keap1.

[0049] VI. Evaluation of the anti-inflammatory activity of hexapeptide LE6

[0050] 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 LE6 (purity >90%).

[0051] The anti-inflammatory activity of solid-phase synthesized hexapeptide LE6 was evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. Specifically:

[0052] (1) Prepare DMEM high-glucose complete culture medium;

[0053] (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.

[0054] (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. 5 cells / mL;

[0055] (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.

[0056] (5) RAW264.7 cells were subjected to different treatments, specifically:

[0057] (i) Control group: 100 μL of DMEM high glucose complete culture medium was added to RAW264.7 cells, 6 replicates;

[0058] (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;

[0059] (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;

[0060] (iv) Low-dose hexapeptide LE6 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 LE6 solution with a concentration of 2M were added to RAW264.7 cells, with 6 replicates;

[0061] (v) High-dose hexapeptide LE6 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 LE6 solution with a concentration of 2M were added to RAW264.7 cells, with 6 replicates;

[0062] (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).

[0063] The results of the detection of inflammatory factors (TNF-α, IL-1β, IL-10) in each group are shown in the figure. Figure 2 .

[0064] Depend on Figure 2 As 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 low-dose (200 mM) and high-dose (400 mM) hexapeptide LE6 significantly reduced the TNF-a content to 367.47±119.2 pg / mL (p<0.001) and 292.53±79.2 pg / mL (p<0.001), respectively, on the basis of the model.

[0065] Depend on Figure 2As 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 LE6 significantly reduced the IL-1β level to 548.81±111.6 pg / mL (p<0.001) and 548.44±53.0 pg / mL (p<0.001) on the basis of the model, respectively.

[0066] 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 LE6 significantly reduced the IL-10 content to 631.96±162.7 pg / mL (p<0.01) and 511.43±99.0 pg / mL (p<0.001) on the basis of the model.

[0067] In summary, in the LPS-induced RAW264.7 macrophage inflammation model, both low-dose (200 mM) and high-dose (400 mM) treatment with hexapeptide LE6 significantly inhibited TNF-α, IL-1β, and IL-10. Among these, hexapeptide LE6 showed better inhibitory effects on TNF-α than the positive control dexamethasone (1 μM), comparable inhibitory effects on IL-1β, and weaker inhibitory effects on IL-10. Since TNF-α and IL-1β are pro-inflammatory factors, while IL-10 is an anti-inflammatory factor, this differential regulation is more beneficial for maintaining immune balance while effectively inhibiting excessive inflammation. Therefore, hexapeptide LE6 has a better anti-inflammatory effect than dexamethasone.

[0068] 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 LE6 with anti-inflammatory activity, characterized in that, The amino acid sequence of the hexapeptide LE6 is LEPGFE.

2. The method for preparing the hexapeptide LE6 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 LE6.

3. The use of the hexapeptide LE6 with anti-inflammatory activity as described in claim 1 in the preparation of anti-inflammatory drugs.

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