Stress Buffering and Longevity Effects of Amber Extract on Caenorhabditis elegans (C. elegans)

Abstract

Amber is a fossilized tree resin historically used in wound healing and stress relief. Unfortunately, there is no concrete scientific evidence supporting such efficacy. Here, the stress buffering and longevity effect of Amber extract (AE) in Caenorhabditis elegans (C. elegans) was investigated. Survival assays, health span assays, Enzyme-Linked Immunosorbent Assay (ELISA), Stress biomarker detection assays, Green Fluorescence Proteins (GFP), Real Time quantitative PCR (RT-qPCR) and C. elegans mutants were employed to investigate the stress buffering and longevity effect of AE. In the study, it was observed that AE supplementation improved health span and survival in both normal and stressed worms. Additionally, AE positively regulated stress hormones (cortisol, oxytocin, and dopamine) and decreased fat and reactive oxygen species (ROS) accumulation. Through the Insulin/IGF-1 signaling (IIS) pathway, AE enhanced the nuclear localization of DAF-16 and the expression of heat shock proteins and antioxidant genes in GFP-tagged worms and at messenger RNA levels. Finally, AE failed to increase the survival of daf-16, daf-2, skn-1 and hsf-1 loss-of-function mutants, confirming the involvement of the IIS pathway. Evidently, AE supplementation relieves stress and enhances longevity. Thus, amber may be a potent nutraceutical for stress relief.

Keywords: C. elegans; DAF-16; amber extract; anti-stress; antiaging; antioxidant; stress hormones.

Figure 1

Effect of AE on lifespan and health span of wild type C. elegans. Worms were cultured at 20 °C on NGM-OP50 plates containing DMSO or AE (5, 25 and 50 µg/mL). (A) Lifespan of worms treated with or without AE (n = 30 worms/group). Statistical differences compared to control (DMSO) were considered significant at * p < 0.05, ** p < 0.01, *** p < 0.005 by log-rank test. (B) Reproduction ability of AE-treated and non-treated worms. The number of worms per group was one and the data of three independent experiments were represented. n = 3. (C) Body length determination of worms fed with or without AE for 96 h (n = 20 worms/group). (D) Motility of AE-treated and non-treated worms (n = 10 worms/group). Statistical differences compared to control (DMSO) were considered significant at * p < 0.05 by two-way ANOVA. Data were represented by mean ± SD. Experiments were performed in triplicate determinations.

Figure 2

Effect of AE treatment on stress tolerance of wild type C. elegans. Worms were cultured on NGM-OP50 plates containing DMSO or AE (50 µg/mL) at 20 °C for 96 h. (A) Heat stress survival of worms exposed to heat at 35 °C for 7 h (n = 30 worms/group) (B) Oxidative stress survival of worms exposed to 0.1% hydrogen peroxide (n = 12 worms/group). Statistical differences compared to control (DMSO) were considered significant at ** p < 0.01 by log-rank test. Data shown are representative of triplicate determinations.

Figure 3

Effect of AE treatment on stress biomarkers in wild type C. elegans. Worms were cultured at 20 °C on NGM-OP50 plates, treated with or without AE (50 µg/mL) for 96 h. (A) Cortisol production was assessed through the 11 beta hydroxysteroid dehydrogenase pathway and its quantity in worm homogenate determined by ELISA (n ≈ 3000 worms). Statistical differences compared to control (DMSO) were considered significant at * p < 0.05, **** p < 0.0001 by one-way ANOVA. Endo represents endogenous cortisol production. Exo represents exogenous cortisol production (cortisone addition). (B) Oxytocin levels in worm homogenate were quantified by ELISA (n ≈ 3000 worms), (C) Dopamine levels in worm homogenate were quantified by ELISA (n ≈ 3000 worms), (D) Fat accumulation was determined by Nile red staining (n = 10 worms/group), (E) Intracellular ROS accumulation was determined by DCFHDA assay after 1 hr incubation time (n = 20 worms/group) and (F) In vitro ROS determination by DPPH assay after 30 min incubation time. Statistical differences compared to control (DMSO) were considered significant at * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.0001 by Student’s t-test. Data were represented as mean ± SD. Experiments were performed in triplicate determinations.

Figure 4

Effects of AE on DAF-16 cellular translocation and the expression of HSP-16.2, SOD-3 and GST-4 in GFP-tagged worms. (A) Subcellular localization of DAF-16::GFP worms treated with or without AE (50 µg/mL) for 96 h (n = 30 worms/group). Subcellular localization was categorized into Cytosolic, Nucleus and Intermediate (between cytosolic and nucleus). (B) HSP-16.2::GFP worms treated or not treated with AE were cultured for 72 h and exposed to heat shock at 35 °C for 2 h (n = 30 worms/group). (C) SOD-3::GFP and (D) GST-4::GFP AE-treated and non-treated worms were cultured for 72 h (n = 30 worms/group). Statistical differences compared to control (DMSO) were considered significant at **** p < 0.001 by Student’s t-test. Data were represented by mean ± SD. Experiments were performed in triplicate determinations and representatives shown. Scale bar for GFP analysis = 200 µm.

Figure 5

Effects of AE on the mRNA expression daf-16 and its target genes in wild type C. elegans. Worms were cultured at 20 °C on NGM-OP50 plates, treated with or without AE (50 µg/mL) for 96 h (n ≈ 500 worms/group). Statistical differences compared to control (DMSO) were considered significant at * p < 0.05, *** p < 0.005 and **** p < 0.0001 by multiple t tests. Data were represented by mean ± SD. Experiments were performed in triplicate determinations.

Figure 6

Effect of AE on the oxidative stress survival of C. elegans mutants. Worms were cultured at 20 °C on NGM-OP50 plates, treated with or without AE (50 µg/mL) for 96 h. (A) daf-2 (e1370) mutants, (B) daf-16 (mgDf50) mutants, (C) skn-1 (tm4241) mutants and (D) hsf-1 (sy441) mutants oxidative stress survival (n = 12 worms/group). Statistical significances of survival curves were assessed by log-rank test. Experiments were performed in triplicate determinations and representative curves shown.