Icariin Improves Stress Resistance and Extends Lifespan in Caenorhabditis elegans through hsf-1 and daf-2-Driven Hormesis

Abstract

Aging presents an increasingly significant challenge globally, driven by the growing proportion of individuals aged 60 and older. Currently, there is substantial research interest in pro-longevity interventions that target pivotal signaling pathways, aiming not only to extend lifespan but also to enhance healthspan. One particularly promising approach involves inducing a hormetic response through the utilization of natural compounds defined as hormetins. Various studies have introduced the flavonoid icariin as beneficial for age-related diseases such as cardiovascular and neurodegenerative conditions. To validate its potential pro-longevity properties, we employed Caenorhabditis elegans as an experimental platform. The accumulated results suggest that icariin extends the lifespan of C. elegans through modulation of the DAF-2, corresponding to the insulin/IGF-1 signaling pathway in humans. Additionally, we identified increased resistance to heat and oxidative stress, modulation of lipid metabolism, improved late-life healthspan, and an extended lifespan upon icariin treatment. Consequently, a model mechanism of action was provided for icariin that involves the modulation of various players within the stress-response network. Collectively, the obtained data reveal that icariin is a potential hormetic agent with geroprotective properties that merits future developments.

Keywords: Caenorhabditis elegans; aging; healthspan; icariin; lifespan; longevity.

Figure 1

Icariin treatment extends the lifespan and enhances the mobility of wild-type C. elegans in contrast to daf-2 mutant strain. (a) Viability test upon icariin treatment (10–200 μM) in N2 and daf-2 C. elegans. The statistical significance of the survival curves was assessed using the Log-rank test. (b) Chemotaxis assay of icariin-treated C. elegans (n = 300–600) from both strains used. (c) Daily and total brood size of icariin-treated N2 and daf-2 nematodes (n = 15). (d) The comparison of the lifespan between icariin-treated worms and the vehicle group was performed using a Kaplan–Meier survival curve represented as a fraction survival (n = 90). (e) Numbers of body bends in 30 s on both day 5 and day 10 in both strains used (n = 45). Data for (b–e) are presented as mean ± SEM, ** p < 0.01 compared to the vehicle group by one-way analysis of variance (ANOVA).

Figure 2

Icariin treatment enhances heat and oxidative stress resistance in C. elegans. Wild-type N2 (a,c,e,g) and daf-2 mutant (b,d,f,h) strains were subjected to treatment with icariin at concentrations of 10, 50, and 100 μM. (a–d) For heat-stress induction, on both the 5th and 10th days of their lifespan, they were incubated at 37 °C for 14 h. The viability was monitored at two-hour intervals until the death of the last worm. (e–h) As an oxidative stressor, on day 5 and day 10 of their lifespan, the worms were exposed to a paraquat concentration of 50 mM. The resistance to oxidative stress was assessed at 24 h intervals until the death of the last worm. The results were represented as percentage survival as mean ± SEM, n = 90, * p < 0.05, ** p < 0.01 (one-way ANOVA).

Figure 3

Icariin supplementation modulates fat metabolism in C. elegans. Representative confocal photographs at 20× magnification (scale bar of 50 μm) of Nile red lipid staining of wild type (a) and daf-2 mutant strain (c) nematodes treated for 24 h with 10, 50, and 100 μM icariin or vehicle. Quantification of lipid accumulation as normalized correlated total cell fluorescence (n = 90), corrected total cell fluorescence (CTCF) expressed as arbitrary units (a.u.) as follows for wild-type (b) and daf-2 (d) nematodes. Values are shown as mean ± SEM, ** p < 0.01 compared to the vehicle group (one-way ANOVA).

Figure 4

Icariin treatment modulates gene expression of evolutionarily conserved pro-longevity pathways in wild-type C. elegans. Normalized relative mRNA expression in arbitrary units (a.u.) of daf-2 (a), daf-16 (b), sir-2.4 (c), sir-2.1 (d), skn-1 (e), hsf-1 (f), jnk-1 (g), and mtl-1 (h) upon treatment with 10, 50, and 100 μM icariin. Data shown are mean ± SEM, n = 9. * p < 0.05, ** p < 0.01 compared to the vehicle group (one-way ANOVA).

Figure 5

Nuclear translocation of DAF-16 was observed upon icariin treatment. Evaluation of nuclear translocation of daf-16(ot971[daf-16::GFP]) mutant strain. (a) Representative fluorescent confocal microphotographs at 20× magnification (scale bar of 50 μm) of vehicle, heat-shocked control (37 °C for 5 min) or icariin-treated C. elegans daf-16(ot971[daf-16::GFP]). Arrowheads indicate increased nuclear localization. (b) Using ImageJ version 1.53t, the fluorescent signal was processed to cell total correlates fluorescence (CTCF), normalized to the vehicle group, and represented in arbitrary units (a.u.). The data are shown as means ± SEM, n = 60, ** p < 0.01, compared to the vehicle (one-way ANOVA).

Figure 6

Mechanism-based model representing the hormetic response of icariin to extend lifespan in C. elegans. At a concentration of 50 μM, icariin inhibits the daf-2 gene that triggers a cascade of events within the IIS pathway. Specifically, DAF-16 translocates to the nucleus, where it activates the expression of mtl-1, a gene responsible for encoding metallothionein, which is a critical player in the organism’s response to stressors. Under stressful conditions, our observations suggest a potential involvement of jnk-1 in facilitating the nuclear translocation of DAF-16, along with possible concurrent activation of sir-2.4. Furthermore, upregulation of skn-1 may potentially contribute to improvements in overall fitness and healthspan. Notably, the hsf-1 is overexpressed by icariin at all treatment concentrations that expose it as a main contributor to the observed lifespan-extending activity.