Nobiletin Delays Aging and Enhances Stress Resistance of Caenorhabditis elegans

Abstract

Nobiletin (NOB), one of polymethoxyflavone existing in citrus fruits, has been reported to exhibit a multitude of biological properties, including anti-inflammation, anti-oxidation, anti-atherosclerosis, neuroprotection, and anti-tumor activity. However, little is known about the anti-aging effect of NOB. The objective of this study was to determine the effects of NOB on lifespan, stress resistance, and its associated gene expression. Using Caenorhabditis elegans, an in vivo nematode model, we found that NOB remarkably extended the lifespan; slowed aging-related functional declines; and increased the resistance against various stressors, including heat shock and ultraviolet radiation. Also, NOB reduced the effects of paraquat stressor on nematodes and scavenged reactive oxygen species (ROS). Furthermore, gene expression revealed that NOB upregulated the expression of sod-3, hsp-16.2, gst-4, skn-1, sek-1, and sir-2.1, which was suggested that anti-aging activity of NOB was mediated most likely by activation of the target genes of the transcription factors including dauer formation (DAF)-16, heat-shock transcription factor (HSF)-1, and skinhead (SKN)-1. In summary, NOB has potential application in extension of lifespan, and its associated healthspan and stress resistances.

Keywords: Caenorhabditis elegans; aging; nobiletin; stress resistance.

Figure 1

Chemical structures of nobiletin (NOB) and its derivatives.

Figure 2

Effect of nobiletin (NOB) on lifespan of C. elegans. N2 worms were treated without NOB (control) or with NOB (3.13, 6.25 or 12.5 µM) from day 0 adult (L4 larvae) to death at 20 °C. The percentage of live animals was plotted against adult days. Detailed parameters are presented in Table 2.

Figure 3

Effect of NOB on motility of (A) day 14, (B) day 18, and (C) day 22 experimental animals. Nematodes were classified into three groups based on motility: group A animals moved spontaneously and freely; group C animals only moved their heads in response to stimulating heads and tails; and group B animals were distributed between these two situations. NOB potently alleviated the decline of in motility in a dose-dependent manner. (n ≥ 40 animals per group for days 14 and 18; n ≥ 25 for day 22). Bars with different letters indicated statistical significance (p < 0.05).

Figure 3

Effect of NOB on motility of (A) day 14, (B) day 18, and (C) day 22 experimental animals. Nematodes were classified into three groups based on motility: group A animals moved spontaneously and freely; group C animals only moved their heads in response to stimulating heads and tails; and group B animals were distributed between these two situations. NOB potently alleviated the decline of in motility in a dose-dependent manner. (n ≥ 40 animals per group for days 14 and 18; n ≥ 25 for day 22). Bars with different letters indicated statistical significance (p < 0.05).

Figure 4

Effect of NOB on body size and lipofuscin accumulation in C. elegans. Wild-type N2 animals were treated without or with 3.13, 6.25, and 12.5 µM NOB for five days at 20 °C. The body size and lipofuscin accumulation were measured using fluorescence microscope, and the results are presented at (A,B). Body length and lipofuscin were quantitated by Image J software (C,D). Bars with different letters indicate statistical significance (p < 0.05).

Figure 5

Effect of NOB on oviposition amount of C. elegans (n = 5 animals per group). There were no significant differences in the number of total progeny between NOB-treated and untreated animals (p < 0.05).

Figure 6

Effect of NOB on stress resistance of C. elegans. The adult nematodes were treated with 3.13, 6.25, and 12.5 µM NOB for five days at 20 °C, followed by three types of stressors. Compared with the control group, NOB significantly raised the resistance of nematodes to (A) heat shock at 35 °C, (B) UV damage, and (C) paraquat-induced oxidative stress. Detailed parameters are presented in Table 3, Table 4 and Table 5.

Figure 7

Inhibition of ROS production in C. elegans. The fluorescence curves against measuring time shown (A) were plotted from three independent trails, and the fluorescence protection area of NOB was shown according to the area under the curve (AUC) of the NOB treatment group and blank group (B).

Figure 8

Effect of NOB on antioxidant enzymes in C. elegans. The activities of relative antioxidant enzymes: SOD (A) and CAT (B), and the malondialdehyde (MDA) content (C) in C. elegans after treatment with indicated concentrations of NOB. Data are shown as mean ± SD, n = 3. Bars with different letters indicated statistical significance (p < 0.05).

Figure 8

Effect of NOB on antioxidant enzymes in C. elegans. The activities of relative antioxidant enzymes: SOD (A) and CAT (B), and the malondialdehyde (MDA) content (C) in C. elegans after treatment with indicated concentrations of NOB. Data are shown as mean ± SD, n = 3. Bars with different letters indicated statistical significance (p < 0.05).

Figure 9

Effect of NOB on antioxidant gene expression in C. elegans. Data are shown as mean ± SD, n = 3. Bars with different letters in each group indicated statistical significance (p < 0.05).

Figure 10

A possible mechanism of action through DAF-16 signalling pathway in C. elegans representing molecular targets for modulation by NOB.