Sea cucumber peptides were provided by Hangzhou Kang Yuan Food Technology Co Ltd (Hangzhou, CHN), Metformin (Met), β-nicotinamide mononucleotide (NMN), Methyl viologen (paraquat), and 5-fluoro-2'-deoxyuridine (FUDR) and were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, CHN). Total superoxide dismutase (SOD) assay kit, catalase (CAT) assay kit and malondialdehyde (MDA) assay kit were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, CHN). Reactive oxygen species (ROS) was obtained from Vigorous Biotechnology Beijing Co., Ltd. (Beijing, CHN).

C. elegans (wild-type N2), and mutant strains CF1038 [daf-16(mu86)I], TJ356 [zIs356 IV (daf-16p::daf-16a/b::GFP + rol-6(su1006))], CB1370 [daf-2(e1370)III], CF1553 [(pAD76) sod-3p::GFP], SN5[old-1(mk1)II], BR2742[pept-1(lg601)X.], RB1159[tyr-3(ok1194)I.], RB1985[acox-1.5(ok2619) III.].were purchased from the Caenorhabditis Genetics Center (CGC). The expression of Green Fluorescent Protein (GFP) and the lifespan of the mutant nematodes were recorded. Escherichia coli OP50 (E. coli OP50) was obtained from SunyBiotech.

Gel exclusion chromatography in HPLC was used to TSK-GEL G2500PWXL as the separation column to analyze the molecular weight distribution of SCPs. The amino acid composition of SCPs was measured by HPLC (Thermo Scientific, Waltham, USA). Briefly, 20 mg of samples were dissolved in 6 M HCl (1 mL), followed by hydrolysis at 150°C for 1.5 h. After the completion of hydrolysis, derivation. After filtration with a 0.22 μm membrane filter, the sample was analyzed by HPLC on the AQ-C18 column (4.6 × 250 mm, Welch Co., Ltd, Shanghai, China) at 254 nm using the mobile phase of A (acetic acid/sodium acetate buffer solution) and B (80% acetonitrile) at 1.0 mL/min.

The L4 stage of nematodes was transferred to NGM plates containing SCPs-L (0.0625 mg/mL), SCPs-M (0.125 mg/mL), SCPs-H (0.25 mg/mL), Metformin (Met, 40 μmol/L), β-nicotinamide mononucleotide (NMN, 0.25 mg/mL) and FUDR (1 mg/mL) to prevent spawning. For the synchronization of C. elegans, put the pregnant adults in the NGM medium coated with OP50 and observe their oviposition status. The pregnant adults are picked out after oviposition, and the eggs are cultured for 48 h to L4 stage adults at 20°C to get the same-aged worms for further experiments (16).

All lifespan assays were carried out at 20°C. The transfer day was designated as day 0. Worms were transferred another day to fresh extracts or control plates until day 7, which was thought to be the adulthood of the C. elegans. During the experiment, worm bags and abnormal dead worms were removed. Triplicate plates were used for each group (30 per plate). During the lifespan test, the movements of the C. elegans were recorded at 18, 20, 22, and 24 days.

Same-aged C. elegans were picked onto the medicated plates and cultivated for 7 days. For the thermotolerance assay, nematodes were placed in 37°C conditions for 13 h and then the number of dead worms was counted. Triplicate plates were used for each group (30 per plate). To test the resistance of C. elegans to paraquat, nematodes were exposed to 140 mM paraquat. The number of surviving worms was counted every 2 h until all the worms died.

To determine the lipofuscin level, on 13 days of treatment, worms treated with SCPs were placed on 1% agarose pads on glass slides and anesthetized with Levamisole Phosphate (6.75 μM). The worms were visualized under a fluorescence microscope (Axio Scope. A1, American), and were measured using Image J 1.8.0 software.

After day 7 of treatment with SCPs, the nematodes were washed with nutrient buffers (M9), and then 100 μL of the prepared ROS fluorescent agent was added for fluorescent excitation at 20°C for 5 h. The worms were visualized under a fluorescence microscope (Axio Scope. A1, American). The fluorescence intensity of the worms was measured using Image J 1.8.0 software (24).

The nematodes treated by SCPs were lysed with double distilled water in an ice bath after 7 days. The activities of SOD, MDA, and CAT content were determined (25).

Total RNAs of nematodes administration (7 days) were isolated using an RNA extraction kit (Aidlab Biotechnologies Co. Ltd, Beijing, China) and cDNAs were synthesized using a reverse transcription kit (Takara, Biotechnology Co. Ltd, Dalian, China). The qPCR reaction was performed on a qTOWER 2.0 PCR system (Light Cycler, Switzerland) using SYBR Green PCR Master Mix (Takara). Relative expression levels of genes were calculated using the 2^−ΔΔCT method, and the gene act-1 was set as the internal reference, shown in Table 1. The best primers for genes of C. elegans were obtained from the qPrimerDB-qPCR Primer Database (26).

Data are reported as means ± standard deviations (SD) or standard errors (SEM). Assays for each experiment were performed independently at least three times. At least three separate assays were run for each experiment. The Kaplan-Meier test was used to plot survival curves using GraphPad Prism 8.0 (GraphPad Software, Inc., CA, USA), and SPSS 21.0 was used to do the log-rank test. One-way analysis of variance (ANOVA) analyses with the Tukey-Kramer test or t-test was carried out to detect the statistical significance with a significant level of α = 0.05 (27).

The molecular weight of SCPs < 1,000 Da accounted for 94.04% of the total, of which molecular weights < 180 Da, 180–500 Da, and 500-1000 Da respectively accounted for 16.39, 53.70, and 23.95%, and molecular weight >2,000 Da only accounted for 0.29%. This shows that sea cucumber peptides are small molecule peptides that are helpful for human digestion and absorption. SCPs are also high in glycine (Gly), proline (Pro), aspartic (Asp), and Alanine (Ala) (Figure 1).

The results showed that the survival rate of the nematodes pretreated with SCPs was much higher than the control group (Figure 2). As shown in Table 2, the mean lifespan of SCPs-L, SCPs-M, and SCPs-H groups was increased by 6.50, 31.46, and 21.30%, compared to the control group. Meanwhile, the maximum lifespan of the three groups was increased by 3.48, 32.17, and 20.00%, compared to the control group. More importantly, after administering SCPs-M, the mean and maximum lifespans were prolonged by 21.09 and 2.86%, 21.74, and 10.43%, compared to the Met and NMN groups.

At 10 h, there was a significant difference between the levels of SCPs-M and positive controls were consistent (Figure 3A). The survival rates of SCPs groups were considerably greater than the control group during paraquat stress and heat stress (Figures 3B,C). At the same time, we detected whether the increased lifespan was accompanied by an improvement in the vitality of the nematodes. We tested the motility of nematodes treated with SCPs for 18, 20, 22, and 24 days, as shown in Figure 3D. The mortality of the SCPs treatment group was lower than the control group, which suggested that SCPs did not affect muscle contraction and other functions of N2. The majority of worms could move on their own by day 18. By day 20, only 40.81% of the worms in the control group possessed class A motility, compared to 42.86, 60.00, and 55.41% in the SCPs-L, M, and H treatment groups, respectively. On days 22 and 24, 24.07 and 30.99% of worms had class A and B motility in the SCPs-M treatment group more than those in the control group. These findings suggested that SCPs could improve C. elegans capacity for mobility and stress tolerance.

The autofluorescence of lipofuscin can be used to estimate the aging of nematodes. As shown in Figures 4A,B, SCPs, Met, and NMN can effectively reduce lipofuscin in nematodes. The lipofuscin content in N2 worms treated with SCPs-L, M, and H was lower by 35.48, 40.84, and 40.41%, respectively, compared to the control group. Increased ROS levels may damage cell structure significantly, so prevention of excessive ROS accumulation has been shown to be an efficient strategy to delay aging. When the nematodes were pretreated with SCPs, the fluorescence of N2 was substantially reduced as compared to the control group. Among these, the SCPs-M group had the lowest ROS content at the same level as the NMN group (Figures 4C,D). The activities of SOD, MDA, and the CAT contents are important indexes of defense against potential oxidative damage in organisms. As shown in Figure 3E, SOD activities were significantly increased by SCPs treatment (by 1.4–1.5-fold in the SCPs-treated groups compared with the control group). The MDA contents were 10.18%-32.44% lower in the SCPs treated groups than in the control group. The CAT activities were significantly increased by SCPs treatment (by 1.6–2.1-fold in the SCP-treated groups compared with the control). These results indicated that SCPs reduced oxidative deposition in the body and enhanced the antioxidant capacity of nematodes.

To explore the mechanisms of these beneficial effects, we analyzed the expression levels of genes related to longevity, stress resistance, apoptosis, or the impact Dauer period (Figure 5). The expression level of genes in the control group was set to 1. In SCP s-treated groups, the relative expression levels of daf-2 ranged from 0.41 ± 0.08 to 0.79 ± 0.11, daf-16 and sod-3 were SCPs-dose-dependent; the relative expression levels of pept-1 ranged from 1.31 ± 0.20 to 2.65 ± 0.57. Those of old-1 ranged from 2.89 ± 0.37 to 6.02 ± 1.78. Other genes showing increased expression levels after SCPs treatment included tyr-3, and acox-1.5. It is hypothesized that pept-1 may regulate daf-16 and its down-regulated genes through daf-2 (28).

The GFP signal was shown as cytosolic, intermediate, and nuclear localization in Figure 6A. The results illustrated a significant rate of higher nuclear location up to 44% and lower cytosolic location down to 57% compared to the control (Figure 6B). Furthermore, SCPs had no significant effect on lifespan in daf-16 null mutants, control (11.28 ± 0.34), SCPs-L (11.67 ± 0.35), SCPs-M (12.22 ± 0.06), SCPs-H (12.19 ± 0.21). These data suggested that the deletion of daf-16 could affect the role of SCPs (Figure 6C). These results conveyed that SCPs might promote DAF-16 translocation to enhance the effects on stress resistance and longevity.

The inhibited daf-2 or activated daf-16 could be the key target for regulating IIS to delay aging in nematodes. SCPs could effectively reduce the accumulation of lipofuscin (Figures 6D,E) and extended lifespan in daf-2 null mutants with no significant difference, control (22.78 ± 0.88), SCPs-L (20.72 ± 0.91), SCPs-M (22.80 ± 0.30), SCPs-H (22.11 ± 0.91), which suggested that daf-2 played an important role in delaying aging in SCPs (Figure 6F). SOD-3 can remove excess superoxide and free radicals in the body (29). In comparison to the control group, SCPs-L, M, and H treatments increased the expression of SOD-3: GFP by 76.21, 90.98, and 26.89%, respectively (Figures 6G,H). SCPs had no significant effect on lifespan in pept-1 null mutants, control (21.35 ± 0.83), SCPs-L (20.43 ± 0.88), SCPs-M (20.77 ± 0.26), SCPs-H (21.08 ± 0.16) (Figure 6I). OLD-1 was transcriptionally regulated by the DAF-16 forkhead transcription factor, and both were expressed in the whole body. SCPs were able to significantly reduce the lifespan of old-1 null mutants compared with the control group, control (14.62 ± 0.55)^a, SCPs-L (13.02 ± 0.23)^b, SCPs-M (11.32 ± 0.12)^c, SCPs-H (12.39 ± 0.25)^bc (Figure 6J). These results suggest that SCPs extend the C. elegans lifespan and improve antioxidant ability via daf-16, daf-2, sod-3, pept-1, and old-1.

SCPs up-regulated tyr-3 and acox-1.5 by other ways of anti-aging, which suggests SCPs might mediate the reduction of apoptosis and regulation of the Dauer phase in nematodes, by validating their corresponding mutants, we found that SCPs extended, respectively the lifespan of tyr-3 null mutants 19.91, 14.72, 10.89% than the control group (Figure 6K). And also, SCPs extended the lifespan of acox-1.5 null mutants, control (14.07 ± 0.21)^b, SCPs-L (16.14 ± 0.20)^a, SCPs-M (16.65 ± 0.11)^a, SCPs-H (16.21 ± 0.13)^a (Figure 6L). This means SCPs are not able to act by inhibiting apoptosis and regulating the Dauer phase.

In summary, the following diagram of the mechanism of SCPs regulating aging was obtained in Figure 7.