Application of oyster peptide in improvement of learning and memory ability and anti-aging

Abstract

The invention belongs to the technical field of biology, discloses application of oyster peptide in improvement of learning and memory ability and anti-aging, and particularly discloses oyster peptide or pharmaceutically acceptable salt thereof. The invention provides an oyster peptide (namely OP-4), which mainly comprises an amino acid sequence of a peptide fragment as shown in SX1X2NYGTMTSM, and in the amino acid sequence, X1 is S or H, and X2 is Y or L. The OP-4 can improve oxidative stress injury, reduce toxicity of senescence-related marker protein, improve learning and memory ability and delay senescence, and the mechanism of the OP-4 is closely related to an IIS signal channel and a P38MAPK signal channel.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of oyster peptides in improving learning and memory abilities and resisting aging. Background Art

[0002] In recent years, domestic and foreign scholars have conducted in-depth research on oysters, and the extracts obtained by different means have a wide range of biological activities, such as lowering blood sugar and blood lipids, anti-aging, anti-oxidation, anti-fatigue, anti-tumor, immune regulation, etc., and have broad prospects in the field of health foods and functional foods. Oysters have multiple biological activities, and their components are mainly water extracts, polypeptide components, enzymatic hydrolysates, etc., but the current research on them is not in-depth and systematic, and there are few reports on their material basis and mechanism of action.

[0003] The decline of physiological functions caused by aging affects people's lives, making the aging of the population more and more serious. Geriatric diseases caused by aging also bring great challenges to families and society. How to avoid or reduce the disadvantages brought by the aging of the population has become a hot issue of concern in today's society. The development and use of new natural anti-aging products can promote the healthy lifespan of the elderly and delay aging, which is of great significance to human society. It is well known that there are rich potential resources of natural anti-aging agents in animals, plants and microorganisms, which can be developed and utilized into functionally active substances.

[0004] In recent years, oysters have received extensive attention due to their multiple biological activities. However, as a high-protein raw material, oysters have a relatively complex composition. Different processing methods (such as water extracts, peptide components, enzymatic hydrolysates, etc.) have different material components, and their activities and effects vary greatly. Studies have reported that the yield group of oyster hydrolysates can delay the lifespan of Caenorhabditis elegans, but the material basis of related research is still unclear, the anti-aging detection indicators are not comprehensive enough, the mechanism of action is not clear, and the changes in the behavior and memory of nematodes have not been studied. Therefore, it is necessary to screen the anti-aging activity of oyster extracts to obtain the best anti-aging activity components. In order to further clarify its material basis, the active ingredients are separated and purified, and the activity is tracked, and the anti-aging effects and molecular mechanisms of the separated components are discussed.

[0005] Therefore, this application is filed. Summary of the invention

[0006] The first aspect of the present invention aims to provide an oyster peptide or a pharmaceutically acceptable salt thereof.

[0007] The second aspect of the present invention aims to provide a fusion protein.

[0008] The third aspect of the present invention aims to provide a biological material related to the oyster peptide of the first aspect of the present invention or the fusion protein of the second aspect of the present invention.

[0009] The fourth aspect of the present invention aims to provide the use of the oyster peptide of the first aspect of the present invention, the fusion protein of the second aspect of the present invention or the biomaterial of the third aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide a product.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides an oyster peptide or a pharmaceutically acceptable salt thereof, wherein the oyster peptide comprises:

[0013] a1) the amino acid sequence is represented by SX1X2NYGTMTSM, wherein X1 is S or H, and X2 is Y or L; and / or

[0014] a2) An amino acid sequence shown in a1) that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0016] In some embodiments of the present invention, the metal salt includes an alkali metal salt or an alkaline earth metal salt.

[0017] In some embodiments of the present invention, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0018] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0019] In some embodiments of the present invention, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0020] In some embodiments of the present invention, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0021] In some embodiments of the present invention, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0022] In some embodiments of the present invention, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine, and ornithine.

[0023] In some embodiments of the present invention, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0024] The second aspect of the present invention provides a fusion protein, wherein the fusion protein comprises the oyster peptide of the first aspect of the present invention.

[0025] In some embodiments of the present invention, the fusion protein further comprises other polypeptides.

[0026] The third aspect of the present invention provides a biomaterial related to the oyster peptide of the first aspect of the present invention or the fusion protein of the second aspect of the present invention, wherein the biomaterial comprises at least one of b1) to b12):

[0027] b1) a nucleic acid molecule encoding the oyster peptide of the first aspect of the present invention or the fusion protein of the second aspect of the present invention;

[0028] b2) an expression cassette containing the nucleic acid molecule described in b1);

[0029] b3) a vector containing the nucleic acid molecule described in b1);

[0030] b4) a vector containing the expression cassette described in b2);

[0031] b5) a recombinant cell containing the nucleic acid molecule described in b1);

[0032] b6) a recombinant cell containing the expression cassette described in b2);

[0033] b7) a recombinant cell containing the vector described in b3);

[0034] b8) a recombinant cell containing the vector described in b4);

[0035] b9) a recombinant microorganism containing the nucleic acid molecule described in b1);

[0036] b10) a recombinant microorganism containing the expression cassette described in b2);

[0037] b11) a recombinant microorganism containing the vector described in b3);

[0038] b12) A recombinant microorganism containing the vector described in b4)

[0039] In some embodiments of the present invention, the vector is an expression vector.

[0040] In some embodiments of the present invention, the expression vector is selected from a plasmid, a phage vector and a viral vector.

[0041] In some embodiments of the present invention, the viral vector can be selected from an adenoviral vector, a parvoviral vector (e.g., an adeno-associated virus (AAV) vector), a retroviral vector (e.g., a lentiviral vector), a cytomegalovirus vector, a Sendai virus vector, an influenza virus vector, and a measles virus vector.

[0042] In some embodiments of the invention, the recombinant cell does not contain propagation material.

[0043] The fourth aspect of the present invention provides the use of the oyster peptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, or the biomaterial according to the third aspect of the present invention in at least one of c1) to c21):

[0044] c1) preparing products for improving learning and memory abilities;

[0045] c2) inhibit / mitigate the toxic effects induced by Aβ protein deposition;

[0046] c3) preparing products that inhibit / mitigate the toxic effects induced by Aβ protein deposition;

[0047] c4) inhibiting the aggregation of polyQ proteins;

[0048] c5) preparing a product for inhibiting the aggregation of polyQ proteins;

[0049] c6) preparing products that extend the lifespan of nematodes;

[0050] c7) preparing products that enhance the motility of nematodes;

[0051] c8) Improve the ability to resist stress response;

[0052] c9) preparing products for improving the ability to resist stress;

[0053] c10) Antioxidant;

[0054] c11) preparing antioxidant products;

[0055] c12) Anti-aging;

[0056] c13) preparing anti-aging products;

[0057] c14) inhibit the expression of daf-2 gene / age-1 gene;

[0058] c15) preparing a product for inhibiting the expression of daf-2 gene / age-1 gene;

[0059] c16) promotes the expression of skn-1 gene / gst-4 gene / clk-1 gene / sek-1 gene;

[0060] c17) preparing a product for promoting the expression of skn-1 gene / gst-4 gene / clk-1 gene / sek-1 gene;

[0061] c18) preparing products that modulate the insulin / IGF-1 signaling pathway;

[0062] c19) preparing products of P38MAPK signaling pathway;

[0063] c20) promotes the translocation of DAF-16 / SKN-1 from the cytoplasm to the nucleus;

[0064] c21) preparing a product that promotes the translocation of DAF-16 / SKN-1 from the cytoplasm to the nucleus.

[0065] In some embodiments of the present invention, the antioxidant activity in c10) and c11) includes achieving the purpose of antioxidant activity by at least one of scavenging free radicals, increasing SOD activity, increasing CAT activity, and reducing MDA content.

[0066] In some embodiments of the present invention, the stress response in c8) and c9) includes oxidative stress damage and heat stress damage.

[0067] In some embodiments of the present invention, the product includes at least one of a reagent, a kit, a cosmetic, a food, and a medicine.

[0068] The fifth aspect of the present invention provides a product, comprising the oyster peptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, or the biomaterial according to the third aspect of the present invention.

[0069] In some embodiments of the present invention, the product further comprises other pharmaceutically acceptable excipients or other second active ingredients.

[0070] In some embodiments of the present invention, the excipients include the pharmaceutically acceptable excipients including at least one of fillers, disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0071] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin or polyvinyl pyrrolidone; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium hydroxymethyl cellulose; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micro-powder silica gel, talc or magnesium stearate; the suspending agent is selected from micro-powder silica gel, beeswax, cellulose, solid polyethylene glycol : The wetting agent is selected from glycerol, Tween-80, hydrogenated castor oil or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blending oil, etc.; the surfactant is selected from sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium; the preservative is selected from at least one of methyl paraben or propyl paraben. The emulsifier includes PEG-100 stearate, glyceryl stearate, PEG-40 stearate, polysorbate-60, methyl glucose sesquistearate, polysorbate-20, C20-22 alcohol phosphate, C12-20 alkyl glucoside, arachidyl glucoside, polyglyceryl-3 methyl glucose distearate, lecithin, hydrogenated lecithin, ceteareth-12, steareth-2, steareth-21, PEG-20 methyl glucose sesquistearate, sorbitan sesquioleate, lauryl PEG / PPG-18 / At least one of 18 polymethicone, lauryl PEG-9 polydimethylsiloxyethyl polydimethicone, PEG-10 polydimethicone, polydimethicone PEG-10 / 15 crosspolymer, cetearyl olivate, sorbitan olivate, PEG-30 dipolyhydroxystearate, cetyl PEG / PPG-10 / 1 polymethicone, polyglyceryl-2 dipolyhydroxystearate, polyglyceryl-3 diisostearate, cetearyl glucoside, sodium stearoyl glutamate, potassium cetyl phosphate, and Span-60.

[0072] The beneficial effects of the present invention are:

[0073] The present invention provides an oyster peptide (i.e., OP-4), whose main peptide amino acid sequence is shown as SX1X2NYGTMTSM, wherein X1 is S or H, and X2 is Y or L. OP-4 can improve oxidative stress damage, reduce the toxicity of aging-related marker proteins, improve learning and memory abilities, and delay aging, and its mechanism is closely related to the IIS signaling pathway and the P38MAPK signaling pathway.

[0074] Specifically, OP-4 can significantly improve learning and memory abilities, improve the motility of nematodes, and has no effect on swallowing frequency. It can prolong the average paralysis time induced by Aβ protein in nematodes CL4176, increase the chemotaxis index of nematodes CL2355, and inhibit the aggregation of polyQ proteins in transgenic nematodes AM141. OP-4 can enhance H2O2-induced oxidative stress damage in nematodes, increase the levels of SOD and CAT in nematodes, and reduce the level of MDA. OP-4 can significantly promote the transfer of DAF-16 and SKN-1 transcription factors from the cytoplasm to the nucleus, thereby activating downstream gene expression and increasing the protein expression of SOD-3. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is the chromatographic separation result of OP.

[0076] Figure 2 This is the primary mass spectrum of OP-4.

[0077] Figure 3 This is the secondary mass spectrum of OP-4.

[0078] Figure 4 This is the SSYNYGTMTSM ion fragment analysis diagram.

[0079] Figure 5 This is the SHLNYGTMTSM ion fragment analysis diagram.

[0080] Figure 6 This is the effect of OP-4 on the paralysis rate of CL4176 nematodes.

[0081] Figure 7 Schematic diagram of the chemotactic behavior of OP-4 towards CL2355 nematodes.

[0082] Figure 8 The effect of OP-4 on the chemotactic behavior of CL2355 nematodes.

[0083] Fig. 9 These are fluorescence images of OP-4 on polyQ aggregation in AM141 nematodes; A is the Control group; B is the 50 μg / mL group; C is the 100 μg / mL group; and D is the 200 μg / mL group. The fluorescence intensity magnification is 10×10.

[0084] Fig.10 The effect of OP-4 on polyQ aggregation in AM141 nematodes.

[0085] Fig.11 The effect of OP-4 on the life span of C.elegans.

[0086] Fig.12The effect of OP-4 on the motility of C.elegans.

[0087] Fig.13 The effect of OP-4 on the swallowing frequency of C.elegans.

[0088] Fig.14 To isolate and purify the components and their effects on the survival rate of C.elegans under heat stress.

[0089] Fig.15 To separate and purify the components and their influence on DPPH free radical scavenging ability.

[0090] Fig.16 The effect of OP-4 on the ability of C.elegans to resist H2O2 oxidative damage.

[0091] Fig.17 The effect of OP-4 on the antioxidant enzyme activities of C.elegans; where A is SOD, B is CAT, and C is MDA.

[0092] Fig.18 This is the effect of OP-4 on the mRNA expression level of related aging genes.

[0093] Fig.19 Shown are the effects of OP-4 on related mutant nematodes.

[0094] Fig. 20 The protein expression of DAF-16 by OP-4; A is cytosolic; B is intermediate; C is nuclear.

[0095] Fig.21 The effect of OP-4 on the nuclear localization of DAF-16::GFP.

[0096] Fig. 22 The expression of SKN-1 protein by OP-4; A is cytoplasmic; B is intermediate; C is nuclear.

[0097] Fig.23 The effect of OP-4 on the nuclear localization of SKN-1::GFP.

[0098] Fig.24 This is the effect of OP-4 on the expression level of SOD-3::GFP protein.

[0099] Fig.25 This is the effect of OP-4 on the expression level of HSP-16.2::GFP protein. DETAILED DESCRIPTION

[0100] The present invention is further described in detail below through specific examples.

[0101] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0102] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0103] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0104] The mutant strains CL4176, CL2355, CL2122, AM141, CB1370 [daf-2 (e1370)], ld1s7 [skn-1b / c:: GFP + rol-6 (su1006)], CF1553 muls [(pAD76) sod-3p:: GFP + rol-6 (su1006)], TJ356 zls356 [daf-16p:: daf-16a / b:: GFP + rol-6 (su1006)], and TJ375 gpIs1 [hsf-16.2p:: GFP] in the examples were purchased from the official website of the Caenorhabditis Genetics Center (CGC) in the United States.

[0105] Glutathione Peroxidase (GSH-Px), Superoxide Dismutase (SOD), Malondialdehyde (MDA) kits, Beijing Box Biotechnology Co., Ltd.; Hieff qPCR SYBR Green Master Mix (no ROX), Shanghai Yisheng Biotechnology Co., Ltd., 11201ES08; HifairIII 1stStrand cDNASynthesis SuperMix for qPCR, Shanghai Yisheng Biotechnology Co., Ltd.

[0106] All experiments in the examples were repeated 3 times, and the results were expressed as Mean±SD. IBM SPSS Statistics 26 software was used to perform significance analysis through one-way ANOVA test, and Graphad Prism 9 software was used for statistical analysis.

[0107] Example 1 Preparation of oyster protein polypeptide enzymatic hydrolysate (OP)

[0108] Fresh oysters were air-dried and crushed, pre-cooled distilled water was added according to a solid-liquid ratio of 1:15, distilled water and papain were added in a mass ratio of 0.5%, enzymolysis was carried out in water at 55°C for 4 hours, and the mixture was allowed to stand at 85°C for 1.5 hours. The supernatant was filtered with a cotton cake filter, 1.5% activated carbon was added and kept at 70°C for 40 minutes for decolorization, the activated carbon was removed by a plate and frame filter, nanofiltration membrane and vacuum falling film were combined for concentration, and the mixture was spray-dried to obtain oyster protein polypeptide hydrolysis products (OP), which were stored at -80°C for later use.

[0109] OP separation and purification: Weigh the OP sample, dissolve it in 15% acetonitrile aqueous solution, and prepare a 20 mg / mL concentration sample for standby use. Use Waters e2695 chromatograph system, chromatographic conditions: C18 (Waters, 5μm, 4.6mm×150mm); injection volume is 30μL, mobile phase A is ultrapure water, B is acetonitrile (≥99%), both contain 0.1% (v / v) trifluoroacetic acid (TFA), flow rate: 0.8mL / min, gradient elution time 35min, gradient elution: 0-8min (80.5% A, 19.5% B); 8-24min (79.4% A, 20.6% B); 24-30min (78.8% A, 21.2% B); 30-35min (50% A, 50% B) column temperature: 30°C, the absorbance of the eluent is detected at 220nm. One tube was collected every 1 minute, and the samples were combined according to the retention time. Tubes 9 to 12 were combined (recorded as OP-1), tubes 13 to 14 were combined (recorded as OP-2), tubes 15 to 18 were combined (recorded as OP-3), and tubes 19 and 29 were combined (recorded as OP-4). The samples were concentrated by rotary evaporation, freeze-dried, and separated components were obtained. The components were refrigerated for later use. It can be seen from the chromatographic separation results that OP-4 has the highest yield and contains relatively simple components ( Figure 1 ).

[0110] OP-4 was further subjected to mass spectrometry structure identification. The details are as follows: Liquid phase conditions are as follows: Chromatographic column: BEH C18 (waters, 1.7μm, 2.1×50mm); Mobile phase: 90% acetonitrile (containing 0.1% formic acid); Constant flow rate: 0.6mL / min elution; Column temperature: 40°C. Mass spectrometry conditions: Positive mode: voltage 3.5kv, ion source temperature 110°C, desolvation temperature 400°C, nitrogen flow rate 800L / h; Negative mode: voltage 3kv, ion source temperature 110°C, desolvation temperature 400°C, nitrogen flow rate 800L / h. Through the analysis of the primary mass spectrum, the signal with m / z of 1241.633 was the strongest and was the only peptide component. The peptide segment was further subjected to secondary fragmentation and detection ( Figure 2 ), 1241.715 is the parent ion signal peak, and the remaining peaks are fragment peaks detected after the parent ion is fragmented by the secondary fragmentation ( Figure 3 In BioTools software, through DeNovo sequencing analysis, the sequence with the highest matching sequence score is SSYNYGTMTSM (SEQ ID NO: 1) ( Figure 4 ) or SHLNYGTMTSM (SEQ ID NO: 2) ( Figure 5 ).

[0111] Example 2 Effect of oyster protein polypeptide enzymatic hydrolysate (OP-4) on learning and memory of Caenorhabditis elegans

[0112] 1. Paralysis phenotype experiment of mutant CL4176 nematodes

[0113] The CL4176 nematodes containing eggs were picked into a culture medium plate containing food, and eggs were laid for 5 hours. After the eggs were cultured at 16°C for 48 hours, they were picked into OP-4 culture medium (containing FUDR) coated with different concentrations of 50μg / mL (low dose), 100μg / mL (medium dose) and 200μg / mL (high dose), respectively. Ultrapure water was used as a blank control, and the temperature was raised to 25°C and cultured until all the nematodes were paralyzed. In this way, the expression of Aβ protein in the muscle cells of CL4176 nematodes was induced, and the number of paralyzed nematodes was recorded every 2 hours. The criterion for judging nematode paralysis is that when the nematode is gently touched with a picking needle, the nematode only moves slightly with its head. The experiment was repeated 3 times and the curve was drawn.

[0114] The paralysis degree of CL4176 nematodes was measured using low, medium and high concentrations of OP-4 (the average paralysis time is the average time from the beginning of paralysis to the end of paralysis of the nematodes). The experimental results showed that low, medium and high concentrations of OP-4 could prolong the average paralysis time of transgenic CL4176 nematodes induced by Aβ toxicity (Table 1). As time went on, the paralysis curve of the nematodes moved significantly to the right ( Figure 6), in a concentration gradient-dependent manner. Compared with the blank control group, the average paralysis time was delayed by 2.02%, 8.95% and 23.18%, respectively (P<0.05), indicating that OP-4 can inhibit the toxicity induced by Aβ protein deposition in CL4176 nematodes, improve its paralysis phenotype, and have a neuroprotective effect.

[0115] Table 1 Effect of OP-4 on paralysis of CL4176 nematodes

[0116]

[0117] 2. Chemotaxis Behavior Experiment of Mutant Strain CL2355 Nematodes

[0118] CL2355 containing eggs were picked up to the culture medium plate containing food and laid eggs for 5 hours. After the eggs were cultured at 16°C for 48 hours, they were picked up to OP-4 culture medium (containing FUDR) coated with different concentrations of 50μg / mL (low dose), 100μg / mL (medium dose) and 200μg / mL (high dose), respectively. Ultrapure water was used as a blank control. The temperature was raised to 23°C and cultured for 48 hours to measure the chemotaxis behavior of the nematodes (see the schematic diagram of the chemotaxis behavior of CL2355 nematodes for details). Figure 7 ). Collect them into 2mL EP tubes respectively, and wash them repeatedly with M9 solution until the E. coli is completely removed. Preparation method of chemotaxis agar plate: prepare an agar plate with a diameter of 60mm, divide it into two sides evenly, and add equal volumes of 10% butanone and 95% ethanol on each side. Add equal volumes of 0.1mol / L hydrochloric acid tetramisole solution to both sides of the agar plate to anesthetize the nematodes, place 100 nematodes in the center of the agar plate, continue to culture in a 23℃ incubator for 1h, and count the nematodes on both sides of the agar plate under a microscope. Chemotaxis index (CI). CI = (number of nematodes in the butanone position - number of nematodes in the ethanol position) / total number of nematodes in the agar medium. The experiment was repeated 3 times and the curve was drawn.

[0119] In order to further observe the effect of OP-4 on learning and memory in nematodes, the AD model CL2355 nematodes were used as experimental subjects. The results showed that compared with the chemotaxis index of the blank control group (0.065±0.001), the chemotaxis index of CL2355 nematodes significantly increased under low, medium and high concentrations of OP-4 treatment, which were 0.12±0.014, 0.145±0.001 and 0.175±0.001, respectively (P<0.05) ( Figure 8 ). That is, OP-4 delayed the paralysis induced by Aβ protein in the muscles of CL4176 nematodes, significantly prolonged the survival time of CL4176 nematodes, and improved the defect of chemotaxis behavior of CL2355 nematodes caused by overexpression of Aβ protein. This suggests that OP-4 can reduce the toxicity of Aβ protein, have neuroprotective effects and improve learning and memory abilities.

[0120] 3. Polyglutamine (polyQ) aggregation experiment in the mutant AM141 nematode

[0121] AM141 nematodes were synchronized and cultured to the L4 stage. They were selected into OP-4 medium (containing FUDR) coated with different concentrations of 50μg / mL (low dose), 100μg / mL (medium dose) and 200μg / mL (high dose), and ultrapure water was used as a blank control. After 5 days of culture, the poly Q40:YFP aggregation in the nematodes was measured. They were collected into 2mL EP tubes and washed repeatedly with M9 solution until the E. coli was completely removed. Preparation method of 1% agarose slide: weigh 1g agar powder and dissolve it in 100mL ultrapure water to prepare 1% agar medium, dissolve it in a microwave oven, draw a small amount of agar medium and drop it in the center of the slide, cover it with a cover glass, gently flatten it, and remove the cover glass after solidification to obtain an agar slide. The nematodes were transferred to the agar pad, anesthetized with hydrochloric acid tetramisole solution, observed and collected pictures using a fluorescence microscope, and the fluorescence intensity was statistically analyzed using Image J. The experiment was repeated three times and the curves were drawn.

[0122] PolyQ aggregation in cells can cause toxicity and even cell death, leading to various related diseases, such as Huntington's. The experimental results showed that when the polyQ aggregation model AM141 nematodes were treated with OP-4, the average number of polyQ40:YFP fluorescent aggregation points in the blank control group was 70.62±0.57. After the nematodes were treated with low, medium and high concentrations of OP-4 for 120 hours, the average number of polyQ40:YFP fluorescent points decreased to 64.26±3.05, 44.16±1.89 and 34.92±1.14, respectively (P<0.05)( Fig. 9 and Fig.10 ), OP-4 can inhibit the aggregation of polyQ proteins in a concentration gradient-dependent manner. This suggests that OP-4 can inhibit the fluorescence expression in transgenic nematodes and effectively prevent neurodegenerative diseases caused by polyQ expansion.

[0123] Example 3 Effect of oyster protein polypeptide enzymatic hydrolysate (OP-4) on anti-aging

[0124] 1. Life test

[0125] C.elegans was synchronized and cultured to the L4 stage, and a sample group and a blank group were set up. The concentrations of the sample groups were 50, 100, 200, 400, and 800 μg / mL, respectively, and ultrapure water was set up as a blank control group (each culture medium contained 150 mmoL / L FUDR). The nematodes were picked up and placed in each group of culture medium, each culture medium contained 30 nematodes, and 3 parallels were set up and placed in a 20°C incubator for culture. The number of nematode survival was counted from the date of transfer, and the day of transfer was recorded as day 0. The nematode status was observed every other day, and the survival time was recorded. The nematode survival judgment was based on Xu A (Xu A, Zhang Z, Ko SH, et al. Microtubule regulators act in the nervous system to modulate fatmetabolism and longevity through DAF-16 in C.elegans [J]. Aging cell, 2019, 18 (2): 12884). The life span curve was drawn.

[0126] The results showed that compared with the blank control group, there was no significant difference in the average lifespan under low and medium concentrations of OP-4 treatment. Under high concentration treatment, OP-4 shifted the lifespan curve of C.elegans to the right, and the average lifespan was significantly extended by 11.2±1.21d (P<0.05), and the lifespan percentage increased by 36.6%, with the longest lifespan being 17d ( Fig.11 and Table 2 ).

[0127] Table 2 Effect of OP-4 on the life span of C.elegans

[0128]

[0129] 2. Exercise capacity test

[0130] Nematode culture and lifespan experiment. The movement ability of C.elegans at 3d, 7d, and 11d was measured respectively. Measurement method: Place the nematodes in a blank plate containing culture medium, drop a drop of M9 solution (preparation method: weigh 0.5g NaCl, 1.512g Na2HPO4, 0.025g MgSO4, 0.3g KH2PO4, add 100mL deionized water, sterilize at 121℃ for 20min, cool and set aside), let it adapt for 30s, and record the movement ability of the nematodes within 60s. Draw a curve.

[0131] The results are as follows Fig.12The results showed that on the third day of OP-4 treatment, compared with the blank control group, the nematodes were all in the A-level movement state, accounting for 100%; on the seventh day of OP-4 treatment, the proportion of nematodes in the A-level movement state decreased significantly. Compared with 18.89% of the nematodes in the blank group, the proportion of nematodes in the A-level movement state increased to 33.27%, 44.11% and 50.0% at low, medium and high concentrations (P < 0.05), compared with 42.22% of the nematodes in the blank group in the B-level movement state, the proportion of nematodes in the B-level movement state increased to 46.20%, 44.11% and 45.1% at low, medium and high concentrations. % (P<0.05), compared with the 3rd day, the proportion of nematodes in the C-level movement state increased significantly in the 7th day; on the 11th day of OP-4 treatment, compared with the 4.89% of nematodes in the blank group in the A-level movement state, at low, medium and high concentrations, the proportion of nematodes in the A-level movement state increased to 9.99%, 17.24% and 38.9% (P<0.05), and the proportion of nematodes in the B-level movement state was not significantly different from that on the 7th day. Compared with the blank group C-level movement of 50%, at low, medium and high concentrations, the proportion of nematodes with C-level movement level decreased to 45%, 36.76% and 21% respectively (P<0.05). In summary, with the extension of treatment time, the overall movement of nematodes decreased, and compared with the blank, the proportion of nematodes in the A-level movement state increased significantly, indicating that OP-4 can significantly enhance the movement ability of nematodes and improve the movement state of nematodes in the aging state.

[0132] 3. Swallowing frequency test

[0133] Nematode culture and lifespan experiment. Place in a 20℃ incubator for 5 days, observe and record the swallowing frequency of the nematodes within 60 seconds under a microscope (Chen Li, Zhang Shaobo, Huang Dian, et al. Study on the characteristics of active substances produced by deep-sea Dokdo Bacillus A493 and optimization of fermentation conditions [J]. Chemistry and Bioengineering, 2012, 29(09): 26-31). Draw a curve.

[0134] Nematodes swallow food, so the swallowing frequency is used as a measure of the food intake of Caenorhabditis elegans. The swallowing frequency of nematodes was measured, and the results were as follows: Fig.13 As shown, compared with the blank control group, there was no significant difference in the swallowing frequency of nematodes (P>0.05), indicating that OP-4 could not change the food intake of nematodes and had no toxic effect.

[0135] 4. Heat stress survival rate experiment, DPPH free radical scavenging rate experiment

[0136] Heat stress survival rate experiment: Cultivation of nematodes is the same as lifespan experiment. After transferring them to a 35℃ incubator to induce heat stress damage for 5 hours, they are placed in a 20℃ constant temperature incubator for 5 hours to recover and record the survival rate of the nematodes. The method for determining death is the same as the lifespan experiment. Draw a curve.

[0137] DPPH free radical scavenging rate experiment: Take 2 mL of DPPH solution (0.4 mmol / L), add the same volume of OP samples with different concentrations (50, 100, 200 μg / mL) and shake well. C The positive group is A1, the sample group replaced by ultrapure water is A0, and the DPPH solution is replaced by ultrapure water is A2. The reaction is protected from light for 30 minutes, and the absorbance OD is detected at a wavelength of 517nm. Each sample is repeated in three groups. The following formula is the calculation method:

[0138] Stress resistance is often used as an important indicator of the anti-aging effect of Caenorhabditis elegans. Fig.14 As shown in the results, OP and the four isolated components significantly increased the survival rate of heat-stressed nematodes compared with the blank control group (P < 0.05). There was no significant difference between OP and OP-4, but the average survival rate of OP-4 was higher than that of OP. Compared with the blank control group, at low and medium concentrations, OP-1, OP-2 and OP-3 had no significant difference (P > 0.05). At high concentrations, OP-1 and OP-3 had significant differences, increasing the survival rate of nematodes by 19.55% and 16.22%, respectively; OP-4 had significant differences at medium and high concentrations, increasing the survival rate of nematodes by 26.06% and 29.86%. Compared with OP-1 and OP-3, OP-4 had the best survival rate effect at high concentrations. It is speculated that OP-4 may be the main active ingredient of OP or its components have high activity intensity.

[0139] The scavenging effect of the four separated components on DPPH free radicals was determined. Fig.15 As shown in the results, compared with the blank control group, at low and medium concentrations, the separated components had no significant effect on the DPPH clearance rate (P>0.05), and at high concentrations, OP-1, OP-2 and OP-3 had no significant difference. OP-4 and OP had differences at medium and high concentrations, among which OP-4 had the best effect, significantly increasing the DPPH clearance rate by 17.95% and 32.3% at medium and high concentrations (P<0.05). This shows that OP-1 to OP-4 active peptides have good in vitro antioxidant activity.

[0140] Example 4

[0141] Further research on the molecular mechanism of OP-4 was conducted, mainly by measuring the expression levels of related genes in nematodes, and exploring the important signal pathways and transcription factors in the aging process of nematode models based on different genotypes.

[0142] The details are as follows:

[0143] 1. Acute oxidative stress injury experiment

[0144] The nematode culture was the same as in Example 3. After 3 days of culture, the nematodes were placed in a 96-well plate, with 15 nematodes in each well. 200 μL of 2% H2O2 solution was added to each well. The survival rate was observed after culturing in a 20°C incubator for 5 hours. A curve was drawn.

[0145] The accumulation of oxidative damage can cause aging of nematodes, and vice versa. Example 3 shows that OP-4 has the effect of scavenging free radicals in vitro and alleviating heat stress in nematodes, further clarifying whether OP-4 can improve oxidative stress damage in nematodes, thereby alleviating the oxidative damage mechanism of aging. The results showed that OP-4 increased the survival rate of nematodes induced by H2O2. There was no significant difference between the low concentration and the blank control (P>0.05); the medium and high concentrations can significantly increase the survival rate of nematodes (P<0.05), which were 46.67% and 70%, respectively, and increased by 18.5% and 41.9%, respectively ( Fig.16 ). This indicates that OP-4 exhibits antioxidant activity both in vivo and in vitro, which may be one of its anti-aging mechanisms.

[0146] 2. Determination of Antioxidant Enzyme Activity in Vivo

[0147] The nematode culture was the same as in Example 3. After 3 days of nematode culture, the nematodes of each group were collected in a 2 mL EP tube, washed several times with M9 buffer, treated with a sample and an extract at a volume ratio of 500:1, ultrasonically broken in an ice bath for 3 seconds, with an interval of 10 seconds, repeated 30 times, centrifuged at 8000 r / min and 4°C for 10 minutes, and the supernatant was placed on ice for testing. The superoxide dismutase (SOD) and catalase (CAT) activities and malondialdehyde (MDA) content were determined according to the method described in the kit.

[0148] Compared with the blank control group, the activities of SOD and CAT in the nematodes were significantly increased, and the content of MDA was significantly decreased ( Fig.17). At medium and high concentrations, SOD activity increased significantly by 40.33% and 98.18% (P < 0.05), and there was no significant difference at low concentration (P > 0.05). CAT increased significantly by 52.3% only at high concentration (P < 0.05). After treatment with low, medium and high concentrations, the MDA content in nematodes was significantly reduced, which was 9.55%, 2.76% and 1.67% respectively (P < 0.05), and there was no significant difference between low and medium concentrations. Therefore, the mechanism of OP-4 in alleviating oxidative stress damage is related to increasing the level of antioxidant enzymes in nematodes, while reducing the level of lipid peroxidation, which has a protective effect on cells. The results show that OP-4 can enhance the activity of SOD and CAT, reduce the accumulation of MDA and the peroxidation of unsaturated fatty acids, reduce oxidative damage in nematodes, eliminate excessive free radicals in the body, and improve the ability of nematodes to resist the influence of adverse external environment, thereby prolonging the life span of nematodes.

[0149] 3.q-PCR detection

[0150] The nematode culture was the same as in Example 3. After 3 days of culturing, the nematodes were collected into 2 mL EP tubes, washed repeatedly with M9 solution until the E. coli was completely removed, and the nematode RNA was extracted and reverse transcribed into cDNA. The expression levels of hsp-16.2, skn-1, daf-16, daf-2, akt-1, hsf-1, jnk-1, age-1, sod-3, gst-4, clk-1 and sek-1 in the nematode genome were detected by q-PCR, with Actin as the internal reference gene.

[0151] The results showed that compared with the blank control group, high concentration of OP-4 significantly downregulated the mRNA expression level of daf-2 gene to 0.68±0.05 (P<0.05), the mRNA expression level of age-1 gene was significantly downregulated to 0.87±0.06 (P<0.05), and the mRNA expression level of daf-16 gene was significantly upregulated to 1.28±0.05 (P<0.05). There was no significant difference in akt-1 and jnk-1 (P>0.05), indicating that OP-4 inhibited the IIS signaling pathway and activated the transcription factor daf-16 of the pathway. sek-1 is the upstream of skn-1 in the P38MAPK signaling pathway, gst-4 is the downstream regulator of skn-1, sod-3 is the downstream regulator of daf-16, daf-16 plays a role in the IIS signaling pathway of nematodes, and hsf-16.2 is the downstream transcription factor of hsf-1, which plays a role in the stress response of nematodes. At high concentrations, the mRNA expression levels of hsf-1 and hsp-16.2 genes did not change significantly (P>0.05). This shows that the molecular mechanism of anti-aging in nematodes after OP-4 treatment is basically unrelated to the signaling pathway in which the heat stress response is located. At high concentrations, the mRNA expression levels of skn-1, gst-4, clk-1, and sek-1 were significantly upregulated to 1.15±0.04, 1.43±0.2, 1.87±0.22, and 2.03±0.46, respectively (P<0.05), and the mRNA expression level of the downstream stress gene sod-3 was significantly increased to 1.37±0.04 (P<0.05) ( Fig.18 ).

[0152] 4. Lifespan Test of Mutant C. elegans

[0153] The nematode culture was the same as in Example 3. The nematodes were transferred to an agar pad and anesthetized with tetramisole hydrochloride solution. The nematodes were observed and images were collected using a fluorescence microscope. The fluorescence intensity was statistically analyzed using Image J. The nematodes with functional loss, E1370 (daf-2), TJ356 (daf-16), LD1 (skn-1), and TJ375 (hsp-16.2), were selected.

[0154] The insulin / IGF-1 signaling pathway (IIS signaling pathway) is one of the most important signaling pathways for aging in Caenorhabditis elegans. From the above research results, it can be seen that OP-4 can regulate the gene expression of the IIS signaling pathway. In order to further explore the mechanism of delaying aging in nematodes, the functionally deficient nematodes E1370 (daf-2), TJ356 (daf-16), LD1 (skn-1) and TJ375 (hsp-16.2) were selected to further study the relationship between key genes related to lifespan and the lifespan-extending effect of OP-4. The results showed that under high concentrations, compared with the blank control group, the average lifespan of daf-2, daf-16 and skn-1 mutant nematodes was extended by 0.43±0.05d, 0.51±0.07d and 0.61±0.05d, respectively, and the relative lifespan percentage was extended by 4.6%, 5.3% and 6.4%, respectively, and there was no significant difference in the maximum lifespan of the three mutant nematodes compared with the blank control group (P>0.05); the average lifespan of hsp-16.2 mutant nematodes was extended by 1.11±0.17d (P<0.05), the relative lifespan percentage was extended by 10.9%, and the maximum lifespan reached 17d, which was significantly different from the blank control group (P<0.05) (Table 3 and Fig.19 ). This indicates that the lifespan extension effect mediated by OP-4 may be related to the mutant genes daf-2, daf-16, and skn-1, but not to hsp-16.2, which is consistent with the results of mRNA expression levels of related aging genes. This suggests that the mechanism of action of OP-4 mainly includes the IIS signaling pathway of daf-2 / daf-16 and the P38MAPK pathway where skn-1 is located, to regulate the lifespan of nematodes, thereby delaying nematode aging and thus conducting intracellular energy metabolism.

[0155] Table 3 Effects of OP-4 on related mutant nematodes

[0156]

[0157] 5. Nuclear localization experiment of mutant nematodes

[0158] The nematode culture was the same as in Example 3. The nematodes were transferred to an agar pad and anesthetized with tetramisole hydrochloride solution. The nematodes were observed and images were collected using a fluorescence microscope, and the fluorescence intensity was statistically analyzed using Image J. Among them, the nematodes TJ356 (daf-16) and LD1 (skn-1) with functional loss were selected, and the nuclear localization of DAF-16::GFP and SKN-1::GFP was determined.

[0159] DAF-16 and SKN-1 are important nuclear transcription factors. Their nuclear migration activates the expression of their respective downstream genes, causing a series of physiological changes in the body's cells. The activation of related signaling pathways can be determined by observing their nuclear migration. Mutant nematodes with fluorescent markers are a common method. The results showed that under normal growth conditions, DAF-16::GFP is mainly located in the cytoplasm. Under OP-4 intervention, compared with the blank control group, the localization of DAF-16::GFP in the cell nucleus reached 20%, an increase of 6.67%, with significant differences (P < 0.05); DAF-16::GFP located in the cytoplasm was significantly reduced by 11.66% (P < 0.05), and DAF-16::GFP located in the interstitial matrix was significantly increased by 5% (P < 0.05) ( Fig. 20 and Fig.21 ). This indicates that OP-4 can significantly promote the transfer of DAF-16::GFP from the cytoplasm to the nucleus. Compared with the blank control group, the localization of SKN-1::GFP in the nucleus reached 31.66%, an increase of 11.66%, with a significant difference (P < 0.05); the localization of SKN-1::GFP in the cytoplasm was significantly reduced by 19.54% (P < 0.05), and the localization of SKN-1::GFP in the interstitial matrix was significantly increased by 7.88% (P < 0.05) ( Fig. 22 and Fig.23 ). This indicates that OP-4 can significantly promote the transfer of SKN-1::GFP from the cytoplasm to the nucleus. In summary, OP-4 promoted the transfer of DAF-16::GFP and SKN-1:GFP from the cytoplasm to the nucleus, regulated the stress resistance of nematodes, and significantly increased the protein expression level of SOD-3::GFP, suggesting that the lifespan extension effect mediated by OP-4 is related to daf-2, daf-16 and skn-1, but not to hsp-16.2.

[0160] 6. Protein expression experiment of mutant nematodes

[0161] The nematode culture was the same as in Example 3. (The mutant nematode protein expression experiment did not use daf-16 nematodes.) The nematodes were transferred to an agar pad, anesthetized with tetramisole hydrochloride solution, observed and captured using a fluorescence microscope, and the fluorescence intensity was statistically analyzed using ImageJ. The expression levels of HSP-16.2 and SOD-3 proteins were measured.

[0162] High concentration of OP-4 significantly increased the expression level of SOD-3::GFP in nematodes (32.75%) (P<0.05) ( Fig.24). This indicates that OP-4 can directly increase the level of SOD-3 in nematodes without inducing oxidative stress damage. Compared with the expression of blank histones, at high concentrations, the expression level of HSP-16.2::GFP increased by 9.25%, with no significant difference (P < 0.05) ( Fig.25 ). The results showed that OP-4 could not increase the expression level of HSP-16.2::GFP protein in mutant nematodes. In summary, OP-4 significantly increased the protein expression level of SOD-3::GFP, but had no significant effect on the expression level of HSP-16.2::GFP, indicating that the anti-aging effect of OP is related to directly increasing the level of antioxidant enzymes in the body, but has nothing to do with hsp-16.2 heat shock protein, that is, the HSF1 signaling pathway.

[0163] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An oyster peptide or a pharmaceutically acceptable salt thereof, the oyster peptide comprising: a1) The amino acid sequence is shown as SX1X2NYGTMTSM, wherein: X1 is S or H, X2 is Y or L; and / or a2) An amino acid sequence shown in a1) that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

2. A fusion protein comprising the oyster peptide according to claim 1.

3. A biomaterial related to the oyster peptide according to claim 1 or the fusion protein according to claim 2, wherein the biomaterial comprises at least one of b1) to b12): b1) a nucleic acid molecule encoding the oyster peptide according to claim 1 or the fusion protein according to claim 2; b2) an expression cassette containing the nucleic acid molecule described in b1); b3) a vector containing the nucleic acid molecule described in b1); b4) a vector containing the expression cassette described in b2); b5) a recombinant cell containing the nucleic acid molecule described in b1); b6) a recombinant cell containing the expression cassette described in b2); b7) a recombinant cell containing the vector described in b3); b8) a recombinant cell containing the vector described in b4); b9) a recombinant microorganism containing the nucleic acid molecule described in b1); b10) a recombinant microorganism containing the expression cassette described in b2); b11) a recombinant microorganism containing the vector described in b3); b12) A recombinant microorganism containing the vector described in b4).

4. The biomaterial according to claim 3, characterized in that The vector is an expression vector; Preferably, the expression vector is selected from the group consisting of a plasmid, a phage vector and a viral vector.

5. Use of the oyster peptide or a pharmaceutically acceptable salt thereof according to claim 1, the fusion protein according to claim 2, or the biomaterial according to claim 3 or 4 in at least one of c1) to c21): c1) preparing products for improving learning and memory abilities; c2) inhibit / mitigate the toxic effects induced by Aβ protein deposition; c3) preparing products that inhibit / mitigate the toxic effects induced by Aβ protein deposition; c4) inhibiting the aggregation of polyQ proteins; c5) preparing a product for inhibiting the aggregation of polyQ proteins; c6) preparing products that extend the lifespan of nematodes; c7) preparing products that enhance the motility of nematodes; c8) Improve the ability to resist stress response; c9) preparing products for improving the ability to resist stress; c10) Antioxidant; c11) preparing antioxidant products; c12) Anti-aging; c13) preparing anti-aging products; c14) inhibit the expression of daf-2 gene / age-1 gene; c15) preparing a product for inhibiting the expression of daf-2 gene / age-1 gene; c16) promotes the expression of skn-1 gene / gst-4 gene / clk-1 gene / sek-1 gene; c17) preparing a product for promoting the expression of skn-1 gene / gst-4 gene / clk-1 gene / sek-1 gene; c18) preparing products that modulate the insulin / IGF-1 signaling pathway; c19) preparing products of P38MAPK signaling pathway; c20) promotes the translocation of DAF-16 / SKN-1 from the cytoplasm to the nucleus; c21) preparing a product that promotes the translocation of DAF-16 / SKN-1 from the cytoplasm to the nucleus.

6. The use according to claim 5, characterized in that: The antioxidant activity in c10) and c11) includes achieving the antioxidant purpose by at least one of scavenging free radicals, increasing SOD activity, increasing CAT activity, and reducing MDA content.

7. The use according to claim 5, characterized in that: The stress responses in c8) and c9) include oxidative stress damage and heat stress damage.

8. A product comprising the pharmaceutically acceptable salt of the oyster peptide according to claim 1, the fusion protein according to claim 2, or the biomaterial according to claim 3 or 4.

9. The product according to claim 8, characterized in that The product includes at least one of a reagent, a kit, a cosmetic, a food and a medicine.

10. The product according to claim 9, characterized in that The product may also include other pharmaceutically acceptable excipients or other second active ingredients.

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