Mutant and wild-type C. elegans strains used in this study were acquired from the Caenorhabditis Genetics Center (CGC). Worms were cultured using standard methods as previously described (28). Strains were fed with OP50 Escherichia coli and cultured at 20°C unless otherwise indicated. Strains used in this study: wild-type: N2, CF1038: daf-16(mu86), AA10: daf-12(rh286), BX160: fat-7(wa36) fat-5(tm420), CB6738: lys-7(ok1384), BX275: fard-1(wa28), RB908: pmp-1(ok773), and RB2102: hacd-1(ok2776).

The designated parts of the target genes were cloned into Vector L4440 and expressed in an RNAi bacterial strain (HT115). A double-stranded target RNAi (vector, L4440) strain was cultured and used to inactivate the target gene function (29). Eggs from worms fed with the target RNAi bacteria were transferred to fresh NGM plates containing the same bacteria and allowed to grow at 15°C for 3 days with or without the presence of VK2. Stage 4 larva were then transferred to the same RNAi NGM plates with or without VK2 to perform the indicated experiments. A total of 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) was used in the RNAi NGM plates to induce the double-stranded RNA. The L4440 carrying an empty RNAi plasmid was used as a negative control in the RNAi experiments (29).

Oil Red O staining is the standard method to test fat content in worms, and performed as previously reported (30–32). Synchronized eggs were cultured on OP50 plates with or without 0.1 μM VK2 treatment for 3 days at 20°C. Worms were then collected and washed in M9 three times and then fixed with the addition of 600 μL of 40% isopropanol to the worm pellet and rocked at room temperature for 3 min. Subsequently, the worms were stained in Oil Red O working solution for 2 h at room temperature with rotating at 30 rpm. After washing, the worms were mounted and imaged with a color camera outfitted with DIC optics. The Oil red O intensity was then quantified. At least 90 animals were imaged in at least three separate experiments using a Zeiss Axioplan microscope (Zeiss). The red intensity was captured and quantified using Image J software (NIH).

Synchronized young adults were transferred to NGM plates seeded with OP50 and cultured on plates with or without 0.1 μM VK2 at 20°C. The worms were scored every day by counting the number of worms that were moving (alive) until the day that all the worms had died. To compare the survival rates between the different treatments, mean survival rates were calculated using the Kaplan-Meier method, and the significant difference in the overall survival rates was determined using the log-rank test (33, 34).

For the pathogen infection assay, NGM plates with or without 0.1 μM VK2 were seeded with Pseudomonas aeruginosa (PA14) and cultured overnight before use. The young adults were then transferred to NGM plates seeded with Pseudomonas aeruginosa (PA14) and cultured at 20°C. The worms were assessed every 12 h as dead or alive (33). Worms were considered dead when they failed to respond to a touch with a platinum wire. At least three independent repeat experiments were performed.

For heat tolerance assays, worms at day 5 were transferred to NGM plates with or without 0.1 μM VK2 and incubated at 37°C. The animals were then scored as dead when they did not respond to a gentle touch with a platinum wire and were assessed every 2 h. At least three independent repeat experiments were performed.

For oxidative stress induced by H₂O₂, synchronized L1 worms were cultured in M9 with or without 0.1 μM VK2 at 20°C for 3 days. Then, H₂O₂ was added into the M9 to a final working concentration of 10 mM. About 50–100 μL M9 with at least 50 worms were selected to be scoured as alive or dead every 2 h. The survival percentages were then calculated. At least three independent repeat experiments were performed.

Synchronized young adults were cultured on NGM plates with or without 0.1 μM VK2 and incubated at 20°C for 7 days. The worms were transferred to new NGM plates with or without VK2 every day to get rid of the progeny and then collected. Total RNA quality was measured using a NanoDrop ND-1000 (Agilent Technologies). Library preparation was performed using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina), and the quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Transcriptome alignment and quantification were performed using Hisat2 software. C. elegans genome version WBcel235 was used as the reference. Differentially expressed genes were identified using the Ballgown package of R project to calculate the Fragments Per Kilobase of gene/transcript model per million mapped fragments (RPKM). The newest version of KEGG pathway enrichment and Gene Ontology were performed using R project or Python developed by Aksomics Inc. The heat maps were generated using Group FPKM (The average of log scaled FPKM: log₂(FPKM + 1) of the genes in the groups).

The synchronized L1 worms were cultured on plates with or without 0.1 μM VK2 at 20°C for 3 days. A single worm was picked to new plates for each treatment and transferred onto fresh plates, respectively, every day until no eggs were produced. After transferring, the plates containing the eggs yielded by one single worm were put at 25°C for 2 days, and the hatched worms and dead eggs were scored for each plate.

Synchronized young adult worms were cultured on plates with or without 0.1 μM VK2 at 20°C for 7 days. The worms were then transferred to new NGM plates for each treatment, respectively, every day to remove the progeny. The worms were then collected, and total RNA was extracted using RNAiso Plus (Takara) and converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and an ABI 7500 system. The relative expression levels of the genes were assessed using the 2^−ΔΔCT method and normalized to the expression of the cdc-42 gene. P-values were calculated using a two-tailed t-test (32–34). The primers used are listed in Supplementary Table 1.

The role of VK2 on aging-related diseases has been reported in several prior studies (22, 35); however, the effect of VK2 on lifespan has not been addressed. We use C. elegans as a model to test the effect of VK2 on lifespan. It was observed that the low-dose VK2 (0.01–10 μM) treatment extended the lifespan of wild-type worms, with the 0.1 μM VK2 treatment having the strongest effect on lifespan extension (Figure 1A), demonstrating about a 20% mean lifespan extension and a maximum lifespan extension from 22 days to 29 days (Table 1). These experiments also showed that VK2 (0.1 μM) could prolong the reproductive span of wild-type worms, with the VK2-treated worms producing more progeny at day 3 and day 4 relative to the control worms (Figure 1B).

Vitamin K has been considered an anti-oxidant in relation to the aging process (35). As such, we tested the function of VK2 on stress resistance in worms. Our results showed that the survival of worms treated with VK2 was significantly improved under heat (37°C) stress (Figure 1C) and H₂O₂-induced inner oxidative conditions (Figure 1D).

A recent study also suggested that VK2 might have an effect on immunity (36). C. elegans is normally propagated on agar plates seeded with the Escherichia coli (E. coli) strain OP50 (28). To address the role of VK2 on immunity in worms, we cultured wild-type worms on a lawn of pathogenic bacteria, Pseudomonas aeruginosa (PA14) (P. aeruginosa), to test the role of VK2 on resistance to pathogen infection. The results show that N2 worms treated with VK2 demonstrated a significantly enhanced survival compared with control worms (Figure 1E), suggesting that VK2 also influenced the innate immune signaling response.

VK2 levels might impact body fat levels. Interestingly, we found that VK2-treated worms indeed had a significantly lower fat level compared with control worms (Figure 1F). Thus, by using C. elegans as a model to study the bioactive effects of VK2 in vivo, our results demonstrated that low-dose VK2 treatments have the ability to prolong longevity, increase stress and immunity resistance, and lower body fat content.

In order to assess how the observed changes in VK2-treated worms were related to gene expression, we performed RNA-sequencing (RNA-seq) analysis to assess differential gene expression patterns. The RNA-seq analysis indicated that 1,550 genes were up-regulated by at least 1.5 times in VK2-treated worms compare with the control, with only 602 genes down-regulated more than 1.5 times (Figure 2A). Pathway enrichment analyses were performed on the up-regulated gene set, and the data showed that six genes, including daf-12, fard-1, fat-5/6/7, and lipl-4, were enriched among the genes in the longevity pathway in worms. Based on the RNA-seq data, the heat-map analysis indicated that these genes were up-regulated by VK2 treatment (Figure 2B). To confirm the observed changes in gene expression levels, real-time PCR was used to quantify these changes in expression. The results confirmed that all six of these genes were up-regulated in VK2-treated worms compared with the control worms and the expression levels of fat-5 and lipl-4 were up-regulated more than the other four genes (Figure 2C). The fard-1 gene has been reported to be a target gene of DAF-12 (37). As such, the longevity extension function of VK2 was tested on daf-12 mutants. The results showed that the lifespan extension activity of VK2 was significantly reduced in daf-12 mutants (Figure 2E). The daf-16 mutants were treated with VK2, and it was found that the lifespan extension effects of VK2 were significantly diminished in the absence of daf-16 gene function (Figure 2D). To further confirm that VK2 acted via these genes to extend the lifespan of wild-type worms, the effects on VK2 were tested in fat-5 mutants and lipl-4 RNAi worms. The results showed that VK2 had almost no effect on fat-5/7 mutants (Figure 2F) and that VK2 treatment did not significantly extend the lifespan of lipl-4 RNAi worms relative to that of control worms (Figure 2G).

As VK2 can improve the ability of worms to survive in harsh environments, such as those encountered during heat stress and infection, GO enrichment was performed on the set of VK2 up-regulated genes. Forty-five genes were determined to be involved in the biological process of defense and immune response. Among these, 16 genes were identified to have a clear biological function according to the data available on Wormbase. The expression levels of these 16 genes affected by VK2 treatment were analyzed via heat-map analysis (Figure 3A) and further confirmed using real-time PCR (Figure 3B). The results showed that all 16 of these genes were up-regulated, with two genes (frk-1 and lys-7) significantly up-regulated by more than five times in VK2-treated worms compared with the control (Figure 3B). frk-1, an ortholog of human FER tyrosine kinase, is considered to be involved in several processes, including the innate immune response (38). lys-7 is also reported to be involved in the defense response in worms (39, 40). To verify that these two genes played important roles in the VK2-improved stress response, we treated frk-1 knock-down worms and lys-7 mutants with VK2 under conditions of heat and infection stress. The results showed VK2 failed to significantly improve the survival of frk-1 RNAi worms (Figure 3C) or lys-7 mutants (Figure 3D) at 37°C. Additionally, VK2-treated frk-1 and lys-7 mutants were cultured on P. aeruginosa to test their survival ability. We found that VK2 also failed to significantly improve the survival of frk-1 RNAi worms (Figure 3E) and lys-7 mutants (Figure 3F). These results implied that frk-1 and lys-7 gene expression were boosted in worms when treated with VK2 to provide protection in these harsh environments.

The RNA-seq results show that the peroxisome signaling pathway was the second most significantly enriched pathway among the 1,550 genes up-regulated by VK2 (Figure 4A). There were 18 peroxisome-related genes significantly up-regulated, and the expression levels of these genes are shown using a heat-map in Figure 4B. The expression levels of these genes were further confirmed using real-time PCR (Figure 4C). All 18 of these genes were up-regulated by VK2 treatment in wild-type worms, with acox-1.2, acs-18, and pmp-1 up-regulated more than five times compared with the control (Figure 4C). fard-1 is involved in longevity and is also related to the function of peroxisomes. We cultured fard-1 mutants under H₂O₂-induced (10 mM) oxidative conditions, and the results showed that the VK2 treatment failed to significantly improve the survival of fard-1 mutants (Figure 4D).

Peroxisomes are oxidative organelles that contain digestive enzymes for breaking down toxic materials in the cell and play an important role in the decomposition of reactive oxygen species (ROS) (41–43). The human orthologs of acox-1.2 have been implicated in peroxisomal acyl-CoA oxidase activity and are involved in fatty acid β-oxidation (Wormbase). Fatty acids are broken down by fatty acid β-oxidation in peroxisomes to produce energy (44, 45). acs-18 is predicted to activate long-chain fatty acid-CoA ligase, and pmp-1 is predicted to be a peroxisomal membrane protein and has been shown to enable long-chain fatty acid transporter activity (Wormbase). Because all three of these genes are related to fatty acid metabolism via fatty acid β-oxidation in peroxisomes, we tested the effect of VK2 on these mutants. It was found that VK2 treatment failed to improve the oxidative stress resistance induced by H₂O₂ in acox-1.2 RNAi worms (Figure 4E), acs-18 RNAi worms (Figure 4F), or pmp-1 mutants (Figure 4G). These results indicated that these three genes play important roles in the fatty acid digestion signaling cascade within peroxisomes and that VK2 can reduce the expression levels of these genes to enhance oxidant resistance.

Based on the above results, it was considered that the longevity, stress resistance, and immune response genes invoked by VK2 treatment almost all function in fatty acid metabolism. More importantly, the RNA-seq results showed that the genes up-regulated by VK2 in worms were significantly enriched in fatty acid metabolism and degradation pathways (Figure 5A). It was speculated that the main function of VK2 in regard to improving the health of the animals was mainly dependent on its ability to enhance fat digestion. To test this hypothesis, we carefully assessed the up-regulated genes induced by VK2 treatment that were enriched in fatty acid related pathways (Figure 5A). It was found that 17 VK2 up-regulated genes were involved in fatty acid metabolism and also played important roles in fat degradation and unsaturated fatty acid biosynthesis (Figure 5A). Among these, 15 genes have defined functions in fat digestion, and their roles in the fat digestion pathway are summarized in Figure 5B. These genes regulate fatty acid elongation and desaturation as well as fatty acid-CoA synthesis. Fatty acid-CoA can be degraded and digested via fatty acid β-oxidation, which is consistent with our findings that VK2 can enhance the fatty acid β-oxidation function of peroxisomes. Interestingly, among them, acox-1.2, acs-18, and fat-5 were confirmed to be involved in regulating longevity (Figures 2B,C) and stress resistance (Figures 4B,C) and also enriched in the fat metabolism pathways (Figures 5C,D). The other three genes related to fat metabolism significantly regulated by VK2 were also analyzed (Figure 5C) and further confirmed using real-time PCR. The results showed that hacd-1, acdh-8, and elo-2 were also significantly up-regulated in VK2-treated worms (Figure 5D). Next, the fat content was assessed in acox-1.2 RNAi, acs-18 RNAi, fat-5, and hacd-1 mutants. The results showed that the fat mass in the body of acox-1.2 RNAi worms (Figure 5E) and acs-18 RNAi worms (Figure 5F) as well as fat-5 (Figure 5G), hacd-1 (Figure 5H) mutants was not significantly reduced compared to controls following VK2 treatments. These results further confirmed that VK2 could enhance fat digestion mechanisms, especially fatty acid β-oxidation, to improve the survival of worms.