Introduction

Marine algae are a rich source of biologically active compound, some of which includes carotenoids, fatty acids, polysaccharides, proteins, and peptides. Protein and peptides present in these marine algae are reported to have various biomedical applications (Lordan et al. 2011) due to presence of plenty of essential amino acids in their constitution (Taboada et al. 2010). Phycobiliproteins (PBPs) constitute around 60 % of the total protein content and 20 % of the dry cell weight of cyanobacteria (Soni et al. 2008). Till date, they have been reported as potent pharmacological and medicinal agent having hepato-protective (Vadiraja et al. 1998), anti-inflammatory (Romay et al. 2003), anti-cancerous (Zhang et al. 2011), anti-Alzheimer’s disease (Singh et al. 2014), and antioxidant activity (Sonani et al 2014; Soni et al 2008). Moreover, PBPs are also utilized as natural colorants in food products like chewing gums, dairy products, jellies as well as in cosmetics (Rasmussen and Morrissey 2007). Phycoerythrin (PE), a majorly produced PBP, is an oligomeric pigment protein, consisting of two chromophore/s-appended dissimilar polypeptide chain belonging to two different families (α and β) (Parmar et al. 2011). PE, isolated from marine cyanobacterium Lyngbya sp. A09DM, has shown strong and wide range of antioxidant activity in vitro against various reactive oxygen species (Sonani et al. 2014). Moreover, toxicity analysis of purified PE has approved that it is nontoxic and safe for oral consumption (unpublished data).

“Aging” is believed to be the manifestation of two major phenomenona, the accumulation of reactive oxygen species (ROS) and downstream effects of insulin/insulin-like growth factor signaling (IIS) pathway (Blagosklonny 2008). There are many theories proposed for aging, out of which, free radical theory is the most prevalent. It suggests that an accumulation of free radicals and damage cause by them is the major cause of aging (Harman 1998). Several lines of evidences proposed that increase in endogenous antioxidant production or supplement of exogenous antioxidant moderates aging by averting the free radicals (Melov et al. 2000). This influential theory offered doorway to investigate anti-aging effects of various antioxidant compounds using different model organisms (Gems and Doonan 2009). Several studies have reported beneficial effects of exogenous antioxidant supplements like vitamin E (α-tocopherol) (Harrington and Harley 1988), trolox (Ishii et al. 2004), and α-lipoic acid (Benedetti et al. 2008; Brown et al. 2006) on the manifestation of aging. Despite of having such strong lines of evidences, oxidative damage is not accepted as a sole cause for aging due to inefficacy of some antioxidants in averting aging effects (Bjelakovic et al. 2007; Schulz et al. 2007). Reduction in function of any components of the IIS pathway leads to the activation of a series of genes involved in life span control, stress tolerance, and protein misfolding suppression (Ogg et al. 1997; Lin et al. 1997), collectively resulting in life span extension (Kenyon 2010). Finding pharmacological compounds having inhibitory role in the IIS pathway has been a major research motivation for many research groups.

The aging process in C. elegans is quite conserved and portrays similarity to humans in some aspects like decreased motility, decreased cognitive abilities, immunosenescence, decreased rate of reproduction, and increased accumulation of harmful metabolites like lipofuscin (Iwasa et al. 2010). C. elegans is thus considered as a good model to study aging and testing the therapeutic compounds which may curtail aging. Use of PE as a nutraceutical food supplement for its anti-oxidative, anti-inflammatory, immunomodulatory, and antihyperalgesic properties has been reported (Soni et al. 2008; Cian et al. 2012). However, the effect of antioxidant properties of PE on aging is not explored. In this paper, we explored the potential of PE as an anti-aging drug. The effect of PE on life span and signs of aging was tested using wild type (N2 Bristol) and various mutant C. elegans strain. Our results demonstrate that PE treatment enhanced the life span of C. elegans and also decreased the rate of progression of aging-related surrogate physiological markers like decline in the rate of pharyngeal pumping and locomotion. PE treatment also conferred better tolerance to thermal and oxidative stress. As various defensive mechanisms of C. elegans against aging have been hypothesized to occur either via downregulation of DAF-2–AGE-1–DAF-16 insulin signaling pathway (Kenyon 2010; Narasimhan et al. 2009; Samuelson et al. 2007; Murphy and Hu 2013; Cai et al. 2011) or via scavenging of reactive oxygen species, we targeted to track the effect of PE with respect to these two major facets. C. elegans null mutants and easy knockout strategy for various controllers of the IIS pathways facilitate the rational strategy to track out the mode of action of any drug in C. elegans. Null mutants of daf-2(e1370), daf-16(mu86), and age-1(hx546) genes have been employed to reveal the point of action of PE across the IIS pathways of C. elegans. Transcription factor for heat shock (HSF-1) and oxidative stress (SKN-1) response activates expression of a series of essential proteins and molecular chaperons needed to maintain proteostasis under respective stress conditions (Morton and Lamitina 2013; Zhou et al. 2011). In C. elegans, expression of heat shock protein by HSF-1 has been reported to be DAF-16 dependent (Kenyon 2010). SKN-1 is a worm homolog of human nuclear factor E2-related transcription factor (Nrf) and has also been reported to be involved in the IIS pathway (Tullet et al 2008). To investigate whether the protective effect of PE is mediated through HSF-1 and SKN-1, we checked the effect of PE in wild type and CL4176 (C. elegans Alzheimer’s disease model) animals under RNAi-mediated knockout conditions.

Thus, in the present study, we explored the potential of PE as an anti-aging drug. It systematically describes the effect of PE feeding on C. elegans aging. We also explored the possible pathways through which PE might be exhibiting its effects.

Materials and methods

PE preparation

Lyngbya sp. A09DM was isolated from the rocky shore of Okha, Gujarat, India. Enrichment, isolation, and identification of culture were carried out according to protocol described by Shah et al. (2001). Isolated strain was grown in artificial salt nutrient (ASN) III medium (Waterbury and Stanier 1981) under illumination (12:12 h light-dark cycles) of 36 W white fluorescent lamp which provided 130 μmol photons m-2 s-1 flux density at the surface of the flasks. PE was obtained by resuspending cells from 30-day-old culture in the isolation buffer (20 mM Tris, pH 8.0) and further subjecting it to repetitive freeze (−25 °C)–thaw (4 °C) cycles. The brick red supernatant, obtained after centrifugation, was purified by eluting through anion-exchange low-pressure liquid chromatography and gel permeation chromatography. Purity was affirmed by native-PAGE and UV-visible absorption spectroscopy. The resulting fractions were concentrated by ultra-filtration using a 30-kDa MWCO centrifugal device (Macrosep®, Pall Corporation). Concentrated PE was lyophilized at (−100 ° C) (Sentry 2.0, VirTis, SP Scientific) and stored at 4 ° C under dark condition.

C. elegans strains and cultivation

The various C. elegans strains used in this study were N2 Bristol (wild type), CL4176 (dvIs27 [pAF29 (myo-3/Ab 1-42/let UTR) + pRF4 (rol-6(su1006)]), daf-16(mu86), daf-2(e1370), age-1 (hx546), muIs84 pAD76 (sod-3::GFP), and eat-2(ad1116). All of the strains were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, Minneapolis MN. All C. elegans strains were grown at 20 °C (unless specified) on nematode growth medium (NGM) agar plates seeded with the E. coli strain OP50 under standard laboratory conditions as described by Brenner (1974). The E. coli OP50, E. coli HT115(DE3), and RNAi constructs were grown in Luria-Bertani (LB) medium at 37 °C. Grown cultures were spotted (200 μl of 1010 cfu/ml solution) onto NGM plates and incubated at 37 °C for 24 h prior to worm inoculation. Food source including E. coli OP50, E. coli HT115(DE3), and RNAi constructs were also obtained from the CGC. The effect of PE was checked by providing the PE with NGM agar at various doses of 10, 50, and 100 μg ml-1 of media.

RNAi experiments

E. coli HT115 (DE3) expressing dsRNA (of RNAi) for hsf-1 and skn-1 were fed to worms as described previously (Timmons and Fire 1998; Kamath and Ahringer 2003). In brief, L4-stage hermaphrodites were fed with hsf-1 and skn-1 (RNAi) expressing bacteria for 3 days to allow the intestinal ingestion of RNAi, and ingested worms (confirmed phenotypically) are cloned to individual wells for each gene screened. Each of these is allowed to lay eggs for 1 day. Siblings and the progeny obtained from the eggs were used as RNAi mutant/knockout for particular genes.

Worm synchronization and life span assay

To obtain age-synchronized nematodes, 5–10 gravid adults were allowed to lay egg on NGM plates for 3 h and then removed. Plates with eggs were treated with sodium hypochlorite solution (1 N NaOH+ 5 % NaOCl, Sigma-Aldrich). Treated eggs were repeatedly washed in M9 buffer (3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl, 1 ml of 1 M MgSO4, make up the volume to 1 L using H2O). The eggs were further incubated in M9 and allowed to hatch overnight at 20 °C. The resulting synchronized L1-stage worms were put on standard NGM plates with E. coli OP50 and incubated until L4 larval stage. The group of L4-synchronized C. elegans were taken in fresh NGM plates for life span assay (numbers of animal (n) for each group is depicted in supplementary table 1). Experiment was performed in triplicates. The animals were transferred into fresh food plate every alternate day and the number of dead animals was also scored. Response against mechanical stimulus (given by prodding with a platinum pick) and pharyngeal pumping rate were taken as surrogate markers to monitor dead animals (Kenyon et al. 1993). Pharyngeal pumping rate was estimated by manually monitoring an individual animal of 5th and 10th day to count the rhythmic contractions of the pharynx over 20 s by hand counter. The mean pharyngeal pumping rate for various C. elegans groups (5th day—control: n = 165, PE-treated: n = 152; 10th day—control: n = 110, PE-treated: n = 145) was expressed per minute. The plot of fraction survival against time (days) was constructed by taking treatment day as first day and subjected to log-rank test to obtain the mean life span.

Stress resistance assay

For stress resistance assay, N2 worms were subjected to temperature and oxidative stress. For both assays, age-synchronized young adult worms were transferred to NGM plates with and without PE treatment. They were grown till fourth day (post adulthood) and then subjected to temperature and oxidative stress, respectively. For temperature stress, PE-treated and untreated animals were shifted from 20 to 35 °C for 12 h and the number of dead worms was counted at a regular interval of every 2 h until the 12th hour. Oxidative stress was given by washing the animals 3–4 times with M9 buffer followed by soaking them in 10 and 20 mM hydrogen peroxide (H2O2) solution for 2 h as described by Cai et al. (2011). Following 2 h of oxidative stress, the animals were transferred to fresh NGM plates and incubated for 16 h at 20 °C for recovery. After 16 h, live animals were scored for PE-treated and untreated condition.

Locomotion assays

Locomotion assay was performed on the 5th and 10th day adult, respectively by counting the number of body bends observed under a stereo microscope during a 30-s interval (Kumar et al. 2010). A body bend was defined as a change in the reciprocating motion of bending at the mid-body. Assay was performed on PE-treated and untreated worms.

Paralysis assays

CL4176 transgenic animals containing heat-inductive transgene of human Aβ1-42 in muscle cells were transferred to PE-treated and untreated plates and incubated at 16 °C (permissive temperature) for 48 h followed by shifting them to 25 °C (non-permissive temperature) for 20 h, to stimulate amyloid-beta (Aβ) expression whose aggregation in muscle cells leads to paralysis. The number of paralyzed worms was scored using the method described by Link (2003). In brief, after 20 h of incubation at 25 °C, numbers of paralyzed animals were scored at every 2 h interval until all animals were paralyzed. Newly paralyzed worms were distinguished by looking at the “halo” of cleared bacterial lawn produced by them due to movement of only the head while feeding. Therefore, worms with halos were considered as paralyzed. Moreover, worms, not responding to mechanical stimulus, were also counted as paralyzed.

RNA isolation and real time PCR

Synchronized L1 animals of wild type were raised with and without PE at 20 °C. For RNA isolation, 1-day young adult animals were washed 2–3 times by M9 buffer followed by resuspending them in Trizol reagent (Invitrogen). Total RNA was isolated by chloroform extraction, followed by ethanol precipitation and DNase treatment. cDNA was synthesized using standard protocol. Real time PCR was carried out using PCR master mix (Bio-Rad). HSF-1 transcripts levels were quantified by normalizing it to actin (internal control), and relative expression level of transcripts were calculated.

DAF-16::GFP and Sod-3::GFP localization assay

Synchronized population of L1 stage DAF-16::GFP and Sod-3::GFP transgenic worms were grown in PE-treated and untreated plates. Young adult worms were imaged at 10× using a Nikon E-800 fluorescence microscope. The filters 400/30 and 508/20 were used for excitation and emission to examine the GFP transgenic lines, respectively.

Detailed information regarding experimentation methodology and results like total number of animal used, food source used in various assays, and statistical tests are summarized in supplementary table 1 and 2.

Statistical analysis

All results were expressed as mean with standard errors of the mean (SEM). A student’s two-tailed t test was used when comparing two sets of data. p < 0.05 was considered statistically significant. For life span, log-rank tests were performed for survival curves by the Prism 4 software. p < 0.001 was considered statistically significant.

Results

PE purification and characterization

Two successive cycles of freezing and thawing at −25 and 4 °C, respectively, was found optimum for the extraction of crude intracellular content from the cyanobacterial cells. PE purified from crude extract, was assessed for purity and homogeneity by UV-visible spectroscopy and gel electrophoretic analysis. UV-visible spectrum showing peak at 565 nm dominated over the peak at 280 nm (Fig. 1), indicating the absence of other protein impurities. PE preparation, obtained by described protocol, was found to have purity ratio of about 6.75 (A565/ A280, calculated from UV-visible spectrum). Moreover, manifestation of only a single band on the native-PAGE of purified PE stated the purity as well as homogeneity of PE preparation (Fig. 1 inset).

Fig. 1
figure 1

PE purification and characterization. UV-visible absorbance spectrum of purified PE from Lyngbya sp. A09DM. Purified PE and its native-PAGE profile upon silver staining (Inset)

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PE treatment increases life span and delays signs of aging in C. elegans

Effect of PE on life span of C. elegans was examined by growing the L4 animals at 20 °C under different PE concentrations (0—control, 10, 50, and 100 μg/ml). The number of dead animals were scored every second day from young adult stage till complete death of all the animals. The mean life span of control animals was found to be 15 ± 0.1 (mean life span ± standard error of mean) days compared to PE-treated animals showing respective values of 15 ± 0.4 days at 10 μg/ml PE 17.3 ± 0.1 days for 50 μg/ml PE, and 19.9 ± 0.3 days for 100 μg/ml PE concentrations, respectively (p < 0.001, log-rank test) (Fig. 2a). PE treatment showed a significant increase in the life span in a dose-dependent manner; however, the animal’s brood size and health did not show any difference (data not shown).

Fig. 2
figure 2

PE treatment extends life span and improves health span of C. elegans. a Effect of PE treatment on mean life span. PE pre–exposure improved the N2 adult’s survival in dose-dependent manner. Mean survival time- Control, 15 ± 0.1 (mean ± SEM) days; PE (10 μg/ml), 15 ± 0.4 days; PE (50 μg/ml), 17.3 ± 0.1 days; PE (100 μg/ml), 19.9 ± 0.3 days. p < 0.001 (log-rank test). Data is represented as mean ± SEM. b PE treatment delays the progression of decline in pharyngeal pumping rate with aging. Effect of PE on the pharyngeal pumping of C. elegans at 5th and 10th day post adulthood in N2 animals. Pharyngeal pumping at 5th day—control, 120 ± 15.5 min-1, number of animals = 165; PE treated (100 μg/ml), 168 ± 19.5 min-1, number of animals = 152 (***p < 4.5 × 10-11, t test). Pharyngeal pumping at 10th day—control, 37 ± 6.2 min-1, number of animals = 110; PE treated (100 μg/ml), 57.2 ± 11.2 min-1, number of animals = 145 (***p < 3.1 × 10-9, t test). Data is represented as mean ± SEM. c PE treatment delays progression of locomotion decline with aging. Average body bends per minute in control and PE-treated C. elegans scored at 5th and 10th day post adulthood in N2 animals. Body bends per minute—control, 100 ± 5.8 (at 5th day) and 18.00 ± 3.2 (at 10th day); PE treated (100 μg/ml), 111 ± 4.6 (**p < 5.1 × 10-9, t test) (at 5th day) and 29 ± 4.4 (**p < 2.3 × 10-10, t test) (at 10th day). Data represented as mean ± SEM

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To check the efficacy of PE in delaying aging, we measure surrogate physiological markers of aging like pharyngeal pumping rate and rate of worm locomotion in PE-treated (100 μg/ml) and untreated condition in the time course of aging (5th and 10th day post adulthood). The data is represented as mean ± standard error of mean. The rate of pharyngeal pumping for control animals was found to be 120 ± 15.5 and 37 ± 6.2 min-1 for 5th and 10th day, respectively compared to PE-treated animals with respective values of 168 ± 19.5 (p < 4.5 × 10-11, t test) and 57.2 ± 11.2 min-1 (p < 3.1 × 10-9, t test) (Fig. 2b). Effect of PE (100 μg/ml) treatment on worms locomotory behavior with aging was assayed by counting the number of body bends per minute. The number of body bends per minute observed for control animals were 100 ± 5.8 and 18.00 ± 3.2 for 5th and 10th day, respectively compared to PE (100 μg/ml) treated with 111 ± 4.6 (p < 5.1 × 10-9, t test) and 29 ± 4.4 (p < 2.3 × 10-10, t test) values for 5th and 10th day, respectively (Fig. 2c). PE-treated worms showed significantly better pharyngeal pumping rate and locomotory response compared to control worms. Results implied that PE treatment attenuated the aging related depreciation in physiological functions such feeding and locomotion to a certain extent.

PE treatment enhances stress tolerance in C. elegans

To explore the protective role of PE against stress, we assayed PE-treated and untreated worms for heat and oxidative stress resistance. Assay plates were prepared by culturing N2 young adult worms in PE-treated (100 μg/ml) and untreated plates for 4 days at 20 °C. For thermo tolerance assay, both PE-treated and untreated worms were shifted to 35 °C and numbers of dead worms were scored at every 2 h interval until the 12th hour. Worms pre-exposed with 100 μg/ml PE showed significantly higher survival rate compared to untreated worms (Fig. 3a). The mean survival percentage at 35 °C in time span of 12 h was found to be significantly higher for PE-treated (100 μg/ml) worms with mean value of 41.6 ± 2.5 % compared to control animals 22.2 ± 2.5 % (p < 0.0001, t test) (Fig. 3b).

Fig. 3
figure 3

PE treatment-enhanced stress tolerance in C. elegans. a PE treatment-enhanced thermo stress tolerance N2 animals (4th day post adulthood) with and without PE pretreatment of 100 μg/ml were transferred to 35 °C for 12 h. The number of dead animals were scored every 2 h until the 12th hour and depicted as survival curves. b PE treatment-enhanced thermo stress tolerance mean survival percentages of PE-treated and untreated N2 animals (4th day post adulthood) subjected to 35 °C for 12 h. Mean survival time—untreated control, 22.2 ± 2.5 %; PE treated (100 μg/ml), 41.6 ± 2.5 % (***p < 0.0001, t test). Data is represented as mean ± SEM. c PE treatment-enhanced oxidative stress tolerance of PE-treated and untreated N2 animals (4th day post adulthood) exposed to 10 and 20 mM hydrogen peroxide for 2 h at 20 °C. Survival percentage of animals was scored 16 h post recovery from oxidative stress. Mean survival percentage at 10 mM H2O2 exposure is 63.1 ± 6.4 (control) and 30.1 ± 3.2 % (PE-treated) (***p > 0.0001, t test). Mean survival percentage at 20 mM H2O2 exposure is 10.1 ± 0.9 (control) and 11.5 ± 1.3 (PE-treated). Data is represented as mean ± SEM

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Animals were subjected to oxidative stress by treating them with H2O2 (10 and 20 mM) for 2 h and were scored 16 h post recovery. PE-treated (100 μg/ml) animals subjected to 10 mM H2O2 showed significantly higher mean survival percentage compared to control with the respective value of 63.1±6.4 % (PE-treated, p < 0.0001, t test) and 30.1 ± 3.2 % (untreated control). However, no significant difference was observed between PE-treated and untreated control animals when exposed to 20 mM H2O2 (Fig. 3c). Altogether, these results show that PE treatment confers enhanced stress tolerance in C. elegans.

Extension of life span by PE is independent of DAF-2, AGE-1, and DAF-16 signaling

DAF-16 is major effector of IGF-1/insulin signaling pathway, where upstream signaling cascade like DAF-2/AGE-1 result in phosphorylation of DAF-16 resulting in blockage of its nuclear translocation (Ogg et al. 1997; Murphy and Hu 2013). Mutation in daf-2/age-1 permits nuclear translocation of DAF-16 resulting in activation of genes responsible for life span extension and stress resistance (Lin et al. 1997; Ogg et al. 1997; Murphy and Hu 2013). DAF-16 is expressed in the cytoplasm and move into the nucleus to regulate expression of various stress response gene. To check the effect of PE on IGF-1 signaling pathway, we looked at DAF-16 protein expression in PE-treated and untreated C. elegans by probing the nuclear localization of DAF-16 using DAF-16::GFP transgenic animals. Nuclear localization of DAF 16::GFP was induced by subjecting DAF 16::GFP animals to heat shock (35 °C) for 15 min. DAF-16::GFP transgenic animals were found to show more stress tolerance and longevity upon PE treatment irrespective of DAF-16::GFP expression and localization. No significant change in the expression of DAF-16::GFP upon PE treatment was observed, ruling out the role of the DAF-16 pathway in PE-mediated life span extension (Fig. 4a). The same was also examined by observing the effect of PE (100 μg/ml) treatment on the life span of daf-16 null mutant strains, mu86. PE treatment showed increase in the life span of daf-16(mu86) animals with the mean value of 16.6 ± 1.2 days (mean life span ± standard error of mean), compared to untreated control worms, 12.8 ± 0.8 days (p < 0.001, log-rank test) (Fig. 4b). Results thus showed that the life span enhancement by PE treatment is independent of DAF-16, a downstream molecule of insulin/ IGF-1 signaling pathway.

Fig. 4
figure 4

Effect of PE on life span is independent of DAF-2/AGE-1/DAF-16. a Representative image showing nuclear localization of DAF 16::GFP. DAF 16::GFP animals were grown in PE-treated and untreated conditions and subjected to heat shock (35 °C) for 15 min to induce nuclear localization of DAF 16::GFP. Scale bar represents 200 μm. b Effect of PE treatment on life span of daf-16(mu86) mutant. PE exposure increased the mean life span of daf-16(mu86) mutant indicating PE effects are DAF-16 independent. Mean life span—control, 12.8 ± 1.2 days; PE-treated (100 μg/ml), 16.6 ± 0.4 days (p < 0.001, log-rank test). Data is represented as mean ± SEM. c Effect of PE treatment on life span of daf-2(e1370) mutant. PE exposure increased the mean life span of daf-2(e1370) mutant indicating PE effect is independent of DAF-2/IGF-1 receptor. Mean life span of control animals, 30.1 ± 0.9 days; PE-treated (100 μg/ml), 35.2 ± 1.3 days (p < 0.001, log-rank test). Data is represented as mean ± SEM. d Effect of PE treatment on life span of age-1 (hx546) mutant. PE exposure increased the mean life span of age-1 (hx546) mutant indicating PE effect is independent of AGE-1—the key kinase of the IGF-1 pathway. Mean life span of control animals, 29.5 ± 1.5 days; PE-treated (100 μg/ml), 35.4 ± 1.5 days (p < 0.001, log-rank test). Data is represented as mean ± SEM

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We also checked the effect of PE treatment on upstream components like DAF-2/IGF-1 receptor (IGFR) and AGE-1/phosphoinositide 3-kinase (PI3K) of IGF-1 signaling pathways. DAF-2 is C. elegans homolog for mammalian IGF-1 receptor and is the only IGF-1/insulin signaling receptor present in C. elegans whereas AGE-1 is homolog for mammalian PI3K catalytic subunit (Finch and Ruvkun 2001). Mutants of both age-1 and daf-2 have been reported to result in increased life span (Samuelson et al. 2007; Murphy and Hu 2013). PE treatment enhanced the life span significantly for both daf-2(e1370) and age-1(hx546) mutant strains with mean survival time ± standard error of mean value of 35.2 ± 1.3 days (p < 0.001, log-rank test) (untreated control—30.1 ± 2.1 days) for daf-2(e1370) and 35.4 ± 2.6 days (p < 0.001, log-rank test) (untreated control—29.5 ± 1.6 days) for age-1(hx546), respectively (Fig. 4c, d).

To examine further, we also checked the expression of SOD-3, one of the direct targets of DAF-16. No considerable change in expression of SOD-3::GFP expression was observed on PE addition (sup figure 1). To examine the role of dietary restriction (DR) on PE-dependent enhancement in life span, we performed the life span assay in eat-2(ad1116) mutants. Mutation in eat-2 gene causes the reduced pharyngeal pumping rate which results in lesser food intake (Crawford et al. 2007). The mean life span of eat-2(ad1116) increased from 15.9 ± 0.9 to 18.8 ± 1.1 days (p < 0.001, log-rank test) upon PE treatment (100 μg/ml) (sup figure 2), suggesting that the PE-mediated life span expansion is independent of DR. Results thus show that PE treatment effects are DAF-2, AGE-1, and DAF-16 independent.

PE ameliorates protein aggregation and protetoxicity-mediated paralysis phenotype

PE is a potent antioxidant (Soni et al. 2009), and numerous lines of evidence have reported involvement of oxidative stress in occurrence and progression of Alzheimer’s disease and Aβ toxicity (Jomova et al. 2010). We analyzed the effect of PE on Aβ aggregation using C. elegans CL4176, a transgenic model for Alzheimer’s disease. The strain CL4176 (dvIs27[pAF29(myo-3/Ab 1-42/let UTR) + pRF4(rol-6(su1006)]) is engineered to provide temperature-inducible muscle expression of a human β-amyloid peptide (Aβ) transgene, resulting in paralysis phenotype due to Aβ toxicity upon temperature upshift to 25 °C (Link 2003). Synchronized eggs from transgenic worms were seeded on NGM plates with (100 μg/ml PE) and without PE and incubated for 36 h at 16 °C. This was followed by temperature upshift to 22 and 25 °C, respectively to induce Aβ expression. Worms that did not move or only moved their head when gently touched by a platinum loop were scored as paralyzed. A time course of paralysis for CL4176 worms was recorded post temperature upshift for both 22 and 25 °C for total duration of 36–48 h. A significant delay in paralysis phenotype was observed in the PE-treated worms compared with untreated control (Fig. 5a). The reproducibility of paralysis at a given temperature (22 or 25 °C) in individual trials was consistent. We also quantified paralysis time, defined as time interval from the onset of paralysis to the time at which 50 % of the worms were paralyzed. A statistically significant decrease in Aβ-induced paralysis and delay in paralysis time was observed in the worm fed with PE (Fig. 5b, c).

Fig. 5
figure 5

PE is effective in preventing protein aggregation and delaying protetoxicity-mediated paralysis phenotype in CL4176 animals. a Representative image of CL4176 transgenic animals showing paralysis due to Aβ expression. Representative images of control (no temperature shift) at 16 °C and effect of temperature upshift to 25 °C for induction of Aβ expression in control and PE-treated (100 μg/ml) CL4176 transgenic animals. Scale bar represents 1 mm. b–c Effect of PE in suppressing Aβ1-42-associated paralysis phenotype in CL4176 animals. Survival curves (in term of paralysis) after temperature upshift to 25 °C (b) and 22 °C (c) are represented which show significant increase in survival percentage. Data points are represented as mean ± SEM

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PE-mediated effects are dependent on HSF-1

HSF-1 and SKN-1 transcriptional factors have been also reported to be involved in insulin/IGF-1 signaling in response to cellular stress (Chiang et al 2012; Morton and Lamitina 2013; Tullet et al 2008). To investigate whether the protective effect of PE is mediated through transcriptional factor for heat shock (HSF-1) and oxidative stress (SKN-1) responses, we performed the life span and paralysis assay in wild type and CL4176 animals, respectively in PE-treated (100 μg/ml) and untreated conditions when HSF-1 and SKN-1 genes were knocked out using RNAi. The mean life span ± SEM for control animals was 15.9 ± 1.2 days; hsf-1 (RNAi) animals, 13.7 ± 1.4 days; and skn-1 (RNAi) animals, 15.5 ± 1.6 days compared with PE-treated control animals, 18.6 ± 1.3 days (p < 0.001, log-rank test); hsf-1 (RNAi) animals, 14.3 ± 1.5 days (p = 0.33, log-rank test); and skn-1 (RNAi) animals, 18.5 ± 1.9 days, (p < 0.001, log-rank test), respectively (Fig. 6a, b). There was no increment in life span on PE treatment when HSF-1 was knockdown; in contrast, there was significant increase in life span when SKN-1 was knockdown. To confirm the dependency of the life span expansion effect of PE on HSF-1, we examined hsf-1 mRNA levels in N2 Bristol upon PE treatment (100 μg/ml). We found around 1.7-fold increase in the mRNA level in PE-treated worms (sup figure 3). Altogether, it showed that HSF-1 is required for enhancement of life span mediated by PE treatment.

Fig. 6
figure 6

PE-mediated increase in life span show HSF-1 dependence. a Effect of PE treatment on life span of hsf-1 (RNAi). PE exposure does not increase the mean life span of hsf-1 (RNAi) mutant, indicating dependency of PE effect on HSF-1. Mean life span—control vector, 15.9 ± 1.2 days; hsf-1 (RNAi), 13.7 ± 1.4 days; control vector + PE, 18.6 ± 1.3 days (p < 0.001, log-rank test); hsf-1 (RNAi) + PE, 14.3 ± 1.5 days (p = 0.33, log-rank test). Data is represented as mean ± SEM. b Effect of PE treatment on life span of skn-1 (RNAi). PE exposure increased the mean life span of skn-1 (RNAi) mutant, indicating PE effects are independent of SKN-1. Mean life span—control vector, 15.9 ± 1.2 days; skn-1 (RNAi), 15.5 ± 1.6 days; control vector + PE, 18.6 ± 1.3 days (p < 0.001, log-rank test); skn-1 (RNAi) + PE, 18.5 ± 1.9 days, (p < 0.001, log-rank test). Data is represented as mean ± SEM. c Representative images of CL4176 animals showing paralysis at 25 °C when subjected to hsf-1 and skn-1 RNAi. PE-treated (100 μg/ml) and untreated control vector (CV), hsf-1 (RNAi), and skn-1 (RNAi) CL4176 C. elegans animals subjected to 25 °C. Scale bar represents 1 mm. d Fraction of non-paralyzed worms PE-treated (100 μg/ml) and untreated subjected to hsf-1 and skn-1 RNAi. Fraction of non-paralyzed CL4176 worms—control vector, 18.1 ± 2.7; hsf-1 (RNAi), 15.8 ± 4.8; skn-1 (RNAi), 17.7 ± 2.8; control vector + PE, 55.3 ± 3.9 (***p < 6.5 × 10-11, t test); hsf-1 (RNAi) + PE, 22.4 ± 3.5 (p = 0.02, t test); skn-1 (RNAi) + PE, 49.2 ± 4.6 (***p < 2.3 × 10-12, t test). Data represented as mean ± SEM

Full size image

As HSF-1 and SKN-1 are involved in stress response; we also assayed the effect of HSF-1 and SKN-1 on Aβ aggregation. PE-treated CL4176 animals were grown in control and HSF-1 and SKN-1 knockout (RNAi) conditions. Paralysis time was quantified for all the experimental conditions after 36 h of incubation at 25 °C. The mean value of percentage of non-paralyzed worm ± standard error of mean for control animals was 18.1 ± 2.7; hsf-1 (RNAi) animals, 15.8 ± 4.8; and skn-1 (RNAi) animals, 17.7 ± 2.8 as compared to PE-treated control, 55.3 ± 3.9 (p < 6.5 × 10-11, t test); hsf-1 (RNAi), 22.4 ± 3.5 (p = 0.02, t test); and skn-1 (RNAi) 49.2 ± 4.6 (p < 2.3 × 10-12, t test) (Fig. 6c, d). Comparative accounts of mean and maximum life span of control and PE-treated C. elegans strain in various assays are depicted in supplementary table 2. Altogether, these results depicts that PE-dependent increase in life span requires HSF-1.

Discussion

Aging pattern between C. elegans and mammals has considerable degree of similarity. C. elegans has thus turned out to be a potential model organism for aging-related studies to explore potential anti-aging drugs. ROS has been reported as a major cause of aging, and antioxidants have been reported to curtail the aging effects of ROS. Antioxidants reduce the extent of free radicals-mediated oxidative damage and hence results in increased life span and healthy aging. Several studies are being undertaken to explore possible anti-aging wonder drugs. We checked the potency of PE as possible candidate for curtailing aging and age-related disorders. Our results show that exposure to PE which is a chromophore-containing pigment protein, isolated from cyanobacterium Lyngbya sp. A09DM, increases life span and health span of C. elegans. Various physiological functions including pharyngeal pumping, locomotory health span, feeding rate, and sarcopenia in C. elegans are considered as the hallmarks of aging (Huang et al. 2004). The beneficial effect of PE feeding on these hallmarks of aging in C. elegans also revealed the role of PE in improving health span with increasing age. Efficiency of PE in averting the Aβ-peptide mediated paralysis was probed by using CL4176 (C. elegans model for Alzheimer’s disease). CL4176 was constructed by mutating the smg-1 gene (involved in nonsense-mediated mRNA decay) for heat-induced overexpression of Aβ-peptide in their muscle cells, which in turn initiated the oxidative stress-mediated neurotoxicity manifested in paralysis phenotype upon temperature upshift (Dostal and Link 2010). PE treatment was found to delay Aβ-peptide-mediated paralysis, indicating the importance and function of PE’s antioxidant virtue in its anti-paralytic activity. This result suggested that PE can efficiently obviate the oxidative stress by its antioxidant virtue. PE treatment also provided tolerance to oxidative as well as thermo stress. These results confirmed that PE treatment enhances life span and promotes healthy aging in C. elegans. We also tried exploring the possible pathways through which PE might be showing its effect. One possible mechanism behind the PE effect may be the hormetic effect, which is low dose exposure of toxicant enhances the damage repair mechanism and increases life span consequently (Rattan 2008). PE does not show hormetic effect as it does not show lethality at higher doses. Moreover, toxicity analysis exhibited by Parmar et al., (unpublished), also supports PE as nontoxic and non-lethal and thus rejecting the hormetic effect. The beneficial effect of PE might be due to the inhibition of some of the well conserved mechanisms of aging. IIS signaling components and pathway are highly conserved among C. elegans and other mammals. FOXO family transcription factor DAF-16 and their upstream components like DAF-2/IGF-1 receptor (IGFR) and AGE-1/ phosphoinositide 3-kinase (PI3K) are well established as central regulatory elements for longevity and stress-associated responses. Null mutants for daf-16(mu86), daf-2(e1370), and age-1(hx546) cultured under PE treatment showed significant expansion in life span, indicating that PE works independently of these IIS signaling elements. Moreover the expression of DAF-16 in DAF-16::GFP transgenic animal upon PE treatment showed no significant difference in nuclear localization of the DAF-16::GFP protein. All results collectively proved that PE effects are independent of the DAF-2–AGE-1–DAF-16 pathway. SKN-1 is an important component for oxidative stress responses and is required for life span extension by dietary restriction or inhibition of the TORC1 or TORC2 pathways (Robida-Stubbs et al. 2012). Results of PE effect on null mutants for SKN-1 ruled out the involvement of TOR pathways inhibition in extending life span on PE treatment.

On the contrary, HSF-1 controls stress-induced gene expression is found to be essential for PE-mediated life expansion. The recent work of Chiang et al. (2012) showed that HSF-1 activity is being regulated by IIS via formation of a multi-molecular complex containing the conserved proteins DDL-1 and DDL-2 that sequesters HSF-1 in the cytoplasm. Our results show that the PE-mediated effect is HSF-1 dependent but IIS independent. This may be due to some parallel alternative process may be in hold for HSF-1 activity along with the IIS pathway. The exact signaling pathway and mechanism by which PE mediates its effect is still unclear. Though, some possible routes, by which PE may work, are ruled out in the present experimental work. The effects demonstrated by PE on longevity and aging in C. elegans led us to hypothesize that PE is a potent antioxidant and shows beneficial effect on aging and age-related disorders.

Conclusion

The findings of this report reveal a novel role of PE in extending life span and health span in C. elegans. Our results show that PE supplement not only increases life span but also improves quality of life by delaying aging effects like declining locomotion and feeding rate. It also enhances the stress tolerance capacity and decreases Aβ-induced paralysis in C. elegans Alzheimer’s model. We also checked the signaling pathway through which PE treatment would be affecting. As per our results, life span-prolonging activity of PE does not seem to be associated with well conserved IIS pathway, as PE functions even in the absence of some of the IIS pathway components. However, presence of HSF-1 seems to be important in mediating anti-aging effects of PE. Our results using the C. elegans model gives quite strong evidence for PE as a promising anti-aging candidate. Furthermore, research using other model systems needs to be carried out in the future to establish the potential of PE as an anti-aging wonder drug.