Curcumin Acetylsalicylate Extends the Lifespan of Caenorhabditis elegans

Abstract

Aspirin and curcumin have been reported to be beneficial to anti-aging in a variety of biological models. Here, we synthesized a novel compound, curcumin acetylsalicylate (CA), by combining aspirin and curcumin. We characterized how CA affects the lifespan of Caenorhabditis elegans (C. elegans) worms. Our results demonstrated that CA extended the lifespan of worms in a dose-dependent manner and reached its highest anti-aging effect at the concentration of 20 μM. In addition, CA reduced the deposition of lipofuscin or "age pigment" without affecting the reproductivity of worms. CA also caused a rightward shift of C. elegans lifespan curves in the presence of paraquat-induced (5 mM) oxidative stress or 37 °C acute heat shock. Additionally, CA treatment decreased the reactive oxygen species (ROS) level in C. elegans and increased the expression of downstream genes superoxide dismutase (sod)-3, glutathione S-transferase (gst)-4, heat shock protein (hsp)-16.2, and catalase-1 (ctl-1). Notably, CA treatment resulted in nuclear translocation of the DAF-16 transcription factor, which is known to stimulate the expression of SOD-3, GST-4, HSP-16, and CTL-1. CA did not produce a longevity effect in daf-16 mutants. In sum, our data indicate that CA delayed the aging of C. elegans without affecting reproductivity, and this effect may be mediated by its activation of DAF-16 and subsequent expression of antioxidative genes, such as sod-3 and gst-4. Our study suggests that novel anti-aging drugs may be developed by combining two individual drugs.

Keywords: Caenorhabditis elegans; aging; antioxidation; curcumin acetylsalicylate.

Figure 1

CA increases lifespan in C. elegans. (A) Structural diagram and synthesis steps of aspirin curcumin ester to form CA. Wild-type L4 stage worms were treated with 0.1% DMSO (vehicle control) or (B) with CA (10, 20, 40 μM, n > 151 in each group, p = 0.03, 0.02, 0.38) or (C) with rapamycin (2 nM) or CA (5, 10, 20 μM, n > 165 in each group, p < 0.05). Their survival was monitored from the first day of the L4 stage until death, as described in the Methods. Assays were performed for each condition at least three times. A representative trial is shown. The difference was examined with the log-rank test.

Figure 2

Effects of CA on lipofuscin and reproduction of C. elegans. Wild-type worms were treated with DMSO (control) or with rapamycin (2 nM) or CA (5, 10, 20 μM) for 5 days, followed by detection of lipofuscin autofluorescence as shown by the representative images of worms, scale bar = 100 μm. (A) Cumulative data are shown in the bar figure (B) n > 19 worms per group, ** p = 0.0016, *** p = 0.0001, **** p < 0.0001). Egg production rates were recorded (C) n = 10 worms per group, p > 0.05).

Figure 3

Effect of CA on C. elegans lifespan under stress conditions. Wild-type worms, after being synchronized to the L4 stage, were pretreated with DMSO (control) or with rapamycin (2 nM) or CA (5, 10, 20 μM) for 5 days, followed by exposure to 5 mM paraquat (A) n = 86–90, p < 0.05) or 37 °C heat shock (B) n = 62–66, p < 0.05).

Figure 4

Effect of CA on intracellular ROS accumulation in C. elegans. Representative fluorescence images of worms, scale bar = 100 μm. (A) Relative fluorescence levels of ROS under oxidative stress compared with that in control (B) n > 150, **** p < 0.0001). Data were obtained from the Biotek microplate reader.

Figure 5

CA upregulates SOD-3 expression in C. elegans. Transgenic worms harboring SOD-3::GFP were treated without (control) or with rapamycin (2 nM) or CA (5, 10, 20 μM) for 3 days. Representative images (A–E) of SOD-3::GFP expression in worms with different treatments, scale bar = 100 μm. Quantification of SOD-3::GFP fluorescence intensity (F, n = 25, **** p ≤ 0.0001, ** p≤ 0.01, * p ≤ 0.05 vs. control).

Figure 6

Effect of CA on GST-4 expression in C. elegans. Transgenic worms harboring GST-4::GFP were treated with DMSO (control) or with rapamycin (1 nM) or CA (5, 10, 20 μM) for 3 days, followed by imaging with fluorescence microscopy. (A–E) Representative images of GST-4::GFP expression. (F) Quantification of GST-4::GFP fluorescence intensity in worms with different treatments as indicated (n = 25 worms per group). The difference was examined with one-way ANOVA with Dunnett’s multiple comparisons test, **** p ≤ 0.0001, ** p ≤ 0.01 vs. control, scale bar = 100 μm.

Figure 7

Effect of CA on DAF-16 localization in C. elegans. Transgenic worms harboring DAF-16::GFP were treated with DMSO (control) or with rapamycin (1 nM) or CA (5, 10, 20 μM) for 5 days. Representative images of DAF-16::GFP expression (A–C), scale bar = 100 μm. (D) Quantitative data of DAF-16::GFP fluorescence in different regions. Statistics of DAF-16 nucleation translocation (n = 25 worms per group, p < 0.05).

Figure 8

Effect of CA on aging-related gene expression in C. elegans. Wild-type worms were treated with DMSO (control) or with 20 μM CA for 3 days, followed by extraction of total RNA and RT-qPCR, as described in the Methods. The relative mRNA expression of skn-1, ctl-1, daf-16, and hsp-16.2 are presented (n = 250–300 worms per group). The difference was examined with student’s t-test, * p < 0.05, ** p ≤ 0.01 vs. control.

Figure 9

Effect of CA on the lifespan C. elegans with a functional mutation in daf-16. Worms were raised at 20 °C on NGM OP50 plates without (control) or with CA (20 μM). The CA 20 μM group had almost the same lifespan as the control group (n > 216 worms per group, p > 0.05).