Agrimonia procera Wallr. Extract Increases Stress Resistance and Prolongs Life Span in Caenorhabditis elegans via Transcription Factor DAF-16 (FoxO Orthologue)

Abstract

Agrimonia procera is a pharmacologically interesting plant which is proposed to protect against various diseases due to its high amount of phytochemicals, e.g., polyphenols. However, in spite of the amount of postulated health benefits, studies concerning the mechanistic effects of Agrimonia procera are limited. Using the nematode Caenorhabditis elegans, we were able to show that an ethanol extract of Agrimonia procera herba (eAE) mediates strong antioxidative effects in the nematode: Beside a strong radical-scavenging activity, eAE reduces accumulation of reactive oxygen species (ROS) accumulation and protects against paraquat-induced oxidative stress. The extract does not protect against amyloid-β-mediated toxicity, but efficiently increases the life span (up to 12.7%), as well as the resistance to thermal stress (prolongation of survival up to 22%), of this model organism. Using nematodes deficient in the forkhead box O (FoxO)-orthologue DAF-16, we were able to demonstrate that beneficial effects of eAE on stress resistance and life span were mediated via this transcription factor. We showed antioxidative, stress-reducing, and life-prolonging effects of eAE in vivo and were able to demonstrate a molecular mechanism of this extract. These results may be important for identifying further molecular targets of eAE in humans.

Keywords: aging; antioxidant; insulin-like signaling; life span-extending effects; oxidative stress; phytochemicals.

Figure 1

Radical-scavenging and antioxidative effects of eAE. (A) Image of Agrimonia procera plant/dried plant material; (B) Radical scavenging activity (TEAC assay): In this cell-free assay, eAE shows antioxidant properties. Values are mean ± SD, n = 3, one-way ANOVA with Dunnett’s multiple comparisons test vs. control (0 µg/mL), a: ** p ≤ 0.01, b: **** p ≤ 0.0001; (C) Thermally induced increase in ROS (DCF assay): Wild type L4 larvae were treated with different concentrations of eAE or with DMSO (vehicle, 0.4%) for 24 h (20 °C), and were then transferred individually into the wells of a 384-well plate containing 50 µM H₂DCF-DA. The increase in fluorescence was measured at 37 °C. Values are mean ± SD, n = 3 (each 16 individuals per group), two-way ANOVA with Dunnett’s multiple comparisons test vs. control at the correspondent time point, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001; (D) Oxidative stress resistance: Wild type L4 larvae were incubated with eAE or with DMSO (vehicle, 0.4%) for 72 h at 20 °C, and all nematodes were then transferred into extract-free media containing 50 mM paraquat. The survival of the nematodes was surveyed via touch-provoked movement every 24 h. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 40 individuals per group), Log Rank (Mantel-Cox) test, * p ≤ 0.05, ** p ≤ 0.01. Calculated data for oxidative stress resistance is shown in Supplementary Table S3.

Figure 2

Effects of eAE on life span and resistance against thermal and amyloid-β-stress. (A) Modulation of life span: Wild type L4 larvae were incubated with different concentrations of eAE or DMSO (vehicle, 0.4%) at 25 °C. On five days a week, medium was exchanged and survival of the nematodes was tested via touch-provoked movement. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 40 individuals per group), Log Rank (Mantel-Cox) test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; (B) Thermal stress resistance: Wild type L4 larvae were treated with different concentrations of eAE or with DMSO (vehicle, 0.4%) for 24 h at 20 °C, and then transferred individually into the wells of a 384-well plate containing 1 µM SYTOX^® Green. The increase in fluorescence was measured at 37 °C. The graph shows the percentage of viable nematodes. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 16 individuals per group), Log Rank (Mantel-Cox) test, *** p ≤ 0.001; (C) Toxicity of Aβ: Transgenic nematodes of the strain CL4176 were incubated as eggs with different concentrations of eAE, DMSO (vehicle, 0.4%) or caffeine as a positive control at 16 °C. L3 larvae were transferred on agar plates containing a lawn of E. coli var. OP50 and kept at 25 °C. After 26, 28, 30, 32, and 34 h, nematodes were analyzed for occurrence of paralysis. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 40 individuals per group), Log Rank (Mantel-Cox) test, **** p ≤ 0.0001. Calculated data for the experiments are shown in Supplementary Tables S1, S2, and S4.

Figure 3

Effects of eAE on the intracellular localization of DAF-16. (A) Intracellular localization of DAF-16: L4 larvae of the transgenic strain TJ356 (DAF-16::GFP) were incubated with different concentrations of eAE or DMSO (vehicle, 0.4%) for 1 h at 20 °C or kept for five minutes at 37 °C (positive control), and localization of the transcription factor was then examined using fluorescence microscopy. Values are mean ± SD, n = 3 (each 20 individuals per group, one-way ANOVA with Tuckey’s multiple comparisons test vs. control, * p ≤ 0.05; (B) representative image for a nematode with mainly cytosolic localization of DAF-16; (C) representative image for nematodes with mainly nuclear localization of DAF-16.

Figure 4

Requirement of DAF-16 for the protective effects of eAE. (A) Thermally induced increase in ROS (DCF assay): L4 larvae deficient in DAF-16 were treated with different concentrations of eAE or with DMSO (vehicle, 0.4%) for 24 h at 20 °C. After washing, they were transferred individually into the wells of a 384-well plate containing 50 µM H₂DCF-DA. The increase in fluorescence was measured at 37 °C. Values are mean ± SD, n = 3 (each 16 individuals per group), two-way ANOVA with Dunnett’s multiple comparisons test vs. control at the correspondent time point, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; (B) Thermal stress resistance: L4 larvae deficient in DAF-16 were treated with different concentrations of eAE or with DMSO (vehicle, 0.4%) for 24 h at 20 °C. After washing, they were transferred individually into the wells of a 384-well plate containing 1 µM SYTOX^® Green. The increase in fluorescence was measured at 37 °C. The graph shows the percentage of viable nematodes. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 16 individuals per group), Log Rank (Mantel-Cox) test; (C) Oxidative stress resistance: L4 larvae deficient in DAF-16 were incubated with eAE or with DMSO (vehicle, 0.4%) for 72 h at 20 °C. Then, all nematodes were transferred into extract-fee media containing 50 mM paraquat. Every 24 h, the survival of the nematodes was surveyed via touch-provoked movement. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 40 individuals per group), Log Rank (Mantel-Cox) test; (D) Modulation of life span: L4 larvae deficient in DAF-16 were incubated with different concentrations of eAE or DMSO (vehicle, 0.4%). On five days a week, media were exchanged and survival of the nematodes was tested via touch-provoked movement. Kaplan-Meier statistics were used for the comparison of the survival curves, n = 3 (each 40 individuals per group), Log Rank (Mantel-Cox) test, * p ≤ 0.05, ** p ≤ 0.01. Calculated data for thermal and oxidative stress resistance, as well as lifespan, are shown in Supplementary Tables S1–S3.