Objectives Resveratrol (3,5,4'-trihydroxy-trans-stilbene) has been reported to be beneficial in cases of cardiovascular and neurodegenerative diseases owing to its property to reduce oxidative stress. However, we found that resveratrol has no significant effect on extension of life span in Caenorhabditis elegans, which is not consistent with previous reports. This finding has lead us to investigate whether resveratrol at least displays protective effects against oxidative damage.
Methods C. elegans was cultured under standard condition with or without resveratrol, after exposed to juglone-induced acute oxidative stress, survival rate and hsp-16.2::GFP expression were measured. The influence of resveratrol on life span of Caenorhabditis elegans under high-glucose-induced oxidative stress was also recorded.
Key findings Resveratrol did not increase normal life span of C. elegans either in liquid medium or on solid growth culture conditions. However, resveratrol alleviated juglone-induced lethal oxidative stress and significantly prolonged the life span of Caenorhabditis elegans in an acute oxidative damage environment and under high glucose conditions.
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Conclusions Resveratrol, as antioxidants, ameliorated oxidative stress in vivo although while not extending the normal life span of Caenorhabditis elegans, indicating that attenuation of oxidative stress is not sufficient to promote longevity.
Keywords: Resveratrol; Stress Resistance; Life Span; Anti-oxidant properties
Oxidative stress apparently plays a critical role in many diseases such as cardiovascular and neurodegenerative diseases. In cells, intracellular reactive oxidative species (ROS) can cause molecular damage to proteins, DNA and membrane lipids. Superoxide dismutase (SOD) has the capacity to decrease the superoxide anion radicals. According to the "the free radical ageing theory", which is disputed by several authors, SOD might exert an anti-ageing effect. However, experimental evidence suggests that overexpression of SOD increased the life span not by removing free radicals but by activating ageing related transcription factors.
Resveratrol, a polyphenol mainly found in grape skin, has anti-inflammatory, anticarcinogenic and anti-cardiovascular[6,7] properties which appear to be related to its antioxidant properties. Furthermore, resveratrol has been reported to extend the life span of many model organisms including yeast, nematodes and fruit flies in a Sir2-dependent manner.[8-10]
Caenorhabditis elegans is widely used as a model organism to study the effects of anti-ageing, anti-Alzheimer, and antidiabetic drugs[11,12] on account of its easy maintenance, short life cycle, and the availability of thousands mutant strains. We used resveratrol as a positive control in previous life span experiments (Chen and Wink, unpublished); however, we could not observe life span extension. Furthermore, other groups also questioned the Sir2 pathway and did not see an effect of resveratrol on the life span in Drosophila melanogaster.
In this study, we re-evaluated the antioxidative effects of resveratrol in C. elegans. The results demonstrated that resveratrol can increase the stress resistance in C. elegans under oxidative stress induced by juglone in a dose-dependent manner. Under normal conditions resveratrol did not extend the life span; however, under high glucose conditions in the medium, a life span extension could be recorded.
Material and methods
Chemicals and reagents
Resveratrol â‰¥99%, epigallocatechin gallate (EGCG) â‰¥95%, 2,2-diphenyl-1-picrylhydrazyl (DPPHâ€¢), D-glucose and 5-hydroxy-1,4-naphthoquinone (juglone) were bought from Sigma- Aldrich GmbH (Germany). Sodium azide was obtained from Applichem GmbH (Germany). Stock solutions (200 mM) of resveratrol in DMSO were stored at -20 Â°C.
Caenorhabditis elegans strains and culture conditions
Wild type N2, TK22 [mev-1(kn1)], and TJ375 [hsp-16.2::GFP(gpIs1)] strains were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota) and maintained on nematode growth medium (NGM) at 20 Â°C as previously described. Age-synchronized worms were generated in all experiments through the sodium hypochlorite method. Worms were allowed to hatch in 35 x 10 mm Petri dishes (CELLSTARÂ®) in liquid S-medium with concentrated Escherichia coli OP50 as a food resource.
DPPHâ€¢ free radical scavenging activity assay
Radical scavenging activity was determined using DPPHâ€¢ according to the method of Brand-Williams with some modifications. Equal volumes of 0.2 mM DPPHâ€¢ and different concentrations of resveratrol were mixed in 96-well plates up to an amount of 200 ÂµL. The absorbance was measured at 517 nm using a Tecan Safire2â„¢ Microplate Reader (Männedorf, Switzerland) after incubating in the dark for 30 min at room temperature. The inhibition was calculated from three independent experiments using the following equation: Inhibition% = (1- A substance/A control) Ã- 100% (A substance: absorbance of substance, A control: absorbance of control).
Quantification of hsp-16.2::GFP expression
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The hsp-16.2::GFP(gpIs1) strain contains a hsp-16.2 promoter coupled to the reporter gene GFP (green fluorescence protein). Hsp-16.2 is expressed in the pharynx of C. elegans under heat shock or oxidative stress caused by pro-oxidants such as the naphthoquinone juglone. L1 stage worms were treated with resveratrol (50, 100, 200 ÂµM) and DMSO as solvent control for 48 h, then exposed to 20 ÂµM juglone for further 24 h. Then 20 worms per group were paralyzed with 10 mM sodium azide in PBS on a microscopic glass slide. The degree of hsp-16.2::GFP fluorescence was measured by confocal fluorescence microscopy at constant exposure times of 400 ms (Nikon-eclipse 90i(2), Nikon Imaging Center, Heidelberg University). For quantification of the GFP fluorescence, images were analyzed using ImageJ software (NIH).
Life span assay
Life span analyses were performed at 20 Â°C. Synchronized adult worms (three days after hatching) were grown on NGM agar, liquid S-medium, or NGM plates plus 50 mM glucose, to which different concentrations of resveratrol (50, 100, 200 ÂµM) were added, living E. Coli as food resource. DMSO was included as a solvent control. In order to exclude an influence of the progeny, hermaphrodites were transferred daily to new fresh plates (plus ingredients) till the end of egg-laying period, after that a transfer was carried out every two days. Vitality of the worms was analysed daily. Worms were scored as dead if they failed to respond to gentle prods with a platinum wire.
L1 larvae of wild type N2 worms were incubated in S-medium with E. coli OP50 (109 bacteria/mL) containing resveratrol (50, 100, 200 ÂµM) and DMSO as solvent control, respectively. After 72 h, worms were transferred to a new Petri dish containing 400 ÂµM of the pro-oxidant juglone which induces lethal oxidative stress. The vitality of worms was examined every hour. Three independent tests were performed.
One-way analysis of variance (ANOVA) and Tukey's post hoc comparisons between multi groups were executed with the program PRISM (GraphPad Software, Inc., San Diego, CA, USA). Mean life span and log-rank test for comparison of difference were performed with StatView 5.0 sofware (SAS).
Results and discussion
A DPPHâ€¢ assay was employed to evaluate the antioxidant activity of resveratrol in vitro. Polyphenol substances can donate their hydrogens to neutralize DPPHâ€¢ free radical, as indicated by a change of their absorbance at 517 nm. Resveratrol reduces DPPHâ€¢ free radicals in a dose-dependent manner (Figure 1) with an IC 50 (the concentration of compound required to inhibit free radical DPPHâ€¢ by 50%) of 57.1 Â± 1.4 ÂµM. EGCG, a polyphenol from green tea, which was used as a positive control, exhibits an IC 50 of 6.1 Â± 0.3 ÂµM. This points to a strong free radicals scavenging ability of resveratrol in vitro.
To further explore whether resveratrol has anti-oxidative properties in vivo, transgenic hsp-16.2::GFP(gpIs1) nematodes were employed. While no GFP can be detected in hsp-16.2::GFP(gpIs1) worms under normal culture conditions, juglone can increase the intracellular O2- and H2O2 concentrations which in turn induce the expression of hsp-16.2.[21,22] Worms, which were exposed to juglone, showed a strong GFP expression in their pharynx (Figure 2a). The expression of hsp-16.2::GFP was reduced by 25.2%, 38.4% and 40.7% in worms pretreated with 50, 100, 200 ÂµM resveratrol, respectively (Figure 2e). This finding provides evidence that resveratrol has been taken up by the worms and that it is bioavailable. Because GFP expression was suppressed in the resveratrol treated group, this indicates that resveratrol (or metabolites derived from it) can protect worms against oxidative stress in vivo. Other antioxidants such as Ginkgo biloba extract EGb 761 and EGCG have also been reported to attenuate juglone-induced oxidative damages.[23,24]
In another set of experiments, the influence of resveratrol on life span was studied. Our results provide evidence that resveratrol extends the life span of wild type N2 worms (in short-term experiments) which were exposed to lethal juglone stress. L1 stage worms were treated with 50, 100, and 200 ÂµM resveratrol for 72 h and were then exposed to 400 ÂµM juglone. Whereas all worms were dead in the control group after 8 h, 27.6% of worms were still alive in the 50 ÂµM resveratrol group. Compared to the juglone group, the mean life span was significantly increased (Figure 3a and Supplement Table S1). Julonge-induced acute oxidative stress results in a large amount of free radicals accumulating in the cells. Resveratrol diminishes these free radicals, hence improving an organism's general health. In addition, resveratrol has also been reported to protect stroke model rats against oxidative stress and attenuates ethanol-induced oxidative stress in rat liver.[25,26]
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Experimental and clinical evidence suggest that oxidative stress plays a crucial role in the pathogenesis of diabetes. In our long-term life span experiments, 50 mM glucose were added to NGM plates to mimic glucose concentration in C. elegans similar to those in diabetic patients. The results show that resveratrol significantly extends the life span of C. elegans under high-glucose conditions (Figure 3b and Supplement Table S2), demonstrating again that resveratrol is effective against oxidative stress with potential as anti-diabetic drug.
However, we did not observe resveratrol prolonged the life span of wild type N2 and mev-1(kn1) worms in long term experiments (Figure 3c, d, e and Supplement Table S2). Although some researchers had reported dose-dependent benefits of resveratrol for health, our result demonstrated that higher doses of resveratrol exhibit pro-apoptotic effect and can even be toxic to C. elegans (Figure 3d and Supplement Table 2). The mean lifespan of N2 worms treated with 200 ÂµM resveratrol decreased by 24.0% compared to the DMSO solvent control group under the same culture conditions. The mev-1(kn1) strain is sensitive to oxidative stress due to enhanced ROS levels. The lifespan of mev-1(kn1) strain was shortened by only 9.6% after 200 ÂµM resveratrol treatment as compared to N2 worms, indicating resveratrol ameliorated ROS damage to C. elegans.
Although resveratrol has been reported to extend the life span of many model organisms,[9,32] we could not confirm this property in C. elegans. In our life span experiments, we transferred the worms almost daily into freshly prepared medium to prevent the development of progeny instead of adding 5-fluoro-2â€²-deoxyuridine (FUdR). 5-fluoro-2â€²-deoxyuridine (FUdR) can increase the lifespan in some mutant strains  and also affects the metabolism of C. elegans. It is possible that synergistic effects of resveratrol and FUdR influence metabolism signal pathway contributing to the extension of life span. Therefore, FUdR should be used with caution when conducting ageing research in C. elegans.
Recently, the anti ageing effect of resveratrol has been a hotly discussed topic. Viswanathan et al. assumed that resveratrol extends lifespan of C. elegans through sir-2.1 - mediated repression of ER stress genes. However, a recent report raised doubts about the positive effect of sirtuins (sir-2.1) on life span extension in C. elegans. Other researchers found that resveratrol had no effect on the life span of Drosophila. Harman first put forward the free radical theory of ageing postulating that ageing is mainly caused by the free radical reactions. Melov et al. had also claimed that superoxide dismutase might extend the life span of C. elegans. In contrast, Cabreiro et al. reported that superoxide dismutase can increase the lifespan of C. elegans not by removing free radicals but by activating transcription factors in the longevity pathway. In our experiment, resveratrol reduced acute oxidative damage, however had no extension effect on the normal life span of C. elegans indicating that antioxidants themselves probably not adequate for lowering the rate of ageing.
In conclusion, in our experiments we measured the antioxidant properties of resveratrol in vitro and in vivo, and the effect of resveratrol on life span of C. elegans. Resveratrol, which is a powerful anti-oxidant, enhances the resistance of C. elegans against oxidative stress in a dose-dependent manner. Although resveratrol improves the stress resistance in vivo, it has little significant effect on life span extension of C. elegans.
Conflict of interest
The authors declare that they have no conflicts of interest to disclose.
We are thankful to the China Scholarship Council (CSC) for financial support to Wei Chen.
We would like to thank the Nikon Imaging Center (Heidelberg University) for the help and Dr. Sami Abbas for the helpful suggestions and discussion.