rv3 and rv4 polypeptides that have anti-aging function and their applications

Abstract

A polypeptide capable of prolonging the lifespan of Caenorhabditis elegans is provided. The polypeptide has the amino acid sequence TX1X2AA (where X1X2 is any amino acid, preferably AF / KA) or a pharmaceutically acceptable salt thereof; or, based on the polypeptide sequence, after the deletion, substitution, or addition of one or more amino acids, the polypeptide retains its anti-aging activity, or a pharmaceutically acceptable salt thereof. The polypeptide has the functions of prolonging the lifespan of Caenorhabditis elegans and resisting aging, as well as improving physical performance, increasing stress resistance, and mitigating the decline in physical performance during the aging process.

Description

Technical field The present invention relates to the field of biopharmaceuticals, and specifically to a polypeptide that has anti-aging activity, and to the application thereof. Background Aging is a process in which, after a period of sexual maturity, an organism gradually loses its capacity for self-renewal and cellular repair, its tissues and organs degenerate in structure and function, and ultimately dies. It is characterized by a decreased ability to cope with stress, impaired balance, and an increased risk of developing diseases. With the aging of the world's population, the various degenerative diseases affecting older adults and the immense medical expenses they incur have become increasingly serious social problems. Maintaining the health of the elderly population is key to reducing the social and economic burden associated with population aging.Therefore, it is of great importance to explore simple measures and methods to intervene in aging and age-related diseases that are simple, inexpensive, effective, safe, and suitable for widespread adoption. In earlier research on aging, scientists believed that pure research on aging should be strictly separated from research on age-related diseases. Age-related diseases generally refer to those whose incidence increases with age. Currently, age-related diseases are thought to primarily include cardiovascular disease, tumors, rheumatism, osteoporosis, cataracts, type 2 diabetes, hypertension, Alzheimer's disease, and the like. However, with the advancement of aging research, it has been discovered that the onset and development of various age-related diseases are analogous to the aging process itself, and that the aging process is the underlying risk factor for many age-related diseases.Social responsibility has also led biologists to realize that extending life expectancy alone cannot reduce the heavy social and economic burden of aging, and that only extending healthy lifespan is of practical importance. Therefore, how to reduce age-related diseases, improve the quality of life of older adults, and extend healthy life has become a hot topic in aging research. Currently, there is no single, strict indicator for measuring healthy lifespan. In general, it is believed that anti-aging interventions that can increase the body's stress-resistance capacity, reduce the onset and progression of age-related diseases, and alleviate age-related degeneration, thereby extending lifespan, can be termed healthy life extension. As a classic animal model for aging research, Caenorhabditis elegans has the following advantages. First, it has a relatively short lifespan, only 2-3 weeks under normal laboratory conditions, allowing for lifetime survival analysis; second, it is easy to obtain large numbers of genetically identical animals under controlled environmental conditions; third, its transparent body allows us to directly observe how cells and tissues change with aging; and fourth, knowledge of its cells and tissues, neuronal connections, and complete genomes contributes to anti-aging research.A major advantage of the Caenorhabditis elegans model is that biological information can be readily obtained using genetic methods, thereby identifying a large number of genetic mutations that can modify lifespan. Furthermore, the lifespan of Caenorhabditis elegans exhibits remarkable plasticity and is affected by environmental conditions, nutritional status, and genetic mutations. Individual lifespans can change even under controlled conditions, thus revealing the random factors of aging. Therefore, Caenorhabditis elegans can be used as an animal model to study the anti-aging activity of drugs. By far, the predominant anti-aging approach is the use of anti-aging drugs. Most clinically used anti-aging drugs are synthetic. For example, vitamin E can promote cell division and inhibit the generation of oxygen free radicals; procaine preparations can prolong the lifespan of cells; and piracetam can slow brain aging. Aspirin can slow the decline in bodily functions caused by aging by combating oxidative stress, thereby extending the lifespan of Caenorhabditis elegans. Metformin, as an AMP-activated protein kinase (AMPK) activator, can also alleviate cognitive decline and has some effect on slowing aging. Drugs such as PAL-12 (a hexapeptide) and resveratrol analogs also promote anti-aging. Patent CN104402975A relates to a method for preparing recombinant proteins in the field of genetic engineering, specifically a novel anti-aging short peptide and a method for its preparation. The anti-aging short peptide is named G13 and consists of thirteen amino acids. The invention further discloses a fermentation process using Escherichia coli, whereby the industrial mass production of recombinant G13 (abbreviated rG13) can be carried out. Based on multiple biological screenings, rG13 has greater biological activity compared to similar products, can stimulate cells to secrete large amounts of collagen, and has anti-aging properties.The short anti-aging peptide provided by the invention has the advantages of remarkable biological activity, a simple production process, low cost, broad market application prospects, and the skin's anti-aging nourishing products are useful for incorporating into the daily lives of ordinary people. Document W02004031211A2 provides peptides in certain pathogens and / or human or murine proteins that are identified as capable of binding to one or more MHC molecules and inducing an immune response in a system. Compositions comprising one or more of the peptides and methods for inducing an immune response in a system by administering the compositions to the system are also provided. WO9202543A1 provides peptide compositions that bind to MHC molecules of interest and inhibit T-cell activation. The peptides are 4–25 amino acid residues in length and have a core binding region comprising, from the amino to the carboxyl terminus, a hydrophobic L-amino acid or amino acid mimetic, a spacer sequence of 2–6 residues, and Thr or a Thr mimetic. At least one residue is a D-amino acid or amino acid mimetic. The compositions can be used to treat diseases associated with particular DR alleles, including autoimmune diseases such as rheumatoid arthritis. ELBAUM MICHAEL B. ET AL. discloses the peptide Ac-TKAAAAKAAAAKAAGY-NH2 (Figure 4). The structural effects of OGlcNAcylation and phosphorylation on α-helices were investigated in this document (BIOCHEMISTRY, vol. 53, no. 14, April 15, 2014 (2014-04-15), pages 2242-2260). Summary 1. Problem to be solved The present invention provides a polypeptide with anti-aging activity and its application. In the present invention, the polypeptide can effectively prolong the lifespan of Caenorhabditis elegans, exhibiting a good anti-aging effect and great potential for development. 2. Technical solutions To solve the above problem, the technical solutions adopted by the present invention are as follows: A polypeptide having anti-aging activity, wherein the amino acid sequence of the polypeptide is TAFAA or TKAAA. A polypeptide having anti-aging activity is further described, or pharmaceutically acceptable salts thereof are provided, characterized in that it has an amino acid sequence of X-TX1X2AA, where X = H, acetyl or propionyl, and X1 and X2 are any amino acids. For the polypeptide that has anti-aging activity, X1X2 is AF or KA. For the polypeptide that has anti-aging activity, the amino acid sequence of the polypeptide is TAFAA (called RV3) or TKAAA (called RV4). The use of the polypeptide to prepare anti-aging medicines or supplements is described. When necessary, one or more pharmaceutically acceptable adjuvants may also be added to the above drugs, and the adjuvant includes a diluent, filler material, binder, wetting agent, absorption enhancer, surfactant, lubricant, and stabilizer that are conventional in the pharmaceutical field. In the present invention, the drugs can be prepared in different forms: injection, lyophilized powder injection, tablet, or granule. The drugs can be prepared in these different forms using conventional methods in the pharmaceutical field. 3. Beneficial effects Compared to the prior art, the present invention has the following beneficial effects: (1) The polypeptide of the present invention has a novel structure with the basic unit of natural amino acids, which is easy to synthesize, separate and purify; (2) The polypeptide of the present invention can effectively prolong the life expectancy of Caenorhabditis elegans and has anti-aging activity; (3) The polypeptide of the present invention is safe, with few adverse reactions and toxic side effects, does not affect the growth and development of Caenorhabditis elegans, and does not affect the reproductive capacity of Caenorhabditis elegans; and (4) The anti-aging effect of the polypeptide involved in the present invention performs well in the Caenorhabditis elegans model, specifically resulting in a significant improvement in the movement behavior capacity of Caenorhabditis elegans, relief from the decrease in movement capacity in the aging process of Caenorhabditis elegans, prolongation of the time to semi-death of Caenorhabditis elegans, improvement of the anti-stress capacity of Caenorhabditis elegans, and prolongation of the life expectancy of Caenorhabditis elegans. Brief description of the drawings Figure 1 shows a survival curve from the experiment on the effect of the polypeptide on the lifespan of Caenorhabditis elegans. The figure shows that the RV4 polypeptide group has the effect of significantly prolonging the lifespan of Caenorhabditis elegans. Figure 2 shows the results of the effect of the polypeptide on the body length and width of Caenorhabditis elegans. Panel A shows the results for body length, and panel B shows the results for body width. Results are expressed as means ÷ SEM. Compared to the control group, ns represents no significant difference, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. Compared to the control group, the polypeptide groups showed no significant differences, indicating that the RV3 and RV4 polypeptides do not affect the normal growth and development of Caenorhabditis elegans and that the polypeptides are safe. Figure 3 shows the results of the polypeptide effect on the number of offspring of Caenorhabditis elegans. Results are expressed as means ± SEM. Compared to the control group, ns represents no significant difference, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. Compared to the control group, the polypeptide groups showed no significant differences, indicating that the RV3 and RV4 polypeptides do not affect the reproductive capacity of Caenorhabditis elegans and that the polypeptides are safe. Figure 4 shows the results of the polypeptide's effect on the movement behavior of Caenorhabditis elegans. Panel A shows the results for head movements, panel B shows the results for body flexion, and panel C shows the results for pharyngeal pumping frequency. Results are expressed as mean ÷ SEM. Compared to the control group, ns represents no significant difference, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. Compared to the control group, the group treated with the RV4 polypeptide showed a significant difference, indicating that the RV4 polypeptide can significantly enhance the muscle movement of Caenorhabditis elegans and improve the nematode's movement behavior. Figure 5 shows a survival curve from the acute heat stress experiment of Caenorhabditis elegans. Panel A shows a survival curve from the acute heat stress experiment for nematodes on day 4, and panel B shows a survival curve from the acute heat stress experiment for nematodes on day 8. The figure shows that the polypeptide groups RV3 and RV4 can significantly improve the acute heat stress resistance capacity of nematodes, indicating that polypeptide drugs can prolong the life cycle of nematodes in a thermal environment. Figure 6 shows the results of the classification of the movement capacity of Caenorhabditis elegans in the acute heat stress experiment. Panel A shows the classification results for the movement capacity of the nematodes in the acute heat stress experiment on Day 4, and Panel B shows the classification results for the movement capacity of the nematodes in the acute heat stress experiment on Day 8. The figure shows that on Day 4, the proportion of grade A nematodes in the RV3 and RV4 polypeptide groups is higher than in the control group, and on Day 8, the proportion of grade C nematodes in the RV3 and RV4 polypeptide groups is lower than in the control group. This indicates that the polypeptide drugs can enhance the ability of Caenorhabditis elegans to withstand acute heat stress, that is, its ability to move in a thermal environment. Figure 7 shows the detection results of the experiment on acute oxidative stress in Caenorhabditis elegans. Panel A shows the detection results for Day 4, and Panel B shows the detection results for Day 8. Results are expressed as means ± SEM. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. The figure shows that the survival rate of nematodes in the group treated with the RV4 polypeptide is significantly higher than that of the control group, indicating that polypeptide drugs can prolong the life cycle of nematodes in an oxidative environment. Figure 8 shows the results of the classification of the movement capacity of Caenorhabditis elegans in the acute oxidative stress experiment. Panel A shows the classification results for the movement capacity of the nematodes in the acute oxidative stress experiment on Day 4, and panel B shows the classification results for the movement capacity of the nematodes in the acute oxidative stress experiment on Day 8.From the figure, it can be observed that, in the Day 8 acute oxidative stress experiment, the proportion of grade A nematodes in the RV3 and RV4 polypeptide groups is higher than in the control group, indicating that polypeptide drugs can enhance the capacity to withstand acute oxidative stress in Caenorhabditis elegans; and in the Day 4 acute oxidative stress experiment, there is no difference in the ability to move between the polypeptide groups and the control group. Figure 9 shows the results of the classification of the movement capacity of Caenorhabditis elegans at different ages. Panel A shows the classification results for the movement capacity of the nematodes on Day 4, Panel B shows the classification results for the movement capacity of the nematodes on Day 8, and Panel C shows the classification results for the movement capacity of the nematodes on Day 12. The figure shows that, compared to the control group, in the RV3 and RV4 polypeptide groups on Day 8 and Day 12, the proportion of nematodes in grade C is lower than in the control group, indicating that the polypeptide may delay muscle aging in Caenorhabditis elegans and improve the movement capacity of the nematodes, thereby extending their lifespan. Figure 10 shows the results of detecting the motility of Caenorhabditis elegans at different ages. Panel A shows the results of detecting the motility of the nematodes on Day 4, panel B shows the results of detecting the motility of the nematodes on Day 8, and panel C shows the results of detecting the motility of the nematodes on Day 12. Results are expressed as means ± SEM. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference.From the figure, it can be observed that, compared to the control group, the frequency of head movements in the group with the RV4 polypeptide is significantly higher than in the control group, and the frequency of pharyngeal pumping is also higher than in the control group, indicating that the RV4 polypeptide can delay muscle aging in Caenorhabditis elegans, improve the movement behavior capacity of the nematodes and alleviate the decrease in the movement capacity of the nematodes, thereby prolonging the life expectancy of the nematodes. Detailed description The present invention is described in more detail below with reference to specific examples. Polypeptide RV3 (TAFAA) and polypeptide RV4 (TKAAA) were synthesized by the Engineering Research Center of Synthetic Polypeptide Drug Discovery and Evaluation in Jiangsu Province, with a purity of 97.30% and 93.85%, respectively. Example 1 Experiment on the effect of the polypeptide on the lifespan of the Caenorhabditis elegans model 1. Materials E. coli OP50 cultured under the following conditions: an incubation oven with shaking, 220 rpm and 37 °C. Caenorhabditis elegans naturally cultivated under the following conditions: an incubation oven at constant temperature and humidity, 20°C and a humidity of 45-55%. 5-FUDR: 15, 6 µg / ml. NaN3: 0.4 M. H2O2: 30 mM. RV3: Thr-Ala-Phe-Ala-Ala, 10 nM. RV4: Thr-Lys-Ala-Ala-Ala, 10 nM. 2. Method Synchronization of Caenorhabditis elegans: A nematode-rich medium (NGM) plate with a moderate density was selected. L4 larvae were collected using a grafting needle, approximately 20 in total, and transferred to a blank NGM plate. After they matured into adults and laid their first clutch of eggs, all adults were collected. Approximately 12 hours later, the eggs hatched into larvae, resulting in synchronized L1 larvae. Preliminary preparation for the Caenorhabditis elegans lifespan experiment: The NGM was washed with 1 ml of M9 buffer. The resulting buffer, containing L1-stage larvae, was collected in an EP tube, placed in a chromatographic chamber at 4 °C for 5 min, and centrifuged at 1500 × g / 4 °C for 3 min. The supernatant was discarded. After resuspending 100 µl of buffer, the nematodes were counted under a microscope and diluted to a concentration of 30 worms / 10 µl. Ten µl of the diluted nematodes were added to the NGM plate inoculated with E. coli OP50, and the nematodes were counted under a microscope to ensure approximately 30 nematodes were present. The NGM plate was then placed in a constant-temperature incubator for culture. Caenorhabditis elegans Lifespan Experiment: After L1-stage nematodes developed to L4, 100 µl of 15.6 µg / ml 5-FUDR was added to inhibit oviposition. Once the nematodes reached adulthood, administration began, and the nematodes were divided into a control group and a 10 nM dose group, recorded as day 1 of the lifespan experiment. Administration was then performed daily, and the number of nematodes that survived, died, and died unexpectedly was observed and recorded each day, along with their condition, until the last nematode died.The criteria for determining nematode death are that Caenorhabditis elegans does not respond to bright light or tapping on the plate, and does not move its pharyngeal muscles under a high-power microscope. Finally, the nematode's head is tapped with a grafting needle; if there is still no response, the nematode is considered dead. Dead nematodes must be collected from the plate. E. coli OP50 should be added promptly when it is depleted. A Kaplan-Meier statistical analysis was performed on the data, and results are expressed as median ± SE. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results (1) Record of the results of the Caenorhabditis elegans life expectancy experiment: Table 1 Recorded results of the number of remaining Caenorhabditis elegans Time / day Number of nematodes remaining in each group RV3 RV4 Control 0 30 27 33 1 30 27 33 2 30 27 33 3 30 27 33 4 30 27 33 5 30 27 33 6 30 27 33 7 30 27 33 8 30 27 33 9 29 27 33 10 28 27 33 11 28 27 33 12 28 27 33 13 27 26 32 14 23 26 32 15 23 26 32 16 22 26 32 (continuation) Time / day Number of nematodes remaining in each group RV3 RV4 Control 17 22 25 30 18 20 21 29 19 16 14 26 20 15 13 26 21 13 12 25 22 9 11 22 23 8 8 19 24 8 5 16 25 7 4 16 26 6 3 12 27 3 2 9 28 1 2 6 29 0 1 6 30 0 1 4 31 0 0 3 32 0 0 2 33 0 0 1 34 0 0 0 Compared to the control group, the day of death in the RV4 polypeptide group was delayed, and the nematodes had a longer lifespan of 33 days, 5 days longer than the control group. Similarly, compared to the control group, the day of death in the RV3 polypeptide group was delayed, and the nematodes had a longer lifespan of 30 days, 2 days longer than the control group. For further details, see Table 1 and Figure 1. (2) Time to semi-death of Caenorhabditis elegans: Table 2 Results of the detection of time to semi-death of Caenorhabditis elegans Control Group RV3 RV4 Time to 20,000 ± 1,174 20,000 ± 0,865 24,000 ± 1,230** semi-death / day Value of P - 0.726 0.003 Note: The above results are expressed as Median ± SE. Compared to the control group, ***P<0.05 and ****P<0.01. Compared to the control group, the time to half-life in the RV4 group showed a highly significant difference, indicating that the RV4 polypeptide significantly prolongs the lifespan of Caenorhabditis elegans, extending it by 4 days. In contrast, the time to half-life in the RV3 group did not show a significant difference compared to the control group. Referring to Table 2 and Figure 1 for further details, the experimental results are statistically significant. Example 2 Experiment on the effect of the polypeptide on the growth and development of Caenorhabditis elegans 1. Materials Same as in Example 1. 2. Method Synchronization of Caenorhabditis elegans: Same as in Example 1. Experiment on the growth and development of Caenorhabditis elegans: Synchronized L1 stage larvae were collected and seeded on solid NGM inoculated with E. coli OP50. The nematodes were divided into a control group and a 10 nM dose group, and placed in a constant temperature and humidity incubation oven to grow them for 72 h, and the length and width of the body of the adult stage nematodes were detected with a detection method such as the following. Twenty Caenorhabditis elegans were collected from each group and placed in a new solid NGM, and 50 µl of NaN30, 4 M was added dropwise. After most of the nematodes were rigid, the nematodes were photographed and recorded with an inverted microscope, and the body length and width of Caenorhabditis elegans were measured using the Photoshop ruler tool.A one-way ANOVA statistical analysis was performed on the data, and the results are expressed as Mean ± SEM. Compared with the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results Table 3 Effect of the polypeptide on the length and width of the body of Caenorhabditis elegans Control Group RV3 RV4 Body length / µm 1256, 974 ± 9, 316 1275, 948 ÷ 19, 674 1230, 070 ± 21, 279 P value (body length) - 0, 934 0, 798 Body width / µm 63, 647 ± 0, 868 62, 328 ± 0, 892 62, 554 ± 0, 926 P value (body width) - 0, 823 0, 902 Note: The above results are expressed as Means ± SEM. Compared with the control group, ***P<0.05 and ****P<0.01. Compared to the control group, the length and width of the Caenorhabditis elegans body in the polypeptide groups did not differ significantly, meaning that neither the RV3 nor RV4 polypeptides affected the normal growth and development of Caenorhabditis elegans, indicating that these polypeptides are safe. Referring to Table 3 and Figure 2 for further details, the experimental results were statistically significant. Example 3 Experiment on the effect of the polypeptide on the reproductive capacity of Caenorhabditis elegans 1. Materials Same as in Example 1. 2. Method Synchronization of Caenorhabditis elegans: Same as in Example 1. Detection of the reproductive capacity of Caenorhabditis elegans: Synchronized L1 larvae were collected and plated onto solid NGM inoculated with E. coli OP50. The nematodes were divided into a control group and a 10 nM dose group, and placed in an incubator at constant temperature and humidity to culture to L4 larvae. One L4 larva was collected from each group and placed on a fresh solid NGM. The nematode was transferred to a fresh medium daily during the egg-laying period. The culture medium containing the eggs was incubated for 24 h. The larvae on each plate were counted until the end of the nematode's egg-laying period. The data were summed to obtain the total number of nematode offspring. The number of offspring of 10 nematodes from each group was recorded.A one-way ANOVA statistical analysis was performed on the data, and the results are expressed as Mean ± SEM. Compared with the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results Table 4 Effect of the polypeptide on the number of offspring of Caenorhabditis elegans Control Group RV3 RV4 Total number of offspring 289, 70 ± 14, 827 266, 60 ± 12, 125 287, 40 ± 12, 295 P-value - 0.750 1.000 Note: The above results are expressed as Means ± SEM. Compared to the control group, ***P<0.05 and ****P<0.01. Compared to the control group, the total number of offspring of Caenorhabditis elegans in the polypeptide groups was not significantly different, meaning that neither the RV3 nor RV4 polypeptides affected the reproductive capacity of Caenorhabditis elegans, indicating that these polypeptides are safe. Referring to Table 4 and Figure 3 for further details, the experimental results were statistically significant. Example 4 Experiment on the effect of the polypeptide on the movement behavior capacity of Caenorhabditis elegans 1. Materials Same as in Example 1. 2. Method Synchronization of Caenorhabditis elegans: Same as in Example 1. Detection of movement behavioral capacity in Caenorhabditis elegans: Synchronized L1-stage larvae were collected and seeded on solid NGM inoculated with E. coli OP50. The nematodes were divided into a control group and a 10 nM dose group and placed in an incubator at constant temperature and humidity for 48 h. The following three indicators of movement behavioral capacity were then detected: head movement frequency, body flexion frequency, and pharyngeal pumping frequency. For head movement frequency, 20 nematodes were collected from each group and transferred to fresh, clean culture medium for acclimation for 1 h. An appropriate amount of M9 buffer was then added, and the number of times the nematode's head moved from side to side and vice versa was observed and recorded using an inverted microscope for 30 s.For body flexion frequency, 20 nematodes were collected from each group and transferred to a new, clean culture medium for acclimatization for 1 h. The number of times the nematode's body flexed during 30 s was observed and recorded using an inverted microscope, and the distance the nematode crawled forward at one wavelength was recorded as one body flexion. For pharyngeal pumping frequency, 20 nematodes were collected from each group and transferred to a culture medium inoculated with E. coli OP50 for acclimation for 1 h. Under an inverted microscope, pharyngeal pumping was observed at sufficient magnification until clearly visible, with photographs taken for 30 s for each nematode. The number of pharyngeal pumping events was counted by slow-playing video at 0.3 times speed using PotPlayer. A one-way ANOVA was performed on the data, and results are expressed as mean ± SEM. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results Table 5 Effect of the polypeptide on the movement behavior capacity of Caenorhabditis elegans Control Group RV3 RV4 Frequency of head movements / 30 s 56, 60 ± 1, 690 59, 10 ± 1, 474 63, 40 ± 1, 297* P-value (head movements) - 0.755 0.014 Body flexion frequency / 30 s 12.90 ÷ 0.397 12.70 ± 0.411 14.75 ± 0.354* P-value (body flexion) - 0.997 0.015 Pharyngeal pumping frequency / 30 s 162.95 ± 2.318 160.80 ± 2.592 175.15 ± 3.368** P-value (pumping frequency) - 0.635 0.008 pharyngeal) Note: The above results are expressed as Means ± SEM. Compared with the control group, ***P<0.05 and ****P<0.01. Compared to the control group, the group treated with polypeptide RV4 showed a significant difference, indicating that RV4 can significantly enhance muscle movement in Caenorhabditis elegans and improve the nematode's movement behavior. Compared to the control group, the group treated with polypeptide RV3 did not show a significant difference. Referring to Table 5 and Figure 4 for further details, the experimental results are statistically significant. Example 5 Experiment on stress in the Caenorhabditis elegans model 1. Materials Same as in Example 1. 2. Method Synchronization of Caenorhabditis elegans: Same as in Example 1. Preliminary preparation for the Caenorhabditis elegans stress experiment: The same as for the life expectancy experiment in Example 1. Acute Heat Stress Experiment in Caenorhabditis elegans: Caenorhabditis elegans that were continuously fed until day 4 were placed in an incubator at a constant temperature of 35 °C for the stress experiment. The survival or death status of the nematodes was recorded every 4 h until the last nematode died. The criterion for determining whether a nematode died was the same as that used in the life expectancy experiment. In addition, 24 h after the start of the experiment, the movement behavior of surviving Caenorhabditis elegans was classified into three grades A, B, and C according to the following criteria: A for crawling autonomously, C for moving only after the head was touched with a grafting needle, and B for being in between. Nematodes that were continuously fed until day 8 were placed in an incubator at a constant temperature of 35 °C.The survival or death status of the nematodes was recorded every 2 h until the last nematode died. The criteria for determining whether a nematode had died and the classification criteria were the same as those used in the heat stress experiment on day 4. A Kaplan-Meier statistical analysis was performed on the data, and the results are expressed as median ± SE. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. Acute oxidative stress experiment in Caenorhabditis elegans: Caenorhabditis elegans that were continuously administered until day 4 and then soaked in 30 mM H₂O for 4 h on day 8 were transferred to a solid NGM for acclimatization for 24 h. The number of surviving and dead Caenorhabditis elegans was recorded under a microscope, and the movement behavior of the surviving nematodes was classified. The criteria for determining nematode death and the classification criteria were the same as those used in the acute heat stress experiment. A one-way ANOVA was performed on the data, and the results are expressed as mean ± SEM. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results (1) Record of the results of the Caenorhabditis elegans heat stress experiment: Table 6 Recorded results of the number of Caenorhabditis elegans remaining under heat stress on Day 4 Time / h Number of nematodes remaining in each group RV3 RV4 Control 0 25 23 22 4 25 23 22 8 25 23 22 12 22 22 21 16 17 22 20 20 13 19 16 24 8 14 11 28 2 8 4 32 0 2 0 36 0 0 0 Table 7 Recorded results of the number of Caenorhabditis elegans remaining under heat stress on Day 8 Number of nematodes remaining in each group Time / h RV3 RV4 Control 0 21 18 24 2 21 18 24 4 18 17 24 6 13 16 23 8 10 14 17 10 10 14 17 12 4 11 10 14 2 7 7 16 0 2 4 18 0 1 1 20 0 0 0 The results of the acute heat stress experiment on Day 4 show that, compared to the control group, there is no difference in the time to death in the groups treated with polypeptides RV3 and RV4. However, the longest survival time for nematodes in the RV3 group is 32 h, 4 h longer than that of the control group. The results of the acute heat stress experiment on Day 8 show that, compared to the control group, the time to death is delayed in the RV4 group, and the longest survival time for nematodes in both dose groups is 18 h, 4 h longer than that of the control group. See Table 6, Table 7, and Figure 5 for further details. (2) Time to semi-death of Caenorhabditis elegans in the heat stress experiment: Table 8 Results of the detection of time to semi-death of Caenorhabditis elegans with heat stress on Day 4 Control Group RV3 RV4 Time to 24,000 ± 2,073 28,000 ± 1,523** 24,000 ± 1,563 semi-death / h P-value - 0.005 0.104 Note: The above results are expressed as Median ± SE. Compared to the control group, ***P<0.05 and ****P<0.01. Table 9 Results of the detection of time to semi-death of Caenorhabditis elegans with heat stress on Day 8 Control Group RV3 RV4 Time until 8,000 ± 1,526 14,000 ± 1,034** 12,000 ± 1,380* half-death / h Value of P - 0.006 0.011 Note: The above results are expressed as Median ± SE. Compared to the control group, ***P<0.05 and ****P<0.01. The results of the Day 4 acute heat stress experiment show that, compared to the control group, the time to semi-death in the group treated with polypeptide RV3 is significantly different, while no difference is observed in the group treated with polypeptide RV4. The results of the Day 8 acute heat stress experiment show that, compared to the control group, the time to semi-death in both the RV3 and RV4 polypeptide groups is significantly different, indicating that RV3 and RV4 can significantly improve the nematodes' ability to withstand acute heat stress. The time to semi-death in the dosage groups is 4–6 hours longer than that of the control group under 35°C heat stress. See Table 8, Table 9, and Figure 5 for further details; the experimental results are statistically significant. (3) Classification of the movement capacity of Caenorhabditis elegans in the heat stress experiment: Table 10 Results of detection of the classification of the movement capacity of Caenorhabditis elegans under heat stress on Day 4 Proportion of nematodes in the different grades after 24 h / % Grade RV3 RV4 Control A 0, 00 13, 33 23, 08 B 62, 50 40, 00 23, 08 C 37, 50 46, 67 53, 85 Note: A for nematodes capable of crawling autonomously, C for nematodes that begin to move only after touching the head with a grafting needle, and B for nematodes between the two states. Table 11 Results of detection of the classification of movement capacity of Caenorhabditis elegans with heat stress on Day 8 Proportion of nematodes in the different grades after 6 h / % Grade RV3 RV4 Control A 26, 32 28, 00 29, 63 B 42, 11 56, 00 51, 85 C 31, 58 16, 00 18, 52 Note: A for nematodes capable of crawling autonomously, C for nematodes that begin to move only after touching the head with a grafting needle, and B for nematodes between the two states. Based on the Day 4 heat stress mobility classification results, the proportion of nematodes in grade A was higher in the RV3 and RV4 polypeptide groups than in the control group. Based on the Day 8 heat stress mobility classification results, the proportion of nematodes in grade C was lower in the dose groups than in the control group. This suggests that polypeptide drugs may enhance the ability of Caenorhabditis elegans to withstand acute heat stress. See Table 10, Table 11, and Figure 6 for further details. (4) Detection results of the experiment on oxidative stress of Caenorhabditis elegans'. Table 12 Results of the detection of the survival rate of Caenorhabditis elegans with oxidative stress on Day 4 Control Group RV3 RV4 Survival Rate 0, 775 ± 0, 040 0, 831 ± 0, 063 0, 960 ± 0, 040* Value of P - 0.362 0.011 Note: The above results are expressed as Means ± SEM. Compared with the control group, ***P<0.05 and ****P<0.01. Table 13 Results of the detection of the survival rate of Caenorhabditis elegans with oxidative stress on Day 8 Control Group RV3 RV4 Survival Rate 0.664 ± 0.058 0.809 ± 0.072 0.868 ± 0.010* P-value - 0.051 0.011 Note: The above results are expressed as Means ± SEM. Compared to the control group, ***P<0.05 and ****P<0.01. Based on the results of the oxidative stress experiment on Day 4 and Day 8, it appears that, compared to the control group, the survival rate of the nematodes in the group treated with the RV4 polypeptide is significantly different, indicating that RV4 polypeptide drugs can significantly enhance the ability of Caenorhabditis elegans to withstand acute oxidative stress. Referring to Table 12, Table 13, and Figure 7 for further details, the experimental results are statistically significant. (5) Classification of the movement capacity of Caenorhabditis elegans in the oxidative stress experiment: Table 14 Results of detection of the classification of the movement capacity of Caenorhabditis elegans with oxidative stress on Day 4 Proportion of nematodes in the different grades after 24 h / % Degree RV3 RV4 Control A 47, 06 64, 71 52, 00 B 41, 18 23, 53 36, 00 C 11, 76 11, 76 12, 00 Note: A for nematodes capable of crawling autonomously, C for nematodes that begin to move only after touching the head with a grafting needle, and B for nematodes between the two states. Table 15 Results of detection of the classification of movement capacity of Caenorhabditis elegans with oxidative stress on Day 8 Proportion of nematodes in the different grades after 24 h / % Grade RV3 RV4 Control A 0, 00 29, 03 34, 62 B 71, 43 48, 39 46, 15 C 28, 57 22, 58 19, 23 Note: A for nematodes capable of crawling autonomously, C for nematodes that begin to move only after touching the head with a grafting needle, and B for nematodes between the two states. Based on the Day 8 oxidative stress motility classification results, the proportion of nematodes in grade A was higher in the groups treated with polypeptides RV3 and RV4 than in the control group, indicating that polypeptide drugs may enhance the ability of Caenorhabditis elegans to withstand acute oxidative stress. The Day 4 oxidative stress motility classification results showed no difference in motility between the polypeptide groups and the control group. See Table 14, Table 15, and Figure 8 for further details. Example 6 Experiment on the decrease in the movement capacity of the Caenorhabditis elegans model 1. Materials Same as in Example 1. 2. Method Synchronization of Caenorhabditis elegans: Same as in Example 1. Preliminary preparation of the experiment on the decrease in the ability to move of Caenorhabditis elegans: The same as that of the experiment on the life expectancy of Example 1. Experiment on the decreased mobility of Caenorhabditis elegans: Caenorhabditis elegans was continuously administered until day 4. On days 8 and 12, samples were collected to assess the nematodes' mobility behavior, including head movement frequency and pharyngeal pumping frequency. The assessment method was the same as that used for assessing mobility in Example 4. In addition, the mobility of surviving nematodes was classified. The classification criteria were the same as those used for classifying mobility in Example 5. A one-way ANOVA was performed on the data, and the results are expressed as Mean ÷ SEM. Compared to the control group, *P<0.05 represents a significant difference, and **P<0.01 represents a highly significant difference. 3. Experimental results (1) Results of the classification of the mobility capacity of Caenorhabditis elegans with different survival days: Table 16 Results of the classification on the movement capacity of Caenorhabditis elegans at different ages Proportion of nematodes in the different grades after 24 h / % Time Grade RV3 RV4 Control A 97, 30 97, 56 100, 00 Day 4 B 2, 70 2, 44 0, 00 C 0, 00 0, 00 0, 00 A 36, 36 38, 10 60, 00 Day 8 B 18, 18 47, 62 16, 00 C 45, 45 14, 29 24, 00 A 28, 57 44, 44 57, 14 Day 12 B 28, 57 33, 33 14, 29 C 42, 86 22, 22 28, 57 Note: A for nematodes capable of crawling autonomously, C for nematodes that begin to move only after touching the head with a grafting needle, and B for nematodes between the two stages. Based on the results of the nematode mobility classification on Day 8 and Day 12, it appears that in the groups treated with polypeptides RV3 and RV4, the proportion of nematodes in grade C is lower than in the control group; and in the group treated with polypeptide RV4, the proportion of nematodes in grade A is higher than in the control group. This indicates that the polypeptide may delay muscle aging in Caenorhabditis elegans and improve the nematodes' mobility, thereby extending their lifespan. For further details, see Table 16 and Figure 9. (2) Results of the detection of the movement capacity of Caenorhabditis elegans with different survival days: Table 17 Results of the detection of the movement capacity of Caenorhabditis elegans at different ages Day Detection indicator Control RV3 RV4 Frequency of movements of the 59, 400 ± 1, 413 58, 750 ± 1, 372 69, 050 ± 1, 739** head / 30 s Value of P - 0, 750 0, 000 Day 4 Pumping frequency Pharyngeal / 30 s 127, 300 ± 7, 262 108, 650 ± 7, 924 147, 444 ± 6, 132* P-value - 0, 061 0, 049 Frequency of movements of the 32, 200 ± 1, 950 35, 800 ± 2, 174 42, 733 ± 2, 194** head / 30 s Value of P - 0.259 0.001 Day 8 Pumping frequency pharyngeal / 30 s 82, 710 ± 11, 437 89, 180 ± 5, 634 105, 330 ± 6, 346 P value - 0, 595 0, 079 (continued) Day Detection Indicator Control RV3 RV4 Frequency of movements of the 9, 250 ± 1, 161 14, 500 ± 3, 878 23, 750 ± 3, 619** head / 30 s Value of P - 0.262 0.003 Day 12 Pumping frequency Pharyngeal / 30 s 68, 500 ± 2, 901 75, 000 ± 13, 103 96, 500 ± 8, 109 P-value - 0, 641 0, 068 Note: The above results are expressed as Means ± SEM. Compared with the control group, ***P<0, 05 and ****P<0, 01. Based on the results of the nematode movement capacity assessments on Day 4, Day 8, and Day 12, it appears that, compared to the control group, the group treated with polypeptide RV4 has a significant difference in head movement frequency and a significant difference in pharyngeal pumping frequency on Day 4. The pharyngeal pumping frequency is not significantly higher than that of the control group on Day 8 and Day 12, indicating that the polypeptide may delay muscle aging in Caenorhabditis elegans, improve the nematodes' movement capacity, and alleviate the decline in their mobility, thereby prolonging their lifespan. Furthermore, there are no significant differences between the group treated with polypeptide RV3 and the control group. (See Table 17 and Figure 17.)10 For more details, the experimental results are statistically significant.

Claims

1. A polypeptide having anti-aging activity, wherein the amino acid sequence of the polypeptide is TAFAA or TKAAA.

2. The polypeptide according to claim 1 for use as an anti-aging drug or supplement.

3. The polypeptide according to claim 2 for use as an anti-aging drug or supplement, wherein one or more pharmaceutically acceptable adjuvants are added to the polypeptide, and the adjuvant comprises a diluent, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant, and stabilizer that are conventional in the pharmaceutical field.

4. The polypeptide according to claim 3 for use as an anti-aging drug or supplement, wherein the polypeptide is prepared as an injection, a lyophilized powder injection, a tablet, or a granule.

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