Mitochondria-targeted antioxidant sea cucumber polypeptide and application thereof

Abstract

The invention belongs to the field of small molecule polypeptides, and particularly relates to a mitochondria-targeted antioxidant sea cucumber polypeptide and application thereof. The mitochondria-targeted antioxidant sea cucumber polypeptide has an amino acid sequence as follows: NMQTHPTYLGSR, and is a hydrophilic polypeptide; the sea cucumber polypeptide can directly act on mitochondria in a targeting mode, accumulation of active oxygen in the mitochondria is reduced, and the sea cucumber polypeptide has an excellent mitochondria-targeting anti-oxidation function, also has neuroprotective activity and can play a role in preventing neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule polypeptides, and in particular relates to an antioxidant sea cucumber polypeptide targeting mitochondria and an application thereof. Background Art

[0002] As the powerhouse of cells, mitochondria are the main site of bioenergy production and are closely related to oxidative stress response. Many diseases such as Alzheimer's disease and Huntington's disease are related to body oxidation and mitochondrial function. Oxidation is the main cause of mitochondrial dysfunction, and mitochondrial dysfunction and excessive oxidative stress response are the main causes of neurodegenerative diseases. Mitochondrial targeted therapy has accuracy and therapeutic efficiency due to its ability to act directly on mitochondria, and has application prospects in the prevention and treatment of neurodegenerative diseases.

[0003] The main causes of mitochondrial dysfunction include: excessive production of mitochondrial reactive oxygen species (ROS); excessive consumption of activated superoxide dismutase 2 (SOD2); changes in mitochondrial membrane potential (MMP); changes in oxygen consumption and Ca 2+ Increase; mitochondrial fission gene Drp-1 up-regulated and mitochondrial fusion gene Mfn-2 down-regulated. Maintaining mitochondrial health is a dynamic balance process, which is regulated by mitochondrial fission, fusion, and biogenesis genes. When this balance is broken due to aging or external stimulation, and adverse substances such as ROS accumulate to a certain extent, it will cause mitochondrial lesions of varying degrees. Dysfunctional mitochondria show membrane depolarization and rupture, accompanied by the massive production of mitochondrial ROS and Ca 2+ Released into the cytoplasm. In order to prevent the vicious cycle of mitochondrial damage and ROS production, cells protect themselves from damage through mitochondrial autophagy, an intrinsic quality control mechanism. When excessive stimulation causes massive damage to mitochondria or is affected by irreversible aging, the clearance efficiency of self-protection mechanisms such as mitochondrial autophagy cannot keep up with the accumulation rate of substances such as ROS, and exogenous auxiliary regulation is required to maintain the dynamic balance of mitochondria.

[0004] Studies have shown that many peptides have antioxidant potential, can effectively reduce the level of ROS and other pro-oxidants, delay or prevent oxidative stress damage, and thus protect cells and tissues. They have great application potential in the fields of medicine, food, cosmetics, etc. However, although the antioxidant activity of most peptides has been obtained through in vitro tests so far, whether they have antioxidant activity in vivo still needs to be verified, and there are very few antioxidant peptides that precisely target mitochondria, and no natural source peptides have been reported. For example, a tripeptide sequence (Ala-Val-Leu) prepared from Arctic ginseng (Lianzhu Lin et al. Anti-aging effect of sea cucumber (Cucumaria frondosa) hydrolysate on fruitflies and D-galactose-induced aging mice [J]. Journal of Functional Foods, 2018) can prolong the survival time of oxidative stress and has antioxidant activities, but no research related to targeting mitochondria has been involved. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of most antioxidant polypeptides in the prior art, such as the lack of mitochondrial targeting performance, the primary purpose of the present invention is to provide an antioxidant sea cucumber polypeptide targeting mitochondria, which can directly target the mitochondria, reduce the accumulation of reactive oxygen species in the mitochondria, and has excellent mitochondrial targeted antioxidant function. Using it as a drug can improve the efficacy and accuracy; at the same time, the polypeptide also has neuroprotective activity.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned mitochondrial-targeted antioxidant sea cucumber polypeptide.

[0007] Another object of the present invention is to provide the application of the above-mentioned mitochondrial-targeted antioxidant sea cucumber polypeptide.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] An antioxidant sea cucumber peptide HoloPep#405 (twelve peptides) targeting mitochondria, whose amino acid sequence is: NMQTHPTYLGSR;

[0010] The molecular formula of the mitochondrial-targeted antioxidant sea cucumber polypeptide is C 59 H 93 N 19 O 19 S, with an average relative molecular mass of 1404.54 g / mol and a theoretical isoelectric point of pH = 9.84, is a hydrophilic polypeptide;

[0011] The method for preparing the mitochondrial-targeted antioxidant sea cucumber polypeptide comprises the following steps:

[0012] (1) Using sea cucumber as raw material, obtaining the above-mentioned mitochondrial-targeted antioxidant sea cucumber polypeptide by enzymatic hydrolysis, separation, extraction and purification, or directly preparing the above-mentioned mitochondrial-targeted antioxidant sea cucumber polypeptide by solid phase synthesis;

[0013] The enzymatic hydrolysis is preferably carried out using trypsin and papain in sequence;

[0014] The specific operation of the enzymolysis is preferably:

[0015] The sea cucumber freeze-dried powder is mixed with water, and then the pH of the solution is adjusted to 7.5-8.0, and trypsin is added for enzymolysis; then the pH of the solution is adjusted to 6.5-7.0, and papain is added for enzymolysis; after enzymolysis, the enzyme is inactivated to obtain a sea cucumber enzymolysate;

[0016] The specific operation of separation and extraction is preferably:

[0017] After the sea cucumber protein hydrolysate obtained after enzymatic hydrolysis is cooled, 95% ethanol by volume is added and allowed to stand; then vacuum filtration and centrifugation are performed to remove the precipitate, and the supernatant is subjected to vacuum rotary evaporation to remove the ethanol, and freeze-dried to obtain a sea cucumber crude polypeptide dry powder;

[0018] The purification is preferably at least one of ultrafiltration purification and gel chromatography column purification;

[0019] The ultrafiltration purification preferably uses Ultracel-3 filter membrane separation to collect components with a molecular weight less than 3 kDa;

[0020] The specific operation of the gel chromatography column purification is preferably:

[0021] The ultrafiltration purified fraction was desalted and further purified using a Sephadex G-25 dextran gel chromatography column;

[0022] The purification preferably further comprises using reverse phase high performance liquid chromatography or size exclusion chromatography to further separate the components into individual peptides;

[0023] Application of the mitochondria-targeted antioxidant sea cucumber polypeptide in the preparation of antioxidant products;

[0024] Application of the mitochondria-targeted antioxidant sea cucumber polypeptide in the preparation of mitochondria-targeted antioxidant products;

[0025] The use of the mitochondrial-targeted antioxidant sea cucumber polypeptide in the preparation of a product with neuroprotective activity;

[0026] Application of the mitochondrial-targeted antioxidant sea cucumber polypeptide in the preparation of products for preventing and treating neurodegenerative diseases;

[0027] An antioxidant product comprising at least one of the above antioxidant sea cucumber polypeptide, a protein hydrolysate containing the above antioxidant sea cucumber polypeptide, and a hydrolysate containing the above antioxidant sea cucumber polypeptide as an active ingredient;

[0028] A product for preventing and treating neurodegenerative diseases, comprising at least one of the above-mentioned antioxidant sea cucumber polypeptide, a protein hydrolysate containing the above-mentioned antioxidant sea cucumber polypeptide, and a hydrolysate containing the above-mentioned antioxidant sea cucumber polypeptide as an active ingredient;

[0029] Principle of the present invention:

[0030] The invention uses sea cucumber as raw material, extracts, separates and identifies a pure natural sea cucumber polypeptide from sea cucumber through enzymatic separation, ultrafiltration purification, LC-MS / MS identification and other steps, and the amino acid sequence of the sea cucumber polypeptide is: NMQTHPTYLGSR, and the molecular formula is C 59 H 93 N 19 O 19 S, with an average relative molecular mass of 1404.54 g / mol and a theoretical isoelectric point of pH = 9.84, is a hydrophilic polypeptide;

[0031] The present invention further uses the model organism Caenorhabditis elegans as a model and finds that the sea cucumber polypeptide has the ability to target mitochondria and has an antioxidant function; it also has neuroprotective activity and can prevent neurodegenerative diseases, as follows:

[0032] (1) Using the intestinal model nematode SJ4143, a methyl rhodamine ethyl ester (TMRE) fluorescence staining experiment was conducted: the mitochondria of the intestinal cells of the nematode SJ4143 contain a green fluorescent GFP marker. After TMRE staining, green fluorescence and red fluorescence can be stimulated under the blue light and green light channels of the fluorescence microscope respectively; the mitochondrial health level (HMI) can be obtained after the ratio of red fluorescence to green fluorescence is normalized (i.e., TMRE / GFP). Since TMRE dye can only stain healthy mitochondria, the ratio can be used to represent the health level of mitochondria. The present invention proves through experiments that the HMI level of nematodes increases after treatment with the sea cucumber polypeptide, indicating that the polypeptide can improve mitochondrial function and has the ability to target mitochondria.

[0033] (2) Mitochondrial DNA copy number test: The present invention further verifies that the sea cucumber polypeptide can improve the health level of mitochondria by detecting the mitochondrial DNA copy number experiment, thereby verifying the accuracy of the conclusion of the above (1) determining whether the sea cucumber polypeptide has targeting ability, and proving that the sea cucumber polypeptide does have the ability to target mitochondria.

[0034] (3) Antioxidant activity test: The present invention uses DCFH / DA (2',7'-dichlorofluorescein diacetate) probe to detect the level of reactive oxygen species (ROS) in mitochondria, proving that the polypeptide has an antioxidant function targeting mitochondria.

[0035] (4) PolyQ (polyglutamine) aggregation inhibition experiment: This experiment verifies that the targeted antioxidant peptide also has neuroprotective activity.

[0036] In summary, the present invention has proved through the above experiments that the sea cucumber polypeptide has an antioxidant function targeting mitochondria, that is, it can be directly administered to the mitochondria for treatment, and play a role in restoring mitochondrial function or delaying mitochondrial oxidative damage. The advantages of the antioxidant sea cucumber polypeptide targeting mitochondria include efficient pharmacokinetics, easy mitochondrial absorption, and the ability to accumulate specifically in mitochondria, avoiding the non-specific accumulation of drugs in other parts of the cell due to the disadvantage of poor non-targeted specificity, which causes side effects after reaching high concentrations. The sea cucumber polypeptide also has a neuroprotective effect, and this effect is further confirmed by the antioxidant performance of targeting mitochondria, so its effect in preventing or delaying neurodegenerative diseases will be better than the prior art.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] (1) The present invention discloses a novel polypeptide obtained from natural sea cucumbers, the amino acid sequence of which is NMQTHPTYLGSR. The polypeptide can be isolated and purified from sea cucumbers, or obtained through artificial synthesis.

[0039] (2) The sea cucumber polypeptide provided by the present invention has a small molecular weight and is easy to absorb. It has been verified through experiments that it has excellent mitochondrial targeting function and can directly target mitochondria. At the same time, the sea cucumber polypeptide can reduce the accumulation of excessive reactive oxygen in nematodes and has excellent antioxidant capacity. It has antioxidant activity based on its ability to target mitochondria, which increases the accuracy of the antioxidant effect and the therapeutic efficiency.

[0040] (3) The sea cucumber polypeptide provided by the present invention significantly inhibits the aggregation of polyglutamine in the model organism Caenorhabditis elegans, and can prevent neurodegenerative diseases. Because the polypeptide has the ability to target mitochondria, the polypeptide has a precise effect in the treatment of neurodegenerative diseases. In the field of small molecule polypeptide technology, the field of targeted mitochondrial therapy has certain significance.

[0041] (4) The sea cucumber polypeptide provided by the present invention can be further used in the development of products such as food, medicine and health care products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the overlap graph of GFP fluorescence and TMRE fluorescence.

[0043] Figure 2 This is the result analysis diagram of the effects of MitoQ and sea cucumber peptide HoloPep#405 on HMI, and the horizontal axis is the number of independent repeated experiments.

[0044] Figure 3 This is an analysis chart of the effect of sea cucumber polypeptide HoloPep#405 on mitochondrial genome copy number.

[0045] Figure 4 This is the result analysis chart of the changing trend of ROS levels within 2 hours after Caenorhabditis elegans was treated with different conditions.

[0046] Figure 5 This is the result analysis diagram of the effect of sea cucumber polypeptide HoloPep#405 on polyQ aggregation of Caenorhabditis elegans. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0048] The concentrated NA22 bacterial liquid in the embodiment is food for C. elegans, and its preparation method is: inoculate the activated Escherichia coli NA22 into LB liquid culture medium for shaking culture, and then collect the bacteria by centrifugation to obtain the concentrated NA22 bacterial liquid.

[0049] The SJ4143 and AM141 Caenorhabditis elegans models are both from the Caenorhabditis Genetics Center at the University of Minnesota.

[0050] Example 1 Isolation and Identification of Sea Cucumber Polypeptides

[0051] 1. Enzymatic separation

[0052] (1) Take fresh sea cucumbers, clean them, chop them and homogenize them. After freeze-drying the homogenate, weigh 5 g of sea cucumber freeze-dried powder, add deionized water at a solid-liquid ratio of 1:4, stir evenly with a glass rod, and preheat in a 45℃ constant temperature water bath. Then adjust the solution pH to 7.5 with NaOH, add 1 mL of trypsin (15000 U / mL), and react at 45℃ for 4 h; then adjust the solution pH to 6.5 with HCl, add 1 mL of papain (15000 U / mL), and react at 65℃ for 4 h; boil and inactivate to obtain sea cucumber protein hydrolysate.

[0053] (2)After the sea cucumber protease hydrolysate prepared in step (1) is naturally cooled to room temperature, 170 mL of ethanol with a volume fraction of 95% is slowly added under stirring with a glass rod. After standing overnight at room temperature, vacuum filtration and centrifugation (4 °C, 8000 rpm, 5 min) are carried out to remove the precipitate. The supernatant is rotary evaporated under reduced pressure to remove ethanol, and then freeze-dried to obtain the crude sea cucumber polypeptide dry powder.

[0054] 2. Ultrafiltration purification and gel chromatography column purification

[0055] (1)The crude sea cucumber polypeptide dry powder prepared in step 1 is formulated into a solution with a concentration of 20 mg / mL. Ultrafiltration is carried out using a centrifugal filter of Amicon Ultra-2 equipped with Ultracel-3, Ultracel-5, and Ultracel-10 membranes of Millipore to obtain the retention components of the sea cucumber protease hydrolysate with MW > 10 kDa, 5 kDa < MW < 10 kDa, 3 kDa < MW < 5 kDa, and MW < 3 kDa; the components with a molecular weight less than 3 kDa are collected, then passed through a 0.45 μm filter membrane to remove impurities, and the salts in the polypeptide sample are removed using a desalting column.

[0056] (2)The polypeptide components after desalting in step (1) are further separated using a packed Sephadex G-25 dextran gel chromatography column. Elution is carried out with primary deionized water at a flow rate of 1 mL / min, and the fractions of each absorption peak are monitored at 280 nm using a 785 UV / VIS detector and collected simultaneously. A total of 5 polypeptide components are obtained, and these 5 polypeptide components are freeze-dried separately to obtain the corresponding freeze-dried sea cucumber polypeptide powders.

[0057] 3. LC-MS / MS identification

[0058] The polypeptide composition and amino acid sequence of the freeze-dried sea cucumber polypeptide powder obtained in step 2 are identified by LC-MS / MS.

[0059] 4. Database alignment and screening

[0060] According to the information of peptide sequences with antioxidant activity provided in the BIOPEP-UWM database, the sea cucumber peptide sequence sequenced in step 3 was compared with the database for virtual activity screening. The specific steps were as follows: log in to the BIOPEP-UWM official website, select the Bioactive peptides database, click the Analysis option, select Profile of potential biological activity in the tab, and then enter the sea cucumber peptide sequence obtained by liquid chromatography-mass spectrometry (LC-MS) in step 3 for comparison. Sea cucumber peptide sequences containing 2 or more antioxidant amino acid fragments and neuroprotective fragments were selected as sea cucumber peptide sequences with potential antioxidant activity and neuroprotective effects for the following test. After screening, a sea cucumber peptide with the sequence NMQTHPTYLGSR was obtained and named HoloPep #405.

[0061] Example 2 Synthesis of sea cucumber polypeptides by Fmoc solid phase method

[0062] The sea cucumber peptide HoloPep #405 was synthesized by Shanghai Taopu Biotechnology Co., Ltd. The specific method is as follows:

[0063] (1) Weigh triphenylmethyl chloride resin and mix with DCM (dichloromethane) solution, and shake for 30 min to allow the resin to fully swell.

[0064] (2) Remove the DCM solvent by sand core filtration, add 3 times molar excess of Fmoc-(first amino acid at the C-terminus)-OH, 10 times molar excess of N,N-diisopropylethylamine and a small amount of dimethyl fumarate to dissolve, shake for 1 hour to allow the first amino acid to bind to the resin, and then repeatedly use dimethyl fumarate and DCM to wash 6 times alternately. Use a 20% volume fraction of piperidine dimethyl fumarate solution to wash for 5 minutes to remove the protective effect of Fmoc-, and repeat this operation for 15 minutes.

[0065] (3) Drain the solution, take out a dozen resin pellets, wash them three times with ethanol, and then add one drop each of ninhydrin, potassium cyanide, and phenol solution. Heat at high temperature until a positive reaction occurs and a dark blue color appears.

[0066] (4) Add dimethyl fumarate, methanol, and dimethyl fumarate to the reaction tube in proportion and wash twice. Then add 3-fold molar excess of Fmoc-(the second amino acid at the C-terminus)-OH, 3-fold molar excess of O-benzotriazole-tetramethyluronium hexafluorophosphate, and a small amount of dimethyl fumarate to dissolve. Immediately thereafter, add 10-fold molar excess of N,N-diisopropylethylamine and react for 40 min to carry out the condensation reaction of amino acids.

[0067] (5) Repeat the above deprotection, ninhydrin detection and condensation reaction until all target amino acids are connected to the resin in order from right to left and the Fmoc-protection is removed. Use ninhydrin to detect until the result is negative, indicating that the reaction is complete. Finally, add methanol solution to wash the resin three times and evaporate the liquid at room temperature.

[0068] (6) Transfer the resin to a centrifuge tube, add a cutting solution to fully separate the polypeptide from the resin, and oscillate at a constant temperature for 120 minutes to cut the polypeptide from the resin. The cutting solution is prepared according to the ratio of 94.5% trifluoroacetic acid, 2.5% water, 2.5% 3,4-ethylenedioxythiophene and 1% disulfide peptide (all by volume fraction). Use a sand core to filter the above liquid, blow the obtained cleavage solution with nitrogen as much as possible, and then wash it repeatedly with anhydrous ether for 6 times, centrifuge at 4000 r / min for 3 minutes, collect the precipitate, and evaporate it at room temperature to obtain the crude sea cucumber polypeptide; the polypeptide with a purity of more than 95% is obtained by separation and purification by high performance liquid chromatography.

[0069] Example 3 Ability of sea cucumber peptide HoloPep #405 to target mitochondria

[0070] In this example, the biological Caenorhabditis elegans SJ4143 was used as the research object, and the mitochondrial function of the sea cucumber polypeptide HoloPep#405 targeting the nematode was evaluated by quantifying TMRE-GFP, also known as the Healthy Mitochondria Indicator (HMI), a fluorescent dye that can characterize mitochondrial integrity. The mtDNA copy number was used to further verify the ability of the sea cucumber polypeptide HoloPep#405 targeting the mitochondria of the nematode from a molecular biological perspective.

[0071] 1. HMI Method

[0072] (1) After synchronizing the spawning stage Caenorhabditis elegans SJ4143, they were placed in a conical flask and cultured until the L1 stage. The nematodes were then divided into three groups: a blank control group, a MitoQ-treated group, and a sea cucumber polypeptide #HoloPep405-treated group. Each group had a new conical flask. Concentrated NA22 bacterial solution was added to the L1 worm liquid of each group as nematode food. The final OD value of NA22 in the control system was 570The experimental group was given drugs at the same time when NA22 was fed at the L1 stage. MitoQ mother solution (mother solution concentration was 5 mM) was added to the MitoQ-treated group to make its final concentration 5 μM; sea cucumber polypeptide #HoloPep405-treated group was added with sea cucumber polypeptide mother solution (mother solution concentration was 10 mM) to make its final concentration 2 mM, and the blank control group was added with an equal amount of S Medium. Finally, the above-mentioned worm-containing system was placed in a shaker at 20°C and 120 rpm for 42 hours to reach the L4 stage of the larvae.

[0073] (2) When the nematodes in step (1) reach the L4 stage after 42 hours of culture, the three groups of nematodes are collected, washed, and transferred to a 48-well plate. Concentrated NA22 bacterial solution (as nematode food), 5-FUdR (to inhibit nematode egg laying), and AMP solution (to inhibit the growth of other bacteria) are added to each well. The final volume of each well is 500 μL, and the number of nematodes in each well is 100-150, that is, the nematode concentration is 200-300 / mL. The final OD of NA22 in each well is 2.5-3.0. 570 The final concentration of 5-FUdR is 75 μg / mL, and the final concentration of AMP is 100 μg / mL.

[0074] (3) After the nematodes in step (2) have been cultured in the 48-well plate for 3 days, TMRE staining is performed. The specific method is as follows: wash the nematodes 3-4 times with M9 buffer, dilute 5 μM TMRE with buffer to 1 μM, pre-add it to a 24-well plate, then add the washed Caenorhabditis elegans, adjust the worm density to about 20-30 / 10 μL, and evenly shake the staining at 20°C in a shaker at 120 rpm for 6 h; after 6 hours, collect the stained nematodes and wash them 3-4 times with M9 buffer; after staining, place the nematodes in M9 buffer for decolorization according to the conventional method, collect the decolorized Caenorhabditis elegans and wash them again with M9 buffer for 3-4 times.

[0075] (4) The cleaned nematodes were fixed on a 2% agarose pad and photographed using a fluorescence microscope using different fluorescence channels to obtain green (GFP) and red (TMRE) fluorescence images. The obtained fluorescence images were processed using image J software to obtain the mean red TMRE fluorescence intensity (Mean-TMRE) and the green mean GFP fluorescence intensity (Mean-GFP), and then the HMI value (HMI=Mean-TMRE / Mean-GFP) was calculated to characterize the ratio of healthy mitochondria to the total mitochondrial content in C. elegans, thereby evaluating the targeting of the mitochondrial therapeutic effect of the sea cucumber peptide HoloPep#405.

[0076] Figure 1This is a microscope display of SJ4143 Caenorhabditis elegans after being stained with the fluorescent dye methyl rhodamine ethyl ester (TMRE), showing green fluorescence and red fluorescence when excited by blue light and green light channels respectively.

[0077] MitoQ is a mitochondrial positive compound that is recognized to have a positive effect on mitochondrial quality. In this example, MitoQ was used as a positive control to verify whether HMI can intuitively and correctly reflect the improvement of MitoQ on the mitochondria of Caenorhabditis elegans. Mitochondria in the control group and the drug-treated group were dyed red by TMRE under the green light channel. After subsequent data processing, it was found that the HMI value of the drug-treated group, i.e., the TMRE / GFP level, was higher than that of the control group. The results are shown in Figure 2. Figure 2 As shown. The results showed that the HMI value of the MitoQ-treated group was significantly higher than that of the control group, indicating that MitoQ has an improvement effect on mitochondrial quality, or that the HMI value can correctly reflect the improvement effect of MitoQ on the mitochondrial quality level. At the same time, the HMI value of nematodes treated with sea cucumber polypeptide #405 was equivalent to that of MitoQ, which can further illustrate the ability of sea cucumber polypeptide #405 to target mitochondria.

[0078] 2. mtDNA copy number

[0079] (1) The specific method and steps are the same as step 1 (1).

[0080] (2) The specific method and steps are the same as step 1 (2).

[0081] (3) After the nematodes in step (2) have been cultured in a 48-well plate for 3 days, 50-100 μL of worm solution was collected from the blank control group, the 5 μM MitoQ treatment group, and the 2 mM HoloPep#405 treatment group, with a worm density of 40-60 worms / 10 μL. Then, the nematode DNA was extracted according to the instructions of the DNA extraction kit (Quanshijin), and the eluted DNA was temporarily stored on ice. After the concentration was determined, it was stored in a -20°C environment.

[0082] (4) Using the DNA obtained in step (3) as a template, real-time fluorescence quantitative PCR amplification was performed to determine the relative DNA content. The data processing method was based on the reference (Rooney JP, Ryde IT, Sanders LH, et al. PCR based determination of mitochondrial DNA copy number in multiple species[J].Mitochondrial Regulation: Methods and Protocols, 2015: 23-38.) to determine the expression levels of mtDNA and nDNA, respectively.

[0083] (5) The relative mitochondrial genome copy number was expressed as the ratio of mtDNA to nDNA. The mtDNA / nDNA ratio of the blank control group was normalized to 1. The mtDNA / nDNA ratios of the MitoQ-treated group and the HoloPep#405-treated group were compared with those of the blank control group.

[0084] The DNA of nematodes was extracted by lysis method using DNA extraction kit (Quanshijin), and the expression levels of mtDNA and nDNA were determined respectively. The relative mitochondrial genome copy number was expressed as the ratio of mtDNA to nDNA. The results are shown in Figure 3 As shown, the mtDNA copy number in the MitoQ group increased by about 35% compared with the blank control group, showing an improvement in mitochondrial quality. At the same time, the sea cucumber peptide HoloPep#405 increased by about 32% compared with the blank control group, showing the ability to target mitochondrial function and superior mitochondrial dysfunction treatment.

[0085] Example 4 Antioxidant activity of sea cucumber polypeptide HoloPep #405

[0086] In order to verify whether the sea cucumber polypeptide HoloPep #405 prepared by the solid phase synthesis method has antioxidant activity, the antioxidant activity was detected using the model organism Caenorhabditis elegans SJ4143. The specific method is as follows:

[0087] (1) Administration: The nematodes were first cultured synchronously to the L4 stage, and then washed with buffer for 3-4 times, and then added to a 24-well plate for administration according to the following steps: In the 24-well plate, concentrated NA22 bacterial solution was pre-added to each well as food for the nematodes, and 5.0 mg / mL 5-FUdR and 5.0 mg / mL AMP were added to inhibit nematode egg laying and inhibit the growth of miscellaneous bacteria; the experiment was divided into four groups: a blank control group, a modeling group, a HoloPep#405 group, and a modeling+HoloPep#405 group, wherein a certain volume of sea cucumber polypeptide HoloPep#405 was added to the HoloPep#405 group and the modeling+HoloPep#405 group, and the sea cucumber polypeptide administration concentration was the same as in Example 3 above, that is, both groups were pre-added with sea cucumber polypeptide mother solution (10 mM) to a 1000 μL system to make the final concentration of HoloPep#405 2 mM; the blank control group and the modeling group were treated with S Medium to fill the liquid volume in the system, so that the final solution volume in each well is equal; finally, add 200 μL of Caenorhabditis elegans solution cultured to the L4 stage after synchronization treatment to each well (each 10 μL contains 20-30 nematodes), and the final volume of each well reaches 1000 μL, so that the concentration of nematodes in the 24-well plate is 400-600 nematodes / mL. Among them, ensure that the final OD of NA22 in each well 570The final concentration of 5-FUdR was 75 μg / mL, and the final concentration of AMP was 100 μg / mL. Four replicate wells were set for each treatment.

[0088] (2) Modeling: After continued cultivation at 120 rpm and 20°C for 24 h, 10 μL of 200 mM paraquat (PQ) solution (final concentration was 2 mM) was added to the modeling group and the modeling + HoloPep#405 group, and 10 μL of SMedium solution was added to the blank control group and the HoloPep#405 group.

[0089] (3) Extraction of nematode protein by lysis: After continuing to culture each group of nematodes in a shaker at 20°C for 48 h, remove the 24-well plate, collect each group of Caenorhabditis elegans in a 1.5 mL centrifuge tube, wash the nematodes thoroughly and place them on ice for later use. Add 200 μL of PBST lysis buffer to each group of centrifuge tubes to resuspend the Caenorhabditis elegans, and use a mini handheld homogenizer to break the nematodes. After breaking, quickly centrifuge in a refrigerated centrifuge, set the parameters to 12,000 rpm, centrifuge at 4°C for 2 min, take the supernatant, and place it on ice for later use.

[0090] (4) Determination of reactive oxygen species level: Take the sample obtained in step (3) (protein content is 1 mg / mL) and use the Bio-Tech kit (i.e., DCFH / DA probe method) to determine the ROS level in nematodes. The steps are as follows: first, dilute the DCFH / DA probe concentration to 100 μM with PBS, then use a black 96-well plate to detect the sample, add 50 μL of sample and 50 μL of DCFH / DA probe to each well; shake to mix; finally, use a fluorescence microplate reader, set the parameters to an emission wavelength of 538 nm and an excitation wavelength of 480 nm, and measure the fluorescence value, starting from 0 min, measure once every 20 min, and measure 7 times for a total of 120 min.

[0091] The results are as follows Figure 4 As shown, the smaller the DCF fluorescence intensity, the lower the ROS level in the nematode, which means that the substance has an ideal antioxidant effect. It can be seen that compared with the blank control group, the DCF fluorescence intensity detected after paraquat modeling increased significantly, proving that after paraquat modeling, a large amount of ROS accumulated in the body of Caenorhabditis elegans, which proves that the modeling is successful, that is, paraquat can destroy the stability of mitochondria in nematodes. The DCF fluorescence intensity measured in the nematodes administered with only sea cucumber polypeptides was lower than that of the control group. The DCF fluorescence intensity curve of the paraquat modeling + HoloPep#405 group is between the control group and the modeling group, which proves that the sea cucumber polypeptide HoloPep#405 can prevent and delay the mitochondrial oxidative damage caused by paraquat to nematodes, and confirms the targeted mitochondrial antioxidant ability of the polypeptide.

[0092] Example 5 Neuroprotective activity of sea cucumber polypeptide HoloPep#405

[0093] The toxicity of protein aggregation is closely related to the state and degree of its aggregation. Polyglutamine (polyQ) proteins aggregate in cells of patients with Huntington's disease (HD) and other neurodegenerative diseases, and are key pathological signs of neurodegenerative diseases. High levels of protein aggregates in vivo can aggravate the toxicity of HD model C. elegans (e.g. AM141), and become a biomarker of aging in this model C. elegans. Inhibiting protein aggregation is an effective method to alleviate protein toxicity and restore protein homeostasis. The phenotypic characteristics of this model C. elegans strain are that the polyQ40::YFP fusion protein is expressed in the body wall muscle cells and discrete fluorescent aggregation points are gradually increased with the growth and development of C. elegans. In this example, 1 mM sea cucumber polypeptide #405 was fed to C. elegans AM141 larvae, and the number of fluorescent aggregation points in the body wall muscle of C. elegans was counted every 24 h to verify whether the polypeptide can inhibit the aggregation of polyQ in vivo and whether it has neuroprotective activity. The specific method is as follows:

[0094] (1) Synchronize C. elegans AM141 to obtain L1 larvae, add S Medium to adjust the worm density to 20-30 worms / 10 μL; then transfer the C. elegans to a 24-well plate, with 600-700 C. elegans per well, and add E. coli NA22 to each well (the final OD in the system is 570 ≈0.5), as food for nematodes in the well plate; the nematodes were divided into a sea cucumber polypeptide HoloPep#405-treated group and a control group, the sea cucumber polypeptide HoloPep#405-treated group was added with sea cucumber polypeptide HoloPep#405 sample to control its final concentration to 1mM; the control group was filled with S Medium to make up the volume to 1000μL, 3 replicate wells were set for each experimental group, and the prepared 24-well plate was placed in a 20℃ constant temperature culture shaker (120r / min) for culture.

[0095] (2) Weigh agarose into a beaker and prepare a 2% agarose pad according to conventional methods for subsequent observation of fluorescent spots.

[0096] (3) Take 100 μL of C. elegans from each treatment group on a 24-well plate and collect them into a 1.5 mL EP tube. Centrifuge at room temperature (2000 r / min, 1 min), discard the supernatant, and then add M9 Buffer to wash 3 times. Leave about 30 μL of worm liquid in each group. After anesthesia with sodium azide, observe. Select a 10×10 or 10×20 field of view to observe C. elegans, fine-tune the focus of the microscope, and count the total number of bright fluorescent aggregation points on the body wall muscles of C. elegans. The time of drug treatment is recorded as day 0. Starting from day 1, count once a day for 4 consecutive days.

[0097] (4) PolyQ aggregation degree = total number of fluorescent spots / total number of C. elegans counted, and compared with C. elegans treated with no sample (control group), the cumulative changes in the number of fluorescent spots on the body wall muscle cells of C. elegans were observed as the C. elegans grew and developed.

[0098] Results Figure 5 As can be seen from the figure, compared with the control group, the number of polyQ aggregation points in the body wall cells of Caenorhabditis elegans AM141 in the group treated with sea cucumber polypeptide HoloPep#405 was reduced, indicating that sea cucumber polypeptide HoloPep#405 has the activity of inhibiting polyQ aggregation in Caenorhabditis elegans, that is, this polypeptide has neuroprotective activity.

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An antioxidant sea cucumber polypeptide targeting mitochondria, characterized in that Its amino acid sequence is: NMQTHPTYLGSR.

2. The method for preparing the mitochondrial-targeted antioxidant sea cucumber polypeptide according to claim 1, characterized in that The following steps are included: (1) Using sea cucumber as raw material, the mitochondrial targeted antioxidant sea cucumber polypeptide according to claim 1 is obtained by enzymatic hydrolysis, separation, extraction and purification, or the mitochondrial targeted antioxidant sea cucumber polypeptide is directly prepared by solid phase synthesis.

3. The method according to claim 2, characterized in that: The enzymolysis is performed by using trypsin and papain in sequence.

4. The method according to claim 2, characterized in that: The specific operation of the separation and extraction is: The sea cucumber protein hydrolysate obtained after enzymatic hydrolysis was cooled, and 95% by volume ethanol was added and allowed to stand; then, vacuum filtration and centrifugation were performed to remove the precipitate, and the supernatant was subjected to vacuum rotary evaporation to remove the ethanol, and freeze-dried to obtain the sea cucumber crude polypeptide dry powder.

5. The method according to claim 2, characterized in that: The purification is at least one of ultrafiltration purification and gel chromatography column purification.

6. The method according to claim 2, characterized in that: The purification also includes using reverse phase high performance liquid chromatography or size exclusion chromatography to further separate the components into single peptides.

7. Use of the mitochondrial-targeted antioxidant sea cucumber polypeptide according to claim 1 in the preparation of antioxidant products.

8. Use of the mitochondrial-targeted antioxidant sea cucumber polypeptide of claim 1 in the preparation of a product with neuroprotective activity.

9. Use of the mitochondrial-targeted antioxidant sea cucumber polypeptide according to claim 1 in the preparation of products for preventing and treating neurodegenerative diseases.

10. An antioxidant product or a product for preventing and treating neurodegenerative diseases, comprising at least one of the antioxidant sea cucumber polypeptide according to claim 1, a protein hydrolysate containing the antioxidant sea cucumber polypeptide according to claim 1, and a hydrolysate containing the antioxidant sea cucumber polypeptide according to claim 1 as an active ingredient.

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