Rosmarinus officinalis L. increases Caenorhabditis elegans stress resistance and longevity in a DAF-16, HSF-1 and SKN-1-dependent manner

Abstract

Improving overall health and quality of life, preventing diseases and increasing life expectancy are key concerns in the field of public health. The search for antioxidants that can inhibit oxidative damage in cells has received a lot of attention. Rosmarinus officinalis L. represents an exceptionally rich source of bioactive compounds with pharmacological properties. In the present study, we explored the effects of the ethanolic extract of R. officinalis (eeRo) on stress resistance and longevity using the non-parasitic nematode Caenorhabditis elegans as a model. We report for the first time that eeRo increased resistance against oxidative and thermal stress and extended C. elegans longevity in an insulin/IGF signaling pathway-dependent manner. These data emphasize the eeRo beneficial effects on C. elegans under stress.

Figure 1. Effect of ethanolic extract of Rosmarinus officinalis L. (eeRo) on juglone-induced mortality. Survival of wild-type (A) and mutant worms (B) treated with eeRo and exposed to 100 µM juglone for 1 h (Ctrl+). Data are reported as percentage of living worms of 100 worms per group in each experiment from 4 independent assays. A, *P<0.05, **P<0.01, ***P<0.001, compared to the Ctrl+ group (one-way ANOVA). B, *P<0.05 (two-way ANOVA).

Figure 2. Effect of ethanolic extract of Rosmarinus officinalis L. (eeRo) on thermal stress. Survival of wild-type (A) and mutant worms (B) treated with eeRo and exposed to thermal stress (35°C) for 4 h (Ctrl+). Data are reported as percentage of living worms of 100 worms in each experiment from 4 independent assays. A, *P<0.05, **P<0.01, ***P<0.001, compared to the Ctrl+ group (one-way ANOVA). B, *P< 0.05 (two-way ANOVA).

Figure 3. Effect of ethanolic extract of Rosmarinus officinalis L. (eeRo) on reactive oxygen species (ROS) production. Data are reported in arbitrary fluorescence units (AFU) from 3 independent assays (n=3). A, Levels of basal and H₂O₂-induced (25 mM/1 h) ROS production in wild-type (N2) worms. *P<0.001 compared to eeRo-treated basal groups; ^#P<0.001 compared to eeRo-treated induced groups; ^γP<0.01 Ctrl basal compared to Ctrl induced groups. B, Levels of ROS production in wild-type (N2) and mev-1 (TK22) worms. *P<0.001, compared to all other groups; ^#P<0.001, compared to all other groups; ^γP<0.001, 10 µM compared to the 25 µM eeRo treated group; ^$P<0.001, 25 µM compared to the 50 µM eeRo treated group; ^&P<0.001, 10 µM compared to the 50 µM eeRo treated group (one-way ANOVA). C, ROS levels in daf-16, hsf-1 and skn-1 mutants treated or not with the extract. *P<0.05, **P<0.01, ***P<0.001, treated compared to untreated group; ^#P<0.01, compared to the other Ctrl groups; ^γP<0.01, compared to the other treated groups (two-way ANOVA).

Figure 4. Effect of ethanolic extract of Rosmarinus officinalis L. (eeRo) on C. elegans lifespan. Survival curves were significantly extended in (A) wild-type and (B) mev-1 worms treated with 25 µg/mL eeRo (P<0.0001, log-rank (Mantel-Cox) test).

Figure 5. Effect of 25 µg/mL ethanolic extract of Rosmarinus officinalis L. (eeRo) in (A) daf-2, (B) daf-16, (C) hsf-1 and (D) skn-1 mutants lifespans. Survival curves were significantly different in daf-16 (median lifespan) and in daf-2 (maximum lifespan) (P<0.05, log-rank (Mantel-Cox) test).