LB-100

HSF-1 and SIR-2.1 linked insulin-like signaling
is involved in goji berry (Lycium spp.)
extracts promoting lifespan extension of
Caenorhabditis elegans

The anti-cancer, vision-improving, and reproduction-enhancing effects of goji berry have been generally
recognized, but its role in anti-aging is rarely studied in depth. Therefore, two widely-circulated goji
berries, Lycium ruthenicum Murr. (LRM) and Lycium Barbarum. L (LB), were selected to explore their
effects on extending lifespan and enhancing defense against extrinsic stress and to uncover the mecha￾nism of action through genetic study. The results showed that supplementation with high-dose LRM
(10 mg mL−1) and LB (100 mg mL−1) extracts significantly extended the lifespan of Caenorhabditis elegans
(C. elegans) by 25.19% and 51.38%, respectively, accompanied by the improved stress tolerance of
C. elegans to paraquat-induced oxidation, UV-B irradiation and heat shock. Furthermore, LRM and LB
extracts remarkably enhanced the activities of antioxidant enzymes including SOD and CAT in C. elegans,
while notably decreased the lipofuscin level. Further genetic research demonstrated that the expression
levels of key genes daf-16, sod-2, sod-3, sir-2.1 and hsp-16.2 in C. elegans were up-regulated by the
intervention with LRM and LB, while that of the age-1 level was down-regulated. Moreover, the daf-16
(mu86) I, sir-2.1 (ok434) IV and hsf-1 (sy441) I mutants reversed the longevity effect brought about by
LRM or LB, which confirmed that these genes were required in goji berry-mediated lifespan extension.
Therefore, we conclude that HSF-1 and SIR-2.1 act collaboratively with the insulin/IGF signaling pathway
(IIS) in a daf-16-independent mode. The present study indicated goji berry as a potential functional food
to alleviate the symptoms of aging.

1. Introduction
The essence of life is resilience,1 which gives each individual
the ability to maintain the stability of the body, thereby resist￾ing external pressure and internal changes. In the early stages
of life, this self-correction mechanism is sufficient to maintain
the biochemical balance in our body, but in the later stages,
the damage that cannot be repaired continues to accumulate
and exceeds the functional threshold of this elastic mecha￾nism.1 The decline of physical strength and cognitive ability
fully explains the body changes and the incidences of diseases
associated with aging. The aging of different organisms exhi￾bits common hallmarks, such as genome instability,2–4 telo￾mere depletion, epigenetic changes, loss of protein homeosta￾sis, and mitochondrial dysfunction.5–8 A major challenge in
aging research is to analyze the relationship between these
signs of aging and their contribution to aging and to identify
therapeutic targets to improve the health of the human body
during the aging process with minimal side effects. Therefore,
it has become a trend to use diet therapy to delay aging or
reduce the risks of aging-related disorders. Numerous daily
consumed foods such as fruits, vegetables and whole grains
are rich in bioactive substances (e.g. phenolics). As the most
studied secondary metabolites in plants, phenolics exhibit
potential antioxidant effects and the capacity of delaying the
process of aging and enhancing human wellness. Blueberry
polyphenols have been demonstrated to facilitate cellular

tance to heat via the CaMKII pathway and to prolong the life￾span of C. elegans by mediating the expression of osr-1 and
unc-43.
9 Similarly, Jamun, a kind of tropical fruit, can extend
lifespan and alleviate neurodegenerative symptoms caused by
Parkinson’s disease.10 Peng et al. found that apple polyphenols
could increase the mean lifespan of Drosophila melanogaster by
up-regulating genes encoding endogenous antioxidant
enzymes including SOD1, SOD2 and CAT, and could restore
the neurological disorders by promoting the expression of
Rpn11, which is a suppressor for the production of ubiquiti￾nated proteins leading to progressive neurodegeneration.11
Goji berry (Lycium spp.), belonging to the Solanaceae
family, is widely distributed in Northwest China, Europe and
Japan. It has always been an ideal candidate to exert signifi￾cant effects on eyesight, neurodegeneration, cardiovascular
disease, cancer, diabetes and reproductivity as well as nour￾ishment for the kidney and liver.12–16 These multiple func￾tions might be attributed to the abundant bioactive com￾ponents in goji berry, including phenolics, carotenoids and
Lycium Barbarum polysaccharides.17 Chlorogenic acid, kaemp￾ferol-3-O-rutinoside, quercetin-3-O-rutinoside are the domi￾nant phenolics in LB, while anthocyanidin is the main part in
LRM.18,19 A previous finding confirmed that ascorbic acid
could positively regulate the two hallmarks of biological
aging, which were inflamm-aging and immunosenescence.20
Chlorogenic acid has been demonstrated to enhance the resis￾tance of C. elegans to thermal stress by inducing autophagy
and activating the heat-shock transcription factor-1 (HSF-1)
and heat shock proteins (HSPs).21 A previous report also
found that cranberry anthocyanin triggered a 10% increase in
lifespan in fruit flies by down-regulating the expression of the
target of rapamycin (TOR), methuselah (MTH), phosphoenol￾pyruvate carboxykinase (PEPCK) and insulin receptor (InR),
while up-regulating that of copper–zinc superoxide dismutase
(SOD1).22
Caenorhabditis elegans (C. elegans) is considered one of the
most classic aging models due to its short life cycle and
characteristics of easy cultivation. Furthermore, various age￾related pathways are highly conserved in C. elegans, such as
the insulin/insulin-like growth factor signaling (IIS), silent
information regulator 2 (SIR2) and HSF-1 pathways, all of
which are closely associated with metabolism, development,
adaption to extracellular stress and lifespan. DAF-16, a
homolog to a mammalian forkhead transcription factor in
C. elegans, is the central effector in the IIS pathway, which
could modulate the expression of many downstream genes
including mtl-1, sod-3, ctl-1/2 and gst-4 that are directly
involved in regulating stress resistance and longevity. The
most intuitive role of HSF-1 is to mediate heat stress proteins
to cope with endogenous degradation and exogenous pressure
by maintaining protein homeostasis. Additionally, SIR2, a key
regulator in the state of calorie restriction, exhibits the effect
of promoting longevity via the regulation of lipid metabolism
and fat storage. Both HSF-1 and SIR2 have been confirmed to
either function at DAF-16 or act on longevity in parallel to the
IIS pathway independent of DAF-16.23
However, previous investigations on the bioactivities of goji
berry were all aimed at certain diseases or functional dis￾orders. Deficient studies are available concerning the mole￾cular mechanism of goji berry on anti-aging. Therefore, two
goji berry varieties, Lycium ruthenicum Murr. (LRM) and Lycium
Barbarum. L (LB), were used for further exploration toward the
mechanism of longevity in the present study.
2. Materials and methods
2.1 Materials and reagents
The two dried goji berry varieties, LB and LRM, were pur￾chased from Qinghai Friendship Economic and Trade Co., Ltd
(Qinghai, China). The solutions used for worm culture includ￾ing S-complete buffer and M9 buffer were prepared according
to the wormbook.24 Escherichia coli OP50 strain was provided by
Wu’s laboratory in Huazhong University of Science and
Technology (Wuhan, Hubei, China). 5-Fluoro-2-deoxyuridine
(FUDR), carbenicillin, ampicillin and paraquat were obtained
from Sigma-Aldrich (St Louis, MO, USA).
2.2 Preparation of C. elegans strains
The C. elegans strains used in the study were obtained from
CGC (Caenorhabditis Genetics Center, University of
Minnesota, Minneapolis, MN, USA): wild-type (Bristol N2), daf-
16 (mu86) I, sir-2.1 (ok434) IV, daf-16::GFP (zls356 IV), jnk-1
(gk7) IV and hsf-1 (sy441) I. Nematode growth medium (NGM)
plates were served as incubators with OP50 as a food source
for all the strains, and the living temperature was set at 20 °C.
The LB extract and LRM extract stock solutions (400 mg mL−1
in ultrapure water) were diluted into different concentrations
by E. coli OP50 based on the antioxidant capacity of both goji
berries (final concentrations: 20, 50, 100 mg mL−1 for LB; 2, 5,
10 mg mL−1 for LRM). The utilization of water as a substitute
for goji extracts was the control group.
2.3 Sample preparation and determination of total phenolic
and flavonoid contents
The extraction of phytochemicals from goji berry was per￾formed as reported previously.25 In brief, chilled 80% acetone
was used to extract the free phytochemicals in goji berries.
After being homogenized and centrifugated, the supernatants
were collected and evaporated to <10% initial volume by rotary
evaporation. The remaining solution was reconstituted in
10 mL of ultrapure water to obtain a stock solution (400 mg
mL−1). The contents of total phenolic and total flavonoid were
measured by the Folin–Ciocalteu method with gallic acid as a
standard,26 and sodium borohydride/chloranil method with
catechin hydrate as a standard,27 respectively. All data were
presented as mean ± SD with three replications.
2.4 Phytochemical profile analysis by HPLC
Phytochemical profiles were analyzed with an HPLC system
according to the previous study,28 using a Sunfire C18 column
(250 × 4.6 mm, 5 μm) maintained at 35 °C and detected at 254,

9 Similarly, Jamun, a kind of tropical fruit, can extend
lifespan and alleviate neurodegenerative symptoms caused by
Parkinson’s disease.10 Peng et al. found that apple polyphenols
could increase the mean lifespan of Drosophila melanogaster by
up-regulating genes encoding endogenous antioxidant
enzymes including SOD1, SOD2 and CAT, and could restore
the neurological disorders by promoting the expression of
Rpn11, which is a suppressor for the production of ubiquiti￾nated proteins leading to progressive neurodegeneration.11
Goji berry (Lycium spp.), belonging to the Solanaceae
family, is widely distributed in Northwest China, Europe and
Japan. It has always been an ideal candidate to exert signifi￾cant effects on eyesight, neurodegeneration, cardiovascular
disease, cancer, diabetes and reproductivity as well as nour￾ishment for the kidney and liver.12–16 These multiple func￾tions might be attributed to the abundant bioactive com￾ponents in goji berry, including phenolics, carotenoids and
Lycium Barbarum polysaccharides.17 Chlorogenic acid, kaemp￾ferol-3-O-rutinoside, quercetin-3-O-rutinoside are the domi￾nant phenolics in LB, while anthocyanidin is the main part in
LRM.18,19 A previous finding confirmed that ascorbic acid
could positively regulate the two hallmarks of biological
aging, which were inflamm-aging and immunosenescence.20
Chlorogenic acid has been demonstrated to enhance the resis￾tance of C. elegans to thermal stress by inducing autophagy
and activating the heat-shock transcription factor-1 (HSF-1)
and heat shock proteins (HSPs).21 A previous report also
found that cranberry anthocyanin triggered a 10% increase in
lifespan in fruit flies by down-regulating the expression of the
target of rapamycin (TOR), methuselah (MTH), phosphoenol￾pyruvate carboxykinase (PEPCK) and insulin receptor (InR),
while up-regulating that of copper–zinc superoxide dismutase
(SOD1).22
Caenorhabditis elegans (C. elegans) is considered one of the
most classic aging models due to its short life cycle and
characteristics of easy cultivation. Furthermore, various age￾related pathways are highly conserved in C. elegans, such as
the insulin/insulin-like growth factor signaling (IIS), silent
information regulator 2 (SIR2) and HSF-1 pathways, all of
which are closely associated with metabolism, development,
adaption to extracellular stress and lifespan. DAF-16, a
homolog to a mammalian forkhead transcription factor in
C. elegans, is the central effector in the IIS pathway, which
could modulate the expression of many downstream genes
including mtl-1, sod-3, ctl-1/2 and gst-4 that are directly
involved in regulating stress resistance and longevity. The
most intuitive role of HSF-1 is to mediate heat stress proteins
to cope with endogenous degradation and exogenous pressure
by maintaining protein homeostasis. Additionally, SIR2, a key
regulator in the state of calorie restriction, exhibits the effect
of promoting longevity via the regulation of lipid metabolism
and fat storage. Both HSF-1 and SIR2 have been confirmed to
either function at DAF-16 or act on longevity in parallel to the
IIS pathway independent of DAF-16.23
However, previous investigations on the bioactivities of goji
berry were all aimed at certain diseases or functional dis￾orders. Deficient studies are available concerning the mole￾cular mechanism of goji berry on anti-aging. Therefore, two
goji berry varieties, Lycium ruthenicum Murr. (LRM) and Lycium
Barbarum. L (LB), were used for further exploration toward the
mechanism of longevity in the present study.
2. Materials and methods
2.1 Materials and reagents
The two dried goji berry varieties, LB and LRM, were pur￾chased from Qinghai Friendship Economic and Trade Co., Ltd
(Qinghai, China). The solutions used for worm culture includ￾ing S-complete buffer and M9 buffer were prepared according
to the wormbook.24 Escherichia coli OP50 strain was provided by
Wu’s laboratory in Huazhong University of Science and
Technology (Wuhan, Hubei, China). 5-Fluoro-2-deoxyuridine
(FUDR), carbenicillin, ampicillin and paraquat were obtained
from Sigma-Aldrich (St Louis, MO, USA).
2.2 Preparation of C. elegans strains
The C. elegans strains used in the study were obtained from
CGC (Caenorhabditis Genetics Center, University of
Minnesota, Minneapolis, MN, USA): wild-type (Bristol N2), daf-
16 (mu86) I, sir-2.1 (ok434) IV, daf-16::GFP (zls356 IV), jnk-1
(gk7) IV and hsf-1 (sy441) I. Nematode growth medium (NGM)
plates were served as incubators with OP50 as a food source
for all the strains, and the living temperature was set at 20 °C.
The LB extract and LRM extract stock solutions (400 mg mL−1
in ultrapure water) were diluted into different concentrations
by E. coli OP50 based on the antioxidant capacity of both goji
berries (final concentrations: 20, 50, 100 mg mL−1 for LB; 2, 5,
10 mg mL−1 for LRM). The utilization of water as a substitute
for goji extracts was the control group.
2.3 Sample preparation and determination of total phenolic
and flavonoid contents
The extraction of phytochemicals from goji berry was per￾formed as reported previously.25 In brief, chilled 80% acetone
was used to extract the free phytochemicals in goji berries.
After being homogenized and centrifugated, the supernatants
were collected and evaporated to evaporation. The remaining solution was reconstituted in
10 mL of ultrapure water to obtain a stock solution (400 mg
mL−1). The contents of total phenolic and total flavonoid were
measured by the Folin–Ciocalteu method with gallic acid as a
standard,26 and sodium borohydride/chloranil method with
catechin hydrate as a standard,27 respectively. All data were
presented as mean ± SD with three replications.
2.4 Phytochemical profile analysis by HPLC
Phytochemical profiles were analyzed with an HPLC system
according to the previous study,28 using a Sunfire C18 column
(250 × 4.6 mm, 5 μm) maintained at 35 °C and detected at 254,
Paper Food & Function
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280, 320 and 520 nm.29 The gradient mobile phase (A: 0.1%
trifluoroacetic acid in ultrapure water, B: acetonitrile) was
established as followed: 0–5 min (10% B), 5–20 min (10–25%
B), 20–25 min (25–35% B), 25–31 min (35–58% B), 31–34 min
(58–60% B), 34–40 min (60–90% B), 40–50 min (90–10% B),
and 50–60 min (10% B) at the flow rate of 1 mL min−1
. The
final data were presented as milligrams per 100 g dry weight
(mg per 100 g DW, mean ± SD, n = 3).
2.5 Lifespan assay
Determination of the lifespan of C. elegans was conducted as
reported before.24 Briefly, after the synchronous eggs were
hatched, a certain number of L4-stage nematodes were trans￾ferred to NGM plates seeded with OP50 and treated with
different concentrations of goji extracts or water. The spawning
of worms was inhibited by the addition of FUDR (15 mM) to
NGM plates. The day when nematodes were intervened with
goji extracts was recorded as the first day. A gentle-prod by
toothbrush hair was applied to determine the survival, and
dead animals were excluded. The surviving worms were
counted daily and transferred to a new plate every two days to
ensure sufficient food until no worm was alive. Nematodes
that drilled into or escaped from the culture medium were
eliminated from data analysis. All assays were performed in tri￾plicates with at least 90 individuals in each group.
2.6 Heat shock and UV irradiation assays
The thermotolerance assay was performed referring to
Wilson’s method with some modifications.9 Before being incu￾bated at 35 °C for 12 h, all the age-synchronized animals were
treated with goji berry extracts at 20 °C for 5 days. Afterward,
the proportions of the dead worms were calculated according
to the nematodes’ response to a gentle touch. UV radiation
assay was conducted according to Rieckher et al.30 with slight
modifications. Nematodes fed with or without LB and LRM
extracts for 5 days were exposed to UV-B irradiation (120 mJ
cm−2) and the number of survivals was counted daily thrice
until all died. Three replicates were carried out in each assay
using at least 90 nematodes per group.
2.7 Oxidative stress resistance assay
A liquid culture assay was carried out for the determination of
oxidative resistance.24 Following exposure to appropriate
diluted LB or LRM extracts for 5 days, adult worms were col￾lected into a 96-well plate with 5–10 nematodes per well, con￾taining 100 μg mL−1 ampicillin and 50 μg mL−1 carbenicillin
for the resistance of miscellaneous bacteria. Meanwhile, 5 mM
of paraquat was added to induce oxidative stress of worms.
The oxidative stress resistance was measured thrice by count￾ing the fraction of surviving animals until all died. The experi￾ments were conducted thrice with at least 90 worms in each
group.
2.8 Determination of motivity and pumping rate
The locomotivity of C. elegans was divided into three grades,
and the differences in movement between the treatment
groups and the control group were compared according to the
percentage of different grades. The high mobile hermaphrodites
that can move autonomously and leave sinusoidal traces were
defined as motion A. Nematodes in motion B needed external
stimulation such as prodding to help them crawl, while those
almost lost their mobility and only maintained the head swing
were classified as motion C.31 Three replicates were performed
in mobility assay with at least 90 nematodes per group for stat￾istical analysis. The pumping rate was determined thrice con￾taining at least 5 animals per group by counting the pharyngeal
contraction frequency every 5 days with a microscope.32
2.9 Determination of lipofuscin level and body size
After 5 days of intervention with or without goji berry extracts,
at least 15 nematodes were shifted to a microslide coated with
a layer of 1% agarose and then were anesthetized by NaN3
(0.5%) for better observation. The fluorescence intensity of
lipofuscin and body length of C. elegans were monitored by
fluorescence microscopy combined with Image J analysis. All
determinations were conducted in triplicate.
2.10 Determination of progeny production
The synchronized nematodes were transferred to fresh NGM
plates without FUDR before they lay eggs and each plate con￾tained only one nematode. During the whole spawning period,
worms were shifted to new plates every day, and the original
plates were kept for quantifying the number of progenies
hatched out of eggs. Each experiment was repeated three times
involving at least 4 nematodes per group.
2.11 Antioxidant enzyme activity
The activities of antioxidant enzymes in C. elegan can reflect
oxidative stress resistance. Therefore, before the superoxide
dismutase (SOD) and catalase (CAT) activities were detected
with the corresponding assay kits (Beyotime Biotechnology
Company, Shanghai, China) following the manufacturer’s
instructions, adult nematodes (at least 2000 per group) were
treated with or without the goji berry extracts for 5 days and
were collected using M9 buffer. Final data were calibrated to
protein levels using BCA reagent (Meilun Biotechnology Co.,
Ltd, Dalian, China).
2.12 Quantitative real-time PCR (qRT-PCR)
The preparation and treatment of L4 larvae were carried out as
described above (refer to section 2.11). Total RNA was extracted
from more than 1000 nematodes per group using TRIzol
reagent and was reverse-transcribed to cDNA using
PrimeScript™ RT reagent kit. The age-related gene expression
was evaluated by Bio-Rad MiniOption™ Real-Time PCR
Detection System (Bio-Rad, Hercules, CA, USA) with SYBR
Green Super-mix. The sequences of primers (Sangon Biotech,
Shanghai, China) for qRT-PCR are depicted in Table S1.†
2.13 Intracellular localization of DAF-16::GFP
The transfer of the age-related gene daf-16 from the cytoplasm
to the nucleus is the key to lifespan extension. Therefore,
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transgenic mutant TJ356 (daf-16::GFP) was used to determine
the translocation through the localization of the green fluo￾rescent protein (GFP) attached DAF-16 protein. The prepa￾ration of worms in different groups was the same as the lipo￾fuscin assay (section 2.9), and then the immobilized nema￾todes were detected under the laser scanning confocal micro￾scope for observation. Three patterns were designated to
define nuclear localization: “cytosolic”, “intermediate” and
“nuclear”, which indicated the invisible cellular localization,
the fluorescence that mostly concentrated on the head and
tail, and the fluorescent dots all over the body, respectively.
The percentages of three conditions were compared between
the treatment groups and the control group for statistical ana￾lysis. Three independent replicates were conducted with more
than 30 worms per group involved.
2.14 Statistical analysis
All results were presented as mean ± SD, and at least three
replicates were performed for each experiment. Log-rank
(Mantel-Cox) tests were applied to lifespan comparative ana￾lysis using GraphPad Prism 8.0 (GraphPad Software Inc., San
Diego, CA, USA). The one-way analysis of variance (ANOVA) and
Tukey’s multiple range test was performed using SPSS software
21.0 (SPSS Inc., Chicago, IL, USA), and p < 0.05 demonstrated a
significant difference in data.
3. Results
3.1 Total phenolics, total flavonoids and phytochemical
profiles of LRM and LB
According to our previous study,29 the total phenolic and total
flavonoid contents in LRM were 3684.02 ± 182.62 mg gallic
acid equivalents (GAE) per 100 g DW and 3314.84 ± 200.31 mg
catechin equivalents (CE) per 100 g DW, respectively
(Fig. S1B†). However, the levels of total phenolic and total fla￾vonoid in LB were much lower than those of LRM, which were
652.06 ± 83.12 mg GAE per 100 g DW and 740.48 ± 86.68 mg
CE per 100 g DW, respectively. As depicted in Fig. S1A and B,†
ascorbic acid was the fundamental phytochemicals in LB with
the content of 240.87 ± 1.00 mg per 100 g DW, while the domi￾nant phenolic component in LRM was anthocyanin composed
of pelargonidin (538.02 ± 16.12 mg per 100 g DW) and delphi￾nidin (45.10 ± 0.22 mg per 100 g DW). The high amounts of
phenolic and flavonoid in two goji berries (LB and LRM),
strongly suggested their potential biological activities.
Therefore, the anti-aging effects of these goji berries and the
molecular mechanism were explored below.
3.2 LB and LRM increased the lifespan of C. elegans
Six doses, 2, 5, 10 mg mL−1 of LRM extracts and 20, 50,
100 mg mL−1 of LB extracts, were selected to explore the
effects of goji berry extracts on the lifespan of C. elegans.
Compared with the control group, the middle (5 mg mL−1)
and high doses (10 mg mL−1) of LRM extracts significantly pro￾longed the average lifespan of C. elegans by 24.28% and
25.19%, respectively (p < 0.05; Fig. 1 and Table 1), while no obvious difference was observed between the control group and the low-dose LRM group (2 mg mL−1 ; p > 0.05). However,
LB extracts exhibited a greater increment of lifespan extension
in a dose-dependent manner, which were 31.18%, 39.26%,
and 51.38% at the doses of 20, 50, and 100 mg mL−1 LB
extracts, respectively (p < 0.05), compared to the control group.
3.3 LRM and LB enhanced the stress resistance to heat
shock, UV and paraquat
A previous study confirmed that the longevity extension of
C. elegans was strongly related to its adaptability and resistance
to external pressure,33 which suggested that goji berry extracts
might pull the “resilience” of C. elegans back to the early
stages of life. To verify our assumption, the nematodes were
exposed to the extreme environment to detect their stress toler￾ance. As shown in the thermal stress assay (Fig. 2A), after the
nematodes were cultured in a 35 °C incubator for 12 h, the
mortality rate in the treatment groups was significantly
reduced compared with the control group (56.94%). The high
dose LRM (10 mg mL−1
) remarkably decreased the mortality
rate of C. elegans by 33.95%, while that of the high-dose LB
group (100 mg mL−1
) was reduced by 30.72%. UVB, the most
harmful UV rays, directly infringe DNA and produces photo￾products (e.g. cyclobutane pyrimidine dimers), further activat￾ing receptors on the cell surface, thus regulating cell prolifer￾ation, differentiation and aging.34 It can be seen in Fig. 2B and
Fig. 1 Lifespan extension effects of Lycium Ruthenicum Murr. (LRM)
and Lycium Barbarum L. (LB) on C. elegans. The log-rank test was used
for statistical analysis and data were expressed as the mean ± SD (n = 3),
indicating there were significant differences between the control group
and the treatment groups.
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Table 2 that 2, 5, 10 mg mL−1 LRM extracts increased the
mean lifespan of nematodes by 13.3%, 20.3% and 24.0%,
respectively, indicating a promoted irradiation tolerance
although no significant difference was found between the
middle-dose and high-dose group. Additionally, in comparison
to the control group, LB extracts exerted significant lifespan
growth of worms in a dose-dependent manner (17.6%, 21.0%
and 37.1%, respectively). The liquid culture was used to assess
the tolerance of C. elegans treated with goji berry extract to
paraquat-induced oxidative stress. After administrating 2, 5
Table 1 Effects of LRM and LB on the lifespan of C. elegans (mean ± SD, n = 3)
Group Number Mean lifespan (days) % of control Maximum lifespan (days)
Control 97 15.62 ± 0.15d 100d 22
Values with different letters in each column were significantly different (p < 0.05).
Fig. 2 Effects of LRM and LB on the fraction survival of C. elegans under 35 °C thermal shock for 12 h (A), 120 mJ cm−2 UV-B irradiation (B), and
paraquat-induced oxidative stress (C). Data were expressed as the mean ± SD (n = 3).
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and 10 mg mL−1 of LRM extracts, the lifespan of C. elegans
increased to 9.38 ± 0.17, 9.66 ± 0.38, and 9.66 ± 0.18 days,
respectively, compared with the control group (6.90 ± 0.07
days; Fig. 2C and Table 2), suggesting a remarkable increment
of oxidative resistance in worms. Meanwhile, 50 and 100 mg
mL−1 LB extracts caused 37.5% and 45.49% increases, respect￾ively, in the mean lifespan of C. elegans, but there was no sig￾nificant difference between the 20 mg mL−1 LB group and the
control group. All results demonstrated that goji berry extracts
exerted potential tolerance of thermal shock, UVB radiation
and paraquat-induced oxidative stress.
3.4 LRM and LB promoted mobility while showed a decline
in the pumping rate
To clarify the effect of goji berry on the entire life cycle of the
C. elegans, we evaluated the movement of nematodes in
different periods (day 5, 10, 15) based on their response to
external stimuli including the swing of the head and tail,
crawling distance and the shape of the trace left behind.
Obviously, in the early stage, most of the worms among the
control group and the treatment groups were in motion A that
showed strong vitality (Fig. 3). Until the 15th day, mid-late
stages of life, the proportion of motion A nematodes in the
control group dropped from 74% to 48%, while that of high
doses of LRM and LB remained above 90%. This result showed
that the nematodes treated with LB and LRM still exhibited
high autonomous locomotivity in the middle-late life, while
more than half of the nematodes in the untreated group could
only maintain the head swing with continuously shrinking the
active area. In addition, the pumping rate of nematodes
should be reduced with the increased age, which means that
their diet is restricted. Interestingly, the nematodes sup￾plemented with LB or LRM showed an even a more rapid
decline in pumping rate compared to the control group
(Fig. 4), which was consistent with the phenomenon we
observed during the lifespan assay. The food (E. coli) consump￾tion rate of the treatment group was lower, and thus the
amount of corresponding remaining food was more than that
of the control group. Therefore, we speculate that the interven￾tion with goji berry extracts could stimulate the calorie restric￾tion pathway in C. elegans to prolong life expectancy.
3.5 LB and LRM-mitigated age pigment accumulation and
showed no effect on body size
Generally, aging is always accompanied by the accumulation of
senile pigment. Therefore, lipofuscin, a type of age pigment, is
a vital indicator of aging. It can be seen in Fig. 6 that the lipo￾fuscin levels of C. elegans in the 20, 50, and 100 mg mL−1 LB
groups showed a remarkable diminution, which were 25.32%,
40.04% and 52.32%, respectively, compared with the control
group (p < 0.05). As depicted in Fig. 5, a smaller dose-depen￾dent decrease in lipofuscin intensity was found in the LRM
treated groups (16.2%, 25.06%, 35.03%, respectively) com￾pared with the control group. For the body size of the
C. elegans, except for the 20 mg mL−1 LB treatment group, no
significant differences were observed between the control
group and other treatment groups (Fig. 5 and 6).
3.6 LRM and LB promoted the progeny of C. elegans
Evidently, lifespan prolongation has been confirmed to be
positively associated with fertility restriction. Therefore, the
number of eggs laid during the spawning period of the
C. elegans was counted in our study. The results showed that
goji berry extracts exhibited no inhibition of the reproductive
ability of nematodes, but played a role in accelerating it,
which might be attributed to the promotion effect of
goji berry on the reproductive system (Fig. 7). Specifically,
the brood size of worms in the 10 mg mL−1 LRM and
100 mg mL−1 LB groups increased remarkably from 147 to
202 and from 147 to 219, representing 37.41% and 48.98%
increments, respectively.
3.7 LB and LRM enhanced the activities of SOD and CAT
As important members of the antioxidant enzyme system in
the biological system, SOD plays a crucial role in scavenging
free radicals within the body to resist external oxidative stress,
Table 2 Effects of LRM and LB on the tolerance to UV-B irradiation and paraquat in C. elegans (mean ± SD, n = 3)
Stress Group Number Mean lifespan (h) % of control Maximum lifespan
UV-B Control 100 6.33 ± 0.14c 100.00c 8
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while CAT is responsible for degrading excessive hydrogen per￾oxide within the body to protect cells from damage. As illus￾trated in Fig. 8B, the contents of SOD in LB groups were dose￾dependently enhanced by 1.88, 2.46, and 2.52 folds, respect￾ively, compared with the control group, and that of CAT activity
was also promoted significantly by 2.00, 2.25, and 2.10-fold,
respectively. Similar results can also be seen in the LRM treat￾ment groups (Fig. 8A). The contents of SOD and CAT in
C. elegans were increased by 2.52 and 3.61-fold, respectively,
after fed with 10 and 2 mg mL−1 LRM. These results suggested
that goji berry extracts could greatly facilitate the antioxidant
enzyme activity in C. elegans.
Fig. 3 Effects of LRM (A) and LB (B) on the motility of C. elegans. Where Motion A, Motion B, and Motion C refer to nematodes that move autono￾mously and leave sinusoidal traces, those that crawl with the help of external stimulus, and those that lose their mobility and only maintain the head
swing, respectively.
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3.8 Effects of LB and LRM on age-related gene expression
Many signaling pathways are highly conserved in C. elegans
and exist strong relevance to longevity, which involves IIS, JNK,
AMPK, and SIRT pathways, etc.
32 Therefore, the effects of goji
berries on the expression levels of several key genes in the
C. elegans were detected to elucidate the underlying mecha￾nism. daf-16 plays a key role in regulating the development,
metabolism, stress resistance and lifespan of nematodes via
the IIS pathway. The overexpression of upstream genes such as
insulin-receptor daf-2, age-1, and akt-2 could cause the inhi￾bition of daf-16. Moreover, the nuclear translocation of daf-16
also triggers the expression of downstream genes, including
sod-3, gst-4 and hsp-16.2 and thereby increasing lifespan. As
shown in Fig. 9A, the relative expression levels of daf-16, sod-2,
sod-3, and hsp-16.2 were up-regulated to 2.54 ± 0.13, 6.68 ±
0.61, 4.57 ± 0.45, and 6.71 ± 0.41 fold, respectively, under the
treatment of high-dose LRM, while that of the age-1 level was
down-regulated to 0.67 ± 0.03 fold. However, in the high-dose
LB treatment group, the expression of daf-16 only augmented
to 1.60 ± 0.27 fold, and correspondingly, the increments of the
expression levels of downstream genes regulated by daf-16,
such as sod-2 and hsp-16.2 (1.67 ± 0.16, and 2.64 ± 0.21 fold,
separately; Fig. 9B), also demonstrated a declining trend com￾pared with those in the LRM treatment group. sir-2.1, a type of
longevity gene, controls the response of animals to calorie
restriction, thus preventing aging-related diseases. The life￾span of nematodes containing a duplication of sir-2.1 can be
extended by 50%.35 In comparison to the control group, the
expression of sir-2.1 in the LRM and LB treatment groups
increased significantly, which were 2.93 ± 0.32 fold of the
control group at a dose of 10 mg mL−1 LRM, and 4.22 ± 0.27
fold of the control group at a dose of 100 mg mL−1 LB, respect￾ively (Fig. 10). In addition, the relative expression of daf-12
ascended to 3.61 ± 0.57 and 2.51 ± 0.32 fold, respectively, after
treating with high doses of LRM and LB. daf-12, involved in the
formation of the dauer period, plays an essential role in repro￾duction and lipid metabolism. It also shows a strong associ￾ation with the prolongation of lifespan caused by germline abla￾tion and operates synergistically with daf-16 to promote long￾evity. Additionally, jnk-1 encodes Jun-N-terminal kinase which
can be activated by different stressors, further reprogramming
metabolic system or intervening with the IIS pathway, thus regu￾lating cell proliferation and proteostasis and extending life￾span.36 Significant change can be found in the expression of
jnk-1 with a 2–3 fold increase in the LRM treatment groups and
a 5–7 fold rise in the LB treatment groups (Fig. 10A and B).
3.9 LB and LRM enhanced the nuclear localization of DAF-16
daf-16 is a vital gene in many pathways involved in lifespan
regulation, and the nuclear localization of daf-16 is the key to
activate downstream genes. Therefore, GFP was used to reflect
this translocation in the present study. According to our pre￾vious qRT-PCR results (Fig. 9), the high-dose LB and LRM
groups maximized the expression of daf-16, and thus these
two concentrations were selected for nuclear localization
experiments. Compared to the control group, DAF-16 in half of
the nematodes transferred into nuclear with 24% still in the
state of “cytosolic” after 10 mg mL−1 LRM treatment, and
more than 70% of nematodes in the 100 mg mL−1 LB treat￾ment group fulfilled this translocation with only 8% stayed in
the original state (Fig. 10). The phenomenon showed that goji
berry extracts could effectively promote the nuclear localization
of DAF-16 and increase the lifespan of nematodes.
3.10 Genetic requirements for increased lifespan from LRM
and LB treatment
On the basis of qRT-PCR data (Fig. 9), we propose that LB and
LRM might interfere with the expression of the life-related
genes to increase the survival of C. elegans. Therefore, further
study was conducted to validate whether the genes that were
promoted or suppressed by LRM or LB are necessary to
prolong lifespan. The hypothesis of the assay was that the life￾span extension effect of goji berry extracts was inhibited or
even deprived after a specific gene was muted. Four mutant
strains, mu86 (daf-16), ok434 (sir-2.1), gk7 ( jnk-1) and sy441
(hsf-1), were chosen based on our previous results of gene
expression. After intervention with 10 mg mL−1 LRM or
100 mg mL−1 LB, the mean lifespan of daf-16, sir-2.1 and hsf-1
mutant animals changed little and showed no significant
difference from the control group (Fig. 11 and Table 3),
demonstrating that the three genes were required for longevity
from the LB or LRM treatment. Logically, in the treatment
group, the mutant strain lacking the jnk-1 gene should exhibit
no increase in the lifespan due to the sharply increased gene
Fig. 4 Effects of LRM (A) and LB (B) on the pumping rate of C. elegans.
Data were expressed as the mean ± SD (n = 3).
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expression of jnk-1 in the qRT-PCR assay (Fig. 10).
Interestingly, the life expectancy of jnk-1 mutant strain in the
high doses of LRM and LB groups increased by 19.05% and
29.70%, respectively, compared with the control group (Fig. S2
and Table S2†), which suggested that jnk-1 was not involved in
LB or LRM-mediated longevity.
4. Discussion
Aging is not only the deterioration of body functions or the
disorder of homeostasis but also results in the risks of
suffering from a variety of chronic diseases such as cardio￾vascular disease, diabetes and even cancer. Moreover, people’s
expectancy for a healthy living continues to ascend, thus, the
concept of anti-aging has emerged and aroused much
concern. Nowadays, traditional medicines are usually
accompanied by certain adverse effects. Hence, diet therapy
has gradually triggered the attention of the public because of
the security and a more acceptable taste. Goji is also known as
a “super-plant” exerting multiple benefits in various aspects
such as anti-cancer, eyesight promoting, fertility enhancement
and lowering hypertension. Although there are some studies
on the longevity of Lycium barbarum polysaccharides, only a
few studies are available regarding the anti-aging effects of
phenolics in goji berry.
Phenolics could scavenge and inhibit oxidative stress,
which is closely related to the onset and development of aging
and other metabolic complications.37 We found that the
amount of total phenolics in LRM was even higher than that
in some “super fruits” such as apples or blueberry.38 In
addition, the total phenolics of LB also reached 817.45 mg per
100 g DW, although the polysaccharides of LB have always
been deeply explored because of their abundant content and
favorable bioactivity. Therefore, we selected the most widely￾circulated varieties of goji berry, LB and LRM, to investigate
their ability to resist extreme stress and the mechanisms of
their anti-aging effects. The results showed that both LB and
LRM possessed the capability to prolong the lifespan of
C. elegans, but it is hard to conclude which one is better. The
high-dose LB (100 mg mL−1
) extended the average lifespan of
nematodes by more than 50%. However, during the process of
screening effective concentrations, we found that LB at a con￾centration of below 20 mg mL−1 had no significant effect on
Fig. 5 Effects of LRM on the accumulated lipofuscin level and the body size of C. elegans. (*A, control group; B, 2 mg mL−1 LRM group; C, 5 mg
mL−1 LRM group and D, 10 mg mL−1 LRM group.) Lipofuscin intensity and body size were analyzed with image J software. Data were expressed as
the mean ± SD (n = 3).
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the lifespan of nematodes, while the LRM could elicit its life￾span-extending effect at a smaller concentration (e.g. 5 mg
mL−1 LRM conferred a 24.28% increase). LB and LRM not only
increased the survival rate of nematodes in the later stage but
also improved their mobility, indicating it is an improved phys￾iological function of C. elegans that LB and LRM triggered
Fig. 7 Effects of LRM and LB on the progeny of C. elegans and all the data were expressed as the mean ± SD (n = 3).
Fig. 6 Effects of LB on the accumulated lipofuscin level and the body size of C. elegans. (A, control group; B, 20 mg mL−1 LB group; C, 50 mg
mL−1 LB group and D, 100 mg mL−1 LB group.) Lipofuscin intensity and body size were analyzed with image J software. Data were expressed as the
mean ± SD (n = 3).
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during the aging process. Moreover, the stress resistance of
C. elegans in adversity has also been greatly augmented by goji
berries compared with untreated animals. This study tested
the survival status of nematodes in three detrimental environ￾ments, namely high temperature, ultraviolet irradiation and
paraquat treatment. Both paraquat and UV-B radiation can
induce the production of reactive oxygen species (ROS),
thereby leading to intracellular lipid peroxidation, DNA frag￾mentation and protein destruction. Therefore, the increased
production of antioxidant enzymes may be the reason for the
improved survival rate of nematodes under these two threats,
which was in close agreement with the results of enzyme
activity assays and the contents of SOD and CAT in the treat￾ment groups were 2–3 times higher than those of the control
group.
Heat shock is the major inducer for the expression of low
molecular weight heat shock proteins (HSPs) in nematodes.
Previous studies have shown that the down-regulating of the
hsp-16.2 level could improve the thermotolerance of C. elegans
by reducing the nematode’s sensitivity to heat.9,39 However,
supplementation with LB and LRM significantly increased the
expression of hsp-16.2 instead of restraining its level. We
suggest that neither LB nor LRM directly interfered with the
expression of hsp-16.2, but by activating heat shock transcrip￾tion factors (HSFs) to up-regulate the expression of heat shock
protein, thus encoding molecular chaperones to assist in
protein folding and keeping the integrity of protein from
damage,40 and eventually caused increased resistance to heat
shock.
Life-related signaling pathways play a pivotal role in aging.
In order to explore the genes that LB and LRM targeted in
extending lifespan, the IIS, HSF and SIR2.1/SIRT signaling
pathways were investigated in this study. The most common
way for the IIS to regulate lifespan is to activate AGE-1 after the
insulin receptor (DAF-2) binds to insulin-like peptides, further
enhancing the content of phosphatidylinositol-3,4,5-trispho￾sphate (PIP3) and then transmitting it to the PIK3-AKT
pathway, resulting in the phosphorylation of DAF-16, the
ortholog of Forkhead box and Class O (FOXO) transcription
factors in C. elegans, thus inhibiting its migration and localiz￾ation in sub-cells.41 When the IIS was down-regulated, daf-16
presented a state of dephosphorylation and transferred from
the cytoplasm to the nucleus, thereby regulating the nema￾tode’s ability to adapt to extrinsic stimulus and extending life
expectancy. Our results showed that both LB and LRM inhib￾ited the expression of age-1 to varying degrees, thereby up-reg￾ulating the daf-16 level. Moreover, it can be seen in the green
fluorescent protein fused in DAF-16 that most of the nema￾Fig. 8 Treatment with LRM (A) and LB (B) promoted the activities of SOD and CAT indicating the antioxidant effect played a significant role in
extending lifespan of C. elegans. At least 3 replicates were carried out involving more than 2000 animals for each experiment.
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todes in the treatment groups achieved daf-16 nuclear translo￾cation. To further verify its necessity, daf-16 mutants were used
in the lifespan assay. We found that a slight lifespan extension
effect still existed with 3.47% and 11.12% increases in the
high-dose LRM and LB groups, respectively, in the absence of
daf-16. In contrast with the effect of these two goji berries on
wild-type nematodes, the daf-16 deficiency showed a negative
regulation of the efficacy of both LRM and LB based on the
sharp decreases in their effects on extending the lifespan of
mutants, which indicated that daf-16 might be still indispens￾able in the function of goji berry extracts but as an extra essen￾tial existence. Hence, we suspect that there are other pathways
that synergistically function with daf-16 to regulate the life
cycle of C. elegans.
A previous study has confirmed that additional HSPs copies
can significantly extend the lifespan of nematodes,9 which is
compatible with our results. The expression of hsp-16.2 was
remarkably augmented in the treatment groups with extended
lifespan. Moreover, the expression level of hsp-16.2 is under
the control of DAF-16 and HSF-1. HSF regulates protein
homeostasis and longevity by activating downstream heat
shock protein genes, which encode proteases and molecular
chaperones in response to the external stress especially heat
shock. Invertebrates such as C. elegans usually only have one
type of HSF, but four different isoforms (HSF-1 to 4) that could
mediate the transcription of hsp genes through binding to the
DNA of the heat shock element (HSE) in promoters of hsp.
42 It
has been reported that the transcriptional activity of HSF-1 is
Fig. 9 Effects of LRM (A) and LB (B) on the expression of age-related genes including age-1, daf-16, sod-2, sod-3, sir-2.1, hsp-16.2, jnk-1, daf-12 in
C. elegans. Values with different letters in each column were significantly different (p < 0.05).
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reversely controlled by a complex composed of protein DDL-1,
DDL-2 (daf-16-dependent longevity genes) together with heat￾shock factor binding protein-1 (HSB-1), and this complex is
directly modulated by the IIS pathway in a DAF-16-indepen￾dent way,23 which means that the knockdown of daf-16 could
not abolish the lifespan extension brought by the elevated
HSF-1 activity. In this study, LB and LRM promoted the daf-16
expression level to activate downstream genes including sod-2
and sod-3, thus alleviating the symptoms of aging to a certain
degree by exerting antioxidant effects. Meanwhile, the
expression of hsp-16.2, the target gene of hsf-1, was also signifi￾cantly up-regulated by goji berries, so we utilized the hsf-1
mutant to elaborate its impact on the regulation of lifespan. As
expected, after the knockout of hsf-1, the longevity effects of
the two goji berry extracts on nematodes were reversed with no
significant differences between the high-dose treatment
groups and the control group, which emphasized the signifi￾cance of hsf-1 for the effects of LB and LRM on anti-aging and
resisting diverse forms of stress.
SIR2, belonging to the NAD+-dependent protein deacetylase
family, is another highly conserved effector in regulating long￾evity. The SIR-2.1 was reported as the homolog to mammalian
SIRT1 and yeast SIR2, which affect the lifespan of C. elegans in
combination with the IIS pathway.43 Tissenbaum et al. found
that increased copies of sir-2.1 promoted the life expectancy of
C. elegans by 50%; they also confirmed that the combination
Fig. 10 Effects of LRM and LB on the translocation of DAF-16::GFP inside the body of C. elegans. (*2A, control group; 2B, 2 mg mL−1 LRM group;
2C, 5 mg mL−1 LRM group and 2D, 10 mg mL−1 LRM group. 3A, control group; 3B, 20 mg mL−1 LB group; 3C, 50 mg mL−1 LB group and 3D, 100 mg
mL−1 LB group.)
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of DAF-16 and SIR-2.1 was involved in stress-response and
anti-aging since the transcription of sod-3, a target gene of
DAF-16, increased along with the overexpression of sir-2.1 in a
daf-16-dependent way.35 Furthermore, no further lifespan
extension was conferred in the daf-2 mutant with the increased
dosage of sir-2.1, and thus sir-2.1 although might be acting in
parallel to the IIS pathway, eventually converges to the tran￾scription of daf-16.
44 However, it has also been proposed that
sir-2.1 may impact lifespan independence of daf-16 when it
comes to calorie restriction (CR) pathway. Since the 1930s, it
has been claimed that limited food consumption could
increase lifespan through reducing the storage of energy or
might even accelerate the metabolic rate, which is still in
dispute, and the theory is applicable to the majority of species
ranging from yeast to primates.45 Recent genetic study
suggests that CR not only activates the expression of sir2, but
simultaneously, CR might be controlled by SIR2 genes, which
makes it become a regulated process.43 In the present study,
sir-2.1 was significantly up-regulated after treating with LRM
or LB. Furthermore, we found that the swallowing frequency of
nematodes continued to decrease over time, and the declining
rates in the treatment groups were even more rapid. It can be
inferred that the overexpression of sir-2.1 induced by goji berry
extracts, in turn, acts on the physiological processes in
C. elegans including fat storage and metabolic regulation,
thereby making them in a CR state which provides the expla￾nation for the lower pumping rate compared with the control
group. These phenomena indicated that the interaction of sir-
2.1 and CR could promote the longevity of C. elegans and
enhance their resistance to extrinsic stimuli. Similarly, sir-2.1
depleted worms were also used to determine their necessity.
The results showed that no significant differences in the
average life expectancy were found between the treatment
groups and the blank group with the absence of sir-2.1, indi￾cating that it elicited the lifespan extension effect as an inte￾gral part. It has been confirmed that the germline signaling
pathway is also involved in lifespan extension. Longevity can
be achieved by the removal of germline or knocking out genes
that affect the proliferation and differentiation of germ cells.46
Hence, we conducted the LB-100 progeny assay to assess the reproduc￾tive ability of C. elegans supplemented with goji berry extracts.
However, no suppressive sign was observed in their genital
system compared with the control group, which suggested that
the germline signal pathway was not involved in the LB or
LRM-mediated longevity of C. elegans.
Table 3 Effects of LRM and LB on the lifespan of daf-16 (mu86), hsf-1 (sy441) and sir-2.1 (ok434) mutants (mean ± SD, n = 3)
Genotype Group Number Mean lifespan (days) % of control Maximum lifespan (days)
daf-16 (mu86) Control 100 12.73 ± 1.28a 100.00a 19
Values with different letters in each column were significantly different (p < 0.05).
Fig. 11 Effects of LRM and LB on the lifespan of daf-16 (mu86) (A), hsf-
1 (sy441) (B) and sir-2.1 (ok434) (C) mutants. The survival percentage of
the mutants indicated that daf-16, hsf-1 and sir-2.1 were required for the
effect of lifespan extension from LRM and LB.
Paper Food & Function
Food Funct. T
5. Conclusion
This study showed that goji berry extracts (LRM and LB) could
prolong the lifespan and ameliorate the deterioration of aging￾related physical functions such as decreased mobility and
increased lipofuscin level in C. elegans. In addition, sup￾plementation with LRM and LB extracts enhanced the tolerance to external pressure including oxidative stress, heat
shock and ultraviolet radiation via up-regulating the activities
of antioxidant enzymes (SOD and CAT). Consequently, the life￾span extension of C. elegans mediated by LRM and LB was
closely related to the expression of age-related genes including
age-1, daf-16, sod-2, sod-3, hsp-16.2 and sir-2.1. Furthermore,
intervention with LRM and LB accelerated the translocation of
daf-16 into the nucleus in the nematodes. The mutant investi￾gation further demonstrated that mutant strains of daf-16, hsf-
1 and sir-2.1 abolished the effects of LRM and LB on the life￾span extension. In summary, we suggest that goji berry
extracts might promote the life extension of nematodes colla￾boratively with the IIS pathway through hsf-1 and sir-2.1 in a
daf-16-independent form. The present exploration would
provide a guideline to  reveal the specific anti-aging mecha￾nisms of functional food interfering with lifespan and ameli￾orating stress.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors acknowledged the 111 Project (B17018), the
Guangzhou Innovation Leading Talent Project, the
Guangdong Basic and Applied Basic Research Foundation
(2020A1515011376), and the Innovative Leading Talents
Project of Guangzhou Development Zone for financial
support.
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