Artemisia annua increases resistance to heat and oxidative stresses , but has no effect on lifespan in Caenorhabditis elegans

The free radical theory of aging suggests that the age-dependent accumulation of oxidative damage induced by free radicals is the main cause of normal aging (Harman, 1956). Reactive oxygen species (RDS) are produced as a byproduct of normal cellular metabolism and main free radicals in cells. To prevent RDS-induced oxidative damage, cells have evolved anti-oxidant defense systems. Cellular anti-oxidant defense systems are composed of enzymatic and non-enzymatic defense systems. Enzymatic defense systems utilize cellular anti-oxidant enzymes, including catalase (CAT), superoxide dismutase (SDD), glutathione peroxidase, and so on, to transform hyperactive RDS into stable compounds (Sohal et al., 1995; Wei & Lee, 2002). In addition, cellular anti-oxidants scavenge cellular RDS non-enzymatically (Miquel, 2001). However, RDS that escape anti-oxidant defense systems can cause oxidative damage in cellular macromolecules, such as DNA, proteins, and lipids. The accumulation of oxidative damage is positively associated with an organism’s chronological age and known to be involved with the incidence of many age-related diseases (Sohal & Weindruch, 1996).


Introduction
The free radical theory of aging suggests that the age-dependent accumulation of oxidative damage induced by free radicals is the main cause of normal aging (Harman, 1956).Reactive oxygen species (RDS) are produced as a byproduct of normal cellular metabolism and main free radicals in cells.To prevent RDS-induced oxidative damage, cells have evolved anti-oxidant defense systems.Cellular anti-oxidant defense systems are composed of enzymatic and non-enzymatic defense systems.Enzymatic defense systems utilize cellular anti-oxidant enzymes, including catalase (CAT), superoxide dismutase (SDD), glutathione peroxidase, and so on, to transform hyperactive RDS into stable compounds (Sohal et al., 1995;Wei & Lee, 2002).In addition, cellular anti-oxidants scavenge cellular RDS non-enzymatically (Miquel, 2001).However, RDS that escape anti-oxidant defense systems can cause oxidative damage in cellular macromolecules, such as DNA, proteins, and lipids.The accumulation of oxidative damage is positively associated with an organism's chronological age and known to be involved with the incidence of many age-related diseases (Sohal & Weindruch, 1996).
Studies have shown that the genetic or nutritional interventions modulating cellular anti-oxidant defense systems can affect the health, as well as lifespan of many experimental organisms.In Drosophila melanogaster, transgenic animals overexpressing both SDD and CAT have increased resistance to oxidative stress and lifespan (Drr & Sohal, 1992).Induction of the Cu/Zn SDD transgene expression also confers longevity phenotype in Drosophila melanogaster (Sun & Tower, 1999).In mice, overexpression of CAT induces increased resistance to hydrogen peroxide (Chen et al., 2004).In contrast, heterozygote knockout of SDD2 leads to increased oxidative damage and premature induction of apoptosis in mice (Kokoszka et al., 2001).Supplementation with anti-oxidants also modulates the response to oxidative stress and lifespan in model organisms.Supplementation of N-acetyl-L-cysteine confers increased resistance to various environmental stressors and longevity phenotype in C. elegans (Dh et al., 2015).Interestingly, worms grown in media prepared with electrolyzed reduced water, known to have a strong anti-oxidant activity, have increased resistance to oxidative stress and extended lifespan (Park et al., 2012;Park & Park, 2013).Treatment with vitamin E prevents age-related decline of cognitive function and improves mitochondrial function, but fails to extend lifespan in mammals (Fukui et al., 2002;Lipman et al., 1998;Navarro et al., 2005).Therefore, the effects of anti-oxidants on response to stressors and lifespan seems to depend on the in vivo activity of each anti-oxidant.
suggested that the polyphenol components of A. annua are responsible for those pharmacological activities.Since polyphenols are strong anti-oxidants, a number of studies focus on revealing of health-promoting activities of plant polyphenols.In the present study, we examine the effects of dietary supplementation of A. annua on health and lifespan using C. elegans as a model system.Response to environmental stressors, including heat stress, ultraviolet (UV) irradiation, and oxidative stress, was monitored in vivo.The effects of A. annua on the expression of stress-responsive genes was determined using green fluorescent protein (GFP).Then, we measured the effects of A. annua on the lifespan of C. elegans.

Preparation of A. annua extract
40 g of A. annua were extracted using hot water extraction with 1.8 L of distilled water at 80 °C for 30 min.Cooled extract were then filtered through a 185 mm-filter paper (Advantec, Tokyo, Japan).The second filtering was performed using a 0.2 μm bottle-top filter (Nalgene Rapid-Flow Bottle Top Filter, Thermo Scientific, Waltham, USA). A. annua extract were aliquoted and stored at 4 °C.

Thermotolerance
Five L4/young adult worms were transferred to a fresh NGM plate and permitted to lay eggs for 5 h.After removing the five adult worms, the eggs were maintained at 20 °C for 3 days.Sixty age-synchronized worms were transferred to a fresh NGM plate containing 100 μL of different concentrations of A. annua (0, 0.1, 1, 10, and 100% of extract) in 5 mL NGM.5-fluoro-2'-deoxyuridine (12.5 mg/L; Sigma-Aldrich, St. Louis, USA) was also added to prevent internal hatching.After 24 h, the worms were exposed to heat stress for 10 h by transferring plates to a 35 °C incubator.Then, the worms were transferred back to 20 °C.Survival rates after heat stress were monitored 24 h after.

Response to UV irradiation
Sixty age-synchronized worms were cultured in NGM plates containing different concentrations of A. annua for 24 h, as previously mentioned.Then, the plates were incubated in a 254 nm-UV crosslinker (BLX-254, VILBER Lourmat Co., Torcy, France) for 1 min at 20 J/cm 2 /min.After UV irradiation, the plates were transferred back to the 20 °C incubator.Alive and dead worms were scored every day until all worms were dead.

Resistance to oxidative stress
Age-synchronized young adults worms were treated with A. annua extract in NGM containing 5-fluoro-2'-deoxyruridine for 24 h.Then, the worms were exposed to 2mM hydrogen peroxide in S-basal without cholesterol (5.85 g sodium chloride, 1 g potassium phosphate dibasic, and 6 g potassium phosphate monobasic in 1 L sterilized distilled-water).The number of dead nematodes was scored after 6 h of exposure to hydrogen peroxide.

GFP expression of stress-responsive genes
Age-synchronized CL2070 and CF 1553 worms were placed in NGM plates supplemented with A. annua extract at 20 °C for 9 days.Then, the worms were mounted on a slide glass coated with 2% agarose and anaesthetized with 1M sodium azide.After covering the slide with a coverslip, expression of each reporter gene was observed using a confocal microscope (Dlympus FV10i, Dlympus, Tokyo, Japan).Fluorescence intensity of a randomly selected single worm was quantified with a fluorescence multi-reader (Infinite F200, Tecan, Grodig, Austria).

Lifespan assay
Sixty age-synchronized 3-day-old worms were transferred to fresh NGM plates containing A. annua extract and 5-fluoro-2'-deoxyruridine. Thereafter, worms were transferred to fresh NGM plates with A. annua extract and 5-fluoro-2'-deoxyruridine every other day.The number of living and dead worms was recorded every day.

Statistical analysis
We employed the log-rank test to analyze the lifespan data.The log-rank test is a non-parametric Mantel-Cox test widely used to compare two time-course survival curves (Peto & Peto, 1972).Statistical significance in the other experiments was assessed with the standard two-tailed Student's t-test.A p-value lower than 0.05 was regarded as significant.

Effect of A. annua on response to heat shock and UV irradiation
Most common environmental stressors encountered during the life cycle includes heat shock and UV irradiation.To reveal the effects of A. annua on response to environmental stressors, we examined the survival of worms after heat stress or UV irradiation.Supplementation of A. annua extract extended the survival after heat shock.In the wild-type control group, 73.3 ± 1.92% (mean of three independent experiments ± SEM) of worms survived after 10 h of 35 °C heat shock.The mean percent survival of worms pre-treated with 100% A. annua extract significantly increased (81.1 ± 1.47%, p = 0.032).However, the dilution of A. annua extract failed to show a significant effect on thermotolerance in C. elegans (Figure 1).We also measured the change in resistance to UV irradiation with different concentrations of A. annua extract.Unlike the increased survival after heat shock, A. annua extract did not affect to resistance to UV irradiation at any concentration of A. annua extract (Figure 2).Independent replicative experiments also showed no effects on survival after UV irradiation (Table 1).Previous findings show that supplementation of Acanthopanax sessiliflorus extract exhibits an increased resistance to heat stress and UV irradiation in C. elegans (Park et al., 2014).N-acetyl-L-cysteine, a sulfur-containing cysteine derivative with an acetyl group attached to the nitrogen of cysteine, has anti-oxidant and anti-cancer activity (Cai et al., 1999;Yedjou & Tchounwou, 2007).Survival of worms after heat shock or UV irradiation significantly increases by pre-treatment with 5 mM N-acetyl-L-cysteine (Dh et al., 2015).Dur data showed that A. annua can specifically modulate the response to heat stress, however has no effect on resistance to UV irradiation.Further studies identifying cellular components regulated by A. annua supplementation will reveal the underlying mechanisms involved in in vivo activities of A. annua.

Increased resistance to oxidative stress by A. annua
Environmental cellular oxidative stress cause detrimental damage to DNA, protein, and lipid molecules and lead to the functional decline of damaged cells and aging of tissues (Martin et al., 1996).We next explored whether the supplementation of A. annua affects the oxidative-stress response in C. elegans.Having observed increased resistance to heat stress with 100% A. annua extract, we compared the survival under oxidative-stress conditions between the untreated wild-type control group and worms pre-treated with 100% A. annua extract.After 6h of hydrogen peroxide treatment, 16.7 ± 2.36% (mean of three independent experiments ± SEM) of worms survived.Supplementation of A. annua extract increased the survival under the same oxidative-stress conditions up to 32.5 ± 3.70% (p = 0.011) (Figure 3).These findings demonstrate the anti-oxidant activity of A. annua in vivo for the first time.Anti-oxidant activity of natural compounds have been reported in various studies and suggest that it may be a possible therapeutic agent for many diseases, in which oxidative stress is a major causal factor.Resveratrol, a polyphenol compound found in red wine, has   strong anti-oxidant activity and is effective in protecting against cancer, atherosclerosis, and neurodegeneration (Ferguson, 2001;Jang et al., 1997).Curcumin is a major ingredient in yellow curry and has an anti-oxidant activity (Martin-Aragon et al., 1997).
Curcumin has been used as a traditional medicine in India for the treatment of cancer and gastrointestinal diseases (Kitani et al., 2004).Supplementation of Acanthopanax assiliflorus extract confers increased resistance to oxidative stress in C. elegans (Park et al., 2014).Acanthopanax assiliflorus is a native plant grown in Korea, Japan, and China and is used in diabetes, tumor, and rheumatoid arthritis treatment (Fujikawa et al., 1996).Taken together, our findings indicate that A. annua can work as a strong anti-oxidant in vivo and suggest that it may be used to develop novel natural therapeutics for diseases generated by oxidative stress.

Induction of stress-responsive genes by A. annua
Based on our data showing increased resistance to heat and oxidative stress by A. annua extract, we monitored the expression level alteration of heat-shock-responsive and anti-oxidant genes caused by A. annua extract supplementation.The expression level of hsp-16.2 is positively correlated with increased resistance to heat-shock stress and lifespan in C. elegans (Rea et al., 2005).We observed a significant up-regulation of hsp-16.2 in worms treated with 100% A. annua extract (Figure 4a).The relative expression of hsp-16.2increased to 138.9 ± 13.94% in A. annua-treated worms, compared to the untreated wild-type control (100.0 ± 7.30%) (Figure 4b).sod-3 is an anti-oxidant gene involved in the cellular enzymatic defense system against reactive oxygen species (Sánchez-Blanco & Kim, 2011).As expected from increased resistance to oxidative stress, the expression of  sod-3 was induced by A. annua extract (Figure 4b).There was about 1.89 fold increase in expression of sod-3 in worms treated with 100% A. annua extract, compared to the untreated worms (p < 0.001) (Figure 4b).A recent study shows that dietary supplementation of N-acetyl-L-cysteine results in extended survival time under heat-and oxidative-stress conditions, as well as reduced susceptibility to stresses that accompany the up-regulation of stress-responsive genes (Dh et al., 2015).Gene expression data obtained here support our hypothesis that supplementation of A. annua extract confers increased resistance to heat and oxidative stress in vivo and suggests that the underlying mechanisms may be involved with the induction of stress-responsive genes, including heat shock proteins and anti-oxidant genes.

Effect of A. annua on lifespan of C. elegans
Free radical theory of aging suggests that oxidative stress caused by free radicals is one of major factors leading to aging (Harman, 1956).Having observed increased resistance to oxidative stress by A. annua extract, we examined the effect of A. annua supplementation on lifespan in C. elegans.As shown in Figure 5, there is no significant difference in both mean and maximum lifespan between the wild-type control and A. annua-treated worms.The mean lifespan in the wild-type control group and worms supplemented with 100% A. annua extract was 15.8 and 15.3 days, respectively (p = 0.336).Independent repeated experiments also failed to show significant effects of A. annua on lifespan (Table 1).The effects of genetic or nutritional interventions modulating cellular anti-oxidant defense systems on lifespan remains controversial.In Drosophila melanogaster, the neuronal expression of human SDD-1 extends lifespan (Parkes et al., 1998).Simultaneous over-expression of Cu/Zn SDD and CAT increases lifespan in short-lived strains, but not in long-lived strains (Drr et al., 2003;Drr & Sohal, 1992).Dver-expression of anti-oxidant genes, including SDD and CAT, does not induce a longevity phenotype in mice (Chen et al., 2004).Supplementation of Acanthopanax sessiliflorus extract increases resistance to oxidative stress and extends lifespan in C. elegans (Park et al., 2014).Anti-oxidant polyphenols extracted from green tea also show lifespan-extending effects in mice (Kitani et al., 2004).However, dietary intervention with strong anti-oxidants, such as coenzyme Q 10 and α-lipoic acid, fails to increase lifespan, although it reduces tumor incidence (Lee et al., 2004).

Figure 1 .
Figure 1.The effects of different concentrations of A. annua extract on thermotolerance in C. elegans.Pre-treatment of 100% A. annua extract significantly increased resistance to heat stress.Values are the mean ± SEM of three independent experiments (n = 60).* Indicates a significant difference from the control (p < 0.05).

Figure 2 .
Figure 2. Time-course survival of worms after UV irradiation.In all experimental groups supplemented with different concentrations of A. annua extract, there is no significant difference in survival after UV irradiation compared to the untreated wild-type control.

Figure 3 .
Figure 3. Increased resistance to oxidative stress by A. annua extract in C. elegans.Values are the mean ± SEM of three independent experiments (n = 60).* Indicates a significant difference from the control (p < 0.05).

Figure 4 .
Figure 4.The change in expression of stress-responsive genes by A. annua extract.(a) Total GFP fluorescence of each whole worm was compared between the control and A. annua-treated worms for hsp-16.2and sod-3 genes; (b) GFP fluorescence intensity was quantified with a fluorescence multi-reader (n=20).Values are the mean ± SEM of three independent experiments.* Indicates a significant difference from the control (p < 0.05).

Figure 5 .
Figure 5.The effect of A. annua extract on the lifespan of C. elegans.Both mean and maximum lifespan were unaffected by supplementation with A. annua extract.

Table 1 .
Effects of A. annua extract on response to UV irradiation and the lifespan in C. elegans.
p-value was determined using the log-rank test.