Paederoside Promotes Longevity and Fitness in C. elegans Through Ubiquitination and Degradation of DAF-2/IGF1R, Activating DAF-16/FOXO and SKN-1/NRF2 Transcription Factors

TL;DR

Paederoside extends the lifespan of C. elegans by reducing oxidative stress and activating key longevity genes.

Contribution

This study reveals that paederoside promotes longevity by degrading IGF1R and activating DAF-16 and SKN-1 pathways.

Findings

PSG at 100 μg/mL significantly prolonged the lifespan of C. elegans.

PSG reduced lipofuscin accumulation and increased antioxidant enzyme activity.

PSG induces IGF1R degradation via the ubiquitin–proteasome system in HeLa cells.

Abstract

Paederia scandens (Lour.) Merr is a substance exhibiting medicine–food homology (MFH), commonly used in China. However, the antioxidant and anti-aging effects of paederoside (PSG) have not been thoroughly investigated; therefore, in this study, Caenorhabditis elegans (C. elegans) was treated with PSG to investigate these effects. We found that 50, 80, and 100 μg/mL of PSG could prolong the lifespan of C. elegans, and administration of 100 μg/mL PSG significantly reduced the accumulation of lipofuscin. Under conditions of oxidative stress, RT-qPCR analysis revealed that PSG treatment significantly up-regulated the expression of key antioxidant gene skn-1 and longevity-associated gene daf-16. In addition, PSG increased the activity of the antioxidant enzymes SOD and CAT and reduced the level of MDA. When DAF-2 activity is reduced or inhibited in C. elegans, DAF-16 and SKN-1 are activated and translocate to the nucleus to promote stress resistance and prolong lifespan. Finally, by utilizing HeLa cell models, we demonstrated that the core component of Paederia scandens, PSG, promotes targeted degradation of IGF1R through the ubiquitin–proteasome system. Our results suggest that feeding C. elegans PSG is effective in extending this organism’s lifespan by improving oxidative stress resistance; thus, PSG has significant potential for development as an anti-aging food product and drug.

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1. Introduction

Aging affects the entire human population; it is a multifactorial and pervasive biological process defined by temporally regulated quantitative shifts at the cellular, organ, and systemic levels [1,2,3,4]. Many drugs have been shown to play an important role in slowing down the aging process. In fact, traditional Chinese herbal medicines, comprising bioactive constituents including glycosides, polyphenols, flavonoids, and polysaccharides, elicit anti-aging responses through potent antioxidant and anti-inflammatory mechanisms [5]. Additionally, bioactive small molecules found in these herbs have been found to possess antioxidant abilities. They can enhance antioxidant system function, promote overall health, and potentially slow down the aging process, making them increasingly important targets for the development of anti-aging drugs [6,7].

Paederia scandens (Lour.) Merr, a member of the Rubiaceae family, is a plant exhibiting medicine–food homology (MFH). In the context of traditional medicine, it has indicated its efficacy in dispelling wind, eliminating dampness, promoting blood circulation, relieving pain, and combating aging. Modern pharmacological studies have identified several bioactive compounds in whole plants of Paederia scandens (Lour.) Merr, including paederoside, scandoside, paederoside acid, asperuloside, deacetyl asperuloside, and embelin [8]. These compounds mainly belong to the iridoid glycoside and flavonoid classes and exhibit anti-inflammatory and antioxidant biological functions [9,10]. However, it remains unclear whether paederoside can delay aging by regulating oxidative stress pathways [11,12]. Further investigation is needed to unravel the molecular mechanism underlying its anti-aging effects.

Considering that Caenorhabditis elegans (C. elegans) has characteristics such as a short lifecycle [13], transparent body, and specific genetic background, combined with the fact that the mechanisms of the insulin [14], mTOR [15], and MAPK signaling pathways [16] in aging are becoming increasingly well-understood, we chose C. elegans as our research subject. And, given the central role of the insulin/IGF-1 signaling pathway in governing endocrine-mediated stress responses, diapause, and aging, this pathway serves as the primary focus of our investigation. Meanwhile, molecular docking analysis revealed that paederoside, a characteristic compound in Paederia scandens, exhibits high binding affinity for DAF-2/IGF1R, indicating a potential interaction. Therefore, this study aims to observe physiological indicators in nematodes, including lifespan, fertility, changes in oxidative stress levels, responses to heat stress, and locomotor ability, while exploring the molecular mechanisms of action of paederoside through molecular biology techniques, providing scientific evidence for the development of natural herbal-based anti-aging interventions.

2. Results

2.1. PSG Does Not Affect OP50 Growth

We examined whether PSG exhibited inhibitory effects on E. coli OP50. If significant inhibition is observed, this suggests that the extended lifespan of nematodes might result from restricted bacterial growth and consequent food limitation [17]. As illustrated in Figure 1a,b, compared to the control group, the PSG-treated group exhibited a significantly higher optical density (OD) during the initial 3 h of co-culture. However, by the 5 h time point, no statistically significant difference was observed between the two groups. These results indicate that PSG does not inhibit E. coli OP50 growth and may transiently promote its proliferation in the early culture phase. This suggests that the prolongation of nematode life span by PSG is maybe not caused by dietary restriction and thus insufficient energy supply.

2.2. PSG Prolonged the Lifespan and Increased the Body Size Metrics of C. elegans

To investigate the effects of PSG on the lifespan of C. elegans, we compared the lifespan curves of the three PSG treatment groups (50 µg/mL, 80 µg/mL and 100 µg/mL) to that of the control group; a significant rightward shift was observed (Figure 1c,d), suggesting that PSG treatment extended the lifespan of C. elegans. Exposure to PSG significantly extended the lifespan of C. elegans by 18.9%, 30.9% and 38.2%, respectively, compared to the control group. These data confirm that PSG can prolong the lifespan of C. elegans. As the most significant effect occurred at a concentration of 100 µg/mL, this PSG concentration was selected for the subsequent experiments [18].

In addition, we discovered that PSG at 100 µg/mL significantly increased the body length and area of N2 compared to the control group (Figure 1e,f).

2.3. PSG Improved Locomotion of C. elegans but Did Not Affect Brood Size

In nematode locomotor behavior, the body bending and head bobbing frequencies are the most sensitive physiological indexes, so both were used in this experiment to assess the effect of PSG on nematode locomotor ability. As shown in Figure 2a,b, there were significant differences in the body bending and head bobbing frequencies in the PSG group compared to those in the control group. This result suggests that PSG may delay aging by altering nematode motility.

Worms with reduced pharyngeal pumping ingest fewer bacteria and exhibit numerous dietary-restriction-like characteristics, such as prolonged lifespan. Therefore, we tested whether PSG had an effect on the pharyngeal pumping rate, and found that, compared to the control, 100 µg/mL PSG treatment resulted in a slight down-regulation of pharyngeal pump rates in adults on day 9 (Figure 2d).

Lifespan extension is likely associated with a loss of fecundity. Thus, we investigated the effect of PSG on C. elegans reproduction. We observed no significant difference in total progeny production following PSG treatment (Figure 2c), suggesting that PSG-mediated lifespan extension in C. elegans was not dependent on sacrificial reproduction. All of these results suggest that PSG had no effects on normal growth and development.

2.4. PSG Reduces Lipofuscin Accumulation in C. elegans

Lipofuscin, a naturally occurring fluorescent molecule and biomarker of aging, accumulates with aging in C. elegans. It fluoresces green under light. To understand whether PSG treatment increases C. elegans lifespan, we evaluated the effects of 100 µg/mL PSG on age-associated lipofuscin changes. Overall, C. elegans accumulated less lipofuscin under PSG treatment than in the control group (Figure 2e).

2.5. PSG Improved Stress Resistance in C. elegans

In the oxidative stress resistance assay, PSG administration significantly enhanced nematode survival relative to the control group (Figure 3a). Under thermal stress conditions at 37 °C, nematodes treated with an optimal concentration of PSG exhibited significantly enhanced survival compared to the control group (Figure 3b). In summary, PSG was effective in increasing the tolerance of nematodes to heat and oxidative stress.

2.6. Determination of ROS and MDA Content and Antioxidant Enzyme Activity

The intracellular ROS levels were quantified using DCFH-DA fluorescence assays, and the results demonstrated that treatment with 100 µg/mL PSG significantly attenuated ROS accumulation relative to the control group (Figure 3c). The MDA levels followed the same trend as that of ROS accumulation (Figure 3d). Furthermore, the PSG treatment significantly enhanced the enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) relative to the control (Figure 3e,f).

2.7. PSG Modulated Oxidative-Stress-Related Gene Expression in C. elegans

The expression of daf-2 and age-1 in C. elegans receiving PSG at 100 µg/mL was significantly down-regulated compared with the control group, whereas daf-16 and skn-1 gene expression was significantly up-regulated under PSG treatment (Figure 4). Furthermore, the genes sod-3 and gst-4 are both involved in the oxidative stress response; PSG treatment up-regulates their expression, enhancing resistance to both oxidative and thermal stress.

2.8. The Longevity Extension Caused by PSG Depends on DAF-16/FOXO

DAF-16 is a FOXO transcription factor whose overexpression is associated with increased longevity. Using a DAF-16::GFP fusion, we determined the subcellular distribution of DAF-16 (Figure 5). At 9 days old, the proportion of C. elegans showing DAF-16 nuclear localization was 13.1% in the untreated controls and 37.7% in the PSG-treated worms. Enhanced nuclear localization of DAF-16 was observed after PSG supplementation.

2.9. The Longevity Extension Caused by PSG Depends on SKN-1/NRF2

In addition to DAF-16/FOXO, SKN-1 (the C. elegans NRF2 ortholog) has been reported to regulate lifespan, mainly through the oxidative stress response. Using GFP fused to SKN-1, we determined the subcellular dis-tribution of SKN-1 (Figure 6). At 9 days old, the percentage of C. elegans worms showing SKN-1 nuclear localization was 15.9% in the untreated control group and 38.3% in the PSG-treated groups. Enhanced nuclear localization of SKN-1 was observed after PSG supplementation.

2.10. PSG-Induced Longevity Is Dependent on the IIS Pathway

Since PSG inhibited the IIS pathway, which plays an important role in C. elegans lifespan extension, we further identified the role of the IIS pathway in PSG-treated C. elegans lifespan extension by using daf-2-mutant worms. As shown in Figure 7a, the PSG treatment did not extend the lifespan of the daf-2-mutant worms, indicating that PSG-induced C. elegans lifespan extension was dependent on IIS.

As shown in Figure 7b–d, PSG also failed to extend the lifespan of worms with mutations in age-1, daf-16 and skn-1, which are primary components of the IIS pathway. These results indicate that PSG extended the lifespan of C. elegans by inhibiting the IIS pathway.

2.11. PSG Did Not Affect ROS Levels in Mutant Worms

To further substantiate whether daf-2, age-1, daf-16 and skn-1 play a role in the antioxidant process of PSG, the effects of PSG on ROS levels in CB1370, CF1038, TJ1052 and GR2245 worms were determined. Figure 7e-h show that there were no significant differences in ROS levels between the PSG-treated worms and the control group, regardless of CB1370, CF1038, TJ1052 or GR2245 mutations.

2.12. Molecular Docking Analysis

The molecular docking analysis revealed that paederoside, the bioactive constituent of PSG, stably binds to the IGF1R active site with a favorable binding energy (<−7.5 kcal/mol, Figure 8a), indicating IGF1R as a potential therapeutic target of PSG.

2.13. Western Blot Analysis

The Western blot analysis (Figure 8b,c) showed that the expression level of IGF1R protein showed a statistically significant decrease in the PSG-treated cells compared to the untreated controls. This indicates that PSG inhibits IGF1R protein expression. Given that IGF1R plays a central role in key signaling pathways such as those for cell growth, proliferation and survival, the regulation of its expression level is of great biological significance. Therefore, in order to better understand the molecular mechanisms underlying the role of PSG, we further explored the specific pathways through which PSG mediates the down-regulation of IGF1R protein levels. To investigate the mechanistic basis of protein degradation, we focused on the ubiquitin–proteasome system (UPS), a principal pathway mediating regulated proteolysis in eukaryotic cells. The ubiquitination analysis of IGF1R demonstrated a marked elevation in polyubiquitin modification in the PSG-treated samples compared to the controls.

In order to verify whether the decrease in IGF1R protein levels was indeed achieved through the ubiquitin–proteasome pathway, we introduced the proteasome-specific inhibitor MG132 into the intervention experiments. The experimental results showed that PSG-induced IGF1R protein degradation was effectively blocked when MG132 was administered along with PSG. Meanwhile, a co-immunoprecipitation (Co-IP) assay demonstrated that IGF1R interacts with ubiquitin (UB). This interaction promotes the ubiquitination of IGF1R, which contributes to delayed aging (Figure 8d).

In order to further reveal the specific mechanism by which PSG regulates IGF1R ubiquitination and degradation, and to identify the key active sites on the IGF1R molecule, we constructed and analyzed IGF1R mutants with PSG binding sites. In these IGF1R mutants, significant changes in ubiquitination levels were not observed even after PSG treatment. These experiments reveal that PSG regulates IGF1R stability through a highly specific mechanism: it acts on key structural domains of the IGF1R molecule to enhance its ubiquitination modification level, thereby facilitating its degradation via the ubiquitin–proteasome pathway.

We next sought to verify PSG’s modulation of the IIS pathway in cells. First, Western blot analysis was used to examine changes in key IIS proteins and the nuclear translocation of FOXO3. The data revealed that PSG inhibits the insulin/IGF-1 signaling (IIS) pathway (Figure 8f), which subsequently activates the DAF-16/FOXO transcription factors (Figure 8e). Finally, this activation orchestrates a series of beneficial outcomes, including extended lifespan, enhanced stress resistance, and improved antioxidant capacity.

3. Discussion

In view of the aging global population, delaying the aging process has become increasingly important. In terms of longevity interventions, we should not only focus on extending life expectancy but also aim to improve overall health. Paederia scandens is known for its traditional uses including dispelling wind, promoting blood circulation, alleviating pain, detoxifying, and reducing oxidation. However, whether it can serve as a dietary intervention to delay aging remains unknown. Therefore, this study employs C. elegans as a model organism to investigate the anti-aging mechanisms of its main bioactive component, PSG.

First, as PSG is known to possess anti-inflammatory and antibacterial properties, its inhibitory effect on OP50 was investigated in Paederia scandens, and the results showed that PSG had no inhibitory effect on OP50. These findings suggest that the observed lifespan extension may not be mechanistically linked to the restriction of bacterial growth or the consequent reduction in nutrient availability. Our data indicate that PSG prolongs the lifespan of C. elegans in a concentration-dependent manner and enhances locomotor ability, while not affecting egg laying. At the same time, lipofuscin is also a major critical factor in senescence, and our data showed that pre-treatment with PSG for 8 days reduced lipofuscin deposition, which further supports the anti-aging effect of PSG. In summary, these data suggest that PSG not only increases lifespan but also improves healthspan, indicating its significant potential in the field of anti-aging research.

Oxidative stress constitutes a fundamental mechanistic contributor to the aging process, characterized by a disruption of the equilibrium between reactive oxygen species (ROS) generation and cellular antioxidant clearance [19]. With advancing age, the progressive decline in endogenous antioxidant defense mechanisms facilitates ROS accumulation, thereby inducing oxidative damage in cellular components and promoting functional impairment. Aging is closely related to the redox function system in the body. Oxidative damage is caused by excessive accumulation of ROS, which is affected by superoxide dismutase (SOD) and catalase (CAT). Malon dialdehyde (MDA) is a common index reflecting the level of lipid per oxidation.

Therefore, we first explored the role of PSG in antioxidant activity. Our experimental results indicate that after the PSG treatment, the levels of ROS and malondialdehyde (MDA) in C. elegans were significantly reduced, while the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was significantly increased. Additionally, we investigated whether PSG could enhance the stress resistance of C. elegans, and the results showed that the PSG-treated worms exhibited greater resilience to heat stress and H_2_O_2_-induced oxidative stress. These results clearly demonstrate that PSG enhances the antioxidant defense system in C. elegans.

IIS was the senescence regulatory pathway investigated in our experiments as it is a key regulator of development, metabolism and behavior, processes that are highly conserved in both C. elegans and HeLa cells. In C. elegans, DAF-16 is involved in the IIS pathway. The transcription factor DAF-16, a functional ortholog of mammalian FOXO proteins, is critically involved in regulating stress resistance and longevity. The IGF1R gene encodes a homolog of C. elegans daf-2. When daf-2 activity is reduced or inhibited in C. elegans, daf-16 is activated and translocates to the nucleus to promote stress resistance and prolong the lifespan. The SKN-1 transcription factor, a functional and structural ortholog of mammalian NRF-2, plays a critical role in modulating longevity and oxidative stress response in C. elegans. Both DAF-16/FOXO and SKN-1/NRF-2 are two classical transcription factors well known to be involved in the stress response, reducing oxidative damage and extending the lifespan of C. elegans/HeLa cells.

Using HeLa cell models, this study demonstrates that the core component of Paederia scandens, PSG, promotes targeted degradation of insulin-like growth factor 1 receptor (IGF1R) through the ubiquitin–proteasome system. As a member of the receptor tyrosine kinase family, hyperactivation of IGF1R signaling accelerates aging processes. The molecular docking analysis revealed high-affinity binding (binding energy < −7.5 kcal/mol) between PSG’s bioactive constituents and the kinase domain of IGF1R. This specific interaction enhances DAF-2/IGF1R ubiquitination, ultimately improving antioxidant capacity in C. elegans by alleviating IGF1R-mediated suppression of both the DAF-16/FOXO and SKN-1/NRF-2 pathways. In IGF1R-overexpressing cellular models, PSG-induced IGF1R degradation primarily occurred through the ubiquitin–proteasome system.

In the present study, 100 µg/mL of PSG significantly up-regulated the expression of the skn-1 and daf-16 genes and down-regulated the expression of daf-2. In addition, this result was further validated by the different degrees of regulation of skn-1 and daf-16’s downstream genes by PSG. sod-3, as a target gene of daf-16, was responsible for the regulation of SOD and CAT, the overexpression of which enhances the antioxidant capacity of C. elegans. Under certain conditions, skn-1 accumulates in the nucleus and activates its downstream antioxidant gene gst-4, thus enhancing the antioxidative stress capacity of C. elegans. Also, the induction of DAF-16 and SKN-1 nuclear translocation by PSG provides evidence for this hypothesis.

4. Materials and Methods

4.1. Materials and Reagent

Paederoside (PSG) was sourced from Shanghai Macklin Biochemical (Shanghai, China). The compound, with the molecular formula C18H22O11S and a purity of 98%, was supplied as catalog number P889986-20mg (Lot: 20547-45-9). The configured stock solution concentration was 1 mg/mL. It was dissolved in normal saline for use; for storage, it was kept at −4 °C in the short term and −20 °C in the long term. The cDNA synthesis kit and Reactive Oxygen Species (ROS) Assay Kit were supplied by Beyotime Institute of Biotechnology (Beijing, China). RNA was isolated using a kit from Tiangen Biotech (Beijing, China). The HeLa cells were obtained from the BeNa Culture Collection (Suzhou, China). Bleomycin was obtained from TargetMOI Biotech (Shanghai, China). Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) kits were purchased from Nanjing Jiancheng Biotechnology (Nanjing, China).

4.2. Experimental Nematodes, Culture, and Maintenance

C. elegans strains N2 (wild-type); TJ356 (zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]) [20,21]; LD1 (ldIs7 [skn-1b/c::GFP + rol-6(su1006)]); GR2245 (skn-1(mg570) IV); CB1370 (daf-2(e1370) III); CF1038 (daf-16(mu86) I); and TJ1052 (age-1(hx546) II) were provided by the Caenorhabditis Genetics Center (CGC, University of Minnesota, USA). Gravid adults were collected and treated with an alkaline hypochlorite solution (5 M NaOH, 5% NaClO, 1:1 v/v ratio) to isolate eggs. The digestion mixture was vortexed for 3–5 min at room temperature, and then the eggs were subsequently washed with sterile M9 buffer three times by centrifugation (1500 rpm, 1 min) to remove residual hypochlorite. After hatching, synchronized larvae were cultured on E. coli OP50 at 20 °C until reaching the L4 stage (~48 h). For drug treatment, a control substance or PSG (50 µg/mL, 80 µg/mL or 100 µg/mL) was mixed with E. coli OP50 on an NGM plate. A suspension was prepared and evenly spread onto the surface of NGM plates to form a bacterial lawn containing the drug.

4.3. Bacterial Growth Assay

Three test tubes had 10 mL of LB medium added to them; the first tube was used as a blank group, the second tube was filled with 200 μL OP50 bacterial solution, and the third tube was filled with 200 μL OP50 and 400 μL PSG stock solution. The three tubes were put into an incubator at 37 °C at the same time, and 1 mL of culture medium was mixed and aspirated every 1 h. The tubes were cultured for 6 h at 37 °C, and the OD600 nm values were measured with a microplate reader every hour. The experiment was repeated thrice.

4.4. Lifespan Analysis

Synchronized L4-stage N2, CB1370, CF1038, GR2245, and TJ1052 worms were transferred to NGM plates-either control or PSG-treated. Specifically, worms were transferred to freshly prepared NGM plates every 24 to 48 h. Survival was assessed daily using a touch-provocation assay. Nematodes displaying rigidity and absence of movement upon platinum wire stimulation were scored as deceased. The experiment was performed in triplicate, with 30 nematodes randomly assigned per treatment group [22].

4.5. Locomotion, Brood Size, Body Length, and Pharyngeal Pumping Analysis

N2 worms at stage L4 were exposed to control and PSG treatments for 8 days; then, the worms were washed with M9 buffer and placed onto new NGM plates without E. coli OP50. After 1 min of recovery, the frequency of head thrashes and body bends within 30 s was recorded under a microscope.

Ten worms were picked for measurement of pharyngeal pumping rate per 30 s and treated with 100 µg/mL PSG or normal saline.

For the brood size assay, the synchronized control and PSG-treated N2 worms were transferred to a new NGM plate containing E. coli OP50 and allowed to lay eggs, with one worm per plate. The eggs were counted every day until the worms stopped laying eggs.

Body length was measured from the top of the head to the tip of the tail using tools. L4 worms were randomly transferred to fresh NGM plates treated with or without 100 µg/mL PSG. After 8 days of treatment, the plates were photographed and the body length and body area of C. elegans were measured under a microscope (Olympus IX73, Tokyo, Japan) [23].

4.6. Stress Resistance Assays

Synchronized L4-stage wild-type nematodes (30 per group across three independent replicates, totaling 90 per condition) were cultured on NGM plates with or without PSG (100 μg/mL) at 20 °C for 8 days. For oxidative stress tolerance assessment, pretreated worms were washed three times with M9 buffer and transferred to 24-well plates containing 50 mM hydrogen peroxide (H_2_O_2_). Survival was recorded at 30 min intervals until complete mortality was reached. In parallel, thermotolerance assays were conducted by increasing the temperature of the pretreated nematodes from 20 °C to 37 °C, with subsequent monitoring of survival. The experiments were repeated thrice with 30 randomly selected nematodes per treatment [24,25].

4.7. Lipofuscin Assays

On day 9 post-treatment with or without PSG (100 μg/mL), nematodes were anesthetized with 10 mM levamisole. For every group, 12 nematodes were selected for fluorescence imaging (three independent replicates, totaling 36 nematodes per condition), using an Olympus IX73 fluorescence microscope (Tokyo, Japan) equipped with a 10× objective lens and an excitation/emission filter set at 470/550 nm. Bright-field images were acquired to determine body size. Relative fluorescence intensity in vivo was quantified with ImageJ (version 1.52v), and fluorescence per unit area was calculated. Data were normalized to the fluorescence intensity of the blank control group [26,27].

4.8. Quantification of Reactive Oxygen Species (ROS)

Following 8 days of PSG (100 μg/mL) treatment, nematodes were anesthetized with 10 mM levamisole. For ROS detection, a total of 36 nematodes per condition (12 per group across three replicates) were processed. In brief, the worms were anesthetized with levamisole, incubated in 100 µM H_2_DCF-DA solution in the dark at room temperature for 30 min, and then fixed on a 2% agarose pad on a glass slide. Fluorescence imaging was performed using a blue excitation filter (Ex/Em 470/550 nm) under a fluorescence microscope (Olympus IX73, Tokyo, Japan) with a 10× objective lens. Body size was quantified from bright-field images, and relative fluorescence intensity was measured as integrated density using ImageJ (v1.52v). Fluorescence per unit area was calculated, and results were normalized to the control group.

4.9. Nuclear Localization Assays

Following 8-day PSG (100 μg/mL) treatment, thirty TJ356-mutant and LD1-mutant nematodes per group were anesthetized with 10 mM levamisole (Aladdin, Shanghai, China) and mounted on 2% agarose pads. Cytoplasmic and nuclear subcellular localization of GFP in transgenic strains was visualized using fluorescence microscopy (Olympus IX73, Tokyo, Japan) under blue excitation (Ex/Em 470/550 nm) with a 10× objective lens. Representative images were acquired for each strain, and sample sizes were confirmed by enumeration of nematodes in all experimental groups.

4.10. Enzymatic Activity Assays

Nematodes from the control and PSG-treated (8 days) groups were homogenized and centrifuged at 5000 × g for 5 min. The resulting supernatant was assayed for superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) activity using commercial kits (Nanjing Jiancheng Biotechnology, Nanjing, China). The total protein concentration was quantified via the Bradford method, and enzymatic activity was normalized to the corresponding protein content.

4.11. Quantitative Real-Time PCR

Following 8-day PSG treatment (100 μg/mL), total RNA was extracted from 2000 nematodes per condition using the Eastep^®^ Super RNA Kit (Tiangen Biotech, Beijing, China). cDNA synthesis was performed with a quantitative reverse transcription kit according to the manufacturer’s protocol. Quantitative PCR was carried out on a q225 Real-Time PCR System with TransStart^®^ Top Green qPCR SuperMix (Beijing, China) in a 10 µL reaction volume containing 0.4 µL of gene-specific primers (Table 1) and 1 µL of cDNA template. The thermal cycling conditions consisted of initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s, with a final dissociation step at 65 °C for 30 s. Relative gene expression was quantified using the 2^−ΔΔCT^ method. Data were normalized to cdc-42 and presented as mean ± SEM of three independent experiments.

4.12. Molecular Docking

In this study, rigid molecular docking and binding free energy calculations were performed using AutoDock Vina 1.5.7, while PyMOL 2.3 was utilized to visualize the optimal binding conformations and hydrogen bond interactions between active components and key targets, elucidating their binding sites.

4.13. Cell Culture

HeLa cells procured from the BeNa Culture Collection were cultured in MEM, to which 10% FBS, 100 U/mL penicillin, and 50 µg/mL streptomycin were added. Upon reaching 70–80% confluence, cells were trypsinized, divided in a 1:3 ratio, and further cultivated.

4.14. IGF1R Overexpression and Mutant Plasmid Construction

The human IGF1R sequence was constructed using the Homologous Recombination Kit (Vazymz, Nanjing, China, C115) and cloned into the pcDNA3.1 expression plasmid. The PSG binding site in IGF1R was mutated using 2× Phanta Max Master Mix (Vazymz, Nanjing, China, P515). The plasmid was transfected into HeLa cells using Lipo8000 Transfection Reagent (Beyotime, Shanghai, China).

4.15. Western Blot Assay

Cellular proteins were extracted with RIPA lysis buffer and quantified using the Bradford assay. A total of 20 μg of protein per sample was separated by SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37 °C. Protein bands were visualized using chemiluminescence. Quantitative analysis of the bands, normalized to β-actin and β-Tubulin, was performed with ImageJ software. The antibodies used in this study were as follows: β-actin (HUABIO, Hangzhou, China, 1:20,000, HA722023); β-Tubulin (HUABIO, Hangzhou, China, 1:20,000, EM0103); IGF1R (abcam, Cambridge, MA, USA, 1:1000, ab182408, Figure 8d,f); IGF1R (Proteintech Group, Wuhan, China, 1:1000, 21707-1-AP, Figure 8c); ubiquitin (PTM bio, Hangzhou, China, 1:1000, PTM-7228); AKT3 (Absea Biotechnology, Suzhou, China, 1:1000, OC475); pAKT (Absea Biotechnology, Suzhou, China, 1:1000, RC4352); FOXO3 (abmart, Cambridge, MA, USA, 1:1000, PA5461S); and anti-Rabbit IgG-HRP (HUABIO, Hangzhou, China, 1:20,000, HA1001). Band intensities were quantified using ImageJ and derived from three independent biological replicates (n = 3).

4.16. Co-Immunoprecipitation Analysis

Co-immunoprecipitation was performed using the Thermo Fisher Scientific kit (Waltham, MA, USA). Cell lysates were incubated with the indicated primary antibody for 1-2 h at room temperature (RT). The antigen-antibody complexes were then incubated with protein A/G magnetic beads for 1 h at RT. The beads were washed twice with IP lysis/wash buffer and once with purified water, followed by elution of the bound immune complexes for subsequent analysis.

4.17. Immunofluorescence Analysis

Cells cultured in confocal dishes were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. After fixation, cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 min at room temperature and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature to minimize non-specific binding. The samples were then incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. After three washes with PBS (5 min each), the cells were incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor 594, HUABIO, Hangzhou, China, 1:1000, HA1122) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI (1 μg/mL) for 5 min. Fluorescence images were acquired using a confocal laser scanning microscope (Zeiss, LSM 900, Jena, Germany) and analyzed with ImageJ software.

4.18. Statistical Analysis

All experiments were performed with three independent biological replicates. All data were expressed as mean ± SEM. Results from lifespan and oxidative stress survival analysis were plotted as Kaplan–Meier survival curves, and p values were calculated using the log-rank (Mantel–Cox) test. Variations between the experimental groups were calculated by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The classification scoring criteria were based on the nucleus-to-cytoplasm (N/C) average fluorescence intensity ratio. Comparisons of proportions between groups were performed using Fisher’s exact test. The levels of statistical significance were set at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

5. Conclusions

This study systematically elucidated, for the first time, the molecular mechanism by which paederoside exerts anti-aging effects through regulating DAF-2/IGF1R protein stability. We found that paederoside can act on specific functional domains of DAF-2/IGF1R, influencing its protein stability by modulating ubiquitination levels, thereby activating antioxidant pathways and delaying the aging process. These findings provide conclusive evidence that paederoside’s anti-aging effects are dependent on modulation of the IGF1R-mediated IIS pathway.