Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans methods
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Proanthocyanidin components of blueberries enhance longevity
Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC) or hydroxycinnamic esters, mainly chlorogenic acid (CA). Major components of each of these fractions have been shown to confer significant antioxidant activity and ATC can protect cells against oxidative stress in vitro (; ). To determine which fraction(s) delayed aging, we assayed their effects on C. elegans lifespan. Neither the ATC-enriched fraction nor purified CA had any significant effect on longevity ( ) (control, 12.0 ± 0.34 days; ATC, 11.7 ± 0.36 days, P = 0.96 vs. control; CA, 11.7 ± 0.39 days, P = 0.99). However, treatment with the PAC-enriched fraction increased lifespan to a similar extent as the starting BB polyphenol mixture or the remixed fractions ( ) (PAC, 14.4 ± 0.36 day...
Blueberry polyphenols do not appear to prolong lifespan through antimicrobial effects
One contributor to late-age mortality in C. elegans is the detrimental effect of the bacterial food source (; ). Accordingly, C. elegans lifespan could be increased approximately 30% when bacterial growth was arrested by ampicillin ( ). We observed that lifespan of BB-treated wild-type animals was not further extended by the presence of ampicillin to arrest bacterial growth ( ), indicating that BB may have extended lifespan by relieving microbial stress. To investigate this possibility, we first tested whether the BB polyphenols that prolong lifespan inhibited bacterial growth. We monitored growth of the bacterial lawn under conditions identical to C. elegans aging assays, 11 days on NGM agar at 25 °C, in the presence or absence of BB. During this period, untreated bacterial lawns completed approximately one population doubling, while bacterial growth was arrested after ampicill...
Blueberry polyphenols do not appear to prolong lifespan through antimicrobial effects
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. 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)...
Phenotypic assays
For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed w...
Phenotypic assays
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24 °C, washed and incubated overnight at 4 °C with Alexafluor-546-conjugated goat anti-mouse secondary antibody (1: 100) (#A-11003, Invitrogen, Carlsbad, CA, USA). Stained animals were mounted on 2% agarose pads and fluorescence visualized as for lipofuscin with appropriate filter sets. 4-HNE immunofluorescence was measured in the pharynx terminal bulb and somatic gonad using ImageJ software.
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Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC) or hydroxycinnamic esters, mainly chlorogenic acid (CA). Major components of each of these fractions have been shown to confer significant antioxidant activity and ATC can protect cells against oxidative stress in vitro (; ). To determine which fraction(s) delayed aging, we assayed their effects on C. elegans lifespan. Neither the ATC-enriched fraction nor purified CA had any significant effect on longevity ( ) (control, 12.0 ± 0.34 days; ATC, 11.7 ± 0.36 days, P = 0.96 vs. control; CA, 11.7 ± 0.39 days, P = 0.99). However, treatment with the PAC-enriched fraction increased lifespan to a similar extent as the starting BB polyphenol mixture or the remixed fractions ( ) (PAC, 14.4 ± 0.36 days, P < 0.0001 vs. control; start, 14.8 ± 0.36 days, P < 0.0001; remix, 14.0 ± 0.48 days, P < 0.0001; complete statistics for lifespan trials with PAC-enriched fraction are presented in ). Thus, components of the PAC-enriched fraction of blueberries could extend lifespan in C. elegans.
One contributor to late-age mortality in C. elegans is the detrimental effect of the bacterial food source (; ). Accordingly, C. elegans lifespan could be increased approximately 30% when bacterial growth was arrested by ampicillin ( ). We observed that lifespan of BB-treated wild-type animals was not further extended by the presence of ampicillin to arrest bacterial growth ( ), indicating that BB may have extended lifespan by relieving microbial stress. To investigate this possibility, we first tested whether the BB polyphenols that prolong lifespan inhibited bacterial growth. We monitored growth of the bacterial lawn under conditions identical to C. elegans aging assays, 11 days on NGM agar at 25 °C, in the presence or absence of BB. During this period, untreated bacterial lawns completed approximately one population doubling, while bacterial growth was arrested after ampicillin treatment. In contrast, BB polyphenols had no effect on bacterial population growth at the doses tested in lifespan assays ( ). Furthermore, C. elegans nematodes grown on ampicillin-treated bacteria displayed no increase in thermotolerance, nor did the combination of ampicillin and BB together en...
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. 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...
For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed with JMP analysis software (SAS Institute Inc, Cary, NC, USA). Pharynx pumping rates were scored on adults at room temperature (24 °C) under a Nikon SMZ1500 stereomicroscope (Nikon, Melville, NY, USA).
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24 °C, washed and incubated overnight at 4 °C with Alexafluor-546-conjugated goat anti-mouse secondary antibody (1: 100) (#A-11003, Invitrogen, Carlsbad, CA, USA). Stained animals were mounted on 2% agarose pads and fluorescence visualized as for lipofuscin with appropriate filter sets. 4-HNE immunofluorescence was measured in the pharynx terminal bulb and somatic gonad using ImageJ software.
Machine-readable layer
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