Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans

Abstract

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blueberry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.

Fig. 1

Blueberry polyphenols extend lifespan and slow aging in Caenorhabditis elegans. (A) Treatment with blueberry polyphenols [67 µg mL⁻¹ (orange) or 200 µg mL⁻¹ (blue)] extended mean lifespan in fem-1(hc17) animals grown at 25 °C (untreated control, green). (B) Blueberry polyphenols slowed the decline in pharynx pumping during aging. Open circles, untreated; filled circles, treated with 200 µg mL⁻¹ blueberry polyphenols. Average pumping rate (pumps per minute) in 14 animals scored in two trials; error bars indicate SEM among individual animals scored; t-test, untreated vs. treated, day 8, P = 0.023, day 10, P = 0.004.

Fig. 2

Markers of aging and oxidative damage are reduced in animals treated with BB polyphenols. (A)Intestinal autofluorescence from lipofuscin in representative day 16 animals with 0 µg mL⁻¹ (left) or 200 µg mL⁻¹ blueberry polyphenols (right). (B)Mean fluorescence intensity from intestinal lipofuscin in day 16 adults treated with indicated amounts of blueberry polyphenols; 0 µg mL⁻¹, n = 24 animals; 200 µg mL⁻¹, n = 26 animals. (C)Representative images of pharynxes from BB-treated or control animals on adult day 14 immunostained with antisera specific for 4-HNE. Arrows designate terminal bulb. Similar results were obtained for 4-HNE immunofluorescence in the somatic gonad. (D) Mean fluorescence intensity of 4-HNE immunofluorescence in pharynx terminal bulbs from day 14 adults. Third bar shows background fluorescence measured in animals stained with secondary antibody only. P-values are t-test vs. 200 µg mL⁻¹; NSD, no significant difference; n = 18 animals (0 µg mL⁻¹); 16 animals (200 µg mL⁻¹); n = 4 animals (secondary antibody control).

Fig. 3

BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in populations treated with 200 g/ml of blueberry polyphenol (BB PP) at 25 °C. Graph shows mean of two independent experiments with SEM; **P < 0.01 vs. day 0 within treatment; *P < 0.05 vs. day 0 within treatment.

Fig. 4

A PAC-enriched fraction of BB contained components sufficient to extend Caenorhabditis elegans lifespan. The total blueberry polyphenols were fractionated by C18 and Sephadex LH20 to produce fractions enriched in anthocyanins (ATC), proanthocyanidins (PAC) or chlorogenic acid (CA). Purchased, purified CA was used for CA experiments. Each fraction was assayed for effects on C. elegans longevity at a concentration of 67 µg mL⁻¹, similar to that in the complete extract. (A)Only the PAC-enriched fraction (orange) prolonged lifespan. No longevity benefits were observed for the ATC-enriched fraction (purple) or purified CA (blue); 0 µg mL⁻¹, n = 97 animals, 2 trials; ATC, n = 99, 2; CA, n = 91, 2; PAC, n = 88, 2. Complete statistics for PAC trials are presented in Table 2. (B) The reconstituted extract, produced by mixing equal mass ratios of fractions in (A) also conferred the same lifespan extension as the starting mix of polyphenols (remix, n = 40 animals, 1 trial; start, n = 98, 2).

Fig. 5

Treatment with blueberry polyphenols improved thermotolerance, but not oxidative stress resistance. (A)Fractional survival at 35 °C for day 5 fem-1(hc17) or animals with indicated treatments (0, 67 µg mL⁻¹ or 200 µg mL⁻¹ BB). Shown is average survival in 3–5 experiments with 30–60 animals/experiment; error bars, SEM; total number of animals tested: 173 (0 µg mL⁻¹), 208 (67 µg mL⁻¹), 117 (200 µg mL⁻¹); ***P < 0.0001, t-test. (B) Survival on 10 mm paraquat was not affected by BB polyphenols; n = 74–91 animals. (C) Survival on hydrogen peroxide was not affected by 200 µg mL⁻¹ BB polyphenols; error bars, SEM; n = 59–98 animals in three trials. (D) Effect of BB polyphenol subfractions on thermotolerance in fem-1(hc17) animals. Sixteen-hour (16-h) survival at 35 °C was assessed for animals treated with BB polyphenol subfractions shown in Fig. 4, or untreated control animals, as described in Experimental procedures; n≥ 25 animals/trial; *P ≤ 0.05, t-test, treated vs. untreated controls.

Fig. 6

BB and antimicrobial treatment had different effects on Caenorhabditis elegans thermotolerance and lifespan. (A)BB treatment did not inhibit bacterial growth in aging assays. Bacterial lawn growth was monitored over 11 days under conditions replicating those of C. elegans aging assays. Shown are the average number of colony forming units (cfu) per lawn after indicated treatments in two trials, **P < 0.01. (B) Effects of blueberry polyphenols and ampicillin on C. elegans thermotolerance measured as survivorship at 35 °C after 16 h, as in Fig. 5(A). Ampicillin treatment alone had no effect on thermotolerance. Shown are average survivorships in three trials, with at least 20 animals per trial, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) BB polyphenols (200 µg mL⁻¹) and ampicillin had additive effects on daf-16(mgDf50) lifespan at 25 °C (purple curve). daf-16(mgDf50), untreated control, mean 11.6 days, 0.28 SE, n = 69; 200 µg mL⁻¹ BB polyphenols, mean 13.0 days, 0.27 SE, n = 49; 100 µg mL⁻¹ ampicillin, mean 12.7 days, 0.35 SE, n = 53; ampicillin + BB polyphenols, mean 14.7 days, 0.33 SE, n = 60; P < 0.0001, log-rank, for Amp. vs. BB+Amp and for BB vs. BB+Amp.

Fig. 7

Increased lifespan from blueberry polyphenol treatment is blocked by mutations in the osmotic stress resistance pathway. Lifespan in osr-1(rm1) (A), sek-1(ag1) (B), and unc-43(n1186) (C) was not lengthened by treatment with 200 µg mL⁻¹ blueberry polyphenols. Lifespan survival statistics are contained in Table 1.