Mitochondria-targeted antioxidant sea cucumber polypeptide and application thereof

Abstract

The application belongs to the field of small molecule polypeptides, and particularly relates to a mitochondria-targeting antioxidant sea cucumber polypeptide and application thereof. The mitochondria-targeting antioxidant sea cucumber polypeptide has an amino acid sequence of NMQTHPTYLGSR, and is a hydrophilic polypeptide. The sea cucumber polypeptide can directly target mitochondria, reduce accumulation of active oxygen in mitochondria, has excellent mitochondria-targeting antioxidant function, and also has neuroprotective activity, and can prevent neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of small molecule polypeptides, and particularly relates to an antioxidant sea cucumber polypeptide targeting mitochondria and application thereof. BACKGROUND

[0002] As the power plant of cells, mitochondria are the main place of biological energy production and are closely related to oxidative stress. Many diseases such as Alzheimer's disease and Huntington's disease are related to oxidation and mitochondrial function. Oxidation is the main cause of mitochondrial dysfunction, and mitochondrial dysfunction and excessive oxidative stress are the main causes of neurodegenerative diseases. Mitochondrial targeting therapy has accuracy and treatment efficiency because it can directly act on mitochondria, and has application prospects in the fields of preventing and treating 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); oxygen consumption and Ca 2+ Increase; up-regulation of mitochondrial fission gene Drp-1 and down-regulation of mitochondrial fusion gene Mfn-2. Maintaining mitochondrial health is a dynamic balance process, which is jointly regulated by mitochondrial fission, fusion and biogenesis genes. When this balance is broken due to aging or external stimulation, and the accumulation of undesirable substances such as ROS in the body reaches a certain level, it will cause different degrees of mitochondrial lesions. Dysfunctional mitochondria show membrane depolarization and fragmentation, accompanied by a large amount of mitochondrial ROS production and Ca 2+ Release into the cytoplasm. In order to prevent the vicious cycle of mitochondrial damage and ROS production, cells protect themselves from damage through an intrinsic quality control mechanism, mitochondrial autophagy. When excessive stimulation causes a large amount of damaged mitochondria or irreversible aging, the clearance efficiency of the self-protection mechanism such as mitochondrial autophagy cannot keep up with the accumulation speed of ROS and other substances in the body, and exogenous auxiliary regulation is needed to maintain the dynamic balance of mitochondria.

[0004] Studies have shown that many polypeptides have antioxidant potential, can effectively reduce the level of ROS and other pro-oxidants, delay or prevent oxidative stress damage, thereby protecting cells and tissues, and have great application potential in the fields of medicine, food, cosmetics, etc. However, although the antioxidant activity of most polypeptides is obtained by in vitro test so far, whether it has antioxidant activity in vivo still needs to be verified, and there are few polypeptides with precise targeting mitochondria, and no polypeptides from natural sources have been reported. For example, a tripeptide sequence (Ala-Val-Leu) prepared from Cucumaria frondosa (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 under oxidative stress and has antioxidant activity, but it does not involve research related to targeting mitochondria. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of most antioxidant polypeptides in the prior art lacking mitochondrial targeting performance, the primary purpose of the present application is to provide a mitochondria-targeting antioxidant sea cucumber polypeptide, which can directly target mitochondria, reduce the accumulation of reactive oxygen species in mitochondria, has excellent mitochondria-targeting antioxidant function, and can improve efficacy and accuracy when used as a drug. At the same time, the polypeptide also has neuroprotective activity.

[0006] Another purpose of the present application is to provide a preparation method of the above-mentioned mitochondria-targeting antioxidant sea cucumber polypeptide.

[0007] Still another purpose of the present application is to provide an application of the above-mentioned mitochondria-targeting antioxidant sea cucumber polypeptide.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] A mitochondria-targeting antioxidant sea cucumber polypeptide HoloPep#405 (dodecapeptide), which has an amino acid sequence of NMQTHPTYLGSR.

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

[0011] The preparation method of the mitochondria-targeting antioxidant sea cucumber polypeptide comprises the following steps:

[0012] (1) sea cucumber as raw material, by enzymolysis, separation and extraction, purification, to obtain the above-mentioned mitochondria-targeting antioxidant sea cucumber polypeptide, or directly by solid-phase synthesis to prepare the above-mentioned mitochondria-targeting antioxidant sea cucumber polypeptide,

[0013] The enzymolysis preferably uses trypsin and papain in sequence for enzymolysis;

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

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

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

[0017] After cooling the sea cucumber protease hydrolysate obtained after enzymolysis, 95% ethanol by volume is added, and then the solution is adjusted to pH 7.5-8.0, trypsin is added for enzymolysis; then the solution is adjusted to pH 6.5-7.0, papain is added for enzymolysis; after enzymolysis, inactivation is performed to obtain a sea cucumber protease hydrolysate;

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

[0019] The ultrafiltration purification preferably uses a filter membrane with a molecular weight cut-off of 3 kDa for separation, and the components with a molecular weight less than 3 kDa are collected;

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

[0021] After desalination of the components of the ultrafiltration purification, Sephadex G-25 dextran gel chromatography column is used for further purification;

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

[0023] The mitochondria-targeting antioxidant sea cucumber polypeptide in the preparation of an antioxidant product;

[0024] The mitochondria-targeting antioxidant sea cucumber polypeptide in the preparation of a mitochondria-targeting antioxidant product;

[0025] The mitochondria-targeting antioxidant sea cucumber polypeptide in the preparation of a product with neuroprotective activity;

[0026] The application of the antioxidant sea cucumber polypeptide targeting mitochondria in the preparation of a product for preventing and treating neurodegenerative diseases;

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

[0028] A product for preventing and treating neurodegenerative diseases, which comprises at least one of the antioxidant sea cucumber polypeptide, a proteinase hydrolysate containing the antioxidant sea cucumber polypeptide and a hydrolysate containing the antioxidant sea cucumber polypeptide as an active ingredient;

[0029] Principles of the present application:

[0030] The present application extracts and separates a pure natural sea cucumber polypeptide from sea cucumber through enzymatic separation, ultrafiltration purification and LC-MS / MS identification, wherein the sea cucumber polypeptide has an amino acid sequence of NMQTHPTYLGSR, a molecular formula of C 59 H 93 N 19 O 19 S, an average relative molecular mass of 1404.54 g / mol, a theoretical isoelectric point of pH=9.84 and is a hydrophilic polypeptide.

[0031] The present application further uses the model organism Caenorhabditis elegans as a model to find that the sea cucumber polypeptide has the abilities of targeting mitochondria and antioxidation, and also has the neuroprotective activity and the effect of preventing neurodegenerative diseases, and the specific effects are as follows:

[0032] (1) The intestinal model nematode SJ4143 is used to perform a methyl rhodamine ethyl ester (TMRE) fluorescence staining experiment: the intestinal cell mitochondria of the nematode SJ4143 contain green fluorescent GFP markers, and after TMRE staining, green fluorescence and red fluorescence can be excited under the blue light and green light channels of a fluorescence microscope respectively; the red fluorescence to green fluorescence ratio is normalized (i.e., TMRE / GFP) to obtain the mitochondrial health level (HMI). Since the TMRE staining solution can only stain healthy mitochondria, the ratio can represent the health level of mitochondria. The present application proves through experiments that the HMI level of the nematode is increased after the sea cucumber polypeptide treatment, which indicates that the polypeptide can improve the function of mitochondria and has the ability of targeting mitochondria.

[0033] (2) Mitochondrial DNA copy number experiment: the present application further verifies that the sea cucumber polypeptide can improve the health level of mitochondria through a mitochondrial DNA copy number experiment, thereby verifying the accuracy of the conclusion of whether the sea cucumber polypeptide has the targeting property in (1) above and proving that the sea cucumber polypeptide indeed has the ability of targeting mitochondria.

[0034] (3) Anti-oxidation activity test: the polypeptide has the mitochondrial-targeted anti-oxidation function, which is proved by the test using DCFH / DA (2', 7'-dichlorofluorescin diacetate) probe to detect the level of reactive oxygen species (ROS) in mitochondria.

[0035] (4) polyQ (polyglutamine) aggregation inhibition test: the test proves that the polypeptide has the neuroprotective activity.

[0036] In summary, the sea cucumber polypeptide has the mitochondrial-targeted anti-oxidation function, which is proved by the above tests, that is, the polypeptide can be directly administered to mitochondria for treatment, thereby recovering the function of mitochondria or delaying the oxidative damage of mitochondria. The advantages of the sea cucumber polypeptide include high pharmacokinetics, easy absorption by mitochondria, and specific accumulation in mitochondria, thereby avoiding the side effects caused by the non-specific accumulation of drugs in other parts of cells at high concentrations due to the poor specificity of non-targeting. The sea cucumber polypeptide also has the neuroprotective effect, and the effect is further proved by the mitochondrial-targeted anti-oxidation performance, so the effect of preventing or delaying neurodegenerative diseases is better than that of the prior art.

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

[0038] (1) The polypeptide obtained from natural sea cucumber has the amino acid sequence of NMQTHPTYLGSR, and can be separated and purified from sea cucumber or obtained by artificial synthesis.

[0039] (2) The sea cucumber polypeptide has small molecular weight and excellent mitochondrial targeting function, can be directly targeted to mitochondria, and can reduce the accumulation of excess active oxygen in nematodes, so it has excellent anti-oxidation ability and anti-oxidation activity based on the mitochondrial targeting ability, which increases the accuracy and treatment efficiency of the anti-oxidation effect.

[0040] (3) The sea cucumber polypeptide significantly inhibits the aggregation of polyglutamine in the model organism Caenorhabditis elegans, and can prevent neurodegenerative diseases. Because the polypeptide has the mitochondrial targeting ability, the polypeptide has precise effects in the treatment of neurodegenerative diseases in the field of small molecule polypeptide technology and mitochondrial targeting treatment.

[0041] (4) The sea cucumber polypeptide can be further used for the development of food, medicine and health products, and has wide application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a superimposed image of GFP fluorescence and TMRE fluorescence.

[0043] Figure 2 is a result analysis diagram of the influence of MitoQ and sea cucumber polypeptide HoloPep#405 on HMI, and the horizontal coordinate is the number of independent repeated experiments.

[0044] Figure 3 is a result analysis diagram of the influence of sea cucumber polypeptide HoloPep#405 on the copy number of mitochondrial genome.

[0045] Figure 4 is a result analysis diagram of the ROS level change trend within 2 h after the Caenorhabditis elegans is treated under different conditions.

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

[0047] The application will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0048] The concentrated NA22 bacterial solution in the embodiments is the food of Caenorhabditis elegans, and the preparation method is as follows: after the activated Escherichia coli NA22 is inoculated into the LB liquid medium and is oscillation cultured, the bacterial body is collected by centrifugation, and the concentrated NA22 bacterial solution is obtained.

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

[0050] Example 1: Isolation and identification of sea cucumber polypeptide

[0051] 1. Enzymatic separation

[0052] (1) Fresh sea cucumber was washed and chopped, and then homogenized. After the homogenate was freeze-dried, 5 g of the freeze-dried powder of sea cucumber was weighed, deionized water was added at a solid-liquid ratio of 1:4, and the mixture was stirred uniformly with a glass rod, and then preheated in a 45℃ constant temperature water bath. Then, the pH of the solution was adjusted to 7.5 with NaOH, 1 mL of trypsin (15000 U / mL) was added, and the mixture was reacted at 45℃ for 4 h. Then, the pH of the solution was adjusted to 6.5 with HCl, 1 mL of papain (15000 U / mL) was added, and the mixture was reacted at 65℃ for 4 h. Finally, the mixture was boiled to inactivate, and sea cucumber protease hydrolysate was obtained.

[0053] (2) After the sea cucumber protease hydrolysate prepared in step (1) is naturally cooled to room temperature, 170 mL of 95% ethanol by volume is slowly added under stirring with a glass rod, and the mixture is left to stand at room temperature overnight, followed by reduced-pressure filtration and centrifugation (4°C, 8000 rpm, 5 min) to remove the precipitate. The supernatant is subjected to removal of ethanol by reduced-pressure rotary evaporation, and then freeze-drying to obtain sea cucumber crude polypeptide dry powder.

[0054] 2. Ultrafiltration and gel chromatography column purification

[0055] (1) The sea cucumber crude polypeptide dry powder prepared in step 1 is formulated into a 20 mg / mL solution, which is subjected to ultrafiltration using an Amicon Ultra-2 centrifugal filter equipped with Millipore Ultrafiltration Membrane Ultracel-3, Ultracel-5, and Ultracel-10. Ultrafiltration is used to obtain sea cucumber protease hydrolysate cutoff components with MW > 10 kDa, 5 kDa < MW < 10 kDa, 3 kDa < MW < 5 kDa, and MW < 3 kDa. The component with a molecular weight less than 3 kDa is collected, then filtered through a 0.45 μm filter to remove impurities, and a desalting column is used to remove salts in the polypeptide sample.

[0056] (2) The polypeptide component after desalting in step (1) is further separated using a Sephadex G-25 dextran gel chromatography column. Deionized water is used to elute at a flow rate of 1 mL / min, and a 785 UV / VIS detector is used to monitor at 280 nm while collecting fractions of each absorption peak. A total of 5 polypeptide components are obtained, which are freeze-dried to obtain corresponding sea cucumber polypeptide freeze-dried powder.

[0057] 3. LC-MS / MS identification

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

[0059] 4. Database comparison screening

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

[0061] Example 2 Fmoc solid phase method for synthesizing sea cucumber polypeptide

[0062] The sea cucumber polypeptide HoloPep #405 was synthesized by Shanghai Taup Biological Technology Co., Ltd. The specific method is as follows:

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

[0064] (2) Remove the DCM solvent by sand core filtration, add 3 times the molar excess of Fmoc- (the first amino acid at the C terminal) -OH, 10 times the molar excess of N,N-diisopropyl ethylamine and a small amount of dimethyl fumarate for dissolution, shake for 1 h to make the first amino acid bind to the resin, and then repeatedly use dimethyl fumarate and DCM alternately for 6 times. Use a volume fraction of 20% piperidine dimethyl fumarate solution to clean for 5 min to remove the protection of Fmoc-, and then repeat the operation for 15 min.

[0065] (3) After the solution is drained, take out a dozen resin particles, wash them with ethanol for 3 times, and then add each one drop of ninhydrin, potassium cyanide and phenol solution in turn, heat at high temperature until a positive reaction appears, showing a deep blue color.

[0066] (4) Add dimethyl fumarate, methanol, dimethyl fumarate to the reaction tube in proportion in turn, clean 2 times, then add 3 times the molar excess of Fmoc- (the second amino acid at the C terminal) -OH, 3 times the molar excess of O-benzotriazol-tetramethyluronium hexafluorophosphate and a small amount of dimethyl fumarate for dissolution, and then immediately add 10 times the molar excess of N,N-diisopropyl ethylamine to fully react for 40 min for amino acid condensation reaction.

[0067] (5) Repeat the above deprotection, ninhydrin detection and condensation reaction operations in turn until all the target amino acids are sequentially connected to the resin from right to left and the Fmoc-protection is removed. Use ninhydrin for detection until the result is negative, indicating that the reaction is complete. Finally, wash the resin with methanol solution 3 times, and evaporate the liquid at room temperature.

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

[0069] Example 3 Sea cucumber polypeptide HoloPep #405 targeting mitochondria ability

[0070] In this embodiment, the biological Caenorhabditis elegans SJ4143 is used as the research object, the fluorescence dye methyl rhodamine ethyl ester (TMRE) which can characterize the integrity of mitochondria is used to quantify TMRE-GFP, that is, the Healthy Mitochondria Indicator (HMI), and the sea cucumber polypeptide HoloPep #405 targeting nematode mitochondria function is evaluated; and the mtDNA copy number is further verified from the molecular biology point of view to verify the sea cucumber polypeptide HoloPep #405 targeting nematode mitochondria ability.

[0071] I. HMI method

[0072] (1) After the oviposition period of Caenorhabditis elegans SJ4143 is synchronized, add it to a conical flask for liquid culture to L1 stage, then divide the nematodes into 3 groups, namely blank control group, MitoQ administration group and sea cucumber polypeptide #HoloPep405 administration group, one new conical flask for each group, add concentrated NA22 liquid as nematode food in the L1 worm liquid of each group to control the final OD 570about 0.5, the experimental groups were dosed at the same time when feeding NA22 in L1 stage, wherein, the MitoQ dosing group was added with MitoQ mother liquor (mother liquor concentration was 5 mM) to make the final concentration 5 μM; the sea cucumber polypeptide #HoloPep405 dosing group was added with sea cucumber polypeptide mother liquor (mother liquor concentration was 10 mM) to make the final concentration 2 mM, and the blank control group was added with the same amount of S Medium, finally the above-mentioned worm-containing systems were placed in a shaker at 20℃, 120 rpm to continue culturing for 42 h to reach L4 stage of the larvae.

[0073] (2) When the nematodes in step (1) were cultured for 42 h to reach L4 stage, the three groups of nematodes were collected, washed and transferred to 48-well plates, and concentrated NA22 bacterial solution (as nematode food), 5-FUdR (to inhibit nematode egg laying) and AMP solution (to inhibit the growth of other bacteria) were added to each well, wherein the final volume of each well system was 500 μL, the number of nematodes in each well system was 100-150, i.e. the worm concentration was 200-300 pieces / mL, the final OD 570 of NA22 in each well system was about 0.5, the final concentration of 5-FUdR was 75 μg / mL, and the final concentration of AMP was 100 μg / mL.

[0074] (3) After the nematodes in step (2) were cultured in the 48-well plate for 3 days, TMRE staining was performed, the specific method was as follows: the nematodes were washed with M9 buffer for 3-4 times, 5 μM TMRE was diluted to 1 μM with buffer, and was added to a 24-well plate in advance, then the washed nematodes were added, the worm density was adjusted to about 20-30 pieces / 10 μL, and the staining was performed in a shaker at 120 rpm and 20℃ for 6 h; after 6 h, the stained nematodes were collected, washed with M9 buffer for 3-4 times; after staining, the nematodes were subjected to decolorization in M9 buffer according to the conventional method, and the decolorized nematodes were washed with M9 buffer for 3-4 times again.

[0075] (4) The washed nematodes were fixed on a 2% agarose soft pad, and photographed using a fluorescence microscope with different fluorescence channels to obtain green (GFP) and red (TMRE) fluorescence pictures; the obtained fluorescence pictures were processed using image J software to obtain the average red TMRE fluorescence intensity (Mean-TMRE) and the average green GFP fluorescence intensity (Mean-GFP), respectively, then the HMI value (HMI=Mean-TMRE / Mean-GFP) was calculated, which represented the ratio of healthy mitochondria to total mitochondria content in the nematodes, and further evaluated the targeting of sea cucumber polypeptide HoloPep#405 to the mitochondrial treatment effect.

[0076] Figure 1Figure 2 is a microscope display diagram of SJ4143 C. elegans after being dyed with fluorescent dye methyl rhodamine ethyl ester (TMRE), showing green fluorescence and red fluorescence under blue light and green light excitation, respectively.

[0077] MitoQ is a recognized positive compound for mitochondrial quality, and in this embodiment, MitoQ is used as a positive control to verify whether HMI can intuitively and correctly reflect the improvement of MitoQ on the mitochondria of C. elegans. The mitochondria in the control group and the drug group are dyed red by TMRE under the green light channel, and after subsequent data processing, it is found that the HMI value, i.e., the TMRE / GFP level, of the drug group is higher than that of the control group, as shown in Figure 2 The results show that the HMI value of the MitoQ drug group is significantly higher than that of the control group, indicating that MitoQ has an improvement effect on mitochondrial quality, or in other words, the HMI value can correctly reflect the improvement of MitoQ on the level of mitochondrial quality. At the same time, the HMI value of the sea cucumber polypeptide #405 administered to the nematodes at the same period is comparable to that of MitoQ, which further indicates the mitochondrial targeting ability of sea cucumber polypeptide #405.

[0078] II. mtDNA copy number

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

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

[0081] (3) After the nematodes in step (2) are cultured in the 48-well plate for 3 days, 50-100 μL of nematode liquid is taken from the blank control group, the 5 μM MitoQ drug group, and the 2 mM HoloPep #405 drug group, with a worm density of 40-60 worms / 10 μL. Then, according to the DNA extraction kit (full type gold) instructions, the nematode DNA is extracted, the eluted DNA is temporarily stored on ice, and after the concentration is determined, it is stored in a -20℃ environment.

[0082] (4) The DNA obtained in step (3) is used as a template for real-time fluorescent quantitative PCR amplification to determine the relative DNA content. The data processing method is performed according to 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 by the ratio of mtDNA to nDNA, and the mtDNA / nDNA ratio of the blank control group was normalized to 1. The mtDNA / nDNA ratios of the MitoQ administration group and the HoloPep #405 administration group were compared with those of the blank control group.

[0084] The DNA extraction kit (full type gold) was used to extract nematode DNA by lysis method. The expression levels of mtDNA and nDNA were determined, and the relative mitochondrial genome copy number was expressed by the ratio of mtDNA to nDNA. As shown in Table 2, compared with the blank control group, the mtDNA copy number of the MitoQ administration group increased by about 35%, showing an improvement in mitochondrial quality. At the same time, compared with the blank control group, the sea cucumber polypeptide HoloPep #405 increased by about 32%, showing targeted mitochondrial function and superior mitochondrial dysfunction treatment ability. Figure 3

[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 above solid-phase synthesis method has antioxidant activity, the model organism Caenorhabditis elegans SJ4143 was used to detect its antioxidant activity, and the specific method was as follows:

[0087] (1) Drug administration: The nematodes were first synchronized and cultured to the L4 stage, and then washed 3-4 times with buffer. The following steps were used to add drugs to the 24-well plate: 5.0 mg / mL 5-FUdR and 5.0 mg / mL AMP were added to each well of the 24-well plate in advance to inhibit nematode egg production and inhibit the growth of miscellaneous bacteria. The test was divided into four groups: a blank control group, a modeling group, a HoloPep #405 group, and a modeling + HoloPep #405 group. The HoloPep #405 group and the modeling + HoloPep #405 group were added with a certain volume of sea cucumber polypeptide HoloPep #405, and the sea cucumber polypeptide administration concentration was the same as that in Example 3 above, i.e. 2 mM. The blank control group and the modeling group were supplemented with S Medium to make the liquid volume in the system equal, and the final solution volume in each well was equal. Finally, 200 μL of nematode liquid (containing 20-30 nematodes per 10 μL) synchronized and cultured to the L4 stage was added to each well, and the final volume in each well reached 1000 μL, so that the concentration of nematodes in the 24-well plate was 400-600 worms / mL. Among them, the final OD 570 ​The final concentration of 5-FUdR was about 0.5, the final concentration of 5-FUdR was 75 μg / mL, and the final concentration of AMP was 100 μg / mL; 4 replicates were set for each treatment.

[0088] (2) Modeling: After 24 h of continuous culture at 20°C at 120 rpm, 10 μL of 200 mM paraquat (PQ) solution (final concentration of 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) Lysis of nematode protein extraction: After 48 h of continuous culture of each group of nematodes in a 20°C shaker, the 24-well plate was removed, and each group of C. elegans was collected in a 1.5 mL centrifuge tube. After washing the nematodes thoroughly, they were placed on ice for standby. 200 μL of PBST lysis solution was added to each centrifuge tube to resuspend the C. elegans, and a mini handheld homogenizer was used to break the nematodes. After breaking, the nematodes were quickly centrifuged in a refrigerated centrifuge, with the parameters set at 12000 rpm, 4°C, and 2 min. The supernatant was collected and placed on ice for standby.

[0090] (4) Determination of active oxygen level: The sample obtained in step (3) (protein content of 1 mg / mL) was used to determine the ROS level in the nematodes using the Biyun Tian kit (i.e., DCFH / DA probe method), with the following steps: 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 emission wavelength of 538 nm and excitation wavelength of 480 nm, and measure the fluorescence value. Start from 0 min, measure every 20 min for a total of 7 times for 120 min.

[0091] The results are shown in Figure 4 The smaller the DCF fluorescence intensity, the lower the ROS level in the nematodes, indicating that the substance has an ideal antioxidant effect. As can be seen, 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 C. elegans, which proves that the modeling was successful, i.e., paraquat can destroy the stability of nematode mitochondria. The DCF fluorescence intensity measured in the nematodes treated with only sea cucumber polypeptide was lower than that in the control group. The DCF fluorescence intensity of the paraquat modeling + HoloPep#405 group was between that of the control group and the modeling group, which proved that the sea cucumber polypeptide HoloPep#405 could prevent and delay the mitochondrial oxidative damage caused by paraquat to the nematodes, confirming the targeted mitochondrial antioxidant capacity 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 aggregation. The aggregation of polyQ protein in the cells of patients with Huntington's disease (HD) and other neurodegenerative diseases is a key pathological marker of neurodegenerative diseases. High levels of protein aggregates in vivo can exacerbate the toxicity of HD model C. elegans (e.g., AM141) and become a biomarker of aging in the model C. elegans. Inhibiting protein aggregation is an effective method to alleviate protein toxicity and restore protein homeostasis. The phenotype of this model C. elegans strain is that the expression of polyQ40::YFP fusion protein in the body wall muscle cells of C. elegans shows discrete fluorescent aggregation points that gradually increase with the growth and development of C. elegans. In this embodiment, C. elegans AM141 larvae were fed with 1 mM sea cucumber polypeptide #405, 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 in vivo aggregation of polyQ and whether it has neuroprotective activity. The specific method is as follows:

[0094] (1) Synchronize C. elegans AM141 to obtain L1 stage 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, 600-700 C. elegans per well, and at the same time add E. coli NA22 (final OD 570 ≈0.5) in the system as food for the C. elegans in the plate; divide the C. elegans into sea cucumber polypeptide HoloPep#405 administration group and control group, add sea cucumber polypeptide HoloPep#405 sample to the sea cucumber polypeptide HoloPep#405 administration group to control the final concentration at 1 mM; the control group is supplemented with S Medium to 1000 μL, 3 replicates are set for each experimental group, and the prepared 24-well plate is placed in a 20°C constant temperature incubator shaker (120 r / min) for culture.

[0095] (2) Weigh agarose in a beaker and prepare a 2% agarose soft pad according to the conventional method for subsequent observation of fluorescent points.

[0096] (3) Take C. elegans from the 24-well plate, collect 100 μL from each treatment group at a time into a 1.5 mL EP tube, centrifuge at room temperature (2000 r / min, 1 min), discard the supernatant, and wash with M9 Buffer for 3 times, leaving about 30 μL of worm liquid for each group, and observing after sodium azide anesthesia. Select 10x10 or 10x20 fields of view to observe C. elegans, adjust the focal length of the microscope, and count the total number of bright fluorescent aggregation points on the body wall muscle of C. elegans. The time of administration treatment is recorded as day 0, and the counting is performed once a day from day 1, 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 without sample treatment (control group), to observe the accumulation change of the number of fluorescent spots on the body wall muscle cells of C. elegans with growth and development.

[0098] The results are shown in 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 C. elegans AM141 in the sea cucumber polypeptide HoloPep#405 administration group is reduced, indicating that the sea cucumber polypeptide HoloPep#405 has the activity of inhibiting the aggregation of polyQ in C. elegans, that is, the polypeptide has the neuroprotective activity.

[0099] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

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-targeting antioxidant sea cucumber polypeptide according to claim 1, characterized in that... It includes the following steps: (1) Using sea cucumber as raw material, the antioxidant sea cucumber polypeptide targeting mitochondria as described in claim 1 is obtained by enzymatic hydrolysis, separation and extraction and purification, or the above-mentioned antioxidant sea cucumber polypeptide targeting mitochondria is prepared directly by solid phase synthesis.

3. The method according to claim 2, characterized in that: The enzymatic hydrolysis was carried out sequentially using trypsin and papain.

4. The method according to claim 2, characterized in that: The specific operations for separation and extraction are as follows: After cooling the sea cucumber protein hydrolysate obtained by enzymatic hydrolysis, 95% ethanol was added and allowed to stand. Then, vacuum filtration and centrifugation were performed to remove the precipitate. The supernatant was then subjected to vacuum rotary evaporation to remove the ethanol and freeze-dried to obtain crude sea cucumber polypeptide 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 process also includes further separating the components into individual peptides using reversed-phase high-performance liquid chromatography or size exclusion chromatography.

7. The use of the mitochondrial-targeting antioxidant sea cucumber polypeptide of claim 1 in the preparation of antioxidant drugs.

8. The use of the mitochondrial-targeting antioxidant sea cucumber polypeptide of claim 1 in the preparation of a drug for the prevention and treatment of Huntington's disease.

9. An antioxidant drug or a drug for preventing and treating Huntington's disease, comprising the antioxidant sea cucumber polypeptide of claim 1.

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