Hexapeptide LE6 with anti-inflammatory activity and preparation method and application thereof

Abstract

The invention discloses a hexapeptide LE6 with anti-inflammatory activity and a preparation method and application thereof, and belongs to the technical field of small molecule peptides. The amino acid sequence of the hexapeptide LE6 is LEPGFE, and the hexapeptide LE6 can be prepared through a solid-phase synthesis method and an enzymolysis method. The hexapeptide LE6 is identified from polysiphonostegia protein enzymatic hydrolysate and has potential interaction with Keap1, LPS-induced RAW264.7 cell tests find that the levels of cell TNF-alpha, IL-1beta and IL-10 can be remarkably reduced through treatment of a low dose (200mM) and a high dose (400mM) of the hexapeptide LR6, the TNF-alpha reducing effect of the hexapeptide LR6 is superior to that of positive control dexamethasone, and the hexapeptide LR6 can be used for preparing anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of small molecule peptides, and particularly relates to a hexapeptide LE6 with anti-inflammatory activity and a preparation method and application thereof. BACKGROUND

[0002] Chronic inflammation is the common pathological basis of 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 by activated immune cells such as macrophages are the key mediators driving the inflammatory response, and interleukin-10 (IL-10) as an important anti-inflammatory factor, its balance is crucial to control the moderate development of inflammation. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-related factor 2 (Nrf2) signaling pathway is the central regulator of cellular antioxidant stress response, and by interfering with this pathway to regulate the balance of the inflammatory factor network, it has become an important target for developing new anti-inflammatory strategies.

[0003] Although dexamethasone widely used in clinical practice has a strong non-specific anti-inflammatory effect, its long-term or large-dose use can cause adverse reactions such as immunosuppression and metabolic disorders. Studies have shown that dexamethasone has a broad inhibitory effect on TNF-α, IL-1β and IL-10 and other cytokines, and this indiscriminate immunosuppression may weaken the body's normal immune defense function. In this context, food-derived anti-inflammatory peptides have received widespread attention due to their mild effects, good targeting, and high safety.

[0004] Different sources of proteins can produce active peptide segments with different structures and functions after enzymolysis. Zhang Lidi et al. identified and screened 10 new peptides with molecular weight less than 1300 Da in “Isolation, identification and activity analysis of anti-inflammatory peptides from duck liver protease hydrolysate”. LVYPFPGPI and VIESPPEI dose-dependently inhibited the release of NO in inflammatory cells, with inhibition rates of 48.24% and 56.32%, respectively. Xiang Huan et al. reported in “Anti-inflammatory peptides from snakehead (Channa argus) based on virtual screening, molecular docking and cell model screening” that QWWR and DEECWF isolated from snakehead enzymolysis products can significantly inhibit the excessive secretion of NO and inflammatory factors in cells induced by lipopolysaccharide. Miao Jianying et al. reported in the invention patent “Preparation of shell nacre protein hydrolysis anti-inflammatory peptide and its application in skin repair” (CN202411635295.X) that a polypeptide PDFDNGF with anti-inflammatory activity can inhibit the excessive production of NO and cytokines in RAW264.7 cells induced by lipopolysaccharide, while increasing the level of anti-inflammatory cytokines, showing significant anti-inflammatory activity. Wang Aodong et al. analyzed the research progress of fish-derived anti-inflammatory peptides in “Research progress of types and mechanisms of fish-derived anti-inflammatory peptides” in the past decade, and found that more than 50 fish-derived anti-inflammatory peptides have been confirmed in fish, including anti-colitis peptides, anti-arthritis peptides, anti-nephritis peptides, and anti-dermatitis peptides, which are significantly different from the anti-inflammatory peptides involved in the present invention in sequence.

[0005] Polysiphonia urceolata (Latin name: Polysiphonia urceolata), mainly distributed along the coast of the Yellow and Bohai Seas in China, is a kind of marine red algae. Its protein resources have not been fully developed. In particular, there are few reports on its anti-inflammatory effect through the Keap1-Nrf2 pathway, which makes it a valuable resource for exploring new anti-inflammatory peptides. SUMMARY

[0006] The purpose of the present application is to provide a small molecule peptide with novel sequence structure and strong anti-inflammatory activity identified from Polysiphonia urceolata protease hydrolysate, as well as a preparation method and application thereof.

[0007] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows: A hexapeptide LE6 with anti-inflammatory activity, wherein the amino acid sequence of the hexapeptide LE6 is LEPGFE.

[0008] The preparation method of the aforementioned hexapeptide LE6 with anti-inflammatory activity adopts solid-phase synthesis method, uses Fmoc-protected amino acids as raw materials, and selects polystyrene resin as a solid-phase carrier to synthesize the hexapeptide LE6.

[0009] The application of the aforementioned hexapeptide LE6 with anti-inflammatory activity in the preparation of anti-inflammatory drugs.

[0010] The application has the advantages that the hexapeptide LE6 is identified from a polypipe algae protease solution, it is found through molecular docking that the hexapeptide LE6 has potential interaction with kelch-like ECH-associated protein 1 (Keap1), and it is found through in vitro cell experiments that in a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model, low-dose (200 mM) and high-dose (400 mM) hexapeptide LE6 treatment can significantly inhibit TNF-alpha, IL-1beta and IL-10, wherein the inhibitory effect of the hexapeptide LE6 on TNF-alpha is better than that of the positive control dexamethasone (1 muM), the inhibitory effect on IL-1beta is equivalent to that of the positive control dexamethasone, and the inhibitory effect on IL-10 is not as good as that of the positive control dexamethasone, since TNF-alpha and IL-1beta are pro-inflammatory factors, and IL-10 is an anti-inflammatory factor, such differential regulation is more conducive to maintaining immune balance while effectively inhibiting excessive inflammation, and the hexapeptide LE6 has better anti-inflammatory effect than dexamethasone. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a schematic diagram of the binding mode of the hexapeptide LE6 and Keap1; Figure 2 is a diagram of the effect of hexapeptide LE6 treatment on TNF-alpha, IL-1beta and IL-10, wherein A is a diagram of the effect of hexapeptide LE6 treatment on TNF-alpha, B is a diagram of the effect of hexapeptide LE6 treatment on IL-1beta, and C is a diagram of the effect of hexapeptide LE6 treatment on IL-10, LPS is a model group, PC is a dexamethasone group, LD is a low-dose (200 mM) hexapeptide LE6 group, HD is a high-dose (400 mM) hexapeptide LE6 group, and ** represents p<0.01 and *** represents p<0.001. DETAILED DESCRIPTION

[0012] The application will be specifically described below in combination with the drawings and specific embodiments.

[0013] I. Preparation of polypipe algae protein peptides The method for preparing the polypipe algae protein peptides specifically comprises the following steps: (1) 100 g of polypipe algae is placed in 1000 mL of water, 1 g of yeast is added after being warmed to 40 DEG C, and enzymolysis is carried out at this temperature for 2 h; (2) continue to warm, 3 g of alkaline protease and 2 g of neutral protease are added after being warmed to 48 DEG C, and enzymolysis is carried out at this temperature for 2 h; (3) continue to warm, 2 g of papain is added after being warmed to 58 DEG C, and enzymolysis is carried out at this temperature for 3 h; (4) continue to warm, and keep at 85 DEG C for 30 min; (5) The enzymatic product is precipitated, centrifuged and filtered, and the obtained solution is spray dried to obtain a powdery product, i.e., the polymixa protease peptide.

[0014] II. Obtain polypeptide sequences in polymixa protease peptide The above obtained polymixa protease peptide is subjected to mass spectrometry determination by LC-MS / MS, and the mass spectrometry determination result is analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0015] The LC-MS / MS determination conditions are as follows: (1) Liquid phase method: the chromatographic column is C18, 3 μm, 250 mm x 75 μm (Eksigent), the mobile phase A is water, 0.1% formic acid, the mobile phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nL / min, the injection volume is 1 μL, the 70 min chromatographic gradient, and the specific elution gradient is: 0-55 min, A phase from 95% uniformly reduced to 65%; 55-63 min, A phase from 65% uniformly reduced to 50%, 63-64 min, A phase from 50% uniformly reduced to 0; 64-70 min, keep 0% A phase.

[0016] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, first order resolution is 120000, AGC is set to 310, scanning range is 110-2000 m / z. The MIPS mode is peptide, the valence state is selected to be 1-6, the second order resolution is 17500, and the separation window is 1.6 m / z.

[0017] III. Screen active peptides with peak area > 5.00 x 10 7 and amino acid number ≤ 6 From the above obtained several polypeptide sequences, 38 active peptides with peak area > 5.00 x 10 7 and amino acid number ≤ 6 are finally screened out, and the screening results are shown in Table 1-1, Table 1-2 and Table 1-3.

[0018] Table 1-1 High-abundance active peptides in polymixa protease peptide (I)

[0019] Table 1-2 High-abundance active peptides in polymixa protease peptide (II)

[0020] Table 1-3 High-abundance active peptides in polymixa protease peptide (III)

[0021] IV. Screen active peptides with strong Keap1 binding ability The active peptide sequences in Table 1-1, Table 1-2 and Table 1-3 were respectively subjected to molecular docking with Keap1 by using Discovery Studio software, and the 2D structure of the active peptide was converted into 3D structure by energy minimization before docking, and the active peptide sequences with strong binding ability with Keap1 were screened.

[0022] The 3D structure of Keap1 can be downloaded from RCSB protein database (PDB ID: 6QMK). The docking result is expressed by docking score, and the larger the docking score is, the stronger the binding ability of the active peptide with Keap1 is.

[0023] The molecular docking results of the above 38 active peptides with Keap1 are shown in Table 2-1, Table 2-2 and Table 2-3.

[0024] Table 2-1 Prediction results of interaction between active peptides and Keap1 (I)

[0025] Table 2-2 Prediction results of interaction between active peptides and Keap1 (II)

[0026] Table 2-3 Prediction results of interaction between active peptides and Keap1 (III)

[0027] V. Molecular docking analysis Among the 38 active peptides listed in Table 2-1, Table 2-2 and Table 2-3, the docking scores of AREVY (referred to as pentapeptide AY5, SEQ ID NO: 24) and LEPGFE (referred to as hexapeptide LE6, SEQ ID NO: 6) are the largest, which are 100.0140 kcal / mol and 98.8038 kcal / mol, respectively. Considering that the peak area of pentapeptide AY5 is 9.83 x 10 7 , and the peak area of hexapeptide LE6 is 13.2 x 10 7 , the latter has a larger peak area, so LEPGFE (hexapeptide LE6) is selected for further molecular docking analysis.

[0028] After analysis, the binding mode of hexapeptide LE6 with Keap1 is shown in Figure 1 , and the molecular docking is as follows: Hexapeptide LE6 forms 1 salt bridge interaction, 7 H-H bond interactions, 4 C-H bond interactions and 2 electrostatic interactions with Keap1, and 8 amino acid residues are involved in the interaction between hexapeptide LE6 and Keap1.

[0029] VI. Evaluation of anti-inflammatory activity of hexapeptide LE6 The solid-phase synthesis method was adopted, Fmoc-protected amino acids were used as raw materials, and polystyrene resin was selected as a solid-phase carrier to synthesize the hexapeptide LE6 (purity > 90%).

[0030] The anti-inflammatory activity of the solid-phase synthesized hexapeptide LE6 was evaluated by using a RAW264.7 macrophage inflammation model induced by lipopolysaccharide (LPS). Specifically: (1) Prepare DMEM high-sugar complete culture solution; (2) Place RAW264.7 cells in a T25 cell culture bottle, add 6 mL of DMEM high-sugar complete culture solution, gently shake to mix evenly, and place in a constant temperature incubator at 37°C, 5% CO2; (3) Take well-grown RAW264.7 cells in the logarithmic growth phase, prepare a cell suspension with DMEM high-sugar complete culture solution, and the cell density is 1×10 5 (4) Seed RAW264.7 cells in a 96-well plate at 100 μL per well, and place in a constant temperature incubator at 37°C, 5% CO2, discard the culture solution after 12 h of culture; (5) Different treatments were performed on RAW264.7 cells, specifically: (i) Control group: add 100 μL of DMEM high-sugar complete culture solution to RAW264.7 cells, 6 replicates; (ii) Model group: add 99 μL of DMEM high-sugar complete culture solution and 1 μL of lipopolysaccharide (LPS) solution with a concentration of 100 μg / mL to RAW264.7 cells, 6 replicates; (iii) Dexamethasone group: add 89 μL of DMEM high-sugar complete culture solution, 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 to RAW264.7 cells, 6 replicates; (iv) Low-dose hexapeptide LE6 group: add 89 μL of DMEM high-sugar complete culture solution, 1 μL of LPS solution with a concentration of 100 μg / mL, and 10 μL of hexapeptide LE6 solution with a concentration of 2 M to RAW264.7 cells, 6 replicates; (v) High-dose hexapeptide LE6 group: add 79 μL of DMEM high-sugar complete culture solution, 1 μL of LPS solution with a concentration of 100 μg / mL, and 20 μL of hexapeptide LE6 solution with a concentration of 2 M to RAW264.7 cells, 6 replicates; ​(6) Place the cells in a constant temperature incubator at 37℃ and 5% CO2 for 12 hours and collect the cell culture medium. Use an ELISA kit to detect the content of inflammatory factors (TNF-α, IL-1β, IL-10).

[0031] The results of the detection of inflammatory factors (TNF-α, IL-1β, IL-10) in each group are shown below. Figure 2 .

[0032] 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.

[0033] 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 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.

[0034] 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.

[0035] In summary, in the LPS-induced RAW264.7 macrophage inflammation model, low-dose (200 mM) and high-dose (400 mM) hexapeptide LE6 treatment can significantly inhibit TNF-α, IL-1β and IL-10, wherein the inhibitory effect of hexapeptide LE6 on TNF-α is better than that of positive control dexamethasone (1 μM), the inhibitory effect on IL-1β is comparable to that of positive control dexamethasone, and the inhibitory effect on IL-10 is not as good as that of positive control dexamethasone. Since TNF-α and IL-1β are pro-inflammatory factors, and IL-10 is an anti-inflammatory factor, this differential regulation is more conducive to maintaining immune balance while effectively inhibiting excessive inflammation, so hexapeptide LE6 has a better anti-inflammatory effect than dexamethasone.

[0036] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, those skilled in the art can make other different forms of changes or modifications. It is impossible to enumerate all the embodiments here. Any obvious changes or modifications derived from the technical solutions of the present application are still within the protection scope of the present application.

Claims

1. A hexapeptide LE6 having anti-inflammatory activity, characterized in that, The amino acid sequence of the hexapeptide LE6 is LEPGFE.

2. A process for the preparation of the hexapeptide LE6 having anti-inflammatory activity according to claim 1, characterized by, The hexapeptide LE6 is synthesized by solid phase synthesis method using Fmoc-protected amino acids as raw materials and polystyrene resin as solid phase carrier.

3. Use of the hexapeptide LE6 with anti-inflammatory activity according to claim 1 in the preparation of anti-inflammatory drugs.

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