An Nrf2-activating peptide derived from the collagen of the head of the bighead carp

Abstract

The present invention relates to the field of biotechnology, and particularly relates to an Nrf2-activating peptide derived from the collagens of the head of bighead carp. Pro-Ser-Arg-Ile-Leu-Tyr-Gly, this oligopeptide has significant antioxidant activity and can activate the Nrf2 pathway to protect cells from oxidative damage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an Nrf2 agonist peptide derived from the collagen of the head of the fish Background Art

[0002] When the body is in an oxidative stress state, a large amount of ROS can be released, inducing lipid peroxidation reaction in cells, reducing enzyme activity, and changing the protein structure to cause oxidative damage to cells and tissues. The Nrf2-ARE signaling pathway is one of the most important antioxidant pathways and can regulate the expression of endogenous antioxidant enzymes. Under normal physiological conditions, nuclear factor erythroid 2-related factor 2 (Nrf2) binds to kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and is degraded through ubiquitination to maintain the content of Nrf2 at the physiological required level; when the body is under oxidative stress, ROS can cause the cysteine residues of Keap1 to be modified and dissociate from Nrf2 [ , and Nrf2 is released into the nucleus through deubiquitination and binds to the antioxidant response element (ARE) in the nucleus to up-regulate the expression of antioxidant enzymes and phase II detoxifying enzymes to scavenge excessive free radicals, such as heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), etc. Studies have found that activating the Nrf2-ARE signaling pathway can promote the transcription of cell protection genes, reduce the damage caused by oxidative stress, and play a role in protecting the kidneys. Summary of the Invention

[0003] On the one hand, the present invention provides an Nrf2 agonist peptide (PSRILYG), Pro-Ser-Arg-Ile-Leu-Tyr-Gly, derived from the collagen of the head of the fish, which has significant antioxidant activity and can activate the Nrf2 pathway to protect cells from oxidative damage.

[0004] On the other hand, the present invention provides a preparation method of an Nrf2 agonist peptide (PSRILYG), Pro-Ser-Arg-Ile-Leu-Tyr-Gly, derived from the collagen of the head of the fish, which comprises the following steps:

[0005] a) Enzymatic hydrolysis: Take the collagen of the head of the fish, add it to distilled water and mix evenly, and perform enzymatic hydrolysis with papain at pH 8.0 and a temperature of 55°C for 4 hours. After the enzymatic hydrolysis is completed, boil it in a water bath for 10 minutes to inactivate, and centrifuge to take the supernatant and adjust the pH to neutral;

[0006] b) Filtration: Filter the enzymatic hydrolysate through a 0.22 μm filter membrane, and then filter it through a 1 kDa ultrafiltration membrane. Freeze-dry the filtrate to obtain a freeze-dried powder;

[0007] c) Sephadex G-15 gel chromatography: Dissolve 90 mg of the freeze-dried powder obtained in step b) in 3 mL of distilled water, load it onto a Sephadex G-15 gel column for separation. The eluent is distilled water, the flow rate of the eluent is 0.3 mL / min, the collection time for each tube is 3 min, and every 100 tubes form a cycle. Place the eluent collected in the test tubes in a microcuvette, measure the ultraviolet absorption value at a wavelength of 280 nm, combine several tubes of the collected liquid with the strongest absorbance value, freeze-dry it, measure the DPPH· scavenging rate of each peak, and select the component with the highest scavenging activity for subsequent separation;

[0008] d) Automatic high-pressure preparative separation: Prepare a 1 mg / mL solution of the fraction with the highest activity obtained in step c), filter it through a 0.22 μm filter membrane. The mobile phase is water and acetonitrile (containing 0.1% trifluoroacetic acid), the elution gradient of acetonitrile is 10% - 95%, the injection volume is 5 mL, the flow rate is 3.0 mL / min, the elution time is 90 min, and the detection wavelengths are set at 215 and 280 nm; Collect the elution peaks and freeze-dry them; Determine the oligopeptide sequence obtained by LC-MS / MS analysis to be Pro-Ser-Arg-Ile-Leu-Tyr-Gly.

[0009] In some embodiments, the preparation of the fish head bone collagen in step a) includes the following steps: Cut the fish bones into pieces, remove non-collagenous substances, perform decalcification treatment, defatting treatment, and extract fish collagen steps.

[0010] In some embodiments, the preparation of the dragon head fish bone collagen in step a) includes the following steps: Cut the processed fish bones into pieces, stir the fish bones with 0.1 mol / L NaOH solution at a solid-liquid ratio of 1:20 (g / ml) for 48 h, change the solution 4 times in the middle to remove non-collagen substances, and wash with distilled water until neutral; Then soak the fish bones in 0.5 mol / L disodium ethylenediaminetetraacetate solution at a solid-liquid ratio of 1:20 (g / ml) and stir for 48 h for decalcification treatment, change the solution 4 times midway, and wash several times after treatment; Then stir the fish bones with 15% isopropanol solution at a solid-liquid ratio of 1:20 (g / ml) until the fat in the fish bones is removed, and then wash the isopropanol clean. The whole experimental process is carried out at 4 °C; Add 0.5 mol / L acetic acid solution (containing 0.5% pepsin by weight) at a solid-liquid ratio of 1:30 (g / ml) and stir at 4 °C for one day to extract fish bone collagen. After the extraction, filter with degreased cotton gauze, extract twice repeatedly according to the above method, and combine the filtrates; Add NaCl solution to the filtrate for salting out and stir continuously until no precipitate is washed out. Centrifuge the suspension at 4 °C and 12,000 r / min. The precipitate at the bottom of the centrifuge tube is redissolved in 0.5 mol / L acetic acid solution, and finally loaded into a dialysis bag and dialyzed until neutral, and freeze-dried to obtain fish collagen.

[0011] In some embodiments, the weight-to-volume ratio of the dragon head fish bone collagen to distilled water in step a) is 8:500 g / ml.

[0012] In some embodiments, the addition amount of papain in step a) is 5500 U / g.

[0013] In some embodiments, the weight-to-volume ratio of the freeze-dried powder to distilled water in step c) is 30:1 mg / ml.

[0014] The present invention enzymatically hydrolyzes dragon head fish bone collagen, and then uses techniques such as gel chromatography and liquid chromatography-mass spectrometry to separate and identify a novel oligopeptide Pro-Ser-Arg-Ile-Leu-Tyr-Gly (PSRILYG); This oligopeptide has significant antioxidant activity and can activate the Nrf2 pathway to protect cells from oxidative damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the mass spectrum of the oligopeptide PSRILYG in Example 1.

[0016] Figure 2 It is the effect of the oligopeptide PSRILYG in Example 2 on the CAT enzyme activity of high glucose-induced HepG2 cells.

[0017] Figure 3Effect of oligopeptide PSRILYG on SOD enzyme activity in high glucose-induced HepG2 cells in Example 3.

[0018] Figure 4 Effect of oligopeptide PSRILYG on Nrf2 expression in high glucose-induced HepG2 cells in Example 4. Specific implementation mode

[0019] The following further describes the present invention in detail with reference to the accompanying drawings and embodiments, however, they do not constitute a limitation or definition of the scope of the present invention.

[0020] There is no particular limitation on the solvent used in the present invention, and commercially available conventional solvents can be used.

[0021] Model group: Add 55 mmol / L glucose solution, and the treatment time is 48 h.

[0022] Oligopeptide group: Add 55 mmol / L glucose solution, and then add 100 mg / L oligopeptide solution, and the treatment time is 48 h.

[0023] Control group: Neither glucose solution nor oligopeptide solution is added.

[0024] Preparation of the dragon head fish bone collagen of the present invention: Cut the processed fish bones into pieces, stir the fish bones with 0.1 mol / L NaOH solution at a solid-liquid ratio of 1:20 (g / ml) for 48 h, change the solution 4 times in the middle to remove non-collagen substances, and wash with distilled water until neutral; then soak the fish bones in 0.5 mol / L disodium ethylenediaminetetraacetate solution at a solid-liquid ratio of 1:20 (g / ml) and stir for 48 h for decalcification treatment, change the solution 4 times in the middle, and wash several times after treatment; then stir the fish bones with 15% isopropanol solution at a solid-liquid ratio of 1:20 (g / ml) until the fat in the fish bones is removed, and then wash the isopropanol clean. The whole experimental process is carried out at 4 °C; add 0.5 mol / L acetic acid solution (containing 0.5% pepsin by weight) at a solid-liquid ratio of 1:30 (g / ml) and stir at 4 °C for one day to extract fish bone collagen. After the extraction is completed, filter with degreased cotton gauze, extract twice repeatedly according to the above method, and combine the filtrates; add NaCl solution to the filtrate for salting out and stir continuously until no precipitate is washed out. Centrifuge the suspension at 4 °C and 12,000 r / min. The precipitate at the bottom of the centrifuge tube is redissolved in 0.5 mol / L acetic acid solution, and finally filled into a dialysis bag and dialyzed until neutral, and freeze-dried to obtain fish collagen.

[0025] Example 1

[0026] a) Enzymatic hydrolysis: Take 0.8 g of the processed collagen from the head of the fishbone, add 50 mL of distilled water and mix evenly. Use papain for enzymatic hydrolysis at pH 8.0, temperature 55 °C, and the enzyme dosage of 5500 U / g for 4 h of hydrolysis. After the enzymatic hydrolysis is completed, boil in a water bath for 10 min to inactivate the enzyme, centrifuge and take the supernatant to adjust the pH to neutral;

[0027] b) Filtration: Filter the enzymatic hydrolysate through a 0.22 μm filter membrane, and then filter it through a 1 kDa ultrafiltration membrane. Freeze-dry the filtrate;

[0028] c) Sephadex G-15 gel chromatography: Dissolve 90 mg of the above freeze-dried powder in 3 mL of water and load it onto a Sephadex G-15 gel column for separation. The eluent is distilled water, the eluent flow rate is 0.3 mL / min, the collection time for each tube is 3 min, and every 100 tubes is a cycle. Place the eluent collected in the test tube in a microcuvette and measure the ultraviolet absorption value at a wavelength of 280 nm. Combine the several tubes of collected liquid with the strongest absorbance value, freeze-dry, measure the DPPH· scavenging rate of each peak, and select the component with the highest scavenging activity for subsequent separation;

[0029] d) Automatic high-pressure preparation and separation: Prepare the above highest component into a 1 mg / mL solution, filter it through a 0.22 μm filter membrane. The mobile phase is water and acetonitrile (containing 0.1% trifluoroacetic acid), the elution gradient of acetonitrile is 10% - 95%, the injection volume is 5 mL, the flow rate is 3.0 mL / min, the elution time is 90 min, and the detection wavelengths are set at 215 and 280 nm. Collect the elution peaks and freeze-dry;

[0030] e) LC-MS / MS analysis: Resuspend or dilute with 0.1% formic acid solution, after dissolution, centrifuge at high speed and take the supernatant for use. After desalting with C18 and freeze-drying, perform mass spectrometry detection. Mass spectrometry instrument model: Orbitrap Fusion Lumos (Thermofisher). Mass spectrometry method: Positive ion detection mode, primary resolution is 120,000, AGC is set to 3e6, scanning range is 300 - 1400 m / z. Select 10 ions with the highest intensities in 1 MS spectrum for MS / MS analysis, secondary resolution is 15,000, AGC is set to 2e4, isolation window is 1.6 m / z. Liquid phase method: The chromatographic column is Eksigent C18 (3μm, 250mm×75μm), phase A is water + 0.1% formic acid; phase B is acetonitrile + 0.1% formic acid, flow rate is 300 nl / min, injection volume is 4 μL, and chromatographic gradient elution is for 78 min. The obtained results are subjected to De-novo search and database search using PEAKS software. During the data processing process, first calculate the possible amino acid combinations based on the molecular weight information of the fragments in each MS / MS spectrum, and then compare them to the protein database of the sample source. The sample uses the protein database of Mytilus species in NCBI. The results include two parts. One part of the sequencing results can find the corresponding sequences in the protein database of Mytilus species in NCBI. For some peptides that are not aligned in the database, they can be obtained by denovo analysis. The measured oligopeptide sequence is: Pro-Ser-Arg-Ile-Leu-Tyr-Gly, and its mass spectrometry diagram is as Figure 1 shown.

[0031] Example 2

[0032] 1) The experiments were set as a control group (blank), a model group, and an oligopeptide group. In each well of a 96-well plate, inoculate 2×10 4 HepG2 cells, culture overnight, aspirate the culture medium. In the culture medium of the model group and the oligopeptide administration group, add 55 mmol / L glucose solution. In the oligopeptide group, additionally add 200 μL of the prepared oligopeptide solution (100 mg / L) to each well, and the treatment time is 48 h.

[0033] 2) After the culture is completed, discard the culture medium, add PBS and gently rinse the 6-well plate 2 times. Add 50 μL of pre-cooled cell lysate to each well and place it on ice for lysis. Then use a cell scraper to scrape the cells and transfer them to a pre-cooled 1.5 mL centrifuge tube for lysis for 30 min, shaking once every 5 min. After the lysis is completed, centrifuge at low temperature (4°C, 12,000 r / min, 10 min) to take the supernatant, and use a kit to measure the content of CAT (GuYan Biotech Co., Ltd, product number GOY-0089SJ).

[0034] The activity of intracellular CAT is asFigure 2 As shown, compared with the control group, the activity of CAT in the cells of the model group decreased significantly, indicating that HepG2 cells were damaged under high glucose stimulation; compared with the model group, the activity of CAT in the cells of the oligopeptide PSRILYG administration group increased significantly, showing a protective effect on the oxidative damage of HepG2 cells.

[0035] Example 3

[0036] 1) The experiments were set up with a control group, a model group, and an oligopeptide administration group. 2×10 4 HepG2 cells were seeded in each well of a 96-well plate and cultured overnight. Then the culture medium was aspirated. In the model group and the oligopeptide administration group, a 55 mmol / L glucose solution was added to the culture medium. Additionally, 200 μL of the prepared oligopeptide solution (100 mg / L) was added to each well in the oligopeptide group, and the treatment time was 48 h.

[0037] 2) After the culture was completed, the culture medium was discarded, and the 6-well plate was gently rinsed twice with PBS. 50 μL of pre-cooled cell lysate was added to each well and the cells were lysed on ice. Subsequently, the cells were scraped off with a cell scraper and transferred to a pre-cooled 1.5 mL centrifuge tube for lysis for 30 min, with shaking every 5 min. After lysis, centrifugation was performed at low temperature (4°C, 12,000 r / min, 10 min) to collect the supernatant, and the SOD content was measured using a kit (GuYan Biotech Co., Ltd, product number GOY-0087SJ).

[0038] The activity of SOD in the cells was as Figure 3 shown. Compared with the control group, the activity of SOD in the cells of the model group decreased significantly, indicating that HepG2 cells were damaged under high glucose stimulation; compared with the model group, the activity of SOD in the cells of the oligopeptide PSRILYG administration group increased significantly, showing a protective effect on the oxidative damage of HepG2 cells.

[0039] Example 4

[0040] 1) The experiments were set up with a control group, a model group, and an oligopeptide administration group; 2×10 4 HepG2 cells were seeded in each well of a 96-well plate and cultured overnight. Then the culture medium was aspirated. In the model group and the oligopeptide administration group, a 55 mmol / L glucose solution was added to the culture medium. Additionally, 200 μL of the prepared oligopeptide solution (100 mg / L) was added to each well in the oligopeptide group, and the treatment time was 48 h.

[0041] 2) After the cultivation is completed, discard the culture medium, add PBS and gently rinse the 6-well plate twice. Add 50 μL of pre-cooled cell lysate to each well and place it on ice for lysis. Then, use a cell scraper to scrape off the cells and transfer them to a pre-cooled 1.5 mL centrifuge tube for 30 min of lysis, with shaking every 5 min. After the lysis is completed, centrifuge at low temperature (4 °C, 12,000 r / min, 10 min) and take the supernatant.

[0042] 3) Dilute the sample to an appropriate concentration, add protein loading buffer in a ratio of 1:4, mix well, boil the centrifuge tube containing the mixture in a water bath at 100 °C for 10 min, and centrifuge according to the above centrifugation conditions after cooling. Take out some of the supernatant for electrophoresis, and store the rest at -80 °C for later use.

[0043] 4) Assemble the gel preparation rack, prepare a separating gel with a concentration corresponding to the molecular weight of the target protein, and use a 5% concentration for the stacking gel. After the gel solidifies, electrophoresis can be carried out. Load the processed sample and marker into the comb holes near the middle. Perform electrophoresis at a constant voltage. First, run at 80 V until the bottom of the stacking gel, and then use 120 V until the bands of the pre-stained protein marker are finished running, then stop electrophoresis.

[0044] 5) Take out the gel plate, cut the target protein according to the position of the marker band, cut a PVDF membrane of appropriate size, activate it with methanol for 5 min, and place it in the transfer buffer for equilibration and standby. Stack the required materials according to the "sandwich" method, and carefully remove the air bubbles between the separating gel and the PVDF membrane. Place an ice pack in the transfer tank to keep the temperature of the transfer buffer at a lower temperature, and perform transfer at a constant current of 300 mA.

[0045] 6) Prepare a 5% BSA solution with TBST (containing 0.1% Tween-80) and soak the PVDF membrane for at least 1 h at room temperature. Dilute the primary antibodies used in the experiment according to their respective ratios with the above BSA solution, then put the PVDF membrane that has completed the above steps into an incubation box and add 4 mL of primary antibody solution to fully cover it, and incubate overnight in a refrigerator at 4 °C. The next day, wash the PVDF membrane with TBST solution for 25 min, changing the washing solution every 5 min. Dilute the horseradish peroxidase (HRP)-labeled secondary antibody at a certain ratio and incubate it with the PVDF membrane at room temperature for 1 h. After incubation, wash the membrane by the above method.

[0046] 7) Prepare the ECL hypersensitive chemiluminescent chromogenic solution, mix it evenly and place it in the dark. Place the PVDF membrane in a dark room, use a pipette to aspirate the chromogenic solution to cover the entire membrane, let it stand for 1 min, turn on the gel imager for exposure and imaging, save the required pictures, and then calculate the gray value and analyze the results. The results of the effect of the oligopeptide PSRILYG on the expression of Nrf2 in high-glucose-induced HepG2 cells are as Figure 4As shown in the figure, compared with the control group, the expression of Nrf2 in the model group cells decreased significantly, indicating that HepG2 cells cannot resist the oxidative stress by themselves. Compared with the model group, the oligopeptide PSRILYG can significantly increase the expression level of Nrf2 in cells.

[0047] Finally, it should be noted that the above is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiment, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. An Nrf2-activating peptide derived from the collagen of the fish head of Aristichthys nobilis, characterized in that The sequence of the kinins is: Pro-Ser-Arg-Ile-Leu-Tyr-Gly. This kinin has significant antioxidant activity and can activate the Nrf2 pathway to protect cells from oxidative damage.

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