Active peptide for senescent cells and preparation method thereof

Abstract

The invention belongs to the technical field of functional polypeptides, and particularly relates to an active peptide for senescent cells and a preparation method thereof.The active peptide is obtained through molecular docking simulation screening and optimization by taking a helical structural segment, combined with gp130 protein, in IL-6R protein as a polypeptide template; the functional optimized active peptide contains an FF unit for driving nano self-assembly, a GFLG unit which can be subjected to specific enzyme digestion by lysosomal protease which is highly expressed in a senescence cell microenvironment, and an RYFQKWEKLRNQ unit which generates high affinity with an IL-6 / IL-6R / gp130 compound, and the designed active peptide has anti-aging activity and can also be used for collaborative delivery of drugs. Wide application prospects are realized.

Description

An active peptide for senescent cells and its preparation method Technical Field

[0001] This invention belongs to the field of functional polypeptide technology, specifically relating to an active peptide for senescent cells and its preparation method. Background Technology

[0002] Cellular senescence is a stable state of cell cycle arrest that occurs when cells, under the influence of factors such as stress, damage, or replication exhaustion, are accompanied by phenotypic changes such as metabolic reprogramming, persistent activation of DNA damage responses, mitochondrial dysfunction, and enhanced inflammatory signaling. Senescent cells secrete various pro-inflammatory cytokines, chemokines, and proteases, forming SASP (senescence-associated secretory phenotype), including but not limited to IL-6, IL-8, CCL2, and MMPs. SASP can amplify inflammation through autocrine and paracrine processes, induce cellular senescence, disrupt tissue homeostasis, and promote fibrosis, metabolic disorders, and the development of various age-related diseases. Therefore, precise intervention targeting key nodes in the formation, maintenance, and amplification of senescent cells is an important strategy for delaying aging and improving cellular degeneration caused by pathological factors such as stroke.

[0003] Current anti-aging interventions mainly focus on eliminating senescent cells or inhibiting SASP (Sensitive Aging Pathogens). While small-molecule drugs widely used in clinical practice are convenient to administer, they suffer from issues such as insufficient targeting, systemic side effects, and long-term safety concerns. Antibody drugs have high targeting specificity, but their preparation costs are high, and their in vivo distribution and tissue penetration are limited. Peptides, on the other hand, possess advantages such as designable structures, precisely regulated mechanisms of action, and relatively good tissue penetration, making them promising for specific antagonism of aging maintenance pathways. Furthermore, due to the presence of non-covalent interactions such as hydrophobicity, hydrogen bonding, and conjugation, peptides also have the potential for self-assembly and drug loading, exhibiting both activity and functionality. This could provide a novel and industrially viable technological pathway for delaying aging and intervening in related diseases. Summary of the Invention

[0004] In response to the above situation, the present invention provides an active peptide for senescent cells and a method for preparing the same. The method selects protein targets by taking the key signal axis of SASP self-maintenance in senescent cells as the starting point, obtains antagonistic active peptides that can specifically bind to receptor proteins through molecular docking simulation, and optimizes their sequence and improves their functionality, thereby achieving the comprehensive effects of delaying cell senescence, improving the tissue microenvironment and enhancing repair capabilities.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides an active peptide for senescent cells, the active peptide comprising the following raw materials: RinkAmide AM resin, Phe (phenylalanine F), Gly (glycine G), Leu (leucine L), Arg (arginine R), Gln (glutamine Q), Lys (lysine K), Tyr (tyrosine Y), Trp (tryptophan W), Glu (glutamate E), and Asn (asparagine N).

[0006] Furthermore, the mass ratio of Rink Amide AM resin, Phe, Gly, Leu, Arg, Gln, Lys, Tyr, Trp, Glu and Asn is 10:24.8:9.5:11.3:20.7:19.5:15:7.4:8.4:6.8:9.5.

[0007] Furthermore, the amino acid sequence of the active peptide is H2N-FFGFLGRYFQKWEKLRNQ, as shown in SEQ ID NO.9.

[0008] Furthermore, the active peptide was obtained by molecular docking simulation screening and optimization using the helical segment of IL-6R protein that binds to gp130 protein as a polypeptide template.

[0009] Further, the specific method for screening and optimizing the active peptide through molecular docking simulation is as follows: S1: Based on the SASP conduction mechanism, the IL-6 / IL-6R / gp130 complex was selected as the intervention target. Its high-resolution crystal structure (PDB ID: 1P9M) was obtained by searching protein structure databases. The binding interface between IL-6 and the effector protein gp130, i.e., the Site II region, was analyzed. Hotspot regions, i.e., peptide templates, were determined using molecular dynamics simulations and SASA (solvent accessibility surface area) analysis. S2: Based on the peptide template selected in step S1, eight candidate peptides were designed, numbered P1-P8, with amino acid sequences as shown in SEQ ID. As shown in NO.1-8; S3: Using bioinformatics tools, the physicochemical properties and secondary structure of candidate peptides P1-P8 were evaluated and predicted. The α-helix formation tendency of each candidate peptide in aqueous solution was analyzed, and candidate peptides P1, P2, and P6 with good amphiphilicity, strong stability, and high helical tendency were screened out; S4: The screened candidate peptides P1, P2, and P6 were subjected to computer molecular docking simulation with 1P9M, respectively. The binding conformation and binding ability of each candidate peptide with the IL-6 / IL-6R / gp130 complex were evaluated, the binding free energy was calculated, and the candidate peptide P1 with the strongest binding force and the most stable conformation was screened again; S 5: The candidate peptide P1 obtained from screening was functionally sequenced and modified with the FFDFLG peptide at its N-terminus. The π-π stacking and hydrophobic interaction between the benzene rings in FF facilitates the self-assembly of the peptide into stable nanofibers or particles under physiological conditions, which can stabilize the conformation and properties of the peptide molecules in vivo and in vitro. The GFLG peptide can be specifically recognized and cleaved by lysosomal proteases highly expressed in the microenvironment of senescent cells, realizing the targeted and efficient release of active peptides or loaded drugs. The amino acid sequence of the active peptide for senescent cells was designed as H2N-FFGFLGRYFQKWEKLRNQ, as shown in SEQ ID NO.9.

[0010] Furthermore, the amino acid sequence of the candidate peptide P1 is H2N-RYFQKWEKLRNQ, as shown in SEQ ID NO.1.

[0011] Furthermore, the amino acid sequence of the candidate peptide P2 is H2N-WKRLYEHFQKRN, as shown in SEQ ID NO.2.

[0012] Furthermore, the amino acid sequence of the candidate peptide P3 is H2N-KWEYRFQNRLKK, as shown in SEQ ID NO.3.

[0013] Furthermore, the amino acid sequence of the candidate peptide P4 is H2N-RLFWKQEYNRKK, as shown in SEQ ID NO.4.

[0014] Furthermore, the amino acid sequence of the candidate peptide P5 is H2N-YWKRQEFNRLKK, as shown in SEQ ID NO.5.

[0015] Furthermore, the amino acid sequence of the candidate peptide P6 is H2N-KRWYQEFKLRNQ, as shown in SEQ ID NO.6.

[0016] Furthermore, the amino acid sequence of the candidate peptide P7 is H2N-CRYFQKWEKLRNC, as shown in SEQ ID NO.7, wherein the cysteine ​​C residues at both ends of the candidate peptide P7 can form disulfide bonds and thus undergo cyclization.

[0017] Furthermore, the amino acid sequence of the candidate peptide P8 is H2N-CWKRLYEHFQKRNC, as shown in SEQ ID NO.8.

[0018] This invention also provides a method for preparing active peptides for senescent cells, the specific steps of which are as follows: Step 1: Place Rink Amide AM resin in a peptide synthesis reactor, add DMF (N,N-dimethylformamide) to swell, filter to remove the solvent, and then deprotect and wash with DMF to obtain deprotected resin; Step 2: According to the amino acid sequence from C-terminus to N-terminus of the active peptide sequence H2N-FFGFLGRYFQKWEKLRNQ-COOH, take the first amino acid Gln from the C-terminus, activate it, and add it to the deprotected resin. Shake the reaction to graft Gln(Q) into the deprotected resin. Use the ninhydrin method to determine the reaction endpoint until no color development is observed, indicating successful grafting of the first amino acid. After deprotection and washing with DMF, perform the grafting reaction of the second amino acid. Take according to the active peptide sequence... The remaining amino acids Phe, Gly, Leu, Arg, Lys, Tyr, Trp, Glu, and Asn were subjected to the same activation, shaking reaction, ninhydrin color development, deprotection, and DMF washing process until the grafting of the last amino acid, Phe(F), was completed, yielding a polypeptide resin. Step 3: The polypeptide resin was washed alternately with DMF and DCM (dichloromethane) to perform a cleavage reaction, cutting the polypeptide off the resin and removing the protecting groups of the amino acid side chains, yielding crude polypeptide. Step 4: The crude polypeptide was precipitated using ice-cold ether pre-cooled at 4°C, centrifuged, and the supernatant was discarded to obtain the precipitate. The precipitate was then vacuum-dried to obtain an active peptide for senescent cells.

[0019] Furthermore, the deprotection process uses a DMF solution containing 20% ​​piperidine by volume.

[0020] Furthermore, the activation process in step 2 uses an activation solution, which is prepared by HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine) and DMF in a mass ratio of 10.4:7.4:2.3.

[0021] Furthermore, the cutting reaction process described in step 3 uses a cutting fluid, which is prepared by mixing TFA (trifluoroacetic acid), Tis (triisopropylsilane), and deionized water in a mass ratio of 9.5:0.25:0.25.

[0022] The beneficial effects achieved by this invention are as follows: The active peptide prepared by this invention for senescent cells uses the SASP self-maintained IL-6 / IL-6R / gp130 complex as the intervention target. By binding to the Site II hotspot region where IL-6R and gp130 bind, an antagonistic peptide P1 that can mimic the helical structure of IL-6R is designed. This peptide can specifically occupy the binding site of the gp130 receptor and competitively block the downstream signal transduction induced by IL-6R, thereby achieving the reversal regulation of apoptosis and senescence. This not only effectively inhibits the secretion of pro-inflammatory factors by senescent cells, but also prevents the spread of senescent phenotypes to surrounding healthy tissues, thereby achieving the therapeutic effects of delaying organ aging and promoting damage repair.

[0023] Based on the sequence of the antagonistic peptide P1 obtained through screening, a functionalized bioactive peptide was further constructed. The introduction of bisphenylalanine (FF) and a cleavage linker arm (GFLG) enabled the bioactive peptide to undergo self-assembly, forming a stable nanostructure. This significantly reduced the risk of the peptide drug being easily cleaved by enzymes and having a short half-life in vivo, achieving long-term delivery. Furthermore, the bioactive peptide with the nanostructure can also serve as a carrier for co-delivery of drugs. Simultaneously, the GFLG peptide acts as a smart "switch," specifically recognizing and cleaving lysosomal proteases such as cathepsin B, which are highly expressed in the microenvironment of senescent cells, thus improving the efficiency of in-situ release of the bioactive peptide from senescent cells. Attached Figure Description

[0024] Figure 1 is a schematic diagram of the structure of the active peptide for senescent cells of the present invention; Figure 2 is the molecular structure of the candidate peptides P1-P8 designed in Example 2; Figure 3 is the simulation result of molecular docking of candidate peptides P1, P2, P6 and active peptide with the IL-6 / IL-6R / gp130 complex; Figure 4 is the microstructure examination result of the active peptide for senescent cells prepared in Example 6; Figure 5 is the anti-aging activity examination result of the active peptide for senescent cells prepared in Example 6. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0027] In the following embodiments, the structure of the active peptide for senescent cells is shown in Figure 1. Unless otherwise specified, all methods are conventional. All parts are parts by weight. Unless otherwise specified, all raw materials used in the following embodiments are new materials purchased from the market. All amino acids used are amino acids with the N-terminus protected by the Fmoc (9-fluorenylmethoxycarbonyl) group, which are commercially available products. The degree of substitution of the Rink Amide AM resin used is 0.44 mmol / g.

[0028] Example 1 (Target Analysis and Peptide Template Determination): Based on the core role of senescence-associated secretory phenotype (SASP) in maintaining cellular senescence, the IL-6 / IL-6R / gp130 complex was selected as the intervention target. Analysis of the crystal structure numbered 1P9M in the Protein Structure Database (PDB) revealed that the D-helix region of the IL-6R protein binds to the extracellular domain of the effector protein gp130 at Site II. Molecular dynamics simulations and solvent accessibility surface area (SASA) analysis confirmed that the core binding pocket on gp130 is mainly composed of residues such as Phe191, Val189, and Tyr190. Therefore, the corresponding D-helix segment from the IL-6R sequence was extracted as the original peptide template, and a candidate peptide library was designed through amino acid substitution. Example 2 (Construction of Candidate Peptide Library): Based on the peptide template selected in Example 1, eight candidate peptides were designed, namely P1: H2N-RYFQKWEKLRNQ, as shown in SEQ ID NO. P1: H2N-WKRLYEHFQKRN, as shown in SEQ ID NO.2; P3: H2N-KWEYRFQNRLKK, as shown in SEQ ID NO.3; P4: H2N-RLFWKQEYNRKK, as shown in SEQ ID NO.4; P5: H2N-YWKRQEFNRLKK, as shown in SEQ ID NO.5; P6: H2N-KRWYQEFKLRNQ, as shown in SEQ ID NO.6; P7: H2N-CRYFQKWEKLRNC, as shown in SEQ ID NO.7; P8: H2N-CWKRLYEHFQKRNC, as shown in SEQ ID NO.As shown in Figure 8, the cysteine ​​C residues at both ends of candidate peptides P7 and P8 can form disulfide bonds and are prone to cyclization, which is used to examine the binding affinity of cyclized peptides in the IL-6 / IL-6R / gp130 complex; Example 3 (initial screening of candidate peptides): The physicochemical properties and secondary structures of candidate peptides P1-P8 were predicted using ExPASy ProtParam and PEP-FOLD3 tools. Since the 1P9M binding interface is a typical helical-groove binding mode, a higher helical percentage can significantly reduce the entropy loss during binding, thereby improving the affinity. The molecular weight, isoelectric point, GRAVY (average hydrophobicity), and α-helical tendency of the candidate peptides were obtained, and the results are shown in Table 1. Based on the peptide property prediction results, candidate peptides P1, P2, and P6 with isoelectric points in the non-physiological pH range, good hydrophilicity, and high α-helical degree were screened; Example 4 (molecular docking simulation and binding ability evaluation): AutoDock was used to simulate the binding affinity of candidate peptides P1-P8. Using the Vina tool, candidate peptides P1, P2, and P6 obtained from the initial screening were subjected to molecular docking simulations with the IL-6 / IL-6R / gp130 complex. Binding free energy ΔG, van der Waals force energy, electrostatic interactions, number of hydrogen bonds, and interfacial contact area were recorded. The results are shown in Table 2. P1, the candidate peptide with the lowest binding free energy (i.e., the most stable energy) and the largest interfacial contact area, was selected as the template for the anti-aging active peptide sequence. Example 5 (Functional Sequence Optimization and Self-Assembly Evaluation): To address the shortcomings of candidate peptide P1, such as easy degradation and difficulty in local enrichment under complex physiological environments, this example fused an FF-GFLG module peptide to the N-terminus of P1, obtaining the active peptide H2N-FFGFLGRYFQKWEKLRNQ, as shown in SEQ ID NO. 9. Its binding ability, self-assembly ability, enzyme responsiveness, and physiological environment stability were simultaneously evaluated.

[0029] Example 6: This example provides an active peptide for senescent cells. The active peptide comprises the following raw materials in parts by weight: 1 part Rink Amide AM resin, 2.48 parts Phe, 0.95 parts Gly, 1.13 parts Leu, 2.07 parts Arg, 1.95 parts Gln, 1.5 parts Lys, 0.74 parts Tyr, 0.84 parts Trp, 0.68 parts Glu, and 0.95 parts Asn. The Phe, Gly, Leu, Arg, Gln, Lys, Tyr, Trp, Glu, and Asn used are all amino acids protected by an N-terminal Fmoc group, and some amino acid side chains contain protecting groups, specifically Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, and Fmoc-Arg(Pbf)-O. H, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Asn(Trt)-OH; the activation solution used was prepared from 62.4 parts HBTU, 44.4 parts DIPEA and 13.8 parts DMF; the cleavage solution used was prepared from 9.5 parts TFA, 0.25 parts Tis and 0.25 parts deionized water; this embodiment also provides a method for preparing active peptides for senescent cells, the specific steps are as follows: Step 1: Weigh Rink One part of Amide AM resin was placed in a peptide synthesis reactor, 5 mL of DMF was added to swell for 20 min, the solvent was removed by filtration, and then deprotected with 5 mL of 20% piperidine DMF solution for 10 min, washed with 20 mL of DMF, and dried with a nitrogen pump to obtain deprotected resin; Step 2: According to the amino acid sequence from C-terminus to N-terminus of the active peptide sequence H2N-FFGFLGRYFQKWEKLRNQ-COOH, 0.975 parts of the first amino acid Gln at the C-terminus was taken, dissolved in 5 mL of activating solution, activated for 2 min, and then added to the deprotected resin. The reaction was shaken at 100 rpm for 1.5 h. The reaction endpoint was determined by the ninhydrin method. No color development was observed, indicating successful grafting of the first amino acid Q. The resin was then deprotected with 5 mL of 20% piperidine DMF solution for 10 min, washed with 20 mL of DMF, and dried with a nitrogen pump to obtain deprotected resin. After washing with mL of DMF, the grafting reaction for the second amino acid was carried out. The following amino acids were taken in sequence according to the active peptide amino acid order: Asn 0.95 parts, Arg 1.035 parts, Leu 0.565 parts, Lys 0.75 parts, Glu 0.68 parts, Trp 0.84 parts, Lys 0.75 parts, Gln 0.975 parts, Phe 0.62 parts, Tyr 0.74 parts, Arg 1.035 parts, Gly 0.475 parts, Leu 0.565 parts, Phe 0.62 parts, Gly 0.475 parts, Phe 0.62 parts, and Phe 0.Sixty-two portions were dissolved in 5 mL of activating solution, activated for 2 min, and then added to deprotected resin. The above shaking reaction, ninhydrin color development, deprotection, and DMF washing process were repeated until the last amino acid Phe(F) grafting and deprotection were completed, yielding peptide resin. Step 3: The peptide resin was washed three times each with 5 mL DMF and 5 mL DCM, dried with a nitrogen pump, and then 10 portions of cleavage solution were added for a cleavage reaction for 30 min to cleave the peptide from the resin. At the same time, the protecting groups of the amino acid side chains were removed, yielding crude peptide. Step 4: The crude peptide was precipitated using ice-cold diethyl ether pre-cooled at 4°C (volume ratio of ice-cold diethyl ether to crude peptide was 10:1). After centrifugation and discarding the supernatant, the precipitate was obtained and vacuum dried to obtain active peptides for senescent cells.

[0030] Table 1 Physicochemical properties and α-helix structure of candidate peptides

[0031] Table 2. Molecular docking simulation results parameters for candidate peptides P1, P2, and P6.

[0032] Molecular structure of candidate peptides: The secondary structures of candidate peptides P1-P8 were analyzed using the PEP-FOLD3 tool in the RPBS Web Portal. The results are shown in Figure 2.

[0033] Molecular docking simulation: The candidate peptides P1, P2, P6 and the active peptide obtained by screening were docked with the IL-6 / IL-6R / gp130 complex to investigate their structure and binding ability in protein binding. The results are shown in Figure 3.

[0034] Microscopic morphology characterization: 1 mg of the active peptide prepared in Example 6 was dissolved in 1 mL of deionized water, and its microscopic morphology was examined by transmission electron microscopy. The results are shown in Figure 4.

[0035] Anti-aging activity assessment: A stroke-induced senescent cell model was constructed, using SH-SY5Y cells as the in vitro efficacy evaluation target. Cells were incubated with glucose-free Earle's balanced salt solution for 4 h to achieve oxygen and glucose deprivation. Then, simulating clinical stroke ischemia-reperfusion, the oxygen-glucose-deprived cells were removed and replaced with normal culture medium for incubation to achieve oxygen and glucose enrichment. Culture was continued for 5 days to promote the senescence phenotype in SH-SY5Y cells. After the stroke-induced senescent cell model was constructed, 100 nM of the active peptide prepared in Example 6 and 100 nM of rapamycin (positive control drug) were administered as the active peptide group and positive control group, respectively. The groups treated with normal cells + normal culture medium and the SH-SY5Y senescent cell model + normal culture medium were the blank control group and model group, respectively. Cell apoptosis in each group was examined by flow cytometry, and the results are shown in Figure 5.

[0036] The results in Tables 1 and 2 show that candidate peptide P1 has good water solubility, high helicity, and good affinity for the IL-6 / IL-6R / gp130 complex. Therefore, P1 was selected as the template peptide.

[0037] Figure 2 shows that candidate peptides P1-P6 exhibit a helical structure, which meets the expected design goal. P7 has a tendency to form a ring, and the thiol groups in the cysteine ​​residues at both ends of P7 are easily covalently linked to form a cyclic peptide. Although P8 also has cysteine ​​C residues at both ends, its helical conformation is more stable and its ring formation is relatively weak.

[0038] Figure 3 shows the molecular docking simulation results. P1, P2 and P6 can form receptor-ligand structures at the protein-binding interface in the IL-6 / IL-6R / gp130 complex. All of them exhibit a helical tendency, and P1 has the highest degree of helicity and the largest binding area with the complex protein. The active peptide obtained after P1 sequence optimization also retains high binding capacity.

[0039] Figure 4 shows that the active peptide exhibits a long nanofiber morphology in aqueous solution, indicating its strong self-assembly ability. The interior of the fiber can be used for drug loading to achieve synergistic delivery.

[0040] Figure 5 shows that the active peptides have a good therapeutic effect on senescent cells caused by ischemia-reperfusion of nerve cells in stroke, and can significantly inhibit cell apoptosis and reverse the pathological cell senescence process.

[0041] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0042] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. An active peptide for senescent cells, characterized in that, The active peptide comprises the following raw materials in the following mass ratio: Rink Amide AM resin: Phe: Gly: Leu: Arg: Gln: Lys: Tyr: Trp: Glu: Asn = 10: 24.8: 9.5: 11.3: 20.7: 19.5: 15: 7.4: 8.4: 6.8: 9.5; the amino acid sequence of the active peptide is H2N-FFGFLGRYFQKWEKLRNQ, as shown in SEQ ID. As shown in NO.9, the active peptide was obtained by molecular docking simulation screening and optimization using the helical segment of IL-6R protein that binds to gp130 protein as a peptide template. The specific method is as follows: S1: Selecting a peptide template with 1P9M as the intervention target; S2: Designing 8 candidate peptides based on the peptide template, numbered P1-P8 in sequence; S3: Evaluating and analyzing the properties and structure of candidate peptides P1-P8, and screening out candidate peptides P1, P2 and P6; S4: Performing molecular docking simulation with 1P9M on candidate peptides P1, P2 and P6 respectively, and screening out candidate peptide P1; S5: Optimizing the functional sequence of candidate peptide P1 to design an active peptide for senescent cells.

2. The active peptide for senescent cells according to claim 1, characterized in that, The amino acid sequence of candidate peptide P1 is H2N-RYFQKWEKLRNQ, as shown in SEQ ID NO.1; the amino acid sequence of candidate peptide P2 is H2N-WKRLYEHFQKRN, as shown in SEQ ID NO.2; the amino acid sequence of candidate peptide P3 is H2N-KWEYRFQNRLKK, as shown in SEQ ID NO.3; the amino acid sequence of candidate peptide P4 is H2N-RLFWKQEYNRKK, as shown in SEQ ID NO.4; the amino acid sequence of candidate peptide P5 is H2N-YWKRQEFNRLKK, as shown in SEQ ID NO.5; and the amino acid sequence of candidate peptide P6 is H2N-KRWYQEFKLRNQ, as shown in SEQ ID NO.

6.

3. The active peptide for senescent cells according to claim 2, characterized in that, The amino acid sequence of candidate peptide P7 is H2N-CRYFQKWEKLRNC, as shown in SEQ ID NO.7; the amino acid sequence of candidate peptide P8 is H2N-CWKRLYEHFQKRNC, as shown in SEQ ID NO.

8.

4. A method for preparing an active peptide for senescent cells according to any one of claims 1-3, characterized in that, The specific steps are as follows: Step 1: Take Rink Amide AM resin, swell it, filter it, deprotect it, and wash it to obtain deprotected resin; Step 2: Perform amino acid grafting according to the active peptide sequence starting from the C-terminus, and take Phe, Gly, Leu, Arg, Gln, Lys, Tyr, Trp, Glu and Asn respectively for activation, grafting reaction, identification by ninhydrin method, deprotection, and washing to obtain polypeptide resin; Step 3: Cut the polypeptide resin to obtain crude polypeptide; Step 4: Precipitate the crude polypeptide using ice-cold ether, centrifuge it, discard the supernatant, and obtain the precipitate. The precipitate is then vacuum dried to obtain active peptides for senescent cells.

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