Paecilomyces variotii extract increases lifespan and protects against oxidative stress in Caenorhabditis elegans through SKN-1, but not DAF-16

2.1 Preparation of PVE

The P. variabilis SJ1 strain was isolated from wild sea buckthorn (Hippophae rhamnoides) and deposited in China General Microbiological Culture Collection Center (CGMCC) as P. variabilis CGMCCNO.10114. PVE was extracted from P. variabilis SJ1 strain according to Wang et al. (2021a,b). Briefly, the SJ1 hyphal culture by three-stage liquid fermentation was filtered with a 300-mesh filter cloth under pressures of 0.2 and 0.4 MPa, respectively, and the filtrate was mixed with the same volume of 95% alcohol. The mixture was sealed in a container for 14 days to allow extraction of PVE. Then the mixture was injected into a 500- liter tank at 25 °C with a stirring speed of 1000 g/min and a frequency of 20.0 kHz. Then, a filter press was used to remove residue of the mycelia. After drying under vacuum at 40 °C, PVE was obtained from the infiltrate. The average molecular weight of PVE was 758 Da as determined by liquid chromatography electrospray mass spectrometry (LC-ESI-MS) (Wang et al., 2021a,b). In PVE, the organic matter content was 81.5%, with the content of saccharide, protein, amino acid, nucleoside, and lipid of 33.33%, 19.24%, 28.96%, 7.4% and 3.75%, respectively (Wang et al., 2021a,b). PVE at 1.0 mg/mL was prepared in 20% alcohol as stock solution and stored at 4 °C prior to use.

2.2 C. elegans strains and culture conditions

The wild-type Bristol N2, the transgenic nematode strains and Escherichia coli OP50 were provided by the Caenorhabditis Genetics Center (University of Minnesota, USA). The transgenic nematode strains used in this work were: GR1307 [daf-16(mgDF50)], LD1 (ldIs7 [skn-1b/c::GFP + rol-6(su1006)]), EU1 [skn-1(zu67)], and TJ356 (zIs356[daf-16p::daf-16a/b::GFP + rol-6(su1006)]). All worms were cultured at 20 °C in nematode growth medium (NGM) plates with E. coli OP50 as food. C. elegans strains were synchronized according to Brenner (1974). After synchronization, the L4 C. elegans were prepared for further experiments.

2.3 Toxicity assay

PVE was dissolved in M9 buffer to obtain the final concentrations of 1.0, 10, 100, 1000 and 10000 ng/mL. M9 buffer only was served as the solvent control. Each individual well of a 24-well plate was filled with equal volumes (1.0 mL) of PVE at corresponding doses and C. elegans suspension containing 60 synchronized L4 worms at 20 °C. The survival of nematodes was recorded every day and data were expressed as the survival of nematodes after 72 h. The experiment was conducted in independent triplicates.

2.4 Bacterial growth assay

Bacterial growth assay was performed to exclude caloric restriction induced by PVE. Briefly, the final concentration of PVE at 100 ng/mL was added to freshly prepared E. coli OP50 solutions. M9 buffer only was included as the solvent control. E. coli OP50 was cultured for 6 h at 37 °C and the OD₆₀₀ values were measured with a microtiter plate reader (Synergy H1, BioTek, USA). The experiment was conducted in independent triplicates.

2.5 Measurement of lifespan and brood size assays

Sixty L4 synchronized Bristol N2 worms were transferred to fresh NGM plates with or without PVE at 20 °C. 5-fluoro-2-deoxyuridine (Sigma-Aldrich) at 50 μM was used to inhibit progeny growth. Worms were transferred every other day to fresh extracts. The number of living nematodes was counted daily until the last nematode was dead. For the brood size assay, twenty synchronized L4 nematodes were individually transferred to new NGM plates pretreated with PVE at various concentrations or the solvent control and allowed to lay eggs. The number of eggs was counted daily until the worms stopped laying eggs. Both experiments were conducted in independent triplicates.

2.6 Growth, locomotive ability and pharyngeal pumping rate assays

Body length, width and area of wild-type C. elegans were measured following Liu et al. (2022a). Briefly, after treatment with PVE for 24 h, the worms were burned with an alcohol lamp for 10 s and immobilized. The body length and width of C. elegans was measured using an Olympus SZX10 stereoscope. Body area was the product of body length and width. Locomotion assay was performed according to Yu et al. (2021). After exposure, the nematodes were washed with M9 buffer and then placed onto new NGM plates. After 1 min recovery, the frequency of head thrashes and body bends in 30 s was recorded under a stereomicroscope. Pharyngeal pumping rate was measured following Huang et al. (2004). After treatment, worms were washed and transferred to fresh NGM plates without E. coli OP50 and the pharyngeal pumping rate within 30 s was counted after 2 min of exercise. These experiments were conducted in independent triplicates with 20 nematodes for each treatment.

2.7 Measurement of intracellular ROS, lipofuscin and lipid accumulation

The L4 stage wild-type N2, GR1307 [daf-16(mgDf50)] and EU1 [skn-1(zu67)] of nematodes were treated with PVE at 1.0, 10 and 100 ng/mL for 24 h. Then worms were washed three times using M9 buffer. Subsequently, the nematodes were transferred into a 2-mL centrifuge tube, followed by centrifugation at 4000 rpm for 2 min, and the supernatant was removed. Precipitated nematodes were used in the following assays. (1) ROS production: the nematodes were pre-incubated in 1 μM of CM-H₂DCFDA (Beyotime Biotechnology, China) for 2 h at 20 °C. (2) Lipofuscin accumulation: the nematodes were pre-incubated in levamisole at 1 mM. (3) Lipid accumulation: the nematodes were treated by paraformaldehyde (4%) for 1 h. Then the nematodes were stained with Nile red solution (1 μg/mL for 2 h. The worms were washed with S-basal [5.85 g NaCl, 6 g KH₂PO₄, 1 g K₂HPO₄, and 1 mL cholesterol (5 mg/mL in ethanol) in 1 L H₂O, and sterilized by autoclaving]. Fluorescence was detected with a fluorescence microscope (Olympus TH4-200, Japan). Fluorescence intensity was measured using ImageJ. These experiments were conducted in independent triplicates with 20 nematodes for each treatment.

2.8 Determination of MDA content and antioxidant enzyme activities

The content of MDA and activities of antioxidant enzyme assays were determined according to Hui et al. (2020). The nematodes were pretreated with PVE at different concentrations for 24 h at 20 °C. Worms (∼7000) were washed thrice with M9 buffer, and mixed with PBS (phosphate-buffered saline) buffer. The mixture was homogenized in an ice bath, centrifuged at 4000 rpm for 3 min at 4 °C. The supernatant was collected for further analyses. The MDA content and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) were measured using the manufacturer’s protocol (JianCheng, NanJing, China). These experiments were conducted in independent triplicates.

2.9 Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using Trizol reagent, and the cDNA was produced by the Quantscript RT kit (Accurate Biology, China). The qRT-PCR was conducted to test the expression of antioxidant-related genes by using a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, US). The 2^-△△Ct method was used to quantify the relative gene expression, and act-1 was selected as the reference gene (Livak and Schmittgen, 2001). The genes and their PCR primers are listed in Table S1. The experiment was conducted in independent triplicates.

2.10 Intracellular localization of DAF-16 and SKN-1

Intracellular localization of DAF-16 and SKN-1 was analyzed with the transgenic C. elegans strains TJ356 and LD1, respectively. Synchronized mutant nematodes were cultured on NGM plates with PVE (1.0, 10 and 100 ng/mL) for 24 h at 20 °C. After exposure, mutant nematodes were fixed by sodium azide at 20 mM, and a fluorescence microscope was used to observe subcellular distribution. Intracellular location of DAF-16 and SKN-1 was classified into three categories: cytosolic, intermediate and nuclear. The experiment was conducted in independent triplicates with 30 nematodes for each treatment.

2.11 Statistical analysis

Data were expressed as the mean ± SEM and analyzed with SPSS (V16.0, IBM, Chicago, IL). Significant differences were measured using analysis of variance (ANOVA) with Student-Newman-Keuls test at p = 0.05.

3.1 PVE promoted the lifespan in C. elegans and had no obvious effect on bacteria growth and nematodes fecundity

Before conducting other experiments, an in vivo toxicity study was performed. As depicted in Fig. S1, PVE at all tested concentrations were non-toxic to C. elegans. Then lifespan assays were performed and the results showed that PVE at the tested concentrations significantly (p < 0.05) prolonged the mean lifespan of C. elegans as compared to the solvent control (Fig. 1). The mean lifespan of worms treated with PVE at 1, 10, 100, 1000, and 10000 ng/mL was between 12.78 and 15.98 days, while that of the solvent control group was only 10.00 days (Table S2). The result was consistent with previous reports that many extracts had potent lifespan extension properties (Martel et al., 2020). Cuong et al. (2019) reported that lifespan was significantly extended by 35.84% after exposure to extract from Ganoderma lucidum (Basidiomycetes). In our study, exposure to PVE at 1.0, 10 and 100 ng/mL significantly (p < 0.001) extended lifespan by 36.60%, 59.80% and 53.30%, respectively, as compared to the solvent control group. Thus, the concentrations of 1.0, 10 and 100 ng/mL of PVE, which were most effective in improving longevity of C. elegans, were selected for further tests.

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Fig. 1

Effect of Paecilomyces variotii extract (PVE) on the lifespan of C. elegans. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10, 100, 1000 and 10000 ng/ml.

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Previous studies have shown that PVE contained antioxidants, showing antimicrobial and anti-inflammation activities in vitro (Dai et al., 2020; Moreno-Gavíra et al., 2020). It is also suggested that proliferating bacteria adversely affected the nematodes by yielding harmful metabolite (Garigan et al., 2002). It is possible that PVE extended the lifespan via restricting bacterial growth. To determine the influence of PVE on the growth of E. coli OP50, the growth of bacteria in the control and PVE at 100 ng/mL group was assessed (Fig. S2). Results showed that PVE did not inhibit E. coli OP50 growth. Therefore, it is unlikely that PVE extended the lifespan of the nematodes via its antimicrobial effects or restricting bacterial growth.

In a previous study, it was observed that longevity was negatively correlated with fecundity (Arantes-Oliveira et al., 2003). To clarify the effect of PVE on brood size, the number of offspring was measured. Our result showed no significant differences were observed in brood size between PVE-treated and the solvent control groups (Fig. S3). This indicated that PVE had no effect on the reproduction of the nematodes.

3.2 PVE promoted growth but did not influence locomotion behavior and pharyngeal pumping in C. elegans

To determine whether PVE had side effects on the health of C. elegans, we evaluated its effects on growth, locomotion behavior, and pharyngeal pumping of the nematodes. Compared to the solvent control, PVE at 1.0 and 10 ng/mL significantly (p < 0.05) increased the body length of C. elegans (Fig. 2A). There were no significant differences between 100 ng/mL of PVE treatment and the solvent control (p = 0.158). The body width of the nematodes treated with PVE at 10 and 100 ng/mL was significantly (p < 0.001) increased compared to the solvent control (Fig. 2B). There were no significant differences between the nematodes exposed to 1.0 ng/mL of PVE and the solvent control (p = 0.402). Only PVE at 10 ng/mL significantly (p < 0.001) increased the body area of C. elegans compared to the solvent control (Fig. 2C). Body growth is an important indicator for assessing C. elegans health. Our results showed that PVE at 10 ng/mL consistently increased the growth of the worms. This agreed with previous studies, indicating that the worms had larger size after exposure to extracts from G. lucidum or Lentinus edodes (Liu et al., 2022b; Peng et al., 2021).

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Fig. 2

Effect of Paecilomyces variotii extract (PVE) on growth of C. elegans. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. (A) body length; (B) body width; (C) body area. The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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Pharyngeal pumping and locomotion behavior are regulated by complex neural networks and muscles, and motor abnormality reflects function defects in neurological or muscular (Zhen and Samuel, 2015). As shown in Fig. S4, no statistical differences were observed between PVE treatments and the solvent control in locomotion behaviors (head thrash and body bend) and pharyngeal pumping, indicating that PVE treatments had no effects on nematode motility and pharyngeal pumping. This disagrees with a previous study, which showed that Cordyceps bassiana spore (Ascomycetes) extract at 0.145 mg/mL significantly increased the frequency of head thrashes and body bends of the nematodes (Ye et al., 2021). Results from Tiwari et al. (2014) also indicated that no negative effects by an endophytic fungus of Curcuma amada extract were detected on the pharyngeal pumping of the nematodes.

3.3 PVE decreased oxidative stress in C. elegans

Extended lifespan is often accompanied by enhanced stress resistance (Song et al., 2020), so we detected the effect of PVE on oxidative stress and antioxidant enzyme activities. Intracellular ROS, lipofuscin and lipid accumulations are important biomarkers of oxidative stress and aging in the nematodes (Huang et al., 2004). Results from our present study showed that PVE at three tested concentrations significantly reduced the levels of ROS compared to the solvent control, with PVE at 10 ng/mL being the best (Fig. 3A and B). There were no differences in ROS levels between 1.0 and 100 ng/mL of PVE treatment groups (p = 0.660). PVE treatments also significantly reduced the lipofuscin content in the nematodes (p < 0.001) compared to the solvent control (Fig. 3C). A dose-response relationship was found between PVE dose and lipofuscin content. Moreover, compared with the solvent control, PVE at 100 ng/mL significantly (p < 0.001) inhibited the accumulation of lipid (Fig. 3D). PVE at lower doses (1.0 and 10 ng/mL) did not significantly (p = 0.074 and 0.122, respectively) affect the accumulation of lipid in the nematodes.

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Fig. 3

Effect of Paecilomyces variotii extract (PVE) on oxidative stress in C. elegans. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. The results were shown as the percentage relative to the solvent control. (A) Representative images showing accumulations of ROS, lipofuscin and lipid, respectively; (B) Comparison of ROS production; (C) Comparison of lipofuscin accumulation, and (D) Comparison of lipid accumulation. The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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Natural compounds have been well-documented to promote stress resistance in C. elegans (Benedetti et al., 2008). Oxidative stress can be induced by an excessive increase of ROS in C. elegans, and lipofuscin and lipid accumulations are promoted by oxidative stress (Brunk and Terman, 2002). In the present study, PVE decreased accumulation of ROS, lipofuscin and lipid. This indicated that PVE mitigated the damage of intracellular DNA, proteins and other biological macromolecules caused by excessive ROS, reduced lipid peroxidation and enhanced stress resistance, thus improving the survival of C. elegans. The results were agree with Lin et al. (2018) and Cuong et al. (2019), who reported that extract from Cordyceps sobolifera (Ascomycetes) and G. lucidum ameliorated oxidative stress in the nematodes. As for the accumulation of ROS, PVE at 10 ng/mL was lower than that of 100 ng/mL. Our results were in agreement with Azevedo et al. (2019), who reported that an aqueous leaf extract (ALE) from Uncaria tomentosa reduced intracellular accumulations of ROS in N2 worms. The accumulation of ROS in ALE at 40 μg/mL treatment was significantly lower than that of ALE at 80 μg/mL, which might be the reason that ALE at 40 μg/mL switched from an antioxidant to a pro-oxidant behavior. Further investigations are needed to validate if this is the reason for the results obtained in the present study.

3.4 PVE reduced MDA content and enhanced antioxidant enzyme activities in C. elegans

MDA affects the activity of antioxidant enzymes and aggravates the damage to membranes in organisms to accelerate aging process (Lapointe and Hekimi, 2010). Results from our study exhibited that PVE at 1.0, 10, 100 ng/mL significantly (p < 0.05) reduced the level of MDA compared to the solvent control group, with PVE at 10 ng/mL being the best (Fig. 4A). Antioxidant enzymes mitigate biological oxidative damage, and reduce the production of excessive ROS, playing a key role in the defense system (Beckman and Ames, 1998). All PVE treatments significantly increased activities of SOD, CAT and GST in the nematodes, with PVE at 10 ng/mL being the best (Fig. 4B-D). SOD and CAT had a same trend that the highest activity was observed in PVE at 10 ng/mL treatment, followed by PVE at 100 and 1.0 ng/mL with PVE at 1.0 mg/mL being the least. For GST activity, there was no statistically significant difference between PVE at 1.0 and 100 ng/mL (p = 0.165).

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Fig. 4

Effect of Paecilomyces variotii extract (PVE) on MDA content and antioxidant enzyme activities in C. elegans. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. (A) MDA content; (B) SOD activity; (C) CAT activity; (D) GST activity. The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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MDA is the end product of lipid peroxidation, which is considered to be a key indicator to evaluate oxidative damage (Li et al., 2016). SOD, CAT, and GST are antioxidant enzymes that scavenge oxygen free radicals in organisms. For instance, Hu et al. (2021) found that flavonoids from Folium Artemisia Argyi reduced MDA level and improved the antioxidant defense system. The results agreed with ours that PVE treatment enhanced the oxidative stress resistance and improved the antioxidant defense system.

3.5 PVE affected gene expression related to oxidative stress

Our results showed that PVE reduced ROS content and increased lifespan of the nematodes. To explore the pathways involved, the expression of several genes related to oxidative stress in the nematodes was investigated. In the wild-type N2, PVE exposure increased mRNA level of skn-1, mev-1, sod-3, and daf-2, but significantly down-regulated gene expression of nhr-49 and daf-16 (Fig. 5), suggesting that these genes played important roles in mediating oxidative stress in the nematodes. Our results agreed with previous reports, in which extracts from rhodiola and raspberry substantially increased gene expressions of daf-16, sod-3, gst-4, daf-2 and skn-1 (Jiang et al., 2021; Song et al., 2020). Contrary to their results, our results showed that PVE treatment significantly down-regulated gene expression of daf-16. Deng et al. (2020) found that activation of SKN-1 caused strong inhibition of DAF-16 reporters, indicating that SKN-1 may be a negative regulator of DAF-16. This discrepancy could be explained by the complex roles of DAF-16 and SKN-1 in stress resistance and longevity regulation (Deng et al., 2020; Tullet et al., 2017). Havermann et al. (2016) reported that baicalein modulated stress-resistance and lifespan in C. elegans through SKN-1, but not DAF-16. Therefore, whether there is a tradeoff between daf-16 expression and lifespan of C. elegans will need further investigation.

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Fig. 5

Effect of Paecilomyces variotii extract (PVE) on expression of genes related to oxidative stress. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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3.6 PVE induced nuclear translocation of SKN-1::GFP, but not affect DAF-16::GFP

SKN-1 and DAF-16 are important transcription factors that modulate stress response and longevity of C. elegans by natural compounds (Baumeister et al., 2006). In this study, PVE at three concentrations significantly promoted the nuclear localization of SKN-1 (Fig. 6A). Compared with the solvent control, the nuclear localization was significantly (p < 0.05) enhanced by 31.67%, 45.00% and 41.67%, respectively, in the nematodes treated with PVE at 1.0, 10 and 100 ng/mL (Fig. 6B). In contrast to SKN-1, PVE treatments did not affect nuclear translocation of DAF-16 (Fig. 6C). Our results suggested that SKN-1 was the key transcription factor that modulated oxidative stress and lifespan in C. elegans induced by PVE. Most studies indicated that SKN-1 and DAF-16 were activated simultaneously to increase stress resistance and extend lifespan in the nematodes (Deng et al., 2020; Jiang et al., 2021; Song et al., 2020). Contrary to results mentioned above, our results showed that PVE induced nuclear translocation of SKN-1, but not affect DAF-16. Tullet et al. (2017) suggested SKN-1 promoted longevity by a mechanism other than protection against oxidative damage. Thus, a balance between DAF-16 and SKN-1 may appear to promote health in C. elegans with reduced IIS (Park et al., 2021).

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Fig. 6

Effect of Paecilomyces variotii extract (PVE) on intracellular localization of SKN-1 and DAF-16 in C. elegans. L4 stage wild-type N2 nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. (A) The cytosolic localization of SKN-1::GFP and DAF-16::GFP; (B) Intracellular localization of SKN-1; (C) Intracellular localization of DAF-16. The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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3.7 PVE promoted the expression of skn-1 downstream genes, but not affected that of daf-16

To further confirm the role of SKN-1, the expression levels of its downstream genes (gcs-1 and gst-4) were determined. Our results showed that PVE at 10 ng/mL significantly influenced the gene expression of gst-4 and gcs-1 in wild-type C. elegans (Fig. S5). However, these changes disappeared in the skn-1 (zu67) mutant nematodes. Similarly, we detected the effect of PVE on daf-16 downstream genes, including ctl-2 and hsp-16.2. The results showed that PVE at three concentrations significantly (p < 0.05) down-regulated the expression of hsp-16.2 in N2 and GR1307 worms, while, the expression of ctl-2 had no significant differences for all of the PVE treatments and the solvent control (Fig. S6).

3.8 Effects of PVE on the ROS levels of daf-16 and skn-1 mutant strains

Mutant strains lacking daf-16 (GR1307) and skn-1 (EU1) genes were used to quantify the intracellular ROS levels. In GR1307 worms, the ROS levels were significantly lower in all of the PVE treatments compared with the solvent control (Fig. 7C and D). On the other hand, in the EU1 worms, PVE at 1.0 ng/mL showed a prooxidant activity, with the ROS accumulation increasing by 23.40% in comparison with the solvent control (p < 0.001; Fig. 7A and B). No significant differences were observed between PVE treatments at 10 and 100 ng/mL and the solvent control. This result correlates with a study by Tullet et al. (2017) showing that SKN-1 was constitutively active in increasing lifespan independent of DAF-16.

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Fig. 7

Effect of Paecilomyces variotii extract (PVE) on reactive oxygen species (ROS) production of C. elegans. L4 stage mutant nematodes were exposed to PVE at 0 (M9 buffer), 1.0, 10 and 100 ng/ml for 24 h. (A) Representative images showing ROS production in EU1 (skn-1(zu67)); (B) Comparison of fluorescence intensity in EU1 (skn-1(zu67)); (C) Representative images showing ROS production in GR1307 (daf-16(mgDF50)); (D) Comparison of fluorescence intensity in GR1307 (daf-16(mgDF50)). The results were expressed as mean ± standard error of the mean. The statistical significance of difference was analyzed by one-way ANOVA with Student-Newman-Keuls test. Bars with no letters in common are significantly different (p < 0.05).

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