S.Sanghuang was cultured in a 5-litre fermentation tank (Baoxing Bioengineering Equipment Co., Ltd., Shanghai, China) according to the methods used in our laboratory (Guo et al., 2021). One week later, the fermentation liquid was centrifuged to obtain supernatant and mycelial fractions. The mycelia were completely dehydrated in a 60°C oven, and the products were then boiled in water for 2 h. To obtain aqueous extracts, the mixture was centrifuged at 12,000 rpm for 15 min, and the supernatant was separated. The aqueous extracts were freeze-dried with a vacuum freeze-dryer to obtain the dry powder of SSE. The dry powder was stored in a 4°C refrigerator, and solutions with different concentrations were prepared as required during the experiment.

The NGM medium solution was prepared according to the literature (Hou et al., 2019) and then placed into an autoclave for sterilization. It was then poured quickly onto plates and allowed to cool until solidification. The plates were inverted and placed in a 4°C refrigerator until use. OP50 is an uracil trophic deficiency type E. coli, which prevents Escherichia coli from obstructing the growth of nematodes (MacNeil et al., 2013). The OP50 bacteria were streaked onto solid LB medium and then cultured in a 37°C incubator for 24 h. A single colony was selected and placed into 100 mL of LB liquid medium and cultured in a 120 rpm/min 37°C incubator for 8–10 h until the OD600 was approximately 1.5. The bacteria were then stored in a 4°C refrigerator until use.

All strains were obtained from the Caenorhabditis Genetics Center (CGC). Wild-type N2 C. elegans were cultured on NGM media covered with E. coli OP50 as food and incubated in a 20°C incubator. The nematode lysis buffer contained 5% NaClO and 5 M NaOH, and the nematodes that were collected from the M9 buffer were mixed with the lysis buffer. The eggs were centrifuged at 300 rpm/min and cultured on new NGM media without OP50. Two days later, all nematodes were in the Dauer stage (Wong and Roy, 2020) and were then transferred to plates covered with OP50 to obtain age-synchronized nematodes.

OP50 monoclones were placed into 100 mL of LB liquid medium at 37°C and incubated for 12 h. In addition, three bottles of LB liquid medium were prepared, and one of the bottles was used as a blank control. A 100 μL volume of was added OP50 to each of the remaining 2 bottles and an additional 100 µL of SSE was added to one bottle. The cells were cultured in an incubator at 180 rpm/min and 37°C for 8 h, and the OD600 values were measured hourly.

The NGM media of S. The Sanghuang treatment group were covered with 150 μL of SSE solutions with different concentrations and 150 μL of OP50; meanwhile, the control group was covered with 150 μL of M9 buffer solution and 150 μL of OP50. Five-day-old nematodes were placed on media with different concentrations of SSE solution (e.g., 50, 25 and 10 mg/mL), and the pharyngeal pumping rates were observed under a microscope.

Growing synchronized nematodes at the L4 stage were picked from the NGM plates, with 50 nematodes per plate, and at least 3 plates were used for both the control and treatment groups. All of the plates were covered with 150 μL of OP50 and S. angusthuang solutions with different concentrations (e.g., 50, 25 and 10 mg/mL), while the control group used M9 buffer instead of medicine. Then, 50 μM 5-fluoro-20-deoxyuridine (FUDR) was added to each plate to prevent nematode reproduction. The time at which the worms were first treated with SSE was recorded as day 0, and the plates were then placed in a 20°C incubator. The nematodes were transferred to new treatment plates every day to eliminate interference from offspring nematodes. If a needle touched a nematode head several times and there was no response, this nematode was recorded as dead. The number of dead nematodes was recorded until all of the nematodes on each plate were dead, and the survival rates of C. elegans were calculated. The worms that died in the process of selection, became desiccated, were stuck to the sides of plates or experienced other abnormal deaths were excluded from the statistics. GraphPad software was used to plot the survival rates, and the data are reported as the mean ± SD. The experiments were independently repeated at least three times.

Lipofuscin is a marker of senescence in nematodes that can be analysed by using fluorescence microscopy (Kampkotter et al., 2007; Shen et al., 2018). The synchronized L4-stage nematodes were cultured on NGM plates that were covered with SSE solutions of various concentrations. After 10 days of treatment, the worms were first anaesthetized with 100 μL of levamisole (1 M), and the fluorescence intensities of lipofuscin were then observed under a fluorescence microscope and recorded. ImageJ software was used to analyse the relative fluorescence intensities.

During the lifespan assay, we observed the athletic abilities of nematodes on each plate on the 8th, 12th, 16th and 20th days of adulthood. C. elegans individuals were given a mechanical stimulus with a platinum wire and according to the response, it was possible to judge their athletic abilities. “Normal” nematodes moved rapidly without being touched by the wire, “sluggish” nematodes moved only after their heads were touched, and “Immobile” nematodes, even when their heads were touched, moved only slightly. The numbers of nematodes that were in these three states were recorded, and their athletic abilities were calculated.

Synchronized L4-stage nematodes were picked from the control and treatment groups, with only one worm per plate. The worms were cultured under normal conditions, and the time of the first treatment with SSE was recorded as the first day. The worms were transferred to new plates every day and were observed under a light microscope, and the numbers of eggs were calculated. When all of the worms stopped laying eggs, the number of offspring from each worm was counted and summed to obtain the total number of offspring and the reproduction rate was calculated.

Synchronized L4-stage nematodes were picked from the control and treatment groups, with at least 100 nematodes per plate. Ten worms were randomly selected from each plate every other day for microscopic observations, and this procedure was repeated three times to decrease the error levels. We obtained pictures and videos, and ImageJ software was used to determine the body lengths and calculate the number of sinusoidal movements in 30-s intervals.

The transparent bodies of C. elegans enable visualization of fat stores by using dye-labelled imaging (Shen et al., 2018). Here, we used Oil Red O to dye the worm fat. We also cultured synchronized L4-stage C. elegans from the control and treatment groups for 5 days and then collected these nematodes into 1.5-mL tubes. They were then starved for 6 h to burn off excess fat. Paraformaldehyde was used to immobilize the nematodes, which were cleaned with isopropyl alcohol after repeated freeze-thaw cycles. The nematodes were immersed in Oil Red O for 12 h to complete the dying process. The nematodes were then repeatedly rinsed and photographed under a microscope. The fat metabolism speeds were analysed with ImageJ software.

We cultured synchronized L4-stage C. elegans from the control and treatment groups for 5 days. They were transferred to new NGM plates and placed in a 37°C incubator to cause exposure to thermal stress. Every 1.5 h, nematode deaths were observed, and nematodes were determined to dead if they did not respond to repeated stimulation. When all worms were dead, the survival rates under thermal stress were calculated. Each plate contained at least 40 worms. These experiments were repeated at least three times.

The preliminary treatment was the same as for the thermotolerance assay. Then, the worms were transferred to plates that were covered with 100 μL of 3% H2O2. Every 2 h, nematode deaths were observed, and nematodes were determined to be dead if they did not respond to repeated stimulation. We recorded the numbers of dead nematodes and calculated the survival rates. Each plate contained at least 30 worms. These experiments were repeated at least three times.

According to a previous report (Back et al., 2012), we quantitatively analysed the ROS levels in nematodes with a specific fluorescent molecular probe (DCFH-DA) that used a Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). We also cultured synchronized L4-stage C. elegans in the control and treatment groups for 5 days and then collected these nematodes in 1.5-mL centrifuge tubes with M9 buffer. The nematodes were treated according to the ROS Assay kit instructions, and photographs were taken under a fluorescence microscope. ImageJ software was used to analyse the relative fluorescence intensities.

We cultured synchronized L4-stage C. elegans from the control and treatment groups for 7 days, with approximately 500 worms per plate. The nematodes were ground in a mortar filled with liquid nitrogen by using TRIzol reagent to extract total RNA. The RNA qualities and concentrations were determined with a nucleic acid analyser. First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used to obtain cDNA, and the PCR conditions were 42°C for 15 min and then 85°C for 5 s. An Applied Biosystems 7,500 Real-Time PCR System (Applied Biosystems, Waltham, United States) was used to conduct quantitative real-time PCR with SYBR Green Supermix (Takara Biotechnology, Dalian, China). The 2^−ΔΔCT method was used to analyse the gene expression levels.

Wild-type C. elegans treated with 25 mg/mL SSE for seven days were assessed by the APExBIO Company (Shanghai, China). Each sample was about 50mg, over two thousand nematodes, and each group had at least three samples. After pre-processing, the samples were analyzed for inter-group differentiation.

All of the data are presented as the mean values ± SDs that were obtained from at least three replicates. GraphPad Prism 8.02 (GraphPad Software, CA, United States) was used to examine the statistical significance of the differences and to construct the graphics presented. T tests were used to perform the significance tests, and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 were considered to be statistically significant.

Food is one of the most important environmental factors, and dietary restrictions have a great influence on the ageing and other physiological activities of nematodes (Kenyon, 2010; Zhang et al., 2021). One potential concern is that SSE might have limited feeding by affecting the growth of E. coli, so we first verified whether SSE had an effect on E. coli OP50 inhibition. We found that the growth rate of the OD600 value of the SSE groups nearly overlapped with that of the control group (Figure 1A), which indicated that SSE does not interfere with the regular growth of E. coli OP50.

Lifespans provide a key piece of evidence for the ageing of nematodes, so we cultured wild-type C. elegans N2 with different SSE concentrations (e.g., 50, 25, and 10 mg/mL) to investigate whether SSE affected the lifespan of C. elegans. As shown in Figure 1B and Table 1, the survival curves of all nematodes were nearly the same until the 13th day, but after day 14, the survival curves for the SSE treatment groups shifted to the right compared to the control group, and these shifts were especially obvious for the 25 mg/mL group. The mean lifespan for the control group was 16.23 ± 0.36 days, while the 25 mg/mL group survived the longest, reaching 20.52 ± 0.34 days, which was an increase of 26.41%. The lifespans of the 50 and 10 mg/mL groups also increased by 12.28% and 16.27%, respectively. There was also a visible increase in the maximum lifespan. As indicated above, we hypothesized that SSE treatments effectively prolonged the lifespan of nematodes, and other indicators were selected to further explore the influence of SSE on ageing.

Reproduction is one of the most important tasks for living beings; the reproductive capacity is therefore an important indicator of health and ageing. Nematodes generally have higher reproductive capacities in the first 3 days of the L4 stage, and with increasing age, these capacities continuously decrease and stop at approximately the end of the fifth day (Figure 2A). As shown in Figure 2B, the average spawning production of the control group was 239.5 ± 1.4, and there were no significant differences in the total number of eggs produced among the groups that were treated with different SSE concentrations, namely, 215.5 ± 2.9, 254.5 ± 2.6, and 241 ± 3.1 in the 50, 25, and 10 mg/mL groups, respectively. The results showed that the SSE treatments had no effect on nematode reproduction.

Lipofuscin accumulations are an important sign of nematode senescence; the senescence degrees of nematodes can be determined by the fluorescence intensities of lipofuscin determined under a microscope. We found that the nematodes treated with SSE exhibited lighter colours under the microscope, which indicated that the SSE treatments reduced lipofuscin accumulations (Figure 3A). Compared with the control group, the fluorescence intensities decreased by 29.2%, 54.5% and 37.9% at SSE treatment concentrations of 50, 25, and 10 mg/mL, respectively (Figure 3C).

ROS accumulations are considered to be one of the important causes of nematode senescence, and decreasing ROS production can effectively delay ageing. Dichlorofluorescin-diacetate (DCFH-DA) was used to determine the ROS levels in nematodes. Intracellular ROS can oxidize non-fluorescent DCFH into fluorescent DCF; thus, the ROS levels in nematodes can be determined from the fluorescence intensities. As shown in Figure 3B, the green fluorescence of nematodes that were treated with SSE was substantially dimmer than that of the control group. By using the software to process the images, the observed ROS levels were reduced by 83.4%, 87.8% and 56.6% among the SSE groups treated with concentrations of 50, 25, and 10 mg/mL, respectively, compared to the control nematodes (Figure 3D). The results indicated that treatment with SSE effectively reduced ROS accumulations in nematodes.

Body size, movement behaviour, and movement speed are all important indicators of the physiological responses of nematodes; their states are also closely linked to ageing and health. We first evaluated the body sizes of nematodes that were treated with SSE. When the nematodes were just beginning to develop (e.g., on days 4 and 6), the body sizes for the SSE treatment groups were shorter than those of the control group (Figure 4A). However, when the nematodes developed further (e.g., after day 8), the body sizes for the nematodes in the SSE treatment groups was visibly longer than those in the control group. On day 12, the vast majority of nematodes were fully developed, and their body sizes also tended to be stable. The results showed that there were no significant differences in body size among the groups; indeed, at puberty, the SSE treatments even promoted nematode growth.

In addition to measuring body lengths, we also measured the movement speeds of the worms. Obviously, the SSE-treated nematodes exhibited greater speeds than the control nematodes in both the infancy and mature periods. It is worth noting that the 10 mg/mL treatment was associated with greater increases in the early stage, but when the nematodes matured, the 25 and 50 mg/mL SSE groups exhibited more significant effects (Figure 4B). This may be related to the adaptation of nematodes to the SSE treatments. Meanwhile, we observed the motility characteristics of nematodes at different life stages (e.g., on days 8, 12, 16, and 20). All of the worms moved normally in the early stage of the life process, but as the worms aged, the proportions that could move freely decreased towards the end of life. The control group contained almost no functioning nematodes (Figure 4C). However, SSE treatments improved this situation. The worms in the control groups that still exhibited normal movement represented less than 50% of the initial number (on day 16), 70% of the worms treated with 25 mg/mL of SSE moved regularly, and the 50 mg/mL and 10 mg/mL groups also showed varying degrees of improvement. By day 20, the SSE-treated worms still exhibited better movement capabilities than those in the control group; 32% and 15% of the nematodes treated with 25 and 10 mg/mL, respectively, of SSE still moved spontaneously. Last, we compared the pharyngeal pumping rate of worms, either in the presence or absence of SSE. The result indicated that SSE did not affect the pharyngeal pumping rate of the worm (Figure 4D).

Obesity is very detrimental to health, and ageing also leads to fat accumulation, so accelerating the lipid metabolism provides significant health and antiaging benefits. Oil red O is a specific dye for lipid particles, and the brightness of the red colour observed under the microscope indicates the accumulated lipid amounts. As shown in Figure 5A, the control nematodes were deep red, whereas the red colours of nematodes that were treated with SSE became lighter, especially in the 25 mg/mL group, and the nematode bodies were nearly transparent. The average grey value analysis also proved that the SSE groups exhibited brighter red colours than the control group (Figure 5B). The results showed that SSE treatments can decrease lipid contents and accelerate lipid metabolism in C. elegans.

Improved stress resistance in C. elegans is considered to be one of the most important manifestations of life extension. We first measured the survival times of nematodes at 37°C to investigate the effect of SSE treatments on heat shock resistance. As shown in Figure 6A and Table 2, the mean lifespan of the control nematodes was 4.52 ± 0.09 h (maximum of 10 ± 0.70 h), which was clearly lower than those in the SSE treatment groups. The group that survived longest was the 25 mg/mL group, in which the mean lifespan reached 7.97 ± 0.20 h (maximum of 13.5 ± 0 h), an increase of 76.19%. The SSE treatments at concentrations of 50 and 10 mg/mL also had clear effects, and the mean lifespans increased by 34.78% and 49.83%, respectively. These results revealed that SSE treatments could evidently decrease heat shock damage and increase the survival rates of C. elegans.

Subsequently, we investigated whether SSE could improve the antioxidant capacity of C. elegans, and vitamin C was added as a positive control. The survival rates were similar to those obtained from the heat shock assay (Figure 6B; Table 3): the mean lifespan of the control nematodes was only 0.57 ± 0.05 h (maximum of 3.00 ± 1.00 h) in 100 μL of 3% H₂O₂. The 25 mg/mL group exhibited the best effect, with a mean lifespan of 1.64 ± 0.59 h (maximum of 5.67 ± 0.94 h), which represented an increase of 185.32%, and this was very close to that of the positive control group. The 50 and 10 mg/mL groups exhibited increases of 87.09% and 140.45%, respectively. SSE noticeably enhanced the antioxidant capacity of nematodes. Moreover, these results indicated that SSE can improve the survival ability of nematodes in adverse environments.

The above results indicated that SSE can effectively retard ageing in nematodes, and we further explored the possible mechanisms at the transcriptional level. We selected several genes that are associated with ageing and determined their expressions, including daf-16 (Fork-head domain-containing protein), daf-2 (Insulin-like receptor subunit beta), sod-3 (Superoxide dismutase [Mn] 2), ctl-1 (Peroxisomal catalase 1), sir-2.1 (Deacetylase sirtuin-type domain-containing protein), skn-1 (BZIP domain-containing protein), and hsp-16.2 (Heat shock protein hsp-16.2) by using real-time PCR. These results showed that the SSE treatments improved the expressions of daf-16, daf-2, sir-2.1, sod-3 and hsp-16.2 but did not have significant effects on the ctl-1 and skn-1 expressions (Figure 7A).

The forkhead transcription factor, DAF-16, a downstream target of the insulin/IGF-I signaling pathway in C. elegans, is indispensable both for lifespan regulation and stress resistance (Li et al., 2007). Sir-2.1 is critical regulator of FOXO-mediated transcription in response to oxidative stress (Hamilton et al., 2005). RT-PCR results show that the expressions of daf-16 and sir-2.1 increase significantly, so we speculate that these two genes are the key factors in SSE extending the lifespan of C. elegans. We treated daf-16(CF1038) and sir-2.1(VC199) mutant worms with 25 mg/mL SSE. Results shown that the SSE-mediated lifespan extension disappeared, suggesting SSE prolonged the lifespan of nematodes in daf-16 and sir-2.1 dependent manner (Figures 7B, C).

We carried out metabonomics to understand the mechanism of SSE-mediated lifespan extension. Following 7 days of SSE treatment, 49 metabolites were differentially upregulated, and 36 metabolites were downregulated compared to control group (p-value < 0.05 and Fold Change ≥2). Amino acid metabolites, including leucine and isoleucine, were upregulated (Figures 8A, B). Leucine can activate Sirt1 by lowering its KM for NAD⁽⁺⁾, the homolog of mammalian SIRT1 in C. elegans is sir2.1, so SSE may extend the lifespan of C. elegans by activating sir-2.1 through the increase of leucine metabolism (Raynes et al., 2012; Banerjee et al., 2016).

The KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed dramatic changes in the metabolism of amino acid (Figure 8C). It has been reported that the increase of amino acids can prolong the lifespan of nematodes and the extension is dependent on daf-16. SSE increases the rate of amino acid metabolism in C. elegans, which may also be a factor in lifespan extension. Finally, we quantified the linear correlation between the various metabolites (Figure 8D).