Didymin improves UV irradiation resistance in C. elegans

Didymin, a type of flavono-o-glycoside compound naturally present in citrus fruits, has been reported to be an effective anticancer agent. However, its effects on stress resistance are unclear. In this study, we treated Caenorhabditis elegans with didymin at several concentrations. We found that didymin reduced the effects of UV stressor on nematodes by decreasing reactive oxygen species levels and increasing superoxide dismutase (SOD) activity. Furthermore, we found that specific didymin-treated mutant nematodes daf-16(mu86) & daf-2(e1370), daf-16(mu86), akt-1(ok525), akt-2(ok393), and age-1(hx546) were susceptible to UV irradiation, whereas daf-2(e1371) was resistant to UV irradiation. In addition, we found that didymin not only promoted DAF-16 to transfer from cytoplasm to nucleus, but also increased both protein and mRNA expression levels of SOD-3 and HSP-16.2 after UV irradiation. Our results show that didymin affects UV irradiation resistance and it may act on daf-2 to regulate downstream genes through the insulin/IGF-1-like signaling pathway.


INTRODUCTION
As the natural environment deteriorates and the pace of life accelerates, people receive more and more types of stimuli, such as smog, high temperature, radiation, noise, and so on. These stimuli destroy the balance between the body's oxidation and antioxidation systems, which in turn causes the body to produce and accumulate large amounts of reactive oxygen species (ROS), eventually leading to oxidation reactions (Schieber & Chandel, 2014). Stress response could induce DNA damage, cell apoptosis and body's immune response to affect daily behavior and health of the organism, involving many important cell signal transduction processes, such as insulin signaling pathway (Sadagurski & White, 2013). In recent years, the prevention and treatment of biological anti-stress has received increasing attention. Its mechanism research has become the focus of disease treatment and drug development.
The classic definition of oxidation states that oxygen-containing elements or compounds are combined for subsequent oxidation reactions. The term oxide was coined by French chemists Guyton DE Morveau and Antoine Lavoisier, and it is based on oxygen and acid. The oxidation and reduction processes occur together, and therefore they

Lifespan assay
Lifespan assays were performed as previously described (Zhou et al., 2018b). Lifespan assays were carried out by growing wild-type nematodes to the L4 stage on NGM plates containing E. coli OP50 in an incubator set at 20 C. The nematodes were incubated in the presence of didymin (0.05, 0.1, 0.2, 0.5, and one mM) dissolved in 0.2% DMSO for 48 h, whereas the control nematodes were incubated with 0.2% DMSO. The nematodes were then transferred to fresh plates every day. The viable and non-viable nematodes were recorded daily until all animals died. Non-viable nematodes failed to move after gently prodding with a platinum wire. The day of birth was taken as the first day of their lives. Die vulva blowout, bagging or climbing out of the plate were noted. This experiment was performed three times, with at least 100 nematodes per group.

Stress resistance assay
The feeding program for the stress resistance assay was the same as that for the lifespan assay, and the conditions were identical to those in a previous study (Zhou et al., 2017). Wild-type nematodes at the L4 stage were incubated with 0.1 mM didymin for 48 and transferred to new plates without didymin, immediately followed by three kinds of stressors. This experiment was performed three times, with at least 100 nematodes per group.
To assess UV irradiation stress resistance, nematodes were exposed to 254 nm of UV irradiation at a dose of 1,000 J/m 2 and immediately transferred to an incubator set at 20 C. The viable and non-viable nematodes were scored every 12 h until all animals died.
To assess thermal shock stress resistance, the nematodes were transferred from an incubator set at 20 C to one set at 35 C incubator. The viable nematodes were scored every hour until all animals died.
To assess juglone stress resistance, the nematodes were placed in 96-well plates which contained 200 mL S medium (0.01 mM cholesterol, 100 mM NaCl, and 50 mM potassium phosphate, pH 6.0) supplemented with 200 mM juglone in each well. The viable nematodes were observed at 20 C every hour until all animals died.

Determination of ROS level
To measure the intracellular ROS levels, nematodes were cultured in the presence or absence of 0.1 mM didymin for 48 h and transferred to new plates without didymin, immediately followed by exposure to 1,000 J/m 2 UV irradiation. Wild-type C. elegans were then immediately collected and steeped in 200 mM DCFH-DA for 30 min at 20 C. Five hundred nematodes were washed three times with M9 buffer (one mM MgSO 4 , 22 mM KH 2 PO 4 , 42 mM Na 2 HPO 4 , and 85 mM NaCl) and then transferred into a black 96-well plate. A fluorescent microplate reader (Infinite F200 PRO, Tecan, Switzerland) were used to measure ROS-related fluorescence at 485 nm excitation wavelength and 525 nm emission wavelength (Zhang et al., 2018). Relative fluorescence unit was used to reflect relative ROS levels by comparing between control and didymin-treated groups. This experiment was repeated for three times.

Measurement of superoxide dismutase activity
To measure SOD activity, wild-type nematodes at the L4 stage were cultured on NGM plates with or without 0.1 mM didymin for 48 h and transferred to new plates without didymin, immediately followed by 1,000 J/m 2 UV irradiation. The nematodes were collected in M9 buffer and washed three times. Then, they were resuspended in M9 buffer and homogenized in ice (Lee, Xing & Kim, 2017). SOD activity was measured according to a SOD activity detection kit by using Multimode Plate Reader (Infinite F200 PRO, Tecan, Switzerland). This experiment was conducted three times, with at least 500 nematodes per group.

DAF-16 nuclear localization and SOD-3 and HSP-16.2 protein expression
The age-synchronized nematodes TJ356, CF1553, and CL2070 expressed DAF-16::GFP, SOD-3::GFP, and HSP-16.2::GFP fusion protein, respectively. Considering the main localization of DAF-16::GFP in each worm, the distribution of DAF-16 was categorized as cytosolic, intermediate, or nuclear. The nematodes were treated with 0.1 mM didymin under normal conditions at 20 C for 48 h and transferred to new plates without didymin, immediately followed by 1,000 J/m 2 UV irradiation. A total of five mM levamisole was used to anaesthetize worms for 3 min, and then animals were put on a microscope slide which was coated with 2% agar (Rea et al., 2005). GFP was monitored with a fluorescent microscope (DMI 3000 B; Leica, Wetzlar, Germany) which was equipped with a GFP filter at 365 nm excitation wavelength and 420 nm emission wavelength was used to observe GFP (Asthana et al., 2015;Zhang, Lu & Zhou, 2015). Image-Pro Plus Software (Media Cybernetics, Bethesda, MD, USA) was used to quantify fluorescent levels. This experiment was performed three times, with at least 10 nematodes per group.

Statistical analysis
For lifespan and stress resistance assays, Kaplan-Meier method using Prism 5 Software (GraphPad Software, La Jolla, CA, USA) was used to analyze data. Mantel-Cox (log-rank) test was used to analyze statistical significance. One-way analysis of variance was used to analyze all other data. Results were presented as the mean ± standard error of the mean. p-Values < 0.05 were taken as statistically significant ( ÃÃÃ p < 0.001; 0.001 ÃÃ p < 0.01; 0.01 Ã p < 0.05).

Didymin extends the lifespan of nematodes under normal culture conditions
The effect of didymin on the lifespan of wild-type worms was measured. The concentrations of didymin used in this experiment were 0.05, 0.1, 0.2, 0.5, and one mM. Comparing with control worms treated with DMSO, lifespan of nematodes was increased by 0.05, 0.1, and 0.2 mM didymin in a dose-dependent manner. At concentrations of 0.5 and 1 mM, however, didymin decreased the lifespan of nematodes. Because didymin at a concentration of 0.1 mM prolonged the lifespan of nematodes most obviously, this dose was used for the remaining experiments which tested its protective effects on nematodes exposed to different stressors (Fig. 1).
Didymin improves stress resistance of wild-type nematodes exposed to several stressors Many studies have shown that enhancing resistance to stresses can extend the lifespan of C. elegans (Chen et al., 2016;Ogawa et al., 2016). Therefore, we performed several stress resistance assays where wild-type nematodes were exposed to various stressors (i.e., 1,000 J/m 2 of UV irradiation, heat shock at 35 C, and 200 mM juglone), and the lifespan of nematodes was studied. Compared to control nematodes, the resistance to UV irradiation, thermal, and oxidative stresses increased after treatment with 0.1 mM didymin. Based on our results, the effect of 0.1 mM didymin on UV irradiation was the most obvious in these stress resistance. Thus, we used UV irradiation as the stressor for further studies (Fig. 2).

Didymin reduces the ROS levels
The ROS levels are associated with the age-related phenotype (Schaar et al., 2015). ROS levels increase when the body is damaged. So, we tested the ROS level in nematodes treated with or without didymin, followed by UV irradiation. Compared to control nematodes, there was a significant decrease in ROS accumulation after UV irradiation in nematodes treated with 0.1 mM didymin. Thus, the resistance effect of didymin on UV irradiation was related to lower ROS (Fig. 3A).

Didymin increases the SOD activity
Superoxide dismutase is one of the important biological antioxidant enzymes. It eliminates harmful substances during metabolic produces such as ROS (Mi et al., 2018). Compared to control nematodes, we found that SOD activity was higher in nematodes treated with 0.1 mM didymin and exposed to UV irradiation. In other words, SOD activity of didymin might be an important factor in UV irradiation of nematodes (Fig. 3B). Didymin acts through the insulin/IGF-1-like signaling pathway in C. elegans The insulin/IGF-1-like signaling pathway in nematodes includes key components such as DAF-2, AGE-1, AKT-1, AKT-2, and DAF-16 (Evans, Chen & Tan, 2008). Because daf-2 and daf-16 are two important genes that are located upstream and downstream, respectively, in the insulin/IGF-1-like signaling pathway, we judged that didymin-induced lifespan extension after UV irradiation might involve this pathway. We found that didymin certainly failed to enhance resistance to UV irradiation of the daf-2 and daf-16 double genes mutant. Moreover, after UV irradiation, there was no obvious difference in daf-16, akt-1, akt-2, and age-1 single gene mutant exposed to 0.1 mM didymin Figure 3 Didymin decreased the ROS levels and increased SOD activity in C. elegans nematodes exposed to UV irradiation. The adult nematodes were treated with 0.1 mM didymin for 48 h, followed by UV irradiation. The worms were collected in M9 buffer and tested. Compared to control nematodes, (A) the ROS levels decreased and (B) SOD activity increased in nematodes treated with 0.1 mM didymin (n > 10 parallel experiments, p < 0.001). The error bars represent SEM ( ÃÃÃ p < 0.001; 0.001 ÃÃ p < 0.01; 0.01 Ã p < 0.05). Full-size  DOI: 10.7717/peerj.6218/ fig-3 compared to control nematodes. However, 0.1 mM didymin improved UV irradiation resistance of daf-2 mutant. Taken together, results showed that didymin improves resistance of nematodes after UV irradiation through the insulin/IGF-like signaling pathway, and it might act on daf-2 (Fig. 4). An additional study was conducted to determine whether the improvement in anti-UV irradiation stress of C. elegans pretreated with didymin depended on the regulatory activity of daf-16. Didymin's function on DAF-16 translocation after UV irradiation in C. elegans was determined by TJ356 strain, and the expression levels of SOD-3 and HSP-16.2 were detected by CF1553 and CL2070 strains (Meng & Li, 2017;. As we expected, DAF-16::GFP translocated to the nucleus, and the expression of GFP-fused sod-3 or hsp-16.2 was significantly increased after treatment with 0.1 mM didymin (Fig. 5). Figure 4 Didymin acted through the insulin/IGF-1-like signaling pathway in C. elegans. Six kinds of adult mutant nematodes were treated with 0.1 mM didymin for 48 h, followed by UV irradiation. The didymin-treated mutants (A) daf-2 and daf-16 (n > 100, p > 0.05), (B) daf-16 (mu86) (n > 100, p > 0.05), (C) akt-1 (ok525) (n > 100, p > 0.05), (D) akt-2 (ok393) (n > 100, p > 0.05), and (E) age-1 (hx546) (n > 100, p > 0.05) reversed their resistance to UV irradiation. However, the didymin-treated mutant (F) daf-2 (e1371) (n > 100, p < 0.001) failed to reverse its resistance to UV irradiation, indicating that didymin might act on DAF-2 to increase the lifespan of C. elegans nematodes exposed to UV irradiation.
Full-size  DOI: 10.7717/peerj.6218/ fig-4 Moreover, the effect of didymin after UV irradiation on daf-2/daf-16-related target genes was examined by qPCR method. The mRNA levels of sod-3 and hsp16.2 were significantly increased. However, there was no change in the mRNA level of daf-16 compared with the control. In summary, data suggest that didymin improves UV irradiation resistance of C. elegans through the insulin/IGF-1-like signaling pathway, at least partially (Fig. 6).

DISCUSSION
Several animal models of longevity have already been established, including C. elegans, which have a relatively short lifespan, as well as clear genetic and environmental factors Figure 5 Didymin enhanced the nuclear localization of DAF-16 and the expression of SOD-3 and HSP-16.2 after exposure to UV irradiation. Three GFP transgenic nematodes were exposed to 0.1 mM didymin for 48 h, followed by UV irradiation. Didymin enhanced (A-C) the nuclear localization of DAF-16 (n = 10 each group, p < 0.001) and the expression of SOD-3 (D-F) (n = 10 each group, p < 0.001) and HSP-16.2 (G-I) (n = 10 each group, p < 0.001) after UV irradiation. Didymin increased the mRNA expression levels of sod-3 and hsp-16.2, but not that of daf-16 in nematodes exposed to UV irradiation. Adult wild-type nematodes were treated with 0.1 mM didymin for 48 h, followed by UV irradiation. Didymin significantly increased the mRNA levels of (A) sod-3 (p < 0.001) and (B) hsp-16.2 (p < 0.001). However, there was no change in the mRNA level of (C) daf-16 after treatment (p > 0.05). This experiment was performed at least three times, with 500 nematodes per group. The error bars represent SEM ( ÃÃÃ p < 0.001; 0.001 ÃÃ p < 0.01; 0.01 Ã p < 0.05). Full-size  DOI: 10.7717/peerj.6218/ fig-6 ( Kaletta & Hengartner, 2006). With regard to the effects of radiation on nematodes, detailed studies have been conducted at the molecular, cellular, and individual levels for nearly three decades (Sakashita et al., 2010), radiation can damage DNA and produce ROS. Insulin/IGF-1-like signal pathways, which control the lifespan of the nematode, have been discovered and studied in detail. It is found that radiation and oxidative stress can act on the insulin/IGF-1-like signaling pathway, which might lead to accelerated senescence of nematodes and shortened lifespan (Zhou et al., 2018a). Because the signaling system which is related to aging and oxidative stress in nematode cells has been precisely studied, nematodes will provide more help in understanding the mechanisms of higher organisms. Didymin is a commercially available dietary flavonoid glycoside that is derived from citrus fruits (Singhal et al., 2017). This study confirmed the stress resistance activity of didymin in the nematode model system. First, we observed that didymin could significantly prolong the lifespan of wild-type nematodes in a dose-dependent manner. The most effective concentration of didymin was 0.1 mM dissolved in 0.2% DMSO. Thus, we used 0.1 mM didymin for the remaining experiments in this study.
Lifespan and stress resistance are tightly interrelated (Kenyon, 2010;Larsen, 1993). Thus, we next tested the lifespan of nematodes treated with didymin under different stressors. Stress resistance was measured in terms of lifespan after pressure acceptance. We found that the lifespan of nematodes exposed to various stressors was also extended in animals treated with didymin. These results indicated that didymin played a role in stress resistance.
Both environmental damage and normal cellular metabolism can produce ROS, resulting in cell structure breaking or critical cellular functions losing (Fatouros et al., 2012). SOD activity is a pivotal oxidative stress response that serves to protect living cells from ROS Sampayo, Olsen & Lithgow, 2003). Interestingly, we found that nematodes treated with didymin showed low ROS levels and high SOD activity after UV irradiation. Therefore, didymin might resist UV irradiation by reducing ROS levels and increasing SOD activity.
In order to find the relationship between UV irradiation and protein expression, we employed GFP transgene mutants TJ356, CF1553, and CL2070. DAF-16 nuclear translocation can regulate transcription of many genes which are related with stress tolerance and longevity (Su & Wink, 2015). We found that didymin significantly make more DAF-16 moved to nuclear. sod-3 works downstream of daf-16 and it is implicated in detoxification. At the moment, hsp-16.2 is also located downstream of daf-16, and it can hold back the aggregation of unfolded and misfolded proteins. Both sod-3 and hsp-16.2 are essential in stress resistance of worms (Murphy et al., 2003). As our expected, data suggested that both SOD-3 and HSP-16.2 were expressed more after didymin treatment and UV irradiation.
To analyze age-related changes in gene expression, we used qPCR. Our results showed that didymin increased mRNA levels of sod-3 and hsp-16.2. However, the mRNA level daf-16 was unchanged compared with the control. Taken together, these results demonstrated that the effect of didymin on nematodes with regard to UV irradiation stress resistance was at least part through the insulin/IGF-1-like signaling pathway.

CONCLUSION
In summary, we found that didymin improved several stress resistance of nematodes. The effect of didymin on UV irradiation resistance might act via the insulin-like signaling pathway. We would like to further study the mechanism of its role in UV irradiation resistance.