PA (purity≥98%) was purchased from Shanghai Yuanye Bio-Technology Co. Ltd. (Shanghai, China), 5-fluorodeoxyuridine (FUDR), and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and ethanol from Sigma-Aldrich (United States).

All worm strains used in this study were provided by the Caenorhabditis Genetic Center (CGC; University of Minnesota, Minneapolis, MN): N2 (wild-type), CF1038 daf-16 (mu86) I., EU1 skn-1 (zu67)IV., PS3551 hsf-1 (sy441)I., RB754 aak-2 (ok524)X., RB759 akt-1 (ok525)V., VC204 akt-2 (ok393)X., CB4876 clk-1 (e2519)III., CF1553 [(pAD76) sod-3:GFP + rol6 (su1006)], CL2166 dvIs19 [pAF15 (gst-4:GFP::NLS)], SJ4100 (zcIs13 [hsp-6:GFP]), SJ4005 zcIs4V (hsp-4:gfp), SJ4058 zcIs9 [hsp60:GFP + lin-15 (+)], NL5901 [unc54p: alphasynuclein:YFP + unc-119 (+)], DA2123 [lgg-1p:GFP:lgg-1 + rol6 (su1006)], TJ356 [daf-16p:daf-16a/b:GFP + rol-6] and others. Worms were cultured on nematodegrowth media (NGM) at 20°C, except for specific strains requiring alternative conditions.

All strains were cultured on fresh NGM plates for 2–3 generations without starvation. When synchronized larvae reached the L4 stage, worms were transferred to an NGM plate containing PA and FUDR (50 mg/mL, to inhibit nematode reproduction). Dead worms were counted dailywith worms responding to slight touch from the worm pickerrecorded as alive. Worms that were missing, dried or hatched internally were censored from the lifespan count. Experiments were performed with at least 60 nematodes in each group.

Lipofuscin analysis: On the 5th and 10th days of PA treatment, the worms were collected and photographed using a fluorescence microscope (Leica DFC 7000T) at an excitation wavelength of 360–370 nm and an emission wavelength of 420–460 nm to quantifylipofuscin accumulation. At least 30 worms were included in each group, and images of the nematodes were processed using ImageJ.

Body-bending experiment: The worms were synchronized and incubated overnight at 20°C; L1-stage larvae were then incubated on NGM plates until late L4stage.On days 5 and 10, the wormswere transferred to experimental platesadded to water droplets to stabilize for 1 min, and subjected to body-bending analysis under a microscope within 20 s. A minimum of 30 worms were included in each group.

Pharyngeal pumping rate: Following the same preliminary treatment as the body-bending test, the number of pharyngeal pumping events was recorded under a microscope on the 5th and 10th days for the 20 s. A minimum of 30 worms were included in each group.

Mobility assay: This was carried out on days 9, 14, and 18. Sixty worms were observed, and the locomotion of motion A to motion C was quantitatively measured according to previous protocols (Wang H. et al., 2018). All experiments were performed in triplicate.

For the heat shock assay, worms were treated with PA for 7 days from the L4 larvae stage, then transferred to a new NGM plate and incubated at 35°C forheat shock. Dead worms were counted hourly until all worms succumbed. In the oxidative stress assay, worms were treated with PA as described above, then transferred to NGM plates containing paraquat (20 mM, Sigma‒Aldrich). Dead of worms were counted daily. Each group comprised at least 60 nematodes, and the experiment was repeated threetimes.

L4 stage worms were spread on experimental NGM plates containing PA, paraquat (20 mM), or N-acetyl-L-cysteine (1 mM) and incubated at 20°C for 6 days. Afterward, the worms were collected, washed with M9 buffer at least three times, stained with an H2DCF-DA (50 μM) probe and shaken at 35°C for 60 min according to the ROS detection kit (Hui et al., 2020).ROS determination was performed by imaging under a fluorescence microscope. The experiment was independently repeated at least three times, with each experiment involving at least 30 worms.

TJ356 worms cultured to the L4 stage were transferred to the experimental group (200 μM PA)or two control groups.An NGM plate containing TJ356 and OP50 bacteria was subjected to heat shock (37°C, 15 min) as a positive control, while another control plate was placed in a 20°C incubator as a negative control. The localization of DAF-16:GFP was observed every hour under a fluorescence microscope. The green fluorescent nuclear aggregation particles in the TJ356 worms served as the index of the DAF-16 gene in the nucleus (Wang et al., 2021).

Following synchronization, the Subsequently were spread onto experimental plates and grown until adulthood. Approximately 1000 worms from both the experimental and control groups were collected, subjected to multiple washes with PBS to eliminate excess bacterial fluid, and treated with Nile red staining reagent. After fixation with paraformaldehyde (4%) for 25 min, the worms underwent by two washes in PBS+1% Triton buffer solution. Subsequently, Nile red reagent (5 mg/mL) was added under light-protected conditions; after an incubation period of 2 min, the worms were washed three or more times with PBS+1% Triton buffer solution before microscopic examination (Leica DFC7000T). ImageJ software was used for image analysis. Each experiment was repeated three times, utilizing at least 30 worms per repetition.

RNA interference (RNAi) was performed as previously described (Shi et al., 2023). RNAi was conducted by feeding HT115 (DE3) (Fire Lab) bacteria vectors L4440 (control), atg-18, and bec-1, which produce dsRNA against the target gene. The RNAi worm lifespan assay was conducted according to previous methods (Beifuss and Gumienny, 2012).

To detect autophagy in nematodes, the number of GFP-positive foci on the lgg-1 autophagic vesicles was used to evaluate autophagy. DA2123 worms showed diffuse fluorescence in the cytoplasm of various tissues. Through the appearance of fluorescent points, the formation of autophagosome structures can be observed and quantified.

Approximately 3,000 synchronized N2 worms were cultured to late the L4 or early adult stage, transferred to the experimental group (with or without 200 μM PA, containing 20 mM FUDR) and cultured at 20°C for 24 h. Total RNA was extracted using the Steadypure Universal RNA Extraction Kit (Accurate Biology) and reverse-transcribed into cDNA using the PrimeScript™RT reagent kit with gDNA Eraser (Perfect Real Time). mRNA expression was quantified by the SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus) on the QuantStudio 6 Flex system. The relative mRNA expression levels of genes were calculated using the 2^−ΔΔCT method and normalized to the expression of the gene cdc-42 (Yanase, 2020).

The worms were collected on day 6 of the PA intervention. Protein was extracted by homogenization using a sonicator, and protein concentrations were determined by a Bicinchoninic Acid Protein Assay Kit (Beyotime). The experiment was conducted according to the previous protocol (Jeong et al., 2018). ImageJ software was used for image analysis.

Statistical analyses were conducted using SPSS 26.0 Statistics and GraphPad Prism 7.0 software. Fluorescence quantification, oil red quantification, and protein quantification statistics were performed using ImageJ 1.8.0. Lifespan experiments were analyzed using Kaplan-Meier survival analysis. Other data were expressed as the mean ± SD, unless otherwise stated. The p values were determined by two-tailed t-test.A p-value <0.05 was considered a significant difference.

To investigate the impact of PA on lifespan, we treated wild-type N2 worms with various concentrations of PA (Figure 1A). Our results showed that PA at each concentration could prolong the lifespan of C. elegans to a certain extent, while compared with the control, 200 μM PA had the greatest effect and increased the lifespan of C. elegans by 16.7% (Figures 1B–D). Lipofuscin is a yellow‒brown pigment that accumulates with age (Sitte et al., 2000). PA significantly decreased lipofuscin deposition (Figures 1E,F) and improved the movement ability and pharyngeal pump activity of C. elegans (Figures 1G–I) on days 5 and 10 of adulthood. These results indicated that PA could promote the health of C. elegans.

DAF-16, a nematode homolog of the mammalian FOXO transcription factor, plays a crucial role in coordinating diverse biological processes, including stress tolerance, development, reproduction, lipid storage, and longevity (Kim and Webb, 2017). In the insulin signaling pathway, the AKT-1 and AKT-2 kinases act upstream of DAF-16 (Hesp et al., 2015). Our findings demonstrated that PA failed to extend the lifespan of daf-16 (mu86) loss-of-function mutant worms (Figure 2A)and did not significantly prolong the lifespan of Akt-1 or Akt-2 kinase-deficient mutants (Figures 2B,C). Notably, PA enhanced the nuclear accumulation of the DAF-16:GFP fusion protein (Figures 2D,E), and upregulates the mRNA levels of the DAF-16/FOXO downstream target genes sod-3, dod-3, clt-1, and clt-3. However, in daf-16 (mu86) loss-of-function mutants, the expression levels of these genes remained unaltered regardless of the presence or absence of PA (Figure 2F). Collectively, these results suggest that the activation of FOXO/DAF-16 is required for the PA-mediated extension of the C. elegans lifespan.

Heat shock transcription factor (HSF-1) regulates the expression of heat-induced target genes, including small heat shock proteins, and plays a crucial role in longevity regulation and protein toxicity management. It serves as a key downstream transcription factor of the insulin signaling pathway (Brunquell et al., 2017). Our investigation aimed to determine whether hsf-1 also plays a critical role in lifespan regulation within the context of PA treatment targeting the insulin signaling pathway. First, we examined the impact of PA on the lifespan of the HSF-1 mutant PS3551. Our results indicated that PA did not extend the lifespan of this mutant (Figure 3A). Next, we conducted a heat shock experiment on wild-type N2 nematodes and found that PA-treated nematodes exhibited longer survival times then control worms at 35°C (Figure 3B). This suggests that PA confers a certain level of resistance to heat stress. HSP-6 is involved in regulating misfolded protein binding activity in nematodes and participates in mitochondrial unfolded protein responses during heat shock (Govindan et al., 2019). Subsequently, we investigated fluorescence expression using the SJ4005 (hsp-4:GFP), SJ4058 (hsp-60:GFP), and SJ4100 (hsp-6p:GFP) strains to assess the effect of PA treatment. Our results demonstrated an increase in protein expression after PA treatment (Figures 3C–E). Moreover, significant increases in the mRNA levels of the downstream target genes hsp-1, hsp-4, hsp-6, and hsp-60 were detected following PA treatment (Figure 3F). These findings suggest that by inhibiting the IIS signaling pathway, PA can activate downstream mechanisms to regulate longevity through HSF-1 in C. elegans.

Excessive accumulation of free radicals leads to tissue damage and degenerative changes in organs (Blackwell et al., 2015). Our findings demonstrate that PA treatment enhances the survival rate of worms exposed to paraquat and reduces ROS levels (Figures 4A–C). The transcription factor SKN-1/Nrf-2 regulates the expression of numerous antioxidant enzymes and phase I detoxification enzymes. We observed a significant increase in fluorescence intensity in the LD1 mutant strain treated with PA compared with the control group, albeit slightly weaker than that in PQ-treated worms (positive control Figure 4D). However, PA failed to extend the lifespan of skn-1 (zu67) mutants (Figure 4E). SOD and GST-4 play crucial roles in the oxidative stress response; maintaining high levels of SOD-3 and GST-4 expression may contribute to delaying aging (Pohl et al., 2019). Therefore, we treated the sod-3:gfp (CF1553) and gst-4:gfp (CL2166) mutant strains with PA for 7 days, followed by exposure to heat stress for 2 h. Remarkably, PA treatment significantly increased the protein expression of SOD-3 and GST-4 (Figures 4F,G). Additionally, the mRNA levels of skn-1 and its downstream genes skn-1, sod-3, dod-3, and gst-4 in N2 nematodes treated with PA were elevated; however, no alterations were detected in their expression within the skn-1 mutant strain (Figure 4H).

As previously discussed, the longevity of C. elegans is attributed to the antioxidant activity of PA. Mitochondrial dysfunction can lead to oxidative stress and impaired mitochondrial performance due to ROS production (Dilberger et al., 2019). We investigated whether mitochondrial function plays a pivotal role in the lifespan extension induced by PA. Our findings demonstrated that PA fails to extend the lifespan of mutants with mitochondrial dysfunction, namely, isp-1, clk-1, and aak-2 (Figure 5A–C). This finding suggested that PA modulates mitochondrial function in C. elegans. Furthermore, we observed upregulation of the mRNA levels of the mitochondria-associated transcription factors atfs-1, xbp-1, and isp-1 following PA treatment in C. elegans (Figure 5D). Antioxidants have been reported to enhance lipid metabolism (Amorim et al., 2022). Our results indicate that PA has the potential to attenuate Oil Red O staining intensity as an indicator of lipid storage and downregulate the mRNA expression of the target genes fat-1, fat-3, fat-7, and sir-2.1, which are associated with lipid metabolism (Figures 5E–G).

Autophagy is a highly conserved degradation process that, when triggered by stressful conditions, eliminates damaged intracellular macromolecules to maintain cellular homeostasis and promote organismal health and development (Palmisano and Meléndez, 2019). Recent studies have established links between autophagy and various human diseases. Interestingly, many interventions that extend lifespan increase the accumulation of autophagosomes (Peña-Ramos et al., 2022). We examined the fluorescence of BC12921 following PA treatment and evaluated the lifespans of atg-18 and bec-1 mutant nematodes associated with autophagy genes. RNAi targeting autophagy genes, including atg-18 and bec-1, did not definitively prolong lifespan (Figures 6A,B). However, we observed that PA treatment reduced the degradation of the phagocytic substrate SQST-1 in BC12921 (Figures 6C,D) while increasing the fluorescence intensity of the autophagosome marker lgg-1 in DA2123 (Figures 6E,F). These findings suggest that PA can enhance autophagic activity. Furthermore, the mRNA transcription levels of atg-18, lgg-1, unc-51, and vps34 were significantly upregulated upon PA treatment (Figure 6G). Intriguingly, the protein level of S6K, a crucial downstream gene regulated by the mTOR signaling pathway, decreased significantly (Figures 6H,I). This finding implies that the lifespan extension induced by PA may be attributed to enhanced autophagy through activation of the mTOR pathway.