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A Potential Probiotic Lactobacillus plantarum JBC5 Improves Longevity and Healthy Aging by Modulating Antioxidative, Innate Immunity and Serotonin-Signaling Pathways in Caenorhabditis elegans

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Since the hypothesis of Dr. Elie Metchnikoff on lactobacilli-mediated healthy aging, several microbes have been reported to extend the lifespan with different features of healthy aging. However, a microbe affecting diverse features of healthy aging is of choice for broader acceptance and marketability as a next-generation probiotic. We employed Caenorhabditis elegans as a model to understand the potential of Lactobacillus plantarum JBC5 (LPJBC5), isolated from fermented food sample on longevity and healthy aging as well as their underlying mechanisms. Firstly, LPJBC5 enhanced the mean lifespan of C. elegans by 27.81% compared with control (untreated). LPBC5-induced longevity was accompanied with better aging-associated biomarkers, such as physical functions, fat, and lipofuscin accumulation. Lifespan assay on mutant worms and gene expression studies indicated that LPJBC5-mediated longevity was due to upregulation of the skinhead-1 (skn-1) gene activated through p38 MAPK signaling cascade. Secondly, the activated transcription factor SKN-1 upregulated the expression of antioxidative, thermo-tolerant, and anti-pathogenic genes. In support, LPJBC5 conferred resistance against abiotic and biotic stresses such as oxidative, heat, and pathogen. LPJBC5 upregulated the expression of intestinal tight junction protein ZOO-1 and improved gut integrity. Thirdly, LPJBC5 improved the learning and memory of worms trained on LPJBC5 compared with naive worms. The results showed upregulation of genes involved in serotonin signaling (ser-1, mod-1, and tph-1) in LPJBC5-fed worms compared with control, suggesting that serotonin-signaling was essential for LPJBC5-mediated improved cognitive function. Fourthly, LPJBC5 decreased the fat accumulation in worms by reducing the expression of genes encoding key substrates and enzymes of fat metabolism (i.e., fat-5 and fat-7). Lastly, LPJBC5 reduced the production of reactive oxygen species and improved mitochondrial function, thereby reducing apoptosis in worms. The capability of a single bacterium on pro-longevity and the features of healthy aging, including enhancement of gut integrity and cognitive functions, makes it an ideal candidate for promotion as a next-generation probiotic.
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Citation: Kumar, A.; Joishy, T.; Das,
S.; Kalita, M.C.; Mukherjee, A.K.;
Khan, M.R. A Potential Probiotic
Lactobacillus plantarum JBC5 Improves
Longevity and Healthy Aging by
Modulating Antioxidative, Innate
Immunity and Serotonin-Signaling
Pathways in Caenorhabditis elegans.
Antioxidants 2022,11, 268. https://
doi.org/10.3390/antiox11020268
Academic Editor:
Gloria Olaso-Gonzalez
Received: 23 November 2021
Accepted: 30 December 2021
Published: 28 January 2022
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Attribution (CC BY) license (https://
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4.0/).
antioxidants
Article
A Potential Probiotic Lactobacillus plantarum JBC5 Improves
Longevity and Healthy Aging by Modulating Antioxidative,
Innate Immunity and Serotonin-Signaling Pathways in
Caenorhabditis elegans
Arun Kumar 1, Tulsi Joishy 1, Santanu Das 1, Mohan C. Kalita 2, Ashis K. Mukherjee 1,3 and Mojibur R. Khan 1,*
1
Molecular Biology and Microbial Biotechnology Laboratory, Division of Life Sciences, Institute of Advanced
Study in Science and Technology (IASST), Guwahati 781035, Assam, India; arunkumar@iasst.res.in (A.K.);
tulsi.joishy@iasst.res.in (T.J.); santanudas@iasst.res.in (S.D.); director@iasst.gov.in (A.K.M.)
2Department of Biotechnology, Gauhati University, Guwahati 781014, Assam, India; mckalita@gauhati.ac.in
3Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University,
Tezpur 784028, Assam, India
*Correspondence: mojibur.khan@iasst.gov.in
Abstract:
Since the hypothesis of Dr. Elie Metchnikoff on lactobacilli-mediated healthy aging, sev-
eral microbes have been reported to extend the lifespan with different features of healthy aging.
However, a microbe affecting diverse features of healthy aging is of choice for broader acceptance
and marketability as a next-generation probiotic. We employed Caenorhabditis elegans as a model
to understand the potential of Lactobacillus plantarum JBC5 (LPJBC5), isolated from fermented food
sample on longevity and healthy aging as well as their underlying mechanisms. Firstly, LPJBC5 en-
hanced the mean lifespan of C. elegans by 27.81% compared with control (untreated).
LPBC5-induced
longevity was accompanied with better aging-associated biomarkers, such as physical functions, fat,
and lipofuscin accumulation. Lifespan assay on mutant worms and gene expression studies indicated
that LPJBC5-mediated longevity was due to upregulation of the skinhead-1 (skn-1) gene activated
through p38 MAPK signaling cascade. Secondly, the activated transcription factor SKN-1 upregulated
the expression of antioxidative, thermo-tolerant, and anti-pathogenic genes. In support, LPJBC5
conferred resistance against abiotic and biotic stresses such as oxidative, heat, and pathogen. LPJBC5
upregulated the expression of intestinal tight junction protein ZOO-1 and improved gut integrity.
Thirdly, LPJBC5 improved the learning and memory of worms trained on LPJBC5 compared with
naive worms. The results showed upregulation of genes involved in serotonin signaling (ser-1,mod-1,
and tph-1) in LPJBC5-fed worms compared with control, suggesting that serotonin-signaling was
essential for LPJBC5-mediated improved cognitive function. Fourthly, LPJBC5 decreased the fat accu-
mulation in worms by reducing the expression of genes encoding key substrates and enzymes of fat
metabolism (i.e., fat-5 and fat-7). Lastly, LPJBC5 reduced the production of reactive oxygen species and
improved mitochondrial function, thereby reducing apoptosis in worms. The capability of a single
bacterium on pro-longevity and the features of healthy aging, including enhancement of gut integrity
and cognitive functions, makes it an ideal candidate for promotion as a next-generation probiotic.
Keywords:
longevity; lactic acid bacteria; aging biomarkers; SKN-1 transcription factor; p38 MAPK
signaling; behavior; intestinal integrity; mitochondria
1. Introduction
The aging population has a profound impact on the global economy [
1
]. Healthy
aging refers to the “process of developing and maintaining the functional ability that
enables well-being in older age” has been a recent concern [
2
]. The functional abilities
constitute physiological, cognitive, metabolic, and immunological functions in the later
Antioxidants 2022,11, 268. https://doi.org/10.3390/antiox11020268 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 268 2 of 25
stages of life [
3
]. Older age is considered an important risk factor for many comorbidities,
including cancer, diabetes, obesity, neurodegenerative disorders, cardiovascular diseases,
and infections [
4
]. The aging process is influenced by genetics, medical history, pharma-
cological and dietary interventions [
5
8
]. During the past couple of decades, there has
been a significant surge in research on prolongevity and healthy aging. Several studies on
dietary interventions in both invertebrates (worms and flies) and vertebrate hosts (mice
and humans) have shown to promote healthy aging [810].
Caenorhabditis elegans has become a choice as a model for aging research due to its
shorter life cycle (2–3 weeks) and aging-related conserved pathways with humans [
11
].
The significant pathways such as p38 mitogen-activated protein kinase (p38 MAPK) and
DAF-2/DAF-16
pathways regulate longevity, resistance against oxidative stress, and de-
fense against pathogens in worms [
12
14
]. These mechanisms are evolutionarily conserved
in models from worms to mice [
15
]. Many studies have shown that these microbivore
worms (i.e., feeds on microbes) can explain how a microbial diet could influence longevity,
development, reproduction, fat accumulation, cognition, and imparts resistance against
biotic and abiotic stresses [11].
Probiotic microbes are known to improve the host’s health by antimicrobial effect,
nutritional supplementation, immunomodulation, and maintaining the healthy micro-
biota within the host’s gut, thereby also promote healthy aging [
16
]. A century ago,
Dr. Elie Metchnikoff
observed that lactobacilli-containing yogurt consumption led to in-
creased lifespan and better health of Bulgarian farmers [
17
]. It was the first observation on
probiotics-induced longevity. Lactobacillus spp. are the most important bacteria that colo-
nize the gastrointestinal tract of humans since birth [
18
]. Studies suggest that the lactobacilli
population decreases with age, and supplementation of probiotic lactobacilli to older adults
improves their health by modulating gut microbiota composition, enhancing immunity and
gut health [
19
21
]. Previous reports indicated the role of lactobacilli on longevity with dif-
ferent features of healthy aging such as resistance to oxidative stress and toxicity, protection
against pathogenic infections, reducing fat accumulation, improving cognition, alleviating
inflammation, metabolic disorders, and neurodegenerative diseases
[1214,2232]
. How-
ever, a single microbe affecting these diverse features of healthy ageing will increase the
acceptance and marketability as a next generation probiotic.
In our previous research on bacteria of curds prepared using boiled milk and raw
milk from dairy farms of Assam, India [
33
], a potential probiotic bacterium was isolated,
and it was taxonomically characterized as Lactobacillus plantarum strain JBC5 (LPJBC5). In
this work, we have demonstrated that LPJBC5 promotes longevity and diverse aspects of
healthy aging, including delayed age-related physical functions, reduced fat accumulation,
improved resistance to abiotic and biotic stress, gut integrity, cognition functions, mito-
chondrial functions and reduced apoptosis in C. elegans. Our study indicates that bacterium
LPJBC5 affects the diverse features of healthy aging, making it an ideal candidate for
promotion as next-generation probiotics.
2. Materials and Methods
2.1. Materials
All microbiological growth media, including deMan, Rogosa and Sharpe (MRS), Nu-
trient agar and Luria-Bertini (LB) agar were obtained from Himedia, India. The bac-
terial control food E. coli OP50 (OP50), C. elegans Bristol wild-type strain N2 and its
mutants, including DR1572 daf-2 (e1368), AU1 sek-1 (ag1), KU25 pmk-1 (km25), GR1307
daf-16 (mgDf50)
, AU3 nsy-1 (ag3), and EU31 skn-1(zu135) and EU1 skn-1 (zu67) were ob-
tained from Caenorhabditis Genetics Center (CGC), University of Minnesota, USA. All
experiments were performed with self-fertilizing hermaphrodite strains of C. elegans. The
pathogenic strain Staphylococcus aureus MTCC 3160 was obtained from Microbial Type
Culture Collection, CSIR-Institute of Microbial Technology (CSIR-IMTECH), Chandigarh,
India. The epithelial intestinal cell line HT-29 was procured from National Center of Cell
Sciences, Pune, India. The genomic extraction kit was procured from Sigma, Germany.
Antioxidants 2022,11, 268 3 of 25
Total RNA was isolated using RNAeasy mini kit (Invitrogen, Waltham, MA, USA) and
reverse transcribed to cDNA using Verso cDNA synthesis Kit (Thermo Scientific, Waltham,
MA, USA).
2.2. Bacterial Strains and Growth Conditions
LPJBC5 was inoculated and cultured in MRS broth at 30
C for 18 h, whereas OP50
was cultured in Luria-Bertini broth overnight at 37
C. The pathogenic strain S. aureus was
inoculated and cultured in Nutrient broth for overnight at 37
C. All these bacterial strains
were cultured under shaking conditions at 150 rpm.
2.3. Identification for the Presence of Probiotic Marker Genes in LPJBC5
The presence of probiotic marker genes encoding species-specific collagen-binding
protein and bile salt hydrolase was confirmed in LPJBC5, as previously described [
34
]
(Table S1). Additionally, L. plantarum specific sequence and anti-microbial gene (plantaricin-
biosynthetic gene) was amplified and sequenced in LPJBC5 (Table S1) [
35
,
36
]. The genomic
DNA was extracted using a genomic extraction kit, and its concentration was determined
using a NanoDrop
TM
2000c spectrophotometer (Thermo Scientific, USA). The PCR reaction
was set up with a total reaction mixture of 25
µ
L containing 2.5
µ
L of 10
×
Taq buffer, 1.5 U
of Taq DNA polymerase (Sigma, Darmstadt, Germany), 1.5 mM of MgCl
2
, 100
µ
M of dNTP
mixture, 10 pmol of each primer pair and 50 ng of bacterial DNA. The PCR was performed
using the following conditions in a PCR thermal cycler (Eppendorf, Hamburg, Germany):
96
C for 5 min, followed by 35 cycles of 94
C for 30 s, 57
C for 30 s, 72
C for 1 min, and
72
C for 5 min. The amplified product was run on 1.5% agarose gel and imaged under
a UV transilluminator (Vilber, Collégien, France). Sequencing of purified PCR amplified
fragments was performed with Macrogen Inc. (Seoul, Korea). The DNA sequences were
subjected to BLAST analysis and submitted to NCBI (National Center for Biotechnology
Information, Bethesda, Rockville, MD, USA) database.
2.4. Phylogenetic Tree
To determine the phylogenetic relationships of strain LPJBC5, the 16S rDNA gene
sequence was compared with other strains of Lactobacillus and one outgroup genera
Enterococcus faecium
ATCC 19434. The identification of phylogenetic neighbours and calcu-
lation of pairwise 16S rDNA gene sequence similarities and aligned using phylogenetic
tree analysis software (MEGA 7 version) [
37
]. Neighbour-joining algorithms were used for
the reconstruction of the phylogenetic tree. Bootstrap analysis was conducted to determine
the confidence limits of the branching.
2.5. Survival to the Gastrointestinal (GIT) Transit
The simulated gastric juices were prepared by using a method described by
Conway et al. [
38
]. Firstly, the grown LPJBC5 culture (10
9
CFU/mL) was washed twice
with PBS and resuspended in GIT juice (0.3% pepsin) at pH 2.0 and incubated at 37
C for
0 h, 1 h, and 3 h. Secondly, the grown LPJBC5 culture (10
9
CFU/mL) was washed with PBS,
resuspended in intestinal juice (0.1% porcine pancreatin, Sigma, Germany), and further
adjusted to pH 8.0. The treated LPJBC5 cells were incubated at 37
C for 4 h. Thirdly,
the acid tolerance of LPJBC5 was evaluated at different pH range from 1.0, 3.0, and 7.0
(considered as control) of PBS. The LPJBC5 culture (10
9
CFU/mL) was added to 10 mL of
different pH solutions and incubated at 37
C for 4 h. Each experiment was performed in
triplicate, and after completion of incubation time, the viable LPJBC5 cells were counted for
each treatment by spread-plating on MRS agar plates and incubated at 37 C for 24–48 h.
2.6. Assay for Bile Acid Tolerance
The tolerance to Bile salt of the isolate LPJBC5 was measured using a procedure
described by Vinderola and Reinheimer [
39
]. Briefly, the solutions of bile salts were
prepared using Ox-gall bile (Sigma, Darmstadt, Germany) with 0.3% and 1% concentrations,
Antioxidants 2022,11, 268 4 of 25
and PBS served as control. 10
9
CFU/mL cells of grown isolate LPJBC5 were further added
to 10 mL bile salt solutions and incubated at 37
C for 0 h and 4 h. The viable LPJBC5 cells
were counted for each treatment by spread-plating on MRS agar plates and incubated at
37 C for 24–48 h.
2.7. Adhesion to Intestinal Cells
The adhesion ability of LPJBC5 to epithelial intestinal cell line HT-29 was tested using
a procedure discussed in Ayeni et al. [
40
]. The adhesion of isolate LPJBC5 was calculated
as the number of adhered cells to HT-29 cell line after 4 h of incubation in comparison with
the initial population in the DMEM suspension.
2.8. Longevity Assay
The Nematode Growth Medium (NGM) plates (60 mm) were seeded with 10
9
CFU/mL
of OP50 or LPJBC5 suspended in M9 buffer (22 mM KH
2
PO
4
, 42 mM Na
2
HPO
4
, and
86 mM
NaCl) [
41
]. The synchronization of worms were performed by a modified proce-
dure described by Stiernagle [
42
]. “Briefly, the NGM plates with sufficient eggs of self-
fertilizing hermaphrodite worms were washed with deionized water (Millipore, Burlington,
MA, USA
) and pipetted into a 5 mL conical centrifuge tube. The deionized water was
adjusted to 3.5 mL in a conical centrifuge tube and 1.5 mL volume of a mixture of 1 mL
sodium hypochlorite bleach and 0.5 mL of 5 N NaOH was added. The tubes were then
vortexed for approximately 2 min and centrifuged at 7500 rpm to pellet down the released
eggs. The supernatant was thrown, and pellet of worms was washed twice with deionized
water. At last, the remaining pellet was suspended in approximately 0.1 mL of deionized
water and transferred to NGM plate seeded with standard bacterial food E. coli OP50”. The
longevity assays were performed with young adult wild-type N2 and mutant worms. The
longevity experiment was performed in 3 technical replicates in which each NGM plate
contained 50 worms and plated were further incubated at 20
C. The worms were counted
every 24 h and transferred to new NGM plates every two days to maintain sufficient
bacterial food. The worms were excluded from lifespan analysis if it adheres to the wall
of the plate, showing abnormal death due to progeny hatch inside their body and vulva
explosion [
41
]. Survival analysis was conducted using the Kaplan–Meier method in OASIS
2 software, and their mean lifespan was calculated [43].
2.9. Determination of Pharynx Pumping and Locomotor Activity
The pharynx pumps and locomotor activity of worms were determined using a
protocol described by Nakagawa et al. [
12
]. The synchronized worms were initially grown
on OP50 for 2 days and further cultured throughout adulthood for 14 days on OP50 or
LPJBC5. The worms were then transferred to a new OP50-seeded NGM plate 30 min
before recording their pumping rates or locomotor ability. The number of pharynx pumps
rates were measured for 30 s using a stereo zoom microscope (SMZ1270, Nikon, Tokyo,
Japan) [
12
]. The locomotory rate was measured by counting the body bends per minute on
the NGM plate using a stereo zoom microscope [
12
]. Ten worms were assessed for each
bacterial strain, and 3 replicates were used for each bacterium.
2.10. Developmental Rate Assay
The developmental rate of worms fed on both bacterial diets was analyzed using a
procedure described by Soukas et al. [
44
]. About 100 age-synchronized worms were allowed
to lay eggs on OP50-seeded NGM plate for 30 min and synchronized using a bleaching
protocol. Once the worms reached their young adult stage, 20 worms per bacterial strains
were individually transferred to OP50 or LPJBC5 seeded NGM plates in triplicates and
continuously observed under a stereo zoom microscope (SMZ1270, Nikon, Japan) every
30 min until we observed the first laid egg.
Antioxidants 2022,11, 268 5 of 25
2.11. Measurement of Body Size
The body sizes of OP50 and LPJBC5-fed worms were determined as described by
Zanni et al. [
45
]. The age-synchronized worms were initially cultured on OP50 for 3 days
and transferred to OP50 or LPJBC5 (10
9
CFU/mL) seeded plates. The images were captured
using a stereo zoom microscope (SMZ1270, Nikon, Japan) every 24 h until the age of day 7,
and body size was determined using ImageJ software (National Institutes of Health, MD,
USA). Ten worms grown in each bacterial strain were used for measuring the body size,
and for assuring reproducibility, the experiment was performed in triplicate.
2.12. Colonization Efficiency
The synchronized eggs of worms were transferred to NGM-plates seeded with OP50
or LPJBC5. A total of 10 worms per bacterial strain were washed and further lysed to check
their colonization efficiency on the third and eighth days. The whole nematode lysate of
LPJBC5 or OP50-fed worms were plated in triplicates on their respective culture media
containing plates [
46
]. The colony-forming units (CFU) were counted for each bacterial
strain after incubating at their respective cultural conditions.
2.13. Aging Pigment Accumulation
The study on the accumulation of aging pigment or lipofuscin levels of worms was
conducted as described by Kwon et al. [
41
]. Briefly, the worms were cultured on OP50
or LPJBC5 for 14 days, washed, and transferred onto 3% (w/v) agarose pads on a glass
slide for confocal microscopy (TCS SPE, Leica, Wetzlar, Germany). Ten worms were
quantified for each bacterial strain using ImageJ software, and three replicates were used
for each bacterium.
2.14. Brood Size
The brood size of worms was determined as described by Zanni et al. [
45
]. The number
of progenies per nematode was calculated, and the experiment was conducted in triplicate
for both bacterial strains.
2.15. Determination of Fat Accumulation
Age-synchronized young adult worms were cultured on OP50 or LPJBC5 for 14 days.
Further, the accumulation of body fat was examined by Oil Red O (ORO) staining in worms
according to a protocol described by Nakagawa et al. [
12
]. The lipid accumulation was
observed and imaged using a compound microscope (10
×
and 20
×
objective lens) (AX10,
Carl Zeiss, Jena, Germany), and the relative staining intensity was quantitated with ImageJ
software. Ten worms were analyzed to quantify the accumulation of fat for each bacterial
strain, and three replicates were used for each bacterium.
2.16. Food Preference and Learning Memory
The binary choice assay was performed to observe the food preference of worms on
different bacterial diets as described by Bendesky et al. [
47
]. The assay was conducted
on 60 mm NGM plate. A total of 25
µ
L of LPJBC5 or OP50 (10
9
CFU/mL) was seeded
at the opposite sides of the plates and dried for 1 h under laminar airflow. Day 3 worms
(30 worms/plate) were individually transferred onto the center of the NGM plate with
equidistant lawns of OP50 and LPJBC5 and counted worms in both bacterial lawns after
4 h
, and the experiment was conducted in triplicate. The choice index (CI) of bacterial food
preference in worms was calculated as follows:
Choice index (CI) = Number of worms in LPJBC5 Number of worms in OP50/Total
number of worms used in an assay
CI = 1.0 shows complete food preference for control food OP50.
CI = +1.0 shows complete food preference for testing bacteria LPJBC5.
CI = 0.0 shows equal distribution of food preference for both OP50 and LPJBC5.
Antioxidants 2022,11, 268 6 of 25
For training, day 3 worms (n= 30) were cultured onto an NGM plate seeded with
LPJBC5 and cultured for 4 h at 20
C. The worms were further washed thrice with M9
buffer and transferred onto the center of the plate seeded with equidistant lawns of OP50
and LPJBC5. The memory index was calculated as:
Memory index = CI (Trained LPJBC5) CI (Naive OP50)
A total of 30 worms per plate were analyzed in each preference assay, and the experi-
ment was performed in triplicates.
2.17. Thermotolerance and Oxidative Stress-Resistance Assay
Age-synchronized worms were initially cultured on OP50 until the young adult stage,
then individually transferred onto NGM plates seeded with OP50 or LPJBC5 for 3 days
at 20
C. For thermotolerance assay, these cultured plates were maintained at 35
C, and
viable worms were counted every hour until all of them died [48].
For the oxidative stress-resistance assay, day 3 young worms were grown on OP50 or
LPJBC5 for three days and individually picked and transferred to an M9 solution containing
100 mM paraquat (Sigma Aldrich, St. Louis, MO, USA) and incubated at 20
C [
49
]. The
viable worms were counted after 8 h. Fifty worms were transferred to three wells against
each treatment, and the experiment was performed in triplicates for each treatment. The
lipofuscin level was also measured after 8 h of incubation in 100 mM paraquat using
confocal microscopy as described previously (see material and methods 2.13). Ten worms
were quantified for each treatment using ImageJ software, and three replicates were used
for each treatment.
2.18. Determination of Resistance against Pathogenic Bacterial Infections
Age-synchronized young adult worms were initially grown on OP50-seeded plates,
and further individually transferred on OP50 or LPJBC5 seeded NGM plates for 3 days.
These worms were then individually transferred to NGM plates seeded with the pathogenic
bacterium S. aureus (10
9
CFU/mL), and incubated at 20
C [
41
]. Survival of worms was
recorded every 24 h. A total of 50 worms were used in each plate, and the experiment was
performed in triplicate for each bacterium.
2.19. Measurement of Intestinal Integrity against Pathogenic Infection (Smurf Assay)
The effect of LPJBC5 against intestinal barrier infections was evaluated according to
Kim and Moon [
50
]. Briefly, the age-synchronized young adult worms grown on OP50 were
cultured on OP50 or LPJBC5 for five days. The worms were then individually transferred to
NGM plates seeded with pathogen S. aureus for 2 days. To observe intestinal permeability
in response to pathogenic exposure, OP50 was heat-killed at 70
C for 2 h (confirmed by
plating on LB-agar). The heat-killed OP50 culture was centrifuged, and the pellet was
resuspended in blue food dye (5% w/v) and shaken for 3 h at 35 rpm at room temperature.
The worms were then grown in a liquid NGM medium containing heat-killed OP50 stained
with blue food dye for 3 h. The worms were washed several times in M9 buffer, anesthetized
in the same buffer containing 25 mM levamisole (L-025, Sigma, Germany) and imaged with
a compound microscope (AX10, Carl Zeiss, Germany) at 10
×
and 20
×
magnifications. The
blue food dye in the body of worms (n= 10) was quantitatively analyzed using ImageJ
software under 10×magnification.
2.20. RNA Isolation, cDNA Synthesis, and Quantitative Reverse Transcription-Polymerase Chain
Reaction (qRT-PCR)
The age-synchronized worms were grown on OP50 for 2 days at 20
C. The young
adult worms were then transferred to NGM plates seeded with LPJBC5 or OP50 for
24 h
.
Approximately 500 worms per group were collected and washed to extract the total RNA.
About 1
µ
g of extracted RNA was reverse transcribed to cDNA by using Verso cDNA
synthesis Kit. qRT-PCR was performed to measure the expression of the genes using SYBR
Antioxidants 2022,11, 268 7 of 25
Green (Applied Biosystems, USA) in a real-time PCR machine (Applied Biosystem, USA).
The genes include DAF-2/DAF-16 (daf-2 and daf-16), p38 MAPK (nsy-1,sek-1 and pmk-1)
and its downstream genes (skn-1 and skn-1b), FOXA transcription factor pha-4, phase-2
detoxification genes GSTs (gst-4,gst-7 and gst-10), catalases (ctl-1 and ctl2), Thioredoxin-1
(trx-1) and SODs (sod-1,sod-2 and sod-3), heat-shock proteins HSPs (hsp-60 and hsp-70,
hsp-16.1 and hsp-16.2), genes encoding key substrates and enzymes of fat metabolism (fat-5,
fat-6 and fat-7), serotonin signaling genes (ser-1,mod-1, and tph-1), innate immunity genes
[saponin-like proteins (spp-1 and spp-7), lysozymes (lys-1 and lys-8), C-type lectin (CLEC)
domain-containing proteins (clec-60 and clec-85) and antibacterial factor (ABF) (abf-1,abf-2
and abf-3)], tight junction protein zonula occludin zoo-1, mitochondrial DNA (mtDNA)
encoded NADH-ubiquinone oxidoreductase chain 1 (nd-1), anti-apoptotic (ced-9) and pro-
apoptotic (ced-3 and ced-4) genes. The primer sequences of the studied genes are listed in
Table S2. The experiment was performed with three replicates. The relative expression of
each gene was analyzed using 2
∆∆Ct
method [
51
]. The housekeeping gene act-1 was used
to normalize the expression of each gene.
The expression of innate immune genes was studied by pre-culturing the young
adult worms on LPJBC5 or OP50 for 24 h, and then transferred to NGM plates seeded
with
S. aureus
. After 12 h of post-infection, the worms were collected and washed three
times with M9 buffer. RNA isolation, cDNA synthesis, and qRT-PCR were performed as
described above.
2.21. GSH/GSSG Assay
Glutathione Assay Kit (Promega, Madison, WI, USA) was used to individually mea-
sure reduced glutathione (GSH) and oxidized glutathione (GSSG) in day-14 worms grown
on OP50 or LPJBC5 as per the manufacturer’s instructions. It is a luminescent based system
to detect both GSH and GSSG in worms. In brief, the worms were transferred to the
wells of a 96 well flat bottom plate. A 50
µ
L volume of total or oxidized glutathione lysis
reagent was added to each group of well in a 96-well plate, and the plates were shaken
at 3000 rpm for 5 min at room temperature. The luciferin generation reagent was added
(50
µ
L/well) and the plates were incubated for 30 min at room temperature. The luciferin
detection reagent was added (100
µ
L/well) to each well and luminescence was recorded in
a multimode plate reader (Varioskan Flash, Thermo Scientific, USA) at 562 nm. The total
level of luminescence was detected in both LPJBC5-fed and OP50-fed worms and the ratio
of GSH to GSSG was calculated. One hundred worms were used for each bacterial-treated
group, and the experiment was performed in triplicate.
2.22. SOD Activity Assay
SOD Assay Kit-WST (19160, Sigma, USA) was used to measure superoxide dismutase
(SOD) activity in day 14 worms grown on OP50 or LPJBC5 as per the manufacturer’s instruc-
tions. Additionally, the required preparation of protein extracts and further determination
of SOD activity was performed using a procedure described by
Nakagawa et al. [12].
One
hundred worms per group were used to prepare the protein extract, and the experiment
was performed in triplicate.
2.23. Measurement of Intracellular ROS Generation
The fluorescent non-polar probe 2
0
,7
0
-dichlorofluorescein diacetate (H
2
DCFDA) was
employed to determine
in vivo
cytoplasmic ROS in day 14 worms grown on OP50 or
LPJBC5, as described by Yoon et al. [
52
]. Briefly, the protein extract was prepared, and
twenty-five micrograms of protein for each sample were dissolved in PBS to bring the
final volume to 50
µ
L. The protein lysate (25
µ
g/sample) or PBS (control) were further
mixed with 100
µ
L of 50
µ
M chloromethyl-H
2
DCFDA in PBS and transferred to wells
of 96 wells black microplate. The plate was stored at 37
C for 4 h, and fluorescence
intensity was further measured using a multimode reader (Excitation-485, Emission-535)
(Varioskan Flash, Thermo Scientific, USA). The experiment was performed with three
Antioxidants 2022,11, 268 8 of 25
biological replicates, and 100 worms per bacterial treated group were used for fluorescence
measurement. The fluorescence intensity was normalized after subtracting the fluorescence
intensity of the control (without worms).
2.24. Determination of Reactive Oxygen Production and Change in Transmembrane Potential
of Mitochondria
MitoTracker
®
Red CM-H2XRos reagent (M7513, Invitrogen, USA) was employed
to determine mitochondrial ROS in day-14 worms grown on OP50 or LPJBC5 using a
procedure described by Dilberger et al. [
53
]. Briefly, the worms were incubated in 5
µ
M
MitoTracker
®
Red CM-H2XRos reagent (M7513, Invitrogen, USA) at room temperature
for 4 h. Then, the washed worms were visualized under a confocal microscope (excitation
at 525
±
45 nm, and emission at 595
±
35 nm) [
53
]. The experiment was performed in
triplicate, and 10 worms per bacterial-treated group were used for the measurement of
fluorescence intensity.
The JC-1 staining dye (T3168, Invitrogen, USA) was used to determine the mitochon-
drial transmembrane potential in day-14 worms grown on OP50 or LPJBC5 by a previously
described procedure Nakagawa et al. [
12
]. One hundred worms per group were used for
fluorescence measurement, and the experiment was performed in replicates. The fluores-
cence intensity was normalized after subtracting the fluorescence intensity of the control
(without worms).
2.25. Measurement of Intracellular Adenosine Triphosphate (ATP) Concertation
Roche ATP Bioluminescent HSII kit (Merck, Darmstadt, Germany) was used for
bioluminescence-based detection of ATP concentrations as per the manufacturer’s instruc-
tions. Briefly, a standard curve of ATP (1
µ
M–1
×
10
5µ
M) was prepared. Day-14 worms
grown on OP50 or LPJBC5 were washed thrice with PBS, and protein extracts were pre-
pared as described by Nakagawa et al. [
12
]. Bioluminescence of each sample or PBS (sample
blank) was measured against the ATP standard in a multimode plate reader. The ATP
concentration in each sample was calculated using a log-log plot of the standard curve and
the value was expressed nmol of ATP/mg protein. One hundred worms per group were
used to measure ATP concentration, and each experiment was performed in triplicate.
2.26. Quantification of Apoptosis by Tunnel-Assay
In Situ Cell Death Detection Kit, Fluorescein (Merck, Germany) was employed to detect
and quantify apoptosis in day 17 worms grown on OP50 or LPJBC5. The procedure was
followed as per the manufacturer’s instructions. One hundred worms per bacterial-treated
group were used to detect fluorescence intensity, and each experiment was performed
in triplicate.
2.27. Statistical Analysis
All represented data are results of independent three replicates, and every experiment
was repeated twice. The data were represented as mean
±
standard error mean (SEM) of
three determinations. The OASIS 2 program was used for survival analysis of lifespan and
other survival assays. The difference between the survival curve of worms was evaluated
using the log-rank test. All statistical computation analysis was performed using SigmaPlot
version 12.0 (San Jose, CA, USA). The statistical analysis between treatments/groups was
carried out using one-way analysis of variance (ANOVA) and Student’s t-test. A Mann–
Whitney U test was performed if the data were not normally distributed. Between two sets
of data, the p-value of <0.05 was considered statistically significant.
Antioxidants 2022,11, 268 9 of 25
3. Results
3.1. Molecular Taxonomic Characterisation of LPJBC5 and Persistence in In Vitro
Gastrointestinal Conditions
The 16S rDNA sequence of LPJBC5 was submitted to the NCBI database (GenBank
Acc. No. MG824976.1). A rooted phylogenetic tree of 16S rDNA gene sequence analysis
of strain JBC5 showed similarity to the species Lactobacillus plantarum. Its closest relative
species was found to be Lactobacillus plantarum strain CHE37 (MZ571407.1) with 100%
sequence similarity (Figure S1). LPJBC5 was chosen based on the evaluation of probi-
otic attributes by
in vitro
analyses conducted according to the guidelines issued by the
Indian Council of Medical Research and Department of Biotechnology, Govt. of India [
54
].
These parameters are resistance to acidic and bile conditions and the ability to colonize the
intestinal epithelium cell line [
54
] (Table S3). Furthermore, L. plantarum species-specific
PCR assay confirmed the presence of species-specific sequence, probiotic marker genes
(encode bile salt hydrolase (Lpbsh1) and collagen-binding protein (Lpcbp)) and the antimi-
crobial plantaricin-biosynthetic gene (pln), with amplicon lengths 152, 975, 2174 and 231 bp
(
Figure S2A
). All of these sequences have been submitted to the NCBI database (Table S4).
The BLAST analysis of these sequences showed the closest pairwise sequence similarity
to different L. plantarum strains (Table S4). The amplicon size of probiotic markers and
plantaricin genes represented full-length L. plantarum-specific bsh1,cbp, and pln genes
(Table S4).
3.2. LPJBC5 Increases Longevity and Slows the Development of Worms
Feeding of LPJBC5 increased the lifespan of worms by 27.8% compared with those fed
on standard bacterial food, E. coli OP50 (OP50) (*** p< 0.0001, log-rank test) (Figure 1A).
The effect of LPJBC5 on the developmental rate of worms showed that the develop-
mental rate of LPJBC5-fed worms was significantly slower from eggs to reproductive adult
stage (egg to egg) compared with OP50-fed (*** p< 0.001) (Figure 1B). Further, a significant
decrease in body size of the LPJBC5-fed worms was observed compared with the OP50-fed
(*** p< 0.001 on day 4 and 6; ** p< 0.01 on day 5 and 7) (Figure S2B).
3.3. LPJBC5 Is Efficiently Colonized into the Gut of Worms
The bacterial colony forming units (CFU) count increased with age in both LPJBC5
and OP50-fed worms (Figure S2C); however, the CFU count was found to be significantly
higher in LPJBC5-fed worms on both 3rd and 8th days of incubation by 67.11% and 133.47%
compared with OP50-fed worms (*** p< 0.001 for both 3rd and 8th day) (Figure S2C).
Result showed that there was about a two-fold increase in colonization efficiency of LPJBC5
within the gut of worms on 8th day relative to OP50-fed worms.
3.4. Feeding of LPJBC5 Delayed Aging in Worms
The effect of feeding LPJBC5 on age-related biomarkers of worms, such as pharynx
pumping, body bends, and lipofuscin accumulation, was investigated. Feeding of LPJBC5
significantly increased the pharyngeal pumping rates in worms by 179.47% higher on
day-14 compared with OP50-fed (*** p< 0.001) (Figure 1C).
We next counted the number of body bends per minute. It was observed that the fre-
quency of body bending was higher in LPJBC5-fed worms by 148.66% on day-14 compared
with OP50-fed (*** p< 0.001) (Figure 1D). These results indicated that LPJBC5 increased
lifespan and improved the quality of life in the later stage of life.
We also determined the accumulation of lipofuscin on 14-day-old worms fed with
LPJBC5. The results showed that feeding LPJBC5 significantly reduced lipofuscin accumu-
lation by 51.79% of worms compared with OP50-fed (*** p< 0.001) (Figure 1E,F).
3.5. Feeding of LPJBC5 Reduced the Accumulation of Fat in Worms
The results showed that lipid droplet accumulation was reduced by 35.77% in the
LPJBC5-fed worms compared with OP50-fed aged worms (** p< 0.01) (Figure 2A,B).
Antioxidants 2022,11, 268 10 of 25
We next evaluated the number of progenies produced by young adult worms on days
1–5 after transferring to NGM plates seeded with LPJBC5 or OP50. The results showed
no significant difference in the total number of offspring between LPJBC5- and OP50-fed
worms (p> 0.05) (Figure S2D).
Figure 1.
Feeding of probiotic L. plantarum JBC5 (LPJBC5) promotes longevity and age-related
biomarkers of worms. (
A
) Lifespan assay on worms was performed after feeding with the bacteria,
E. coli OP50 (OP50) or L. plantarum JBC5 (LPJBC5) (10
9
CFU/mL). (
B
) Egg-to-egg time was used to
assess the developmental rate after treatment with each bacterium (
C
,
D
) Pharyngeal pumping rate
and locomotor activity were analyzed on the 14th day of bacterial treatments. (
E
,
F
) Aging biomarkers
such as lipofuscin accumulation were assayed on the 14th day of bacterial treatment. Accumulation of
lipofuscin was observed under a confocal microscope at 10
×
magnification (Scale bar, 250
µ
m). Error
bars represent mean ±SEM. Treatment effects were compared using Student’s t-test (*** p< 0.001).
Antioxidants 2022,11, 268 11 of 25
Figure 2.
Treatment with LPJBC5 reduces lipid accumulation and improves the learning and memory
of C. elegans. (
A
,
B
) Lipid accumulation was assayed on the 14th day of bacterial treatment. The images
were observed at 10
×
(Scale bar, 100
µ
m). (
C
) The schematic diagram represents the experimental
design. The experiment was performed on a 60 mm Petri-plate containing NGM medium for
the binary choice assay. (
D
) Observation of results in naive and trained worms for binary choice
assay. (Scale bar, 1 mm). (
E
) Choice index (CI) of food preference for naive and trained worms.
Memory index (CI trained- CI naive) was calculated from the results of the binary choice assay. Error
bars represent mean
±
SEM. Treatment effects were compared using Student’s t-test (** p< 0.01
and *** p< 0.001).
3.6. LPJBC5 Improved Learning and Memory in Worms
A binary choice assay was performed to examine the effect of LPJBC5 on the feeding
behavior of worms. Firstly, the results showed no significant change in the feeding prefer-
ence for LPJBC5-fed worms (CI = +0.12) compared with OP50-fed (p> 0.05) (Figure 2C–E).
Then, we studied the effect of LPJBC5 on the learning ability of worms. Day-3 worms were
trained on LPJBC5 for 4 h, and then a binary choice assay was performed. Trained worms
showed greater feeding preference (CI = +0.56) for LPJBC5 compared with naive worms
(CI = +0.12) (Trained over naive *** p< 0.001) (Figure 2D,E).
Antioxidants 2022,11, 268 12 of 25
Furthermore, the memory index of worms trained on LPJBC5 over naive worms
(Trained–Naive) was calculated. A higher preference of trained worms for LPJBC5 over
OP50 indicates a higher memory index. The results showed that the memory index of the
worms fed on LPJBC5 was significantly higher over naive worms (CI = +0.44) (
*** p< 0.001
)
(Figure 2E). These results indicated that feeding of LPJBC5 improved the learning and
memory of worms.
3.7. Feeding of LPJBC5 Conferred Resistance against Abiotic and Biotic Stress Conditions
The results suggested that the survival of worms against thermal stress was 28.2%
higher in LPJBC5-fed compared with OP50-fed worms (** p< 0.01) (Figure 3A,B). Next, the
effect of LPJBC5 was evaluated on resistance against oxidative stress (100 mM paraquat) on
worms. There was significant increase in the survival rate of LPJBC5-fed worms against
oxidative stress compared with OP50-fed worms (** p< 0.01) (Figure 3C).
Figure 3.
Treatment with LPJBC5 improved the resistance of worms against abiotic stresses. After
treatment with LPJBC5, the survival of worms against heat stress at 35
C (
A
,
B
), oxidative stress
(paraquat-100 mM) as well as accumulation of lipofuscin level in presence and absence of oxidative
stress (
C
,
D
) are presented. Bars represent mean
±
SEM. Treatment effects were compared using
Student’s t-test (* p< 0.05 and ** p< 0.01).
Antioxidants 2022,11, 268 13 of 25
In addition, the results also showed a lower level of lipofuscin (a measure of senes-
cence) in LPJBC5-fed worms compared with OP50-fed worms in both the presence and
absence of oxidative stress conditions (** p< 0.01) (Figure 3D and Figure S3).
Next, we determined whether feeding of LPJBC5 increases the resistance of worms against
pathogenic bacteria S. aureus. The results showed that feeding LPJBC5 increased survival by
25% against S. aureus compared with OP50-fed worms (** p< 0.01) (
Figure 4A,B
). Further-
more, the worms pre-cultured on LPJBC5 showed less distention of the intestinal lumen after
pathogenic exposure compared with pre-cultured worms on OP50 (
Mean ±SEM
,
20.8 ±1.38
for LPJBC5 vs. 38.26 +2.07 for pre-cultured on OP50) (** p< 0.01) (Figures 4C,D and S4B).
Figure 4.
Treatment with LPJBC5 improved the resistance of worms against biotic stresses. The
infection with pathogen Staphylococcus aureus (
A
,
B
). Feeding of LPJBC5 improved intestinal integrity
of worms against pathogen S. aureus (SA) (
C
,
D
). The intestinal cavity was observed in treated groups
(OP50 + SA and LPJBC5 + SA) under a compound microscope at 20
×
(
C
), and area (%) with blue
food dye retained in the intestinal cavity per worm was calculated using ImageJ software (Scale bar,
20
µ
m) (
D
). Error bars represent mean
±
SEM. Treatment effects were compared using Student’s
t-test (** p< 0.01 and *** p< 0.001).
3.8. Understanding the Pathway Involved in Pro-Longevity Effect of LPJBC5-Fed Worms
We firstly investigated the role of DAF-2/DAF-16 mechanism involving two loss-
of-function mutants daf-2 (longer-lived) and daf-16 (shorter-lived) of worms. The results
showed that feeding of LPJBC5 to these two mutant worms extended their longevity (daf-2,
*** p< 0.0001; daf-16, *** p< 0.0001, log-rank test) (Figure 5A,B, Table 1). This suggested that
LPJBC5-induced longevity is not dependent on the involvement of
DAF-2
/
DAF-16 pathway
.
Antioxidants 2022,11, 268 14 of 25
Figure 5.
Elucidation of pathway involved in lifespan enhancement of worms by LPJBC5. Lifespan
assays were performed with mutants (
A
)daf-2 (e1368), (
B
)daf-16 (mgDf50), (
C
)nsy-1 (ag3), (
D
)sek-1
(ag1), (
E
)pmk-1 (km25), (
F
)skn-1 (zu67) and (
G
)skn-1 (zu135). Treatment effects were compared
using log rank test.
Table 1.
The mean lifespan of wild-type and mutant strains of C. elegans fed with OP50 or probiotic
LPJBC5. N.S. corresponds to the non-significant pvalue.
Strain Bacterial Source Mean Life Span ±
SEM (Days)
Total Worms (150) =
Dead/Censored
p-Value (L. plantarum
JBC5 versus E. coli OP50)
N2 (wild-type) OP50 14.56 ±0.34 138/12 p< 0.0001 (***)
LPJBC5 18.61 ±0.48 143/7
daf-2 (e1368) OP50 19.67 ±0.45 141/9 p< 0.0001 (***)
LPJBC5 23.48 ±0.51 142/8
Antioxidants 2022,11, 268 15 of 25
Table 1. Cont.
daf-16 (mgDf50) OP50 12.54 ±0.24 139/11 p< 0.0001 (***)
LPJBC5 15.22 ±0.32 141/9
nsy-1 (ag3) OP50 13.04 ±0.28 134/16 p> 0.05 (N.S.)
LPJBC5 13.50 ±0.26 137/13
sek-1 (ag1) OP50 12.72 ±0.27 138/12 p> 0.05 (N.S.)
LPJBC5 13.13 ±0.22 140/10
pmk-1 (km25) OP50 13.51 ±0.25 142/8 p> 0.05 (N.S.)
LPJBC5 13.94 ±0.30 143/7
skn-1 (zu67) OP50 13.03 ±0.23 140/10 p> 0.05 (N.S.)
LPJBC5 13.49 ±0.27 142/8
skn-1 (zu135) OP50 13.83 ±0.19 139/11 p> 0.05 (N.S.)
LPJBC5 14.37 ±0.28 136/14
We further studied whether LPJBC5-induced longevity is due to the activation of p38
MAPK pathways. Feeding of LPJBC5 to three loss-of-function mutants of worms, i.e., sek-1,
nsy-1 and pmk-1 failed to extend longevity (p> 0.05, log-rank test) (Figure 5C–E, Table 1).
We next asked whether the downstream gene of p38 MAPK pathway, i.e., skn-1 has a role in
LPJBC5-induced longevity in worms. Two skn-1 loss-of-function allele mutants, i.e., skn-1
(zu67) and skn-1 (zu135) were fed with LPJBC5, and determination of their lifespan showed
that feeding of LPJBC5 were unable to extend longevity in either skn-1 allele mutants
(p> 0.05, log-rank test) (Figure 5F,G, Table 1).
3.9. Elucidating the Molecular Mechanisms of LPJBC5-Induced Healthy Aging in Worms
To further understand how LPJBC5 promoted healthy aging in worms, qRT-PCR was
conducted to study the expression of important genes involved in longevity, fat accumula-
tion, learning and memory, stress response, innate immunity, and intestinal integrity.
First, the expression of key genes involved in p38 MAPK and DAF-2/DAF-16 genes
was analyzed. The expression of daf-2 and daf-16 genes was unchanged in LPJBC5-fed
worms in comparison with OP50-fed (p> 0.05 for both daf-2 and daf-16) (Figure S4A). The
expression of genes involved in p38 MAPK signaling, including sek-1,nsy-1 and pmk-1 were
significantly upregulated in LPJBC5-fed worms (* p< 0.05 for sek-1 and nsy-1,
*** p< 0.001
for pmk-1) (Figures 6A and S4A). We further studied whether the feeding of LPJBC5-induced
p38 MAPK signaling activated further downstream target gene skn-1. It was observed
that the expression of skn-1 gene was increased by approximately two-fold in LPJBC5-fed
worms in comparison with OP50-fed (Figure 6A) (** p< 0.01), but there was no significant
change in the expression of the skn-1b gene, which is responsible for dietary restriction
mediated longevity in worms (p> 0.05) (Figure S4A).
Figure 6. Cont.
Antioxidants 2022,11, 268 16 of 25
Figure 6.
Expression of genes involved in longevity, stress resistance, fat accumulation, and learning
and memory (
A
); genes involved in innate immunity and tight junction (ZOO-1) proteins in gut
epithelium against infection with pathogen S. aureus (
B
). mRNA expression was normalized using the
house-keeping gene act-1. Feeding of LPJBC5 improves GSH/GSSG ratio (
C
), SOD activity (
D
) and
intracellular ROS levels (
E
) in worms compared with OP50-fed. Error bars represent
mean ±SEM
.
Treatment effects were compared using Student’s t-test (* p< 0.05, ** p< 0.01 and *** p< 0.001).
Second, the activated transcription factor SKN-1 activates the transcription of genes
involved in phase-2 detoxification. There was increased expression of GSTs (gst-4 and gst-7),
catalases (ctl-1 and ctl2), trx-1 and SODs genes (sod-1 and sod-3) in LPJBC5-fed worms in
comparison with the expression of the same genes in OP50-fed worms (
*** p< 0.001
for
sod-1
,
gst-4 and gst-7; ** p< 0.01 for ctl-1,trx-1 and sod-3; * p< 0.05 for ctl-2) (
Figures 6A and S4A
).
Moreover, there was a two-fold increase in the expression of hsp-60 and hsp-70 genes in the
LPJBC5-fed worms (*** p< 0.001 for hsp-60 and hsp-70) (Figure 6A).
Third, the mRNA expression of genes encoding key substrates and enzymes of fat
metabolism (fat-5,fat-6, and fat-7) was analyzed in LPJBC5-fed worms. Our results sug-
gested that feeding of LPJBC5 significantly decreased the expression of fat5 and fat-7
(
*p< 0.05
for fat-5; *** p< 0.001 for fat-7) (Figure 6A), although the expression of fat-6
gene was unchanged in LPJBC5 in comparison with OP50-fed worms (p> 0.05 for fat-6)
(Figure S4A).
Fourth, the role of serotonin signaling genes involved in the LPJBC5-induced learning
ability of worms, namely ser-1,mod-1, and tph-1, was investigated [
55
]. Consistent with
the results of learning and memory assays, the expression of all three serotonin-signaling
pathway genes were significantly upregulated in LPJBC5-fed worms compared with OP50-
fed (** p< 0.01 for ser-1, * p< 0.05 for mod-1 and tph-1) (Figure 6A).
Fifth, the results showed upregulation of innate immunity genes such as saponin-
like proteins (spp-7), lysozymes (lys-1 and lys-8), CLEC genes (clec-60 and clec-85) and
antibacterial factor (ABF) (abf-2 and abf-3) in LPJBC5-fed worms against infection with
pathogen S. aureus compared with OP50-fed worms (*** p< 0.001 for lys-1,lys-8,clec-60,
clec-85 and abf-2; ** p< 0.01 for spp-7 and lys-1; * p< 0.01 for abf-3) (Figures 6B and S4A).
No significant change in the expression of abf-1 and spp-1 genes was observed between the
LPJBC5- and OP50-fed worm groups (p> 0.05) (Figure S4A).
Lastly, there was a two-fold increase in expression of tight junction protein zonula
occludin zoo-1 in worms pre-cultured on LPJBC5 after exposure against pathogen S. aureus
compared with pre-cultured on OP50 (*** p< 0.001) (Figure 6B).
Antioxidants 2022,11, 268 17 of 25
3.10. Feeding of LPJBC5 Reduced the Production of Reactive Oxygen Species
The effect of LPJBC5 was examined on the ratio of glutathione/glutathione disulfide
(GSH/GSSG) in worms, which indicates an oxidative redox environment in worms. Our
results suggested that feeding of LPJBC5 increased the ratio of GSH/GSSG by approxi-
mately three-fold on day 14 compared with OP50-fed worms (GSSG, ** p< 0.01; GSH, *** p
< 0.001; GSH/GSSG *** p< 0.001) (Figures 6C and S4C). Feeding of LPJBC5 also improved
the SOD activity in day 14 worms by 57.35% compared with OP50-fed worms (** p< 0.01)
(Figure 6D).
Next, 2
0
,7
0
-dichlorofluorescein diacetate (H
2
DCFDA) was employed to detect
in vivo
cytoplasmic ROS levels in worms. There was a 44.12% decrease in the fluorescence intensity
in LPJBC5-fed worms in comparison with OP50-fed (** p< 0.01) (Figure 6E).
3.11. LPJBC5 Improved Mitochondrial Function in Worms
The JC-1 dye was used to investigate the effect of LPJBC5 on the transmembrane
potential of mitochondria. JC-1 penetrates the non-apoptotic cells forming aggregates and
shows red fluorescence, whereas JC-1 present as monomers in the apoptotic cells shows
green fluorescence. Our results suggested that LPJBC5-fed worms showed a significantly
lower level of green fluorescence and a higher level of red fluorescence intensity compared
with OP50-fed (*** p< 0.001 for both red and green fluorescence) (Figure S4D). Additionally,
the ratio of red/green fluorescence intensity was three-fold higher in LPJBC5-fed worms
compared with OP50-fed (*** p< 0.001) (Figure 7A).
Figure 7.
Figure 7.
Treatment with LPJBC5 improves mitochondrial membrane potential (
A
), ATP synthesis (
B
),
reduce mitochondrial ROS levels (
C
,
D
) (Scale bar, 100
µ
m). LPJBC5 treatment altered the expression
of mitochondrial gene and genes involved in apoptosis (
E
). LPJBC5 treatment also supressed the rate
of apoptosis (
F
) in worms. Error bars represent mean
±
SEM. Treatment effects were compared using
Student’s t-test (* p< 0.05, ** p< 0.01 and *** p< 0.001).
Antioxidants 2022,11, 268 18 of 25
We further investigated the effect of LPJBC5 on synthesis of ATP levels in worms.
LPJBC5-fed worms showed a 95.65% higher level of ATP in day-14 worms compared with
OP50-fed worms under same experimental conditions (*** p< 0.001) (Figure 7B). Next,
LPJBC5-fed worms were stained with MitoTracker Red CMXRos to analyze the level of
mitochondrial ROS. There was a 42.09% reduction in mitochondrial ROS in LPJBC5-fed
worms compared with OP50-fed (** p< 0.01) (Figure 7C,D).
In addition, we also studied the change in the expression of mitochondrial DNA
(mtDNA) encoded gene nd-1. The results showed increased expression of nd-1 in LPJBC5-
fed worms in comparison with OP50-fed worms (* p< 0.05) (Figure 7E). Overall, the
feeding of LPJBC5 significantly improved the functioning of mitochondria in the older age
of worms.
3.12. LPJBC5 Retards Programmed Cell Death in Worms
Our results showed that apoptotic cell death was reduced by 37.74% in LPJBC5-fed
worms compared with OP50-fed (** p< 0.01) (Figure 7F). In support, our qRT-PCR results
showed increased expression of anti-apoptotic gene ced-9, whereas expression of pro-
apoptotic genes (ced-3 and ced-4) was significantly reduced in LPJBC5-fed compared with
OP50-fed worms (*** p< 0.001 for ced-9; * p< 0.05 for ced-3; ** p< 0.01 for ced-4) (Figure 7E).
4. Discussion
The rapid progress in medical science has led to increasing lifespans. A recent report
of the United Nations predicts that one in every six individuals will be older than the age
of 65 by 2050 compared with one in every eleven in 2019 [
3
]. The increased life expectancy
is not proportional to the quality of life in the elderly [
3
]. Further, increased lifespan has
been linked with a higher risk of age-associated diseases, such as cancer, obesity, diabetes,
cardiovascular disease, and neurodegenerative disorder [
56
]. It results in a significant
burden on the global economy, as public expenditure has increased substantially with
increased life expectancy [
57
]. Based on the recent developments in the gut microbiome
and fermented foods, scientists are recollecting to understand many ethnic foods and their
microbes for human health. In this study, a potential probiotic bacterium L. plantarum strain
JBC5 isolated from curd was evaluated for its ability to enhance lifespan and promote
healthy aging in worms.
In our study, LPJBC5 persisted in
in vitro
simulated gastrointestinal tests and could
tolerate low pH (pH 1.0, 3.0, and 7.0) and bile acid (0.3–1.0%) without a significant decrease
in viability (Table S3). Our finding corresponds to previous studies and suggests that
LPJBC5 showed good survival ability to simulated gastric juice and bile acid [
58
]. Secondly,
LPJBC5 was also able to adhere to intestinal cell line HT-29 (Table S3). Furthermore, the
potential probiotic genes (collagen-binding protein and bile salt hydrolase) and antimicro-
bial plantaricin gene were confirmed in LPJBC5. The occurrence of collage-binding protein
in LPJBC5 supported its candidature to colonize the host’s intestine efficiently. This hints
that LPJBC5 can tolerate gastrointestinal conditions (stomach, small and large intestine) in
humans and colonize the intestinal mucosa.
Our results suggest that both live and dead probiotic LPJBC5 could extend the lifespan
of worms. Aging is a multifactorial phenomenon regulated by diverse signaling mecha-
nisms; few mechanisms were important in anti-aging process [
5
]. Several mechanisms,
for example, p38 MAPK and DAF-2/DAF-16 pathways, have been identified to extend
longevity in worms [
59
]. The mechanisms of longevity may also regulate the conserved
genes involved in stress resistance, anti-oxidative pathways, immunity, and metabolism [
59
].
In this study, it was observed that in the p38 MAPK pathway mutant worms (sek-1,nsy-1,
and pmk-1), LPJBC5 was unable to extend longevity. The activated p38 MAPK pathway
may also induce the expression of downstream transcription factor SKN-1, which regulates
several processes, such as detoxification, immune response, and metabolism. We confirmed
that the feeding of LPJBC5 to allelic skn-1 mutant worms did not extend longevity. In
contrast, the feeding of LPJBC5 to mutants of DAF-2/DAF-16 pathways (daf-2 and daf-16)
Antioxidants 2022,11, 268 19 of 25
extended the longevity. Our qRT-PCR results also increased the expression of genes in-
volved in p38 MAPK pathway and its downstream target skn-1. Nevertheless, there was
no significant change in gene expression involved in the
DAF-2
/DAF-16 pathway. Thus,
we conclude that the feeding LPJBC5 activated skn-1 through the p38 MAPK signaling
pathway to extend longevity in worms. This result is in accordance to the recent study by
Zhou et al. [60].
Previous reports suggested that longevity is not straightforward, and demonstrated
the fundamental relationship between slowed development and longevity [
61
]. Our study
found that feeding of LPJBC5 compared with feeding of OP50 slowed the developmental
rate and reduced the body size in worms. The smaller body size of worms is often linked
with increased lifespan, which is considered to be as a result of slowed developmental
rate [
41
,
62
]. Generally, longevity is accompanied by positively affecting the aging biomark-
ers in worms, including the rate of pharynx pumping, locomotory activity, and reduction
in lipofuscin accumulation [
63
]. We found that feeding of LPJBC5 improved the rate of
pharyngeal pumping and the number of body bends and showed a decrease in lipofus-
cin accumulation in worms. These results suggested that feeding of LPJBC5 effectively
improved the quality of life in the elderly.
Emerging pieces of evidence suggest that fat metabolism is tightly regulated in the
aging process [
64
]. In worms, the RNA interference (RNAi) and mutant studies have found
that silencing fat genes, including fat-6,fat-5, and fat-7, decreased the fat accumulation
under normal physiological conditions [
65
,
66
]. The over-expression of the fat-7 gene in
worms promoted fat accumulation and reduced the lifespan of transgenic worms [
67
].
Our results suggested that feeding LPJBC5 reduced the fat storage in worms compared
with OP50-fed ones. In support, we found that LPJBC5 reduced the expression of genes
encoding key substrates and enzymes of fat metabolism (fat-5 and fat-7) compared with
OP50-fed worms.
Diet can affect behavior, ranging from feeding, sensory, learning, and memory [
10
].
Our study demonstrated that adult worms had no feeding preference for LPJBC5 over OP50.
Moreover, we trained worms on LPJBC5 to assess its role in learning and memory. We
observed a higher preference of LPJBC5-trained worms for LPJBC5 over OP50, suggesting
worms pre-cultured on LPJBC5 improved their learning and memory for LPJBC5 to choose
LPJBC5 over OP50 compared with naive worms precultured on OP50. Previous studies sug-
gest that serotonin signaling modulates the behavior of worms [
68
]. Our qRT-PCR results
indicated that feeding of LPJBC5 improved the expression of genes involved in serotonin
signaling, thus suggesting a link for communication between the central nervous system
and gastrointestinal tract. The present study only provided proof that pre-conditioned or
trained worms on LPJBC5 improved learning and memory, but furthermore, a mechanistic
approach is needed to comment on its role in the gut–brain axis.
In mammals, the p38 MAPK pathway activates the transcription of genes involved
in inflammatory cytokines and antimicrobials against exposure to lipopolysaccharide
(LPS) [
69
71
]. PMK-1, a vital component of p38 MAPK, is important in in innate immune
defences of worms against pathogens [
72
]. Previous studies have suggested that the gut
pathogen S. aureus primarily colonizes and causes distention of the intestine of worms,
then kills the worms in 5–7 days [
73
]. We observed that LPJBC5 improved the survival
rate of worms against infection with S. aureus. We confirmed that feeding of LPJBC5
over-expressed the pmk-1.
Additionally, LPJBC5 also increased the expression of genes for antimicrobial proteins
and peptides (lys-1,lys-8,clec-60,clec-85,abf-2 and spp-7). The innate immunity of C. elegans
involves antimicrobial peptides (defensin-like peptides) and proteins (lysozymes, caenacins,
saponin like proteins, C-type lectin domain-containing (CLEC) proteins) [
11
]. Lysozymes
cleaves the chemical bond between N-acetylmuramic (NAM) and N-acetylglucosamine
(NAG) acid. In a previous study, lysozyme expression in worms, including lys-1 and
lys-8, limited the accumulation of the pathogen (Serratia marcescens) in their intestine [
74
].
Saponin-like protein (spp.) are N-terminal small lysosomal proteins containing more than
Antioxidants 2022,11, 268 20 of 25
35 amino acid residues which form pores into the pathogenic membranes. Silencing of
Saponin-like protein genes decreased the bacterial load in their intestine [
75
]. CLEC proteins
are carbohydrate-binding protein domain known to perform several functions, especially
cell to cell adhesion and immune response against pathogen. The study conducted by
Miltsch et al. [
76
] reported that knockdown of clec genes (clec-39 and clec-49) reduced
survivability of C. elegans against pathogen Serratia marcescens [
76
]. Antibacterial factor
(abf ) or defensin-like peptides are cysteine-rich cationic proteins, which perform important
role in creating voltage-dependent channel in cell membrane of pathogen and disrupts it.
Knockdown of abf-2 by RNAi decreased the survival of C. elegans by allowing the increase
in microbial growth (Salmonella typhimurium) in the intestine [77].
In support, we observed that LPJBC5 counteracted the disruption of intestinal epithe-
lium caused by the pathogen S. aureus. The impaired intestinal barrier is characterized by
the loss of the epithelial tight junction. In particular, zonula occludin ZOO-1 (human ZO-1
homolog) is a tight junction protein and acts as a representative marker for tight junction
integrity [
78
]. Mechanistically, we found that feeding of LPJBC5 increased the expression
of a gene encoding for tight junction proteins, zoo-1. Some other probiotic bacteria, such as
Bifidobacterium bifidium have also been shown to strengthen the tight junction of intestinal
epithelium in epithelium monolayer models [79].
Previous research suggested that ROS level increases with age and plays a role in
more than 60 diseases, such as rheumatoid arthritis, neurodegenerative diseases, and
gastrointestinal diseases [
80
]. Notably, the cellular redox state is commonly determined
by the level of SOD activity and GSH/GSSG ratio [
81
]. It was observed that there was an
increase in SOD activity and GSH/GSSG ratio in LPJBC5-fed compared with
OP50-fed
worms, thereby reducing the accumulation of ROS levels. In addition, we found that
feeding of LPJBC5 increased the overall survival rate of worms under oxidative and heat-
stressed conditions. In support, the feeding of LPJBC5 upregulated the expression of
essential anti-oxidative genes (sod-1,sod-3, ctl-1,ctl2, gst-4,gst-7, and trx-1) as well as genes
encoding heat-shock proteins (HSPs) (hsp-60 and hsp-70). The over-expressed HSPs may
counteract the protein misfolding due to changes in cellular redox state [82,83].
Mitochondria are the primary source for the production of ROS within cells. The
mitochondrial free radical theory of aging (MFRTA) proposed that an imbalance between
ROS and cellular defense mechanisms may induce mitochondrial dysfunction, resulting in
the production of more ROS, which further leads to aging and cell death [
84
]. The decline
in mitochondrial function has been correlated with developing different pathologies such
as metabolic disorders, type-2 diabetes, and cancer [
85
]. We found that feeding LPJBC5
decreased the production of mitochondrial ROS and improved mitochondrial membrane
potential compared with OP50-fed worms. The mitochondrial electrochemical gradient is a
crucial bioenergetic parameter to access the mitochondrial function that further controls
ATP synthesis. In addition, the feeding of LPJBC5 also upregulated the expression of
mitochondrial DNA (mtDNA)-encoded NADH-ubiquinone oxidoreductase chain 1 (nd-1).
As a result, LPJBC5 increased the level of ATP synthesis compared with OP50-fed worms.
Notably, the higher mitochondrial dysfunction is often correlated with an increased
apoptosis rate or programmed cell death [
86
]. We also confirmed that LPJBC5 reduced
apoptotic cell death in day-17 worms compared with OP50-fed. In support of this obser-
vation, the qRT-PCR results showed that pro-apoptotic gene expression was significantly
reduced, whereas anti-apoptotic gene expression was increased in LPJBC5-fed worms. This
result suggests that LPJBC5 effectively improved mitochondrial function and reduced the
rate of apoptosis in worms.
5. Conclusions
Over a century ago, Dr. Elie Metchnikoff hypothesized that lactic acid bacteria pro-
mote longevity and healthy aging in humans because Bulgarians consumed a sufficient
amount of yogurt in their diet. Recent research suggests that similar longevity and aging
mechanisms operate in C. elegans. Our study suggests that feeding of LPJBC5 activates
Antioxidants 2022,11, 268 21 of 25
the p38 MAPK pathway and its downstream targets to enhance longevity by improving
stress resistance and immunity and other age-associated pathologies in worms (Figure 8).
We believe that LPJBC5 has great potential to promote longevity and healthy aging, and
prevent several age-associated disorders in mammals; thus, it should be promoted as a
next-generation probiotic.
Antioxidants 2022, 11, x FOR PEER REVIEW 21 of 26
Mitochondria are the primary source for the production of ROS within cells. The mi-
tochondrial free radical theory of aging (MFRTA) proposed that an imbalance between
ROS and cellular defense mechanisms may induce mitochondrial dysfunction, resulting
in the production of more ROS, which further leads to aging and cell death [84]. The de-
cline in mitochondrial function has been correlated with developing different pathologies
such as metabolic disorders, type-2 diabetes, and cancer [85]. We found that feeding
LPJBC5 decreased the production of mitochondrial ROS and improved mitochondrial
membrane potential compared with OP50-fed worms. The mitochondrial electrochemical
gradient is a crucial bioenergetic parameter to access the mitochondrial function that fur-
ther controls ATP synthesis. In addition, the feeding of LPJBC5 also upregulated the ex-
pression of mitochondrial DNA (mtDNA)-encoded NADH-ubiquinone oxidoreductase
chain 1 (nd-1). As a result, LPJBC5 increased the level of ATP synthesis compared with
OP50-fed worms.
Notably, the higher mitochondrial dysfunction is often correlated with an increased
apoptosis rate or programmed cell death [86]. We also confirmed that LPJBC5 reduced
apoptotic cell death in day-17 worms compared with OP50-fed. In support of this obser-
vation, the qRT-PCR results showed that pro-apoptotic gene expression was significantly
reduced, whereas anti-apoptotic gene expression was increased in LPJBC5-fed worms.
This result suggests that LPJBC5 effectively improved mitochondrial function and re-
duced the rate of apoptosis in worms.
5. Conclusions
Over a century ago, Dr. Elie Metchnikoff hypothesized that lactic acid bacteria pro-
mote longevity and healthy aging in humans because Bulgarians consumed a sufficient
amount of yogurt in their diet. Recent research suggests that similar longevity and aging
mechanisms operate in C. elegans. Our study suggests that feeding of LPJBC5 activates the
p38 MAPK pathway and its downstream targets to enhance longevity by improving stress
resistance and immunity and other age-associated pathologies in worms (Figure 8). We
believe that LPJBC5 has great potential to promote longevity and healthy aging, and pre-
vent several age-associated disorders in mammals; thus, it should be promoted as a next-
generation probiotic.
Figure 8.
Proposed mechanisms of healthy aging induced by LPJBC5 in worms. Feeding of LPJBC5
activates p38 MAPK signaling cascade (nsy-1-sek-1-pmk-1), which further activates the downstream
SKN-1 transcription factor. The activated SKN-1 further activates the transcription of phase-II
detoxification genes or antioxidative genes. These upregulated antioxidative genes extend longevity,
increasing the resistance against stress (oxidative and heat) and infection against pathogen S. aureus.
The improved antioxidative machinery increases mitochondrial function and ATP synthesis, thereby
reducing apoptosis in worms. Feeding of LPJBC5 also reduces fat storage through downregulation of
genes encoding key substrates and enzymes of fat metabolism, including fat-5 and fat-7. In addition,
the learning and memory of worms were enhanced in LPJBC5-fed worms by upregulating the genes
involved in serotonin signaling. Therefore, feeding of LPJBC5 activates the skn-1 induced pathways
to extend longevity, stress resistance, and immunity, and reduces other age-associated pathologies.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/antiox11020268/s1. Figure S1: Neighbour-joining tree was based
on 16S rRNA gene sequences (1297 bases) of L. plantarum JBC5 (Accession no.: MG824976.1) and
other related species of Lactobacillus. Bootstrap values (expressed as percentages of 1000 replications)
greater than 60% are given at nodes. GenBank accession number are provided in the parentheses
of each strain. Enterococcus faecium ATCC 19434 (Accession no.: DQ411813.1) was used as an out-
group. The evolutionary distances were computed using the Kimura 2-parameter method in MEGA
7 software, representing the number of base substitutions per site. The bar represents 0.01 substi-
tutions per nucleotide position. Figure S2: A. Figure S2 A An agarose gel image of PCR amplified
products of Lactobacillus plantarum specific antimicrobial plantaricin-biosynthetic gene (Pln) PlnF/R
(
lane 1: ~231 bp
) (Acc. no.: MW846638), species-specific segment PlantarumF/R (lane 2: ~152 bp)
(Acc. no.: MW846639), probiotic marker genes (i.e., bile salt hydrolase (Lpbsh1) (lane 3: ~975 bp)
(Acc. no.: MW846636) and collagen-binding protein (Lpcbp) (lane 4: ~2174) (Acc. no.: MW846637)
B. Effect
of LPJBC5 on the body size of worms (*** p< 0.0001, log-rank test). C and D. The coloniza-
tion efficiency and brood size were analyzed after feeding OP50 or LPJBC5. Error bars represent
mean ±SEM.
Treatment effects were compared using Student’s t-test (** p< 0.01 and *** p< 0.001).
Figure S3: Accumulation of lipofuscin (i.e. a measure of senescence) with and without exposure to
oxidative stress (100 mM paraquat) was observed under a confocal microscope at 10
×
magnification
Antioxidants 2022,11, 268 22 of 25
(Scale bar, 100
µ
m). Figure S4: A. qRT-PCR analysis on the expression of genes involved in longevity,
stress resistance, immunity, and fat accumulation. B. The intestinal integrity of worms was observed
in control groups OP50 and LPJBC5 under a compound microscope at 20
×
(Scale bar, 20
µ
m). The
feeding of LPJBC5 improves GSH, reduces GSSG level (C), improves mitochondrial membrane
potential in worms (D). Error bars represent mean
±
SEM. Treatment effects were compared using
Student’s t-test (* p< 0.05, ** p< 0.01 and *** p< 0.001). Table S1: Primers used to characterize the
LPJBC5. Table S2: Primer sets used for qRT-PCR analysis. Table S3: Survival and adhesion of LPJBC5
under
in vitro
simulated gastro-intestinal conditions. Table S4: Closest gene homologs of LPJBC5 in
the NCBI database.
Author Contributions:
Conceptualization, A.K. and M.R.K.; methodology, A.K. and M.R.K.; software,
A.K.; validation, A.K., M.R.K. and A.K.M.; formal analysis, A.K.; investigation, A.K., T.J. and S.D.;
resources, A.K. and M.R.K.; data curation, A.K.; writing—original draft preparation, A.K.; writing—
review and editing, A.K., M.R.K., A.K.M. and M.C.K.; visualization, A.K. and M.R.K.; supervision,
M.R.K., A.K.M. and M.C.K.; project administration, M.R.K.; funding acquisition, M.R.K. All authors
have read and agreed to the published version of the manuscript.
Funding:
This work was funded by institute of advanced study in science and technology (IASST)
and Department of science and technology (DST)-funded ST/SC community development project
(SEED/TITE/2019/103) at IASST.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All relevant data are provided in the manuscript and supplementary
file. Additionally, the DNA sequences including species-specific sequence (Plantarum), probiotic
marker genes (encode bile salt hydrolase (Lpbsh1) and collagen-binding protein (Lpcbp)) and the
antimicrobial plantaricin-biosynthetic gene (pln) of LPJBC5 used in our study are publicly available in
National Center for Biotechnology Information, Bethesda, USA (NCBI) database. The raw fastq files
of these sequences can be accessed through the GenBank accession number MW846636- MW846639
in the link https://www.ncbi.nlm.nih.gov/nuccore/.
Conflicts of Interest: The authors declare no conflict of interest.
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... Some studies have focused on the ability of LAB to protect C. elegans from pathogenic bacteria, such as Salmonella enterica serovar Enteritis (35), Salmonella enterica serovar Typhimurium (32), Yersinia enterocolitica (36), Legionella pneumophila (37), E. coli (38), and Staphylococcus aureus and E. coli O157:H7 (39). Quite recently, Kumar et al. (40), employed C. elegans to explore the probiotic potential of a Lactobacillus plantarum strain (LPJBC5). Those authors concluded that this strain was able to increase worm lifespan, improve stress resistance and promote a series of traits associated with healthy aging, such as physical and cognition performance, fat accumulation, gut integrity, or mitochondrial function. ...
... The observed effects were suggested to be mediated by the activation of the p38 MAPK signaling pathway and downstream targets, such as the SKN-1 transcription factor, upregulating the expression of stress resistance genes. Furthermore, LPJBC5 was also able to downregulate fat-5 and fat-7 genes modulating fat metabolism, as well as to upregulate genes involved in serotonin signaling (ser-1, tph-1, and mod-1) related to improved cognitive function (40). ...
... In earlier studies, it was shown that rather than a direct antioxidant effect, the ability of EC and Quer to improve the resistance against oxidative stress in C. elegans might involve the modulation of transcription factors and genes in molecular pathways related to the endogenous mechanisms of defense, such as the insulin/IGF-1 signaling and MAPK pathways (48, 50, 51). In a recent paper, Kumar et al. (40) reported that L. plantarum LPJBC5 strain was able to increase the lifespan and improve stress resistance in C. elegans through the activation of the p38 MAPK signaling pathway, upregulating the expression of stress resistance genes. The results obtained in the present work seem to suggest that not only the potential ability of L. plantarum to interfere on key molecular pathways but also its interaction with flavonoids could be determining to interpret the effects observed herein regarding resistance against oxidative stress. ...
Article
Full-text available
Introduction Increasing evidence supports the role of gut microbiota in many aspects of human health, including immune, metabolic and neurobehavioral traits. Several studies have focused on how different components of the diet, such as polyphenols, can modulate the composition and function of the gut microbiota leading to health benefits. Methods The effects on the resistance against thermally induced oxidative stress of C. elegans grown in the presence of flavonoids (quercetin or epicatechin) and fed different probiotic strains, namely Lactobacillus plantarum CLC17, Bifidobacterium longum NCIMB 8809 and Enterococcus faecium CECT 410, were explored. Results Feeding C. elegans with the assayed bacteria in the absence of flavonoids did not significantly affect body size and fertility of the worms neither improve their resistance against oxidative stress compared to E. coli controls. However, increased resistance to stress was found when C. elegans was cultivated in the presence of both L. plantarum and flavonoids, but not with B. longum or E. faecium . An exploratory study revealed the presence of glycosylated and sulfated metabolites together with the aglycone in worms treated with quercetin and fed any of the different assayed LAB strains. However, in the assays with epicatechin a differential metabolite, tentatively identified as 5-(4′-hydroxyphenyl)-γ-valerolactone 3′- O -glucoside, was detected in the worms fed L. plantarum but not with the other bacteria. Conclusion The obtained results indicated that the interactions bacteria/polyphenol play a key role in the effects produced in C. elegans regarding resistance against oxidative stress, although those effects cannot be only explained by the ability of bacteria to metabolize polyphenols, but other mechanisms should also be involved.
... Previous studies stated that the population of beneficial bacteria (i.e., lactobacilli) is reducing as the age of people increases, and the administration of lactobacilli provides health outcomes in the aged people by improving gut health and immune-modulatory actions and by regulating the composition of the gut mycobiome [43][44][45]. Probiotics may positively impact healthy aging through several mechanistic ways, such as by reducing oxidative stress, improving cognition and inflammatory responses, reducing fat accumulation, reducing pathogenic protections, and alleviating metabolic illnesses [46]. For example, a recent study reported the positive impact of probiotics on the healthy aging of Caenorhabditis elegans [46]. ...
... Probiotics may positively impact healthy aging through several mechanistic ways, such as by reducing oxidative stress, i