Natural thioallyl compounds increase oxidative stress resistance and lifespan in Caenorhabditis elegans by modulating SKN-1/Nrf

Abstract

Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.

Figure 1. SAC and SAMC increase lifespan and resistance to oxidative- or heat-stress of wild-type C. elegans.

(a) Chemical structures of SAC and SAMC. (b,c) Survival curves of wild-type adults treated with SAC (b) or SAMC (c) at 20 °C. Composites of four replicates are shown respectively, with mean lifespans indicated in parentheses. Statistics are provided in Supplementary Table S1. (d–g) Synchronized day-1 wild-type adults were treated with H₂O (control), SAC or SAMC for 48 hours at 20 °C and then subjected to oxidative stress (250 μM juglone (Jug) for 2 hours at 20 °C) or heat stress (35 °C for 7 hours). (d,e) Survivals after each stress treatment were scored after a 16 hours recovery on NGM agar seeded with E. coli OP50. Data are represented as mean ± SD from three independent experiments. Total number of animals tested: for the oxidative stress assays (Control, n = 218; SAC, n = 211; SAMC, n = 226) and for the heat stress assays (Control, n = 208; SAC, n = 226; SAMC, n = 220). (f,g) Intracellular ROS accumulation in individual animal was measured by using CM-H₂DCFDA. The mean fluorescence intensity of at least 20 animals for each group with or without stress treatment is shown. Error bars represent SEM. ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test).

Figure 2. SAC and SAMC do not affect DAF-16 pathway.

(a) Induction of the sod-3p::GFP or hsp-16.2p::GFP transgene in animals treated with juglone, SAC or SAMC for 24 hours. GFP intensity in pharynx was quantified by ImageJ. Data are represented as relative fluorescence intensity with SEM (n ≥ 16 for each group). (b) Relative mRNA levels of sod-3 (left), hsp-16.2 (middle) and ctl-2 (right) in day-1 wild-type adults treated with juglone, SAC or SAMC for 6 hours (n = 3 of 50 animals) were determined by qRT-PCR. Data are represented as mean ± SEM from three independent experiments normalized to the levels in control. (c) Nuclear localization of DAF-16A::GFP in animals treated with H₂O (control; n = 73), SAC (100 μM; n = 66) or SAMC (100 μM; n = 63) for 24 hour. Juglone (400 μM for 1 hour; n = 63) or heat stress (35 °C for 1 hour; n = 73) were used as positive controls. Nuclear localization of DAF-16A::GFP throughout whole body was classified into High, Medium or Low. ***P < 0.001; NS: not significant (chi² test). (d,e) Survival curves of the daf-16(mgDf47) mutant treated with SAC (d) or SAMC (e) at 20 °C. Composites of three replicates are shown respectively, with mean lifespans indicated in parentheses. Statistics are provided in Supplementary Table S2. *P < 0.05; ***P < 0.001; NS: not significant (one-way ANOVA with Tukey’s post hoc test).

Figure 3. SAC and SAMC modulate SKN-1 pathway.

(a,b) Induction of gst-4p::GFP transgene in day-1 adults of the wild-type background (a) or the skn-1(zu67) mutant (b) treated with juglone, SAC or SAMC (24 h). Data represent relative fluorescence intensity throughout whole body with SD (n ≥ 20). (c,d) Lifespan of the skn-1(zu135) mutant treated with SAC (c) or SAMC (d) at 20 °C. Composites of three replicates with mean lifespans in parentheses. Statistics are provided in Supplementary Table S3. (e,f) Relative mRNA levels of the indicated SKN-1 targets in day-1 wild-type adults treated with SAC (e) or SAMC (f) (24 h). (g) Relative gst-4 mRNA levels in day-1 adults of the sek-1(km4) mutant treated with juglone, SAC or SAMC (24 h). (h) Effect of wdr-23 RNAi on endogenous gst-4 mRNA levels in day-1 wild-type adults treated with juglone, SAC or SAMC (24 h). (e–h) Data represent mean ± SD (n = 3 of 50 animals). (i) Nuclear localization of SKN-1B/C::GFP in L4 animals pretreated with SAC or SAMC from the L4 stage of parental generation, followed by treatment with or without NaN₃. wdr-23 RNAi was used as a positive control. SKN-1B/C::GFP in intestinal nuclei was classified into High, Medium or Low. ***P < 0.001 (for wdr-23 RNAi, n = 106 vs. Control RNAi, n = 102, for the NaN₃ treatment, SAC, n = 142; SAMC, n = 166 vs. Control, n = 151), NS: not significant (without NaN₃, Control, n = 130; SAC, n = 131; SAMC, n = 135) (chi² test). (j) Immunoblotting of endogenous SKN-1. (Left) Whole lysates (4.6 μg/lane) from 300 day-1 adults of the rrf-3(pk1426) mutant treated with control or wdr-23 RNAi, or of the skn-1(zu135) homozygous mutant. (Middle) Whole lysates (15.0 μg/lane) from 1,000 L4 wild-type treated with SAC or SAMC from the L4 stage of parental generation. The blots detected with antibodies against SKN-1 (top) or β-tubulin (bottom). Predicted SKN-1 isoforms (1a–1d) are indicated according to their estimated molecular weights reported in WormBase. (Right) Relative band intensity against β-tubulin of two experiments normalized to the levels in control of each isoform. The blot are data of experiment-1. #: Non-specific band. α: antibody against. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant (one-way ANOVA with Tukey’s post hoc test).

Figure 4. SAC and SAMC do not affect body size and reproduction, but enhance food intake of wild-type C. elegans.

(a) For the reproduction assays, wild-type L4 animals were treated with H₂O (control; n = 17), SAC (n = 19) or SAMC (n = 22) until reproduction period was ceased. Data represent the mean value of daily or total number of progeny from individual animals with SD. (b) The body length of animals treated with H₂O (control; n = 85), SAC (n = 87) or SAMC (n = 91) for 8 days was measured by ImageJ. Data represent mean ± SD. (c) For the food consumption assays, after 8 days of treatment with H₂O (control), SAC, or SAMC, OD 620 nm of liquid medium containing total 50 animals was measured with a spectrophotometer. Data represent mean ± SD (n = 4 of 50 animals). *P < 0.05; **P < 0.01: ***P < 0.001; NS: not significant (one-way ANOVA with Tukey’s post hoc test).