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Article
Comparison of Inhibitory Capacities of 6-, 8-
and 10-Gingerols/Shogaols on the Canonical
NLRP3 Inflammasome-Mediated IL-1β Secretion
Su-Chen Ho * and Yi-Huang Chang
Department of Food Science, Yuanpei University of Medical Technology, Hsinchu 300, Taiwan;
yihuang@mail.ypu.edu.tw
* Correspondence: sche@mail.ypu.edu.tw; Tel.: +886-3-538-1183 (ext. 8496)

Received: 24 January 2018; Accepted: 20 February 2018; Published: 21 February 2018

Abstract: Endogenous noninfectious substances that mediate the nucleotide oligomerization domain
(NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and
interleukin (IL)-1β secretion causes inappropriate sterile inflammation and is implicated in the
pathogenesis of several chronic diseases, such as type 2 diabetes mellitus, gout, atherosclerosis
and Alzheimer’s disease. Consequently, dietary phytochemicals exhibiting capacities to suppress
canonical NLRP3 inflammasome-mediated IL-1β secretion can be a reliable supplement to prevent
such diseases. The purpose of this study was to investigate and compare the inhibitory effects
of ginger phytochemicals, including 6-, 8- and 10-gingerols/shogaols on the canonical NLRP3
inflammasome-mediated IL-1β secretion in THP-1 macrophages with ordered stimulations of
lipopolysaccharide (LPS) and adenosine 50 -triphosphate (ATP). At 20 µM, the 10-gingerol and all the
shogaols significantly inhibited canonical IL-1β secretion. The shogaols had a more potent inhibitory
capacity than that of corresponding gingerols. Increase of alkyl chain length impacted negatively the
inhibitory activity of shogaols. Additionally, these effective ginger phytochemicals not only inhibited
the LPS-primed expression of pro-IL-1β and NLRP3, but also decreased ATP-activated caspase-1.
The results demonstrated that ginger phytochemicals, especially the most potent, 6-shogaol, might
be promising for developing as an inhibitor of the canonical NLRP3 inflammasome-mediated IL-1β
secretion and further applied in prevention of NLRP3 inflammasome-associated diseases.

Keywords: gingerols; shogaols; NLRP3 inflammasome; caspase-1; IL-1β

1. Introduction
Inflammation is an essential innate immunological response of a host to infection and injury.
However, long-term uncontrolled or inappropriate inflammation causes tissue damage, and eventually,
results in disease occurrence. Interleukin (IL)-1β is a master proinflammatory cytokine that exerts
systemic inflammatory or local immunological actions such as severing as a pyrogen to elevate
temperature and enhance leucocyte migration, inducing the expression of acute phase proteins and
adhesion molecules to recruit leucocytes as well as orchestrating the functions and differentiation of
innate and adaptive lymphoid cells, and consequently, to affect all cells and organs [1–4]. The canonical
secretion of IL-1β is rigorously controlled by a unique two-signal process. In response to the first
stimulation of pathogen-associated molecular patterns (PAMPs), such as the lipopolysaccharide (LPS),
macrophages are primed to trigger the intracellular NF-κB signaling cascade that further induces the
expression of immature precursor of IL-1β (pro-IL-1β) and the nucleotide oligomerization domain
(NOD)-like receptor family pyrin domain-containing 3 (NLRP3), a cytosolic pattern recognition
receptor. Upon the second stimulation of PAMPs or damage-associated molecular patterns (DAMPs),
NLRP3 recruits the adaptor molecules—apoptosis-associated speck-like containing CARD (ASC) and

Molecules 2018, 23, 466; doi:10.3390/molecules23020466 www.mdpi.com/journal/molecules


Molecules 2018, 23, 466 2 of 12

pro-caspase-1—to assemble an inflammasome complex; consequently, caspase-1 is activated and


converts pro-IL-1β into mature IL-1β [5]. NLRP3 inflammasome responds to various endogenous
DAMPs such as extracellular ATP, uric acid crystal, cholesterol crystal and β-amyloid plaque,
and produces redundant IL-1β and brings so-called sterile inflammation. Such sterile inflammation
is implicated in the pathogenesis of several noncommunicable diseases including type 2 diabetes,
arthritis, gout, atherosclerosis, and Alzheimer’s disease. Hence, it has been reasonably recognized
as an effective therapeutic strategy to prevent sterile inflammation associated diseases by blocking
the canonical NLRP3 inflammasome-mediated IL-1β secretion [6–8]. Extracellular ATP released from
necrotic cells triggers activation of NLRP3 inflammasome and IL-1β secretion via binding to the
purinergic 2X7 (P2X7) receptor, causes sterile inflammation and contributes to the pathogenesis of
ischemic diseases [9,10]. High concentrations of ATP (5 mM) strongly elicits the processing of pro-IL-1β
into mature IL-1β in LPS-primed macrophages [11]. Consequently, the LPS-primed and ATP-activated
THP-1 macrophage cell model has been widely adopted to evaluate the efficacy of the proposed IL-1β
inhibitors [12–14].
Gingerols and their dehydrated derivatives, shogaols, are identified as the main bioactive
components in fresh and dried gingers, respectively. Gingerols with a thermally labile beta-hydroxy
keto group that can readily undergo dehydration to form the corresponding shogaols with a common
α,β-unsaturated carbonyl moiety (Figure 1a). The structure–activity relationship and the molecular
mechanism underlying the anti-inflammatory capacity of gingerols/shogaols has been deeply explored
by using the LPS-induced cell culture model [15–18], however, the information regarding their
effect on NLRP3 inflammasome-mediated sterile inflammation is still obscure. Consequently, 6-, 8-,
10-gingerols/shogaols were selected to evaluate and compare their suppressing capacities on the
canonical NLRP3 inflammasome-mediated IL-1β secretion in THP-1 macrophages by using LPS
and ATP as first and second stimulations, respectively, to prime inflammatory nuclear factor
(NF)-κB signaling pathway and to activate NLRP3 inflammasome. Additionally, in response to LPS,
macrophages produce great amount of proinflammatory cytokines, including tumor necrosis factor
(TNF)-α, IL-1β, and IL-6. The LPS-induced expression of proinflammatory cytokines is predominantly
regulated at transcriptional level by NF-κB. TNF-α is another master proinflammatory cytokine that
orchestrates the production of a proinflammatory cytokine cascade, affects inflammatory cell activation
and recruitment, and plays a key role in the development of many chronic inflammatory diseases [19].
Thereby, TNF-α secretion and expression was used as a reference herein to observe the effect of3 of
Molecules 2018, 23, 466
ginger
12
phytochemicals on LPS-priming step.

140.0 vehicle 6-gingerol 8-gingerol 10-gingerol


6-shogaol 8-shogaol 10-shogaol
Cell viability (% of vehicle control)

120.0
d d
c
100.0
b b
80.0

60.0
a a

40.0

20.0

0.0
40 μΜ 20 μΜ 10 μΜ 5 μΜ

(a) (b)
Figure 1. Chemical structure (a) and cytotoxic effect (b) of ginger phytochemicals. The phorbol 12-
Figure 1. Chemical structure (a) and cytotoxic effect (b) of ginger phytochemicals. The phorbol
12-myristate 13-acetate (PMA)-differentiated
myristate 13-acetate (PMA)-differentiatedTHP-1THP-1macrophages
macrophageswere weretreated
treatedwith
withginger
ginger
phytochemicals
phytochemicals atatconcentrations
concentrations of
of 5~40
5~40 μM
µMforfor2424h.h.The cellcell
The viabilities were
viabilities thenthen
were assessed with 3-
assessed with
(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Data are expressed
3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Data are expressed as the as the
mean ± SD of triplicate independent experiments. Values of the ginger phytochemicals at
mean ± SD of triplicate independent experiments. Values of the ginger phytochemicals at concentration
concentration of 40 μM with different superscript letters are significant different from one another (p
of 40 µM with different superscript letters are significant different from one another (p < 0.05).
< 0.05).

In order to explore whether ginger phytochemicals inhibit the canonical NLRP3-mediated IL-1β
secretion, THP-1 macrophages were treated with ginger phytochemicals before being LPS-primed
and ATP-activated. Figure 2a shows the effect of pretreatment of ginger phytochemicals on IL-1β
secretion in THP-1 macrophages. As expected, IL-1β secretion dramatically increased after LPS-
priming for 4h and ATP-activation for 3h. All the tested ginger phytochemicals at concentrations of
Cell viability (% of
60.0
a a

40.0

20.0

0.0
Molecules 2018, 23, 466 3 of 12
40 μΜ 20 μΜ 10 μΜ 5 μΜ

(a) (b)
2. Results
Figure 1. Chemical structure (a) and cytotoxic effect (b) of ginger phytochemicals. The phorbol 12-
2.1. Inhibitory
myristate Capacities (PMA)-differentiated
13-acetate of Ginger PhytochemicalsTHP-1
on LPS-Primed and ATP-Activated
macrophages were treated IL-1β Secretion in
with ginger
THP-1 Macrophages
phytochemicals at concentrations of 5~40 μM for 24 h. The cell viabilities were then assessed with 3-
To avoid the cytotoxicity caused
(4,5-dimethyldiazol-2-yl)-2,5-diphenyl by thebromide
tetrazolium overload
(MTT) of method.
tested Data
ginger phytochemicals,
are expressed as the
the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl
mean ± SD of triplicate independent experiments.tetrazolium bromide
Values of (MTT)
the assay
gingerwas first performed at
phytochemicals to
determine the optimal treating concentrations of the ginger phytochemicals. As presented
concentration of 40 μM with different superscript letters are significant different from one another (p in Figure 1b,
10-gingerol
< 0.05). and all shogaols at a concentration of 40 µM had a significant cytotoxicity compared with
vehicle control. On the other hand, at 20 µM, all of the tested ginger phytochemicals did not cause
cellorder
In death.toBased on whether
explore the aboveginger
findings, the modulatoryinhibit
phytochemicals capacities of gingerols/shogaols
the canonical NLRP3-mediated on IL-1β IL-1β
secretion were evaluated at concentrations ≤ 20 µM.
secretion, THP-1 macrophages were treated with ginger phytochemicals before being LPS-primed
In order to explore whether ginger phytochemicals inhibit the canonical NLRP3-mediated IL-1β
and ATP-activated. Figure 2a shows the effect of pretreatment of ginger phytochemicals on IL-1β
secretion, THP-1 macrophages were treated with ginger phytochemicals before being LPS-primed and
secretion in THP-1 macrophages. As expected, IL-1β secretion dramatically increased after LPS-
ATP-activated. Figure 2a shows the effect of pretreatment of ginger phytochemicals on IL-1β secretion
priming
in THP-14hmacrophages.
for and ATP-activation for 3h.
As expected, Allsecretion
IL-1β the tested ginger phytochemicals
dramatically increased after at concentrations
LPS-priming for of
5~20 4h
μM, andexcept 6-gingerolfor
ATP-activation and
3h.8-gingerol, inhibited
All the tested ginger LPS-primed
phytochemicals and at ATP-activated
concentrations of IL-1β
5~20secretion
µM,
in a dose-dependent
except 6-gingerol manner. Notably,
and 8-gingerol, 6-shogaol
inhibited at concentrations
LPS-primed of 10 and
and ATP-activated 20 μM
IL-1β as well
secretion in aas 8-
shogaol at a concentration
dose-dependent manner.ofNotably,
20 μM 6-shogaol
completely blocked IL-1βofsecretion.
at concentrations 10 and 20 µMTheasinhibitory capacity of
well as 8-shogaol
at ginger
tested a concentration of 20 µM was
phytochemicals completely
rankedblocked IL-1β secretion.
as 6-shogaol (102.2%) The inhibitory(95.2%)
> 8-shogaol capacity>of10-gingerol
tested
ginger phytochemicals was ranked as 6-shogaol (102.2%) > 8-shogaol
(63.6%) > 10-shogaol (46.5%) in descending order from high to low. In addition, due (95.2%) > 10-gingerol (63.6%) > to
10-shogaol (46.5%) in descending order from high to low. In addition, due
proinflammatory TNF-α expression/secretion primarily depends on the LPS-primed NF-κB signaling to proinflammatory
TNF-α expression/secretion primarily depends on the LPS-primed NF-κB signaling pathways.
pathways. TNF-α secretion was used as a reference herein to observe the effect of ginger
TNF-α secretion was used as a reference herein to observe the effect of ginger phytochemicals
phytochemicals on LPS-priming step. Apparently, as shown in Figure 2b, except for 6-gingerol, the
on LPS-priming step. Apparently, as shown in Figure 2b, except for 6-gingerol, the other ginger
other ginger phytochemicals diminished dose-dependently TNF-α secretion. These results implied
phytochemicals diminished dose-dependently TNF-α secretion. These results implied that ginger
that phytochemicals,
ginger phytochemicals, especially
especially 6-shogaol, 6-shogaol,
exhibit potent abilityexhibit potent
to inhibit NLRP3ability to inhibit NLRP3
inflammasome-mediated
inflammasome-mediated
IL-1β secretion and thisIL-1β secretion
inhibitory and
capacity thisbeinhibitory
might attributed,capacity might to
at least partially, betheir
attributed, at least
suppressing
partially,
effectto
ontheir suppressingstep.
the LPS-priming effect on the LPS-priming step.

Conc. Inhibition rate (%)


20 μM 100.0 0.0 -7.8 1.8 63.6 102.2 95.2 46.5
10 μM -7.2 7.6 40.1 97.8 47.2 10.1
5 μM 2.8 4.1 28.9 49.1 13.0 -14.7
6000
20 μΜ 10 μΜ 5 μΜ
5000
e
IL-1β secretion (pg/mL)

de d
4000

3000
c
2000
b

1000
a
a a
0

-LPS/-ATP +LPS/+ATP

(a)

Figure 2. Cont.
Molecules 2018, 23, 466 4 of 12
Molecules 2018, 23, 466 4 of 12

Conc. Inhibition rate (%)


20 μM 100.0 0.0 9.6 65.1 99.8 100.1 99.3 97.6
10 μM 14.8 27.9 87.1 95.8 77.9 37.5
5 μM 17.7 15.8 60.8 42.2 55.0 22.7
Molecules 2018, 23, 466 2500 4 of 12
20 μΜ 10 μΜ 5 μΜ

Conc.
2000 c c Inhibition rate (%)

TNF-α secretion (pg/mL)


20 μM 100.0 0.0 9.6 65.1 99.8 100.1 99.3 97.6
10 μM 14.8 27.9 87.1 95.8 77.9 37.5
1500
5 μM 17.7 15.8 60.8 42.2 55.0 22.7

2500
1000 20 μΜ 10 μΜ 5 μΜ

b
2000 c c
TNF-α secretion (pg/mL)

500

1500
a a a a
a
0

1000

b
500
-LPS/-ATP +LPS/+ATP
a a a a
a
0 (b)
Figure 2. Effect of ginger phytochemicals on the LPS-primed and ATP-activated IL-1β (a) and TNF-α
Figure 2. Effect of ginger phytochemicals on the LPS-primed and ATP-activated IL-1β (a) and TNF-α
(b) secretion in THP-1 macrophages.
-LPS/-ATP +LPS/+ATP 13-acetate (PMA)-differentiated THP-
The phorbol 12-myristate
(b) secretion in THP-1 macrophages. The phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1
1 macrophages were treated with ginger phytochemicals (b) for 1 h. Then, the cells were primed with
macrophages were treated with ginger phytochemicals for 1 h. Then, the cells were primed with LPS
LPS for 4h and activated with 5 mM ATP for 3 h. The medium IL-1β and TNF-α concentration was
for 4h and
Figure
assessedactivated
2.
byEffect with 5 mM
of ginger
an enzyme-linked ATP for 3 h.on
phytochemicals
immunosorbent The
themedium
LPS-primed
assay IL-1β
(ELISA) andand TNF-α concentration
ATP-activated
kit. Data IL-1β (a)
are expressed thewas
asand assessed
TNF-α
mean ±
(PMA)-differentiated
SD of triplicate independent experiments. Values of the ginger phytochemicals at concentration of±
by an (b) secretion in THP-1
enzyme-linked macrophages. The
immunosorbent phorbol
assay 12-myristate
(ELISA) kit. 13-acetate
Data are expressed as the THP-
mean 20SD of
1 macrophages
triplicate
μM with differentwere
independent treated with ginger
experiments.
superscript lettersValues phytochemicals
of the ginger
are significant for
different 1 h. Then, the cells
phytochemicals
from were primed with
one anotherat(pconcentration
< 0.05). of 20 µM
LPS for 4h
with different and activated
superscript with are
letters 5 mM ATP for 3different
significant h. The medium IL-1βanother
from one and TNF-α
(p <concentration
0.05). was
assessed by an enzyme-linked immunosorbent assay (ELISA) kit. Data are expressed as the mean ±
2.2. Effect of Ginger Phytochemicals on Pro-IL-1β, NLRP3, Pro-Caspase-1 and Caspase-1 Protein Expression
SD of triplicate independent experiments. Values of the ginger phytochemicals at concentration of 20
in LPS-Primed
2.2. Effect ofμMGinger and ATP-Activatedon
Phytochemicals THP-1 Macrophages
Pro-IL-1β, NLRP3, Pro-Caspase-1 and Caspase-1
with different superscript letters are significant different from one another (p < 0.05). Protein Expression in
LPS-Primed and ATP-Activated
To investigate THP-1phytochemicals
whether ginger Macrophages affect the protein levels of pro-IL-1β, NLRP3, pro-
2.2. Effectand
caspase-1 of Ginger
active Phytochemicals
caspase-1, on Pro-IL-1β,
cells were treated NLRP3, Pro-Caspase-1
with ginger and Caspase-1 Protein Expression
To investigate whether ginger phytochemicals affect the phytochemicals
protein levels before being LPS-
of pro-IL-1β, NLRP3,
in LPS-Primed and ATP-Activated THP-1 Macrophages
primed and ATP-activated. As shown in Figure 3, priming macrophages with LPS significantly
pro-caspase-1 and active caspase-1, cells were treated with ginger phytochemicals before being
enhancedToandthe proteinwhether
investigate expressions
ginger ofphytochemicals
pro-IL-1β and NLRP3. affect theAtprotein
a concentration of 20 μM,NLRP3,
levels of pro-IL-1β, the gingerols
pro-
LPS-primed ATP-activated. As shown in Figure 3, priming macrophages with LPS significantly
and
caspase-1 and active caspase-1, cells were treated with ginger phytochemicals before protein
shogaols, except for 6-gingerol, significantly decreased the LPS-primed pro-IL-1β being LPS- level
enhanced themacrophages.
inprimed
THP-1 protein expressionsThe abilityof pro-IL-1β
of these and NLRP3. At a concentration of 20expression
µM, the gingerols
and ATP-activated. As shown incompounds to suppress
Figure 3, priming pro-IL-1βwith
macrophages protein
LPS significantly was
and shogaols,
ranked except
as 6-shogaol
enhanced for(72.7%)
the protein 6-gingerol, ofsignificantly
and 8-shogaol
expressions pro-IL-1β anddecreased
(70.8%) At athe
≥ 10-gingerol
NLRP3. LPS-primed of 20pro-IL-1β
(62.9%) > 10-shogaol
concentration μM, the(41.0%)protein
gingerols > 8- level
in THP-1
and macrophages.
gingerol (27.2%)except
shogaols, The6-gingerol,
in the for
orderability
from high ofsignificantly
these
to low compounds
(Figure to
thesuppress
3b). Although
decreased pro-IL-1β
the suppressing
LPS-primed pro-IL-1β protein
extent expression
of ginger
protein level
was ranked
in THP-1as macrophages.
phytochemicals6-shogaol
on the (72.7%)
NLRP3 andof8-shogaol
protein
The ability was weaker
these (70.8%)
compounds ≥suppress
thantothose10-gingerol
on the (62.9%)
pro-IL-1β
pro-IL-1β > 10-shogaol
protein,
protein the rankings
expression (41.0%) >
was
ofranked
ability
8-gingerol of 6-shogaol
as
(27.2%) these compounds
in the (72.7%)
order fromto inhibit
and high both
8-shogaol
to low proteins
(70.8%) showed
≥ 10-gingerol
(Figure similar
3b). Although trend.
(62.9%) On the other
the> suppressing
10-shogaol hand,
(41.0%)
extent theginger
> of
8-
pro-caspase-1
gingerol level
(27.2%) inwas
the decreased
order from and
high the
to active
low caspase-1
(Figure 3b). level
Although was
phytochemicals on the NLRP3 protein was weaker than those on the pro-IL-1β protein, the rankings elevated
the after
suppressing LPS-priming
extent of gingerand
ATP
of ability activation.
phytochemicals Pretreatment
on the NLRP3
of these compounds of 6-shogaol
toprotein
inhibitwas and 8-shogaol
weaker
both significantly
than those
proteins attenuated
on the pro-IL-1β
showed similar the decrease
protein,
trend. On of pro-hand,
the rankings
the other
of abilityand
caspase-1 of these compounds
the elevation to inhibit
of active both proteins
caspase-1 inducedshowed similar trend.
by LPS-priming andOn ATPtheactivation
other hand, the
(Figure
the pro-caspase-1 level was decreased and the active caspase-1 level was elevated after LPS-priming
pro-caspase-1 level was decreased and the active caspase-1 level was elevated after LPS-priming and
3d).
and ATPATPactivation. Pretreatment
activation. Pretreatment of 6-shogaol
of 6-shogaol and 8-shogaol
and 8-shogaol significantly
significantly attenuated attenuated
the decrease of thepro-decrease
of pro-caspase-1 and the elevation of active caspase-1
caspase-1 and the elevation of active caspase-1 induced120.0 induced by LPS-priming and
by LPS-priming and ATP activation (Figure ATP activation
e e
(Figure3d).
3d). 100.0
level protein level

d
80.0
120.0 c
60.0 e e ab
b
100.0
pro-IL-1β

a a
40.0 d
80.0
pro-IL-1β protein

c
20.0
60.0
b ab
0.0
40.0 a a

20.0

LPS - + + + + + + +
0.0
ATP - + + + + + + +

(a) (b)
LPS - + + + + + + +
ATP - + + + + + + +

(a) (b)

Figure 3. Cont.
Molecules 2018, 23, 466 5 of 12
Molecules 2018, 23, 466 5 of 12
Molecules 2018, 23, 466 250.0
5 of 12
120.0 pro-caspase-1
e de
de active caspase-1
e

Caspase-1 protein level


100.0 cd 200.0
250.0 de
120.0 pro-caspase-1 cde bcd
NLRP3 protein level
e de bc abc
de active caspase-1
e

Caspase-1 protein level


80.0 100.0 cd 200.0
150.0 de
ab ababc cde bcd
bc c c
NLRP3 protein level ab ab a c bc
60.0 80.0 150.0 ab
bc
100.0 ab
ab a a c c c bc
40.0 60.0 ab bc
100.0
a 50.0 ab
40.0
20.0
a ab
ab
50.0
20.0
0.0 a ab
0.0
0.0 0.0

LPS - + + + + + + + LPS - + + + + + + +
ATP LPS- -+ + + + + + + + + + ++ ++ LPS
ATP - - + + + + + + + + + ++ +
+ +
ATP - + + + + + + + ATP - + + + + + + +

(c)(c) (d)(d)
Figure 3. Effect
Figure of ginger
3. Effect of ginger phytochemicals
phytochemicals on on the protein
proteinlevels
levelsofofpro-IL-1β
pro-IL-1β (b),(b),
NLRP3NLRP3 (c), pro-
(c), pro-
Figure 3. Effect of ginger phytochemicals on the protein levels of pro-IL-1β (b), NLRP3 (c),
caspase-1 (d) and
caspase-1 active
(d) and caspase-1
active caspase-1(d)(d)ininLPS-primed
LPS-primed and andATP-activated
ATP-activated THP-1
THP-1 macrophages.
macrophages. The The
pro-caspase-1 (d) and active caspase-1 (d) in LPS-primed and ATP-activated THP-1 macrophages.
panelspanels
show show a representative
a representative photogramof
photogram of immunoblotting
immunoblotting (a) (a)and
andthethe
quantitative
quantitativeresults of pro-
results of pro-
The panels show a representative photogram of immunoblotting (a) and the quantitative results
IL-1bIL-1b
(b), (b),
NLRP3NLRP3 (c) (c)
as as wellasaspro-caspase-1
well pro-caspase-1 and and active
activecaspase-1
caspase-1 (d),(d),
respectively. The The
respectively. PMA-PMA-
of pro-IL-1b (b),THP-1
differentiated NLRP3 (c) as well
macrophages wereas pro-caspase-1
treated with ginger and active caspase-1 (d), respectively.
differentiated THP-1 macrophages were treated with gingerphytochemicals
phytochemicals (20 μM) for
(20 μM) 1h,forprimed
1h, primed
The PMA-differentiated THP-1 macrophages were treated with ginger phytochemicals
with LPS for 4h and then activated with 5 mM ATP for 3 h. Cytosolic proteins were subjected (20 µM)tofor 1h,
with LPS for 4h and then activated with 5 mM ATP for 3 h. Cytosolic proteins were subjected to
primed with LPS
determine for 4h
protein andof
levels then activated
pro-IL-1β, withpro-caspase-1
NLRP3, 5 mM ATP for and 3 active
h. Cytosolic
caspase-1 proteins were subjected to
by immunoblotting.
determine protein levels of pro-IL-1β, NLRP3, pro-caspase-1 and active caspase-1 by immunoblotting.
determine protein
Data are levelsasofthe
expressed pro-IL-1β,
mean ± SD NLRP3, pro-caspase-1
of triplicate independentandexperiments.
active caspase-1
Valuesbywith
immunoblotting.
different
Data superscript
are expressed asare
the mean ± different
SD of triplicate independent experiments. Values with different
Data are expressed as the mean ± SD of triplicate independent experiments. Values with different
letters significant from one another (p < 0.05).
superscript letters
superscript letters are
are significant
significant different
different from
from one
one another
another (p
(p << 0.05).
0.05).
2.3. Effect of Ginger Phytochemicals on LPS-Primed mRNA Expression of IL-1β, NLRP3 and
2.3. Effect of Ginger Phytochemicals
Inflammasome-Related ComponentsoninLPS-Primed
THP-1 MacrophagesmRNA Expression of IL-1β, NLRP3 and
2.3. Effect of Ginger Phytochemicals on LPS-Primed mRNA Expression of IL-1β, NLRP3 and
Inflammasome-Related
Inflammasome-Related Components
Components
Figure 4 shows inginger
in
the effect of THP-1phytochemicals
THP-1 Macrophages on the mRNA expression of TNF-α, IL-1β,
Macrophages
NLRP3,4 caspase-1,
Figure shows the theASC,
effectand ATP-gated
of ginger
ginger P2X7 receptor
phytochemicals on in
theLPS-primed
mRNA expression THP-1 macrophages,
expression of TNF-α,
TNF-α, IL-1β,
Figure 4 shows effect of phytochemicals on the mRNA of IL-1β,
NLRP3,respectively.
caspase-1,According
ASC, to
andFigure 4,
ATP-gatedLPS stimulation
P2X7 for
receptor 3h markedly
in increased
LPS-primed TNF-α,
THP-1 IL-1β, and
macrophages,
NLRP3, caspase-1,
NLRP3, mildly ASC,
enhancedand P2X7
ATP-gated
receptor. P2X7 receptor
However, in LPS-primed
it did not influenceTHP-1 caspase-1 macrophages, respectively.
and even decreased
respectively.toAccording
According Figure 4, to
LPS Figure 4, LPS stimulation
stimulation for 3h for 3h markedly
markedly increased increased
TNF-α, TNF-α,
IL-1β, IL-1β,
and and
NLRP3,
ASC mRNA expressions. Except for 6-gingerol, all other ginger phytochemicals attenuated LPS-
NLRP3,
mildly mildly
enhanced enhanced P2X7 receptor. However, it did not influence caspase-1 and even decreased
primed TNF-α,P2X7IL-1β, receptor.
and NLRP3 However, it did not influence
mRNA expressions. The effectscaspase-1 and even
of tested ginger decreased ASC
phytochemicals
ASC
mRNA mRNA
on TNF-α, expressions.
expressions.
IL-1β, andExcept Except
NLRP3for mRNAfor 6-gingerol,
6-gingerol,showall all
otherresults
similar other
ginger andginger phytochemicals
phytochemicals
the suppressing attenuated attenuated as LPS-
LPS-primed
ability was sorted
primed
TNF-α, TNF-α,
6-shogaol
IL-1β,and IL-1β,
and and
8-shogaol NLRP3
NLRP3≥ mRNA mRNA
10-gingerol expressions.
and 10-shogaol
expressions. The The
> 8-gingeroleffects
effects ofintested of tested
progressive
gingerginger
order phytochemicals
from high to on
phytochemicals
on TNF-α,
low. IL-1β,
The and
results also NLRP3
indicate mRNA
that the show similar
capability of the results
ginger
TNF-α, IL-1β, and NLRP3 mRNA show similar results and the suppressing ability was and the suppressing
phytochemicals to ability
inhibit was sorted
pro-IL-1β sorted
and as as
6-shogaol
NLRP3 and 8-shogaol
expressions ≥ 10-gingerol
appear to be part and
of 10-shogaol
the molecular > 8-gingerol
mechanism
6-shogaol and 8-shogaol ≥ 10-gingerol and 10-shogaol > 8-gingerol in progressive order from hightoin progressive
explain the order
inhibitory from high
capacity to
low. of these
The phytochemicals
results also indicate on
thatthe
the canonical
capability NLRP3
of the inflammasome-mediated
ginger phytochemicals IL-1β
to inhibitsecretion. In and
pro-IL-1β
to low. The results also indicate that the capability of the ginger phytochemicals to inhibit pro-IL-1β
NLRP3 contrast, 6-shogaol,
expressions 8-shogaol
appear to beand
part 10-gingerol seems tomechanism
increase caspase-1, ASCthe andinhibitory
P2X7 receptor
and NLRP3
mRNA
expressions
expression.
appear to beofpartthe molecular
of the molecular to explain
mechanism to explain capacity
the inhibitory
of these phytochemicals on the canonical NLRP3 inflammasome-mediated
capacity of these phytochemicals on the canonical NLRP3 inflammasome-mediated IL-1β secretion. IL-1β secretion. In
contrast, 6-shogaol,
In contrast, 6-shogaol, 8-shogaol and 10-gingerol seems to increase caspase-1, ASC and P2X7 receptor
160.0 8-shogaol and 10-gingerol seems to increase caspase-1, ASC and P2X7 receptor
mRNA expression. vehicle control LPS control 6-gingerol 8-gingerol
mRNA expression.140.0 10-gingerol 6-shogaol 8-shogaol 10-shogaol
mRNA level (% of LPS control)

cd
120.0 d
160.0
d vehicledcontrolc LPS control e e
6-gingerol 8-gingerol
100.0
10-gingerol 6-shogaol d
8-shogaol 10-shogaol
140.0 c
mRNA level (% of LPS control)

80.0 cd
120.0 d
60.0 d d e e
c
100.0 b cd c
40.0 b
c b
80.0 ab a
a a
20.0 a a a
a a a
60.0 0.0
TNF-α IL-1β b NLRP3 c c
40.0 b
b
(a) ab a
a a
20.0 a a a
a a a
0.0
TNF-α IL-1β NLRP3

(a)

Figure 4. Cont.
Molecules 2018, 23, 466 6 of 12
Molecules 2018, 23, 466 6 of 12

Molecules 2018, 23, 466


600.0 6 of 12
d vehicle control LPS control 6-gingerol 8-gingerol
10-gingerol 6-shogaol 8-shogaol 10-shogaol
600.0
500.0
d vehicle control LPS control 6-gingerol 8-gingerol

of LPS control)
c 10-gingerol 6-shogaol 8-shogaol 10-shogaol
500.0
400.0

(% control) c
400.0
300.0
b
of LPS

d
(%level

300.0 c e
200.0
c c
b b cd d
a b bc
a a ab ab
mRNA

a a a b b
d a
mRNA level

c e
200.0
100.0 c c
a b cd d
a a a a a ab ab b b bc b
a
100.0
0.0
Caspase-1 ASC P2X7R
0.0
Caspase-1 (b) ASC P2X7R

Figure
Figure 4. 4.Effect
Effectof
of ginger
gingerphytochemicals
phytochemicals on the mRNA
on(b)the mRNAexpression of TNF-α
expression of (a), pro-IL-1β
TNF-α (a), NLRP3 (a),
(a), pro-IL-1β
(a), caspase-1 (b), ASC (b) and P2X7R (b) in LPS-primed THP-1 macrophages. The PMA-differentiated
NLRP3
Figure (a), caspase-1
4. Effect (b), phytochemicals
of ginger ASC (b) and P2X7R (b) in LPS-primed
on the mRNA expression of THP-1 macrophages.
TNF-α (a), pro-IL-1β (a), The PMA-
NLRP3
THP-1 macrophages were treated with ginger phytochemicals (20 μM) for 1h and primed with LPS
differentiated THP-1 macrophages were treated with ginger phytochemicals (20 µM)
(a), caspase-1 (b), ASC (b) and P2X7R (b) in LPS-primed THP-1 macrophages. The PMA-differentiatedfor 1h and primed
for 3 h. Total cellular RNA was subjected to determine mRNA levels of target genes by quantitative
withTHP-1
LPS for 3 h. Totalwere
macrophages cellular RNA
treated was
with subjected
ginger to determine
phytochemicals mRNA
(20 μM) for 1hlevels of target
and primed withgenes
LPS by
PCR. Data are expressed as the mean ± SD of triplicate independent experiments. Values with
quantitative PCR.
for 3 h. Total Data are
cellular RNA expressed as thetomean
was subjected ± SD mRNA
determine of triplicate
levelsindependent
of target genesexperiments. Values
by quantitative
different superscript letters are significant different from one another (p < 0.05).
withPCR.
different
Data superscript
are expressed letters aremean
as the significant
± SD different from
of triplicate one another
independent (p < 0.05). Values with
experiments.
different superscript
2.4. Inhibitory Capacities letters are Phytochemicals
of Ginger significant different from one another
on ATP-Activated (p <Secretion
IL-1β 0.05). in LPS-Primed THP-
2.4. 1Inhibitory
MacrophagesCapacities of Ginger Phytochemicals on ATP-Activated IL-1β Secretion in LPS-Primed
THP-12.4. Macrophages
Inhibitory Capacities of Ginger Phytochemicals on ATP-Activated IL-1β Secretion in LPS-Primed THP-
To further explore whether ginger phytochemicals influence the NLRP3 inflammasome-
1 Macrophages
To further
mediated explore whether
maturation process ginger
of IL-1β,phytochemicals
macrophage cells influence the NLRP3
were treated inflammasome-mediated
with ginger phytochemicals
To further
maturation explore whether gingercells
phytochemicals influence the phytochemicals
NLRP3 inflammasome-
after being LPS-primed for 4 h and then activated with ATP for 3 h. The results are shown inafter
process of IL-1β, macrophage were treated with ginger Figurebeing
mediated
LPS-primed maturation
for 4 h and process
then of IL-1β, macrophage
activated with ATP1157 cells
forand were treated
3 h.3209
Thepg/mL with
results ginger phytochemicals
are respectively,
shown in Figure
5a. The LPS-primed THP-1 macrophages secreted IL-1β, under 5a.
after being LPS-primed for 4 h and then activated with ATP for 3 h. The results are shown in Figure
The conditions
LPS-primedofTHP-1 macrophages
without secreted
or with further ATP1157(5andmM) 3209 pg/mL IL-1β,
activation. This respectively,
result indicatedunder conditions
that LPS
5a. The LPS-primed THP-1 macrophages secreted 1157 and 3209 pg/mL IL-1β, respectively, under
stimulation
of without not only
or with affected
further ATP priming
(5 mM) step but alsoThis
activation. contributed partially to
result indicated maturation
that process of
LPS stimulation notIL-only
conditions of without or with further ATP (5 mM) activation. This result indicated that LPS
1β. On
affected the other
priming hand,
step but the
alsoATP stimulation
contributed boosted
partially tomaturation
maturationprocess processandof obviously
IL-1β. Onincreased
the otherthehand,
stimulation not only affected priming step but also contributed partially to maturation process of IL-
secretion
the ATP of IL-1βboosted
stimulation to 2.77-fold of that process
maturation of LPS-primed
and alone. As
obviously presented
increased the in Figure of
secretion 5a,IL-1β
clearly,
to at 20
2.77-fold
1β. On the other hand, the ATP stimulation boosted maturation process and obviously increased the
μM,of
of that 10-gingerol
LPS-primed and all tested
Asshogaols effectively attenuated ATP-stimulated IL-1β secretion
andinallLPS-
secretion of IL-1β to alone.
2.77-fold ofpresented in Figure
that of LPS-primed 5a, clearly,
alone. at 20 µM,
As presented in 10-gingerol
Figure 5a, clearly, at tested
20
primed cells. The concentration of the IL-1β secreted in the 6-shogaol treated cells was even equal to
shogaols effectively attenuated ATP-stimulated IL-1β secretion in LPS-primed cells.
μM, 10-gingerol and all tested shogaols effectively attenuated ATP-stimulated IL-1β secretion in LPS- The concentration
that secreted in the cells without ATP stimulation. At 20 μM, only 6-shogaol and 8-shogaol displayed
of the IL-1βcells.
primed secreted in the 6-shogaol
The concentration treated
of the cells wasineven
IL-1β secreted equal to that
the 6-shogaol secreted
treated in the
cells was evencells without
equal to
a potent inhibitory ability (> 50%) on ATP-activated maturation process of IL-1β.
ATPthat
stimulation.
secreted inAt
the20cells only 6-shogaol
µM,without and 8-shogaol
ATP stimulation. μM, only 6-shogaol
At 20 displayed a potent inhibitory
and 8-shogaolability (>50%) on
displayed
a potent inhibitory
ATP-activated ability
maturation (> 50%) on ATP-activated
Conc.process of IL-1β.
maturation process of IL-1β.
Inhibition rate (%)
20 μM 100.0 0.0 2.7 15.0 36.4 108.0 60.3 28.1
10 μM 3.5 2.3 27.8 91.9 25.4 4.9
Conc.
5 μM -6.8 Inhibition
1.7 rate (%)
18.9 52.4 10.4 -8.7
20 μM 100.0 0.0 2.7 15.0 36.4 108.0 60.3 28.1
6000
10 μM 3.5 2.3 27.8 91.9 25.4 4.9
5 μM -6.8 1.7 18.9 2052.4
μΜ 10 μΜ
10.4 5 μΜ
-8.7
5000
6000
20 μΜ 10 μΜ 5 μΜ
(pg/mL) (pg/mL)

4000
5000
e e de
c cd
IL-1β secretion

3000
4000
e e de b
c cd
IL-1β secretion

2000
3000
a a
b
1000
2000
a a
10000
LPS + + + + + + + +

0 Vehicle Vehicle 6-gingerol 8-gingerol 10-gingerol 6-shogaol 8-shogaol 10-shogaol


LPS
ATP +- ++ ++ ++ ++ ++ ++ ++
Vehicle Vehicle 6-gingerol 8-gingerol 10-gingerol 6-shogaol 8-shogaol 10-shogaol
ATP - + + (a) + + + + +

(a)

Figure 5. Cont.
Molecules 2018, 23, 466 7 of 12
Molecules 2018, 23, 466 7 of 12

250
d d

Active caspase-1 (% of +LPS / -ATP)


200 d

150 c

bc bc ab
100 ab
a

50

0
LPS - + + + + + + + +

ATP - - + + + + + + +

(b)
Figure 5. Effect of ginger phytochemicals on the ATP-activated IL-1β secretion (a) and caspase-1
Figure 5. Effect of ginger phytochemicals on the ATP-activated IL-1β secretion (a) and caspase-1
activity (b) in LPS-primed THP-1 macrophages. The LPS-primed macrophages were treated with
activity (b) in LPS-primed THP-1 macrophages. The LPS-primed macrophages were treated with
ginger phytochemicals for 1 h. Then, the cells were activated with 5 mM ATP for 3 h or 1 h,
ginger phytochemicals for 1 h. Then,
respectively, to determine the cells and
IL-1β secretion were activated
cytosolic with
active 5 mM ATP
caspase-1. Data for
are 3expressed
h or 1 h,as
respectively,
the
to determine
mean IL-1β
± SD secretion and
of triplicate cytosolic active
independent caspase-1.
experiments. Data
Values of are
the expressed as the meanat± SD of
ginger phytochemicals
triplicate independent
concentration of 20experiments. Values
μM (a) and values (b) of thedifferent
with gingersuperscript
phytochemicals atsignificant
letters are concentration of 20 µM
different
from one
(a) and values another
(b) (p < 0.05). superscript letters are significant different from one another (p < 0.05).
with different

Because active caspase-1 plays a key role in IL-1β maturation [20], it is essential to assess the
Because
effect ofactive caspase-1
different plays a key on
ginger phytochemicals role
thein IL-1β
active maturation
caspase-1 [20],
levels. To it is this
address essential to assess the
issue, cellular
effect ofactive
different ginger
caspase-1 phytochemicals
was determined withon the activebound
a covalently caspase-1 levels.inhibitor.
fluorescent To address this issue,incellular
As presented
Figure 5b, upon
active caspase-1 was ATP activation,with
determined the cytosolic level of active
a covalently bound caspase-1 was elevated
fluorescent to 171.5%
inhibitor. of that
As presented in
of the LPS-primed cells. Also shown in Figure 5b, all the tested ginger phytochemicals at 20 μM,
Figure 5b, upon ATP activation, the cytosolic level of active caspase-1 was elevated to 171.5% of that
except 6-gingerol and 8-gingerol, demonstrated significant decrease of active caspase-1. 10-gingerol,
of the LPS-primed cells. Also shown in Figure 5b, all the tested ginger phytochemicals at 20 µM,
6-shogaol, and 8-shogaol completely diminished the activity of ATP-activated caspase-1. Inhibiting
except 6-gingerol and 8-gingerol,
caspase-1 activation demonstrated
appeared to significant
be one of the molecular decrease
mechanisms of activefor
responsible caspase-1. 10-gingerol,
the suppressing
6-shogaol, and of
capacity 8-shogaol completely
these ginger diminished
phytochemicals on NLRP3 the activity of ATP-activated
inflammasome-mediated caspase-1. Inhibiting
IL-1β secretion.
caspase-1 activation appeared to be one of the molecular mechanisms responsible for the suppressing
capacity3.ofDiscussion
these ginger phytochemicals on NLRP3 inflammasome-mediated IL-1β secretion.
Gingerols/shogaols have been demonstrated to attenuate LPS-induced secretion of
3. Discussion
proinflammatory mediators through blocking the NF-κB-governed inflammatory signaling in
macrophages, and thereby possess potent anti-inflammatory activity [16,17,21]. Moreover, 6-shogaol
Gingerols/shogaols
was documented tohave beenmonosodium
alleviate demonstrated to attenuate
urate LPS-induced
crystal-induced secretion
gouty arthritis and of proinflammatory
decrease the
mediators through
lysosomal blocking
enzymatic the NF-κB-governed
activity and the TNF-α level inflammatory
in mice [22]. Itsignaling in macrophages,
might be reasonably expectedand
thatthereby
possess gingerols/shogaols
potent anti-inflammatory
would have activity [16,17,21].
an inhibitory effect Moreover, 6-shogaol
on the canonical was documented to alleviate
NLRP3 inflammasome-mediated
IL-1β secretion. Despite of this, to date, it is still unclear whether
monosodium urate crystal-induced gouty arthritis and decrease the lysosomal enzymatic gingerols/shogaols could inhibitactivity
canonical NLRP3 inflammasome-mediated IL-1β secretion, whose structural feature might impact
and the TNF-α level in mice [22]. It might be reasonably expected that gingerols/shogaols
the inhibitory activity. Our results indicate, for the first time, that 10-gingerol and all the shogaols
would have an inhibitory effect on the canonical NLRP3 inflammasome-mediated IL-1β secretion.
exerted significant inhibitory capacities on LPS-primed and ATP-activated IL-1β secretion at a
Despiteconcentration
of this, to date,
of 20itμM.
is still unclear suppressed
The shogaols whether gingerols/shogaols could inhibit canonical
the canonical NLRP3 inflammasome-mediated IL-NLRP3
inflammasome-mediated
1β secretion more than IL-1βthesecretion, whosegingerols.
corresponding structural feature
This outcome might impact
implied thatthe
theinhibitory
inhibitory activity.
capacity
Our results of shogaols
indicate, for wasthe primarily
first time,attributed to the presence
that 10-gingerol of α,β-unsaturated
and all the shogaolscarbonyl group
exerted in
significant
the chemical structures of these shogaols. Identically, the α,β-unsaturated
inhibitory capacities on LPS-primed and ATP-activated IL-1β secretion at a concentration of 20 µM. carbonyl moiety was
demonstrated to be the most critical determinant of the antioxidant, the anti-inflammatory and the
The shogaols suppressed the canonical NLRP3 inflammasome-mediated IL-1β secretion more than
antiproliferative potencies of shogaols [21,23,24]. Additionally, the increase of alkyl side chain length
the corresponding
weakened thegingerols. This outcome
inhibitory capacity of shogaols implied that the
on canonical NLRP3 inhibitory capacity of shogaols
inflammasome-mediated IL-1β was
primarily attributed
secretion, to theappearing
while presence of enhance those carbonyl
to α,β-unsaturated group in
of gingerols. the chemical structures
Correspondingly, the of
these shogaols. Identically, the α,β-unsaturated carbonyl moiety was demonstrated to be the most
critical determinant of the antioxidant, the anti-inflammatory and the antiproliferative potencies of
shogaols [21,23,24]. Additionally, the increase of alkyl side chain length weakened the inhibitory
capacity of shogaols on canonical NLRP3 inflammasome-mediated IL-1β secretion, while appearing
to enhance those of gingerols. Correspondingly, the antineuroinflammatory, anti-invasive and
antiproliferative activity of shogaols were also negatively impacted by the increase of the alkyl side
chain length of shogaols [21,23,25].
Molecules 2018, 23, 466 8 of 12

The activation of transcription factor NF-κB, which induces proinflammatory genes expression,
is recognized as the early event of canonical IL-1β secretion. In fact, NF-κB signaling is another
predominant inflammatory cascade where the detailed molecular mechanism with regard to the
inhibition effect of gingerols/shogaols on NF-κB governed inflammatory response has been well
established. Gingerols/shogaols could interfere LPS-induced dimerization of toll-like receptor (TLR)-4,
block PI3K/Akt, extracellular signal-regulated kinase 1/2 and p38 mitogen activated protein kinase
(MAPK) signaling, inhibit NF-κB activation and translocation, and consequently, diminish LPS-induced
expression of proinflammatory genes, such as iNOS, COX-2, IL-6, IL-1β and TNF-α [15–18]. In BV2
microglia, the suppressing ability of gingerols/shogaols on the LPS-triggered NF-κB activation was
sorted as 6-shogaol and 8-shogaol > 10-gingerol and 10-shogaol > 8-gingerol. Simultaneously, the NF-κB
suppressing abilities of gingerols/shogaols were parallel with their inhibitory extents on iNOS, IL-1β
and TNF-α expressions [21]. In consistent with the previous study, a similar inhibitory trend of
tested gingerols/shogaols on the LPS-primed expression of TNF-α, pro-IL-1β and NLRP3 was found
herein. These results indicated inhibiting NF-κB governed proinflammatory genes expression at the
priming step contribute to the inhibitory action of 10-gingerol/shogaols on the canonical NLRP3
inflammasome-mediated IL-1β secretion.
On the other hand, in contrast to NF-κB governed pro-IL-1β and NLRP3, the mRNA expression
of caspase-1, ASC and P2X7 receptors were increased by 10-gingerol/shogaols. Lin et al. indicated
that caspase-1 is expressed constitutively in THP-1 macrophages, and its mRNA and protein levels
were not increased by LPS [26]. Based on the functional binding sites existing in the promoter
region, the expression of caspase-1 gene is demonstrated to be activated by several transcriptional
factors, such as IRF-1, signal transducer and activator of transcription (STAT) 1, p53, and p73 and E26
transformation-specific sequence 1 (Ets-1) [27]. On the other hand, 6-shogaol was demonstrated to
induce lung cancer cell apoptosis through activating p53 pathway and to increase the expression of
p21, p27, SOCS1, and IRF1 in prostate cancer cells [28,29]. The inducing effect of these effective ginger
phytochemicals, especially 6-shogaol, on caspase-1 gene expression might be partially attributed to
their activating effect on p53 and IRF1. However, this speculation needs to be proved in the future.
Furthermore, the inducing molecular mechanism of 10-gingerol/shogaols on the ASC and P2X7
receptor mRNA expression is also unclear and should be investigated.
The second stimulation of extracellular ATP induce K+ efflux through an ATP-sensitive P2X7 receptor,
to trigger assembly of NLRP3 inflammasome and, then, caspase-1 is activated by self-cleavage [7].
Our results showed that 10-gingerol, 6-shogaol, and 8-shogaol completely diminished the activity of
ATP-activated caspase-1, however, only 6-shogaol completely decreased ATP-induced IL-1β secretion.
In spite of this, it was demonstrated that inhibiting activation of caspase-1 also contributed to the
inhibitory capacity of these ginger phytochemicals on the canonical NLRP3-mediated IL-1β secretion.
Additionally, IL-1β processing enzyme other than caspase-1, such as caspase-8, might be regulated by
ginger phytochemicals and resulted in increase of IL-1β secretion. This speculation needs to be proved.
Efflux of K+ and generation of reactive oxygen species (ROS) are two intracellular models to
illustrate how the ATP triggers assembly of the NLRP3 inflammasome. [10,30]. However, due to the
elevation effect of the shogaols and 10-gingerol on the mRNA level of P2X7 receptor in the LPS-primed
cells, the blocking of efflux of K+ seems to be the least possible candidate for the mechanistic model to
explain the caspase-1-inhibiting ability of these ginger phytochemicals. It leaves ROS reduction to be
the more plausible model for the explanation of the inhibitory effect of these ginger phytochemicals.
Gingerols/shogaols, particularly the 6-shogaol, possess substantial antioxidant ability and can scavenge
various free radicals, such as superoxide and hydroxyl radical [24]. Additionally, 6-shogaol enhanced the
enzymatic antioxidant defense system through the induction of nuclear factor erythroid 2 (NFE2)-related
factor 2 (Nrf2) [31]. Peng et al., indicated that 6-shogaol conferred protection against oxidative
stress-induced damage by both directly scavenging free radicals and activating endogenous cellular
antioxidant defense system. They proposed that the phenoxyl group of 6-shogaol was responsible for
directly counteracting against free radicals and the α,β-unsaturated carbonyl moiety contributed to
Molecules 2018, 23, 466 9 of 12

activate the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2-ARE signaling pathway by covalently


modification of cysteine residues of Keap1. Oppositely, 6-gingerol lacking the α,β-unsaturated carbonyl
moiety (Michael acceptor moiety) cannot modify Keap1 and thereby fails to shelter PC12 cells from
oxidative stress [32]. Recently, Nrf2-mediated antioxidant signaling pathway has been documented
to be a negative regulator of NLRP3 inflammasome through diminishing ROS production [33,34].
Based on the facts above, interference of NLRP3 inflammasome assembly by ROS reduction might
contribute to the inhibitory activity of shogaols on the canonical NLRP3-inflammasome-mediated IL-1β
secretion. Moreover, the inhibitory activity of shogaols might be partially attributed to their direct
reversible or irreversible modification on cysteine residues of caspase-1 or NLRP3 ATPase [35,36].

4. Materials and Methods

4.1. Chemicals
LPS from Escherichia coli O55:B5, adenosine 5’-triphosphate (ATP), phorbol 12-myristate 13-acetate
(PMA), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) for cell culture experiment were acquired from Sigma-Aldrich (St. Louis, MO, USA). The ginger
phytochemicals, including 6-gingerol (C17 H26 O4 ), 8-gingerol (C19 H30 O4 ), 10-gingerol (C21 H34 O4 ),
6-shogaol (C17 H24 O3 ), 8-shogaol (C19 H28 O3 ), and 10-shogaol (C21 H32 O3 ) with a purity of >99% were
procured from ChromaDex (Irvine, CA, USA). Cell culture medium and reagents were obtained from
Thermo Fisher Scientific (Grand Island, NE, USA). All other chemicals used were analytical grade.

4.2. Cell Culture and Stimulation


Human THP-1 monocytes obtained from the Bioresource Collection and Research Center
(Hsinchu, Taiwan) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine,
25 mM HEPES, 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 U/mL penicillin in a 37 ◦ C
fully humidified incubator with 5% CO2 atmosphere. THP-1 monocytes (1 × 106 cells/mL) were
seeded into 48-well plates, differentiated into macrophages by incubation with 100 nM PMA for 24 h,
and starved further in fresh RPMI 1640 medium overnight. To evaluate the inhibitory effect of ginger
phytochemicals on canonical IL-1β secretion, cells were treated with ginger phytochemicals (5~20 µM)
for 1 h and then primed with 1 µg/mL LPS for 4 h and activated with 5 mM ATP for 3 h. Additionally,
to evaluate the inhibitory effect of ginger phytochemicals on ATP-activated maturation process of
IL-1β, LPS-primed cells were washed with fresh medium and then treated with ginger phytochemicals
(5~20 µM) for 1 h and then activated with ATP for 3 h. The condition media were collected for
IL-1β and TNF-α assay. The MTT assay was adopted to evaluate the cytotoxic effect of tested ginger
phytochemicals on THP-1 macrophages. Briefly, the PMA-differentiated macrophages were treated
with the ginger phytochemicals at concentrations from 5 to 40 µM for 24 h and then incubated with
medium containing 0.5 mg/mL MTT for another 4 h. After which, the formed formazan was dissolved
in DMSO and the absorbance at 550 nm was detected with a multimode reader (Infinite M200 PRO;
Tecan Group Ltd., Männedorf, Switzerland).

4.3. Measurement of IL-1β and TNF-α Secretion


Medium IL-1β and TNF-α levels were determined by using an enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer’s recommendations (BioLegend, San Diego, CA, USA).

4.4. Protein Extraction and Western Blotting


After the in-sequence treatment of ginger phytochemicals, LPS and ATP, THP-1 macrophages were
collected and cytosolic proteins were isolated with a commercial kit (NE-PER nuclear and cytoplasmic
extraction reagents, Thermo Fisher Scientific, Waltham, MA, USA). Target protein expressions were then
determined with immunoblotting. Briefly, total cytosolic proteins were separated on a SDS-PAGE gel,
transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with anti-NLRP3 (NBP1-90207,
Molecules 2018, 23, 466 10 of 12

Novus Biological, Littleton, CO, USA), anti-pro-caspase-1 (sc-56036, Santa Cruz Biotechnology, Dallas, TX,
USA), anti-pro-IL-1β (ab2105, Abcam, Cambridge, MA, USA), or anti-β-actin (3598, Biovision, Milpitas,
CA, USA) antibodies. Following washing, alkaline phosphatase-conjugated secondary antibodies were
used. Immunoreactive protein bands were visualized by chemiluminescence (CDP-Start, PerkinElmer,
Waltham, MA, USA) and the images were captured with a chemiluminescence imager (Fusion solo S;
Vilber Co., Marne-la-Vallée, France). The densitometry of each target protein band was then quantified
using Image J software (National Institutes of Health, Bethesda, MD, USA) and normalized with β-actin.

4.5. RNA Extraction and Quantitative RT-PCR


Cells in six-well plates were harvested after treatment with 20 µM ginger phytochemicals for 1 h and
followed by LPS priming for 3 h. The total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad,
CA, USA) and reverse-transcribed to cDNA using reverse transcription kit (Promega, Madison, WI, USA).
The cDNA was then submitted to quantitative PCR on a StepOneTM real-time PCR system (Applied
Biosystems, Foster City, CA, USA). Relative mRNA expression of target gene was calculated using the
2-∆∆CT method with β-actin as reference gene [37]. The following primers were used: TNF-α-Fw:
50 -CAGAGGGAAGAGTTCCCCAG-30 ; TNF-α-Rv: 50 -CCTTGGTCTGGTAGGAGACG-30 ; NLRP3-Fw:
50 -CTACACACGACTGCGTCTCATCAA-30 ; NLRP3-Rv: 5-GGGTCAAACAGCAACTCCATCTTA-30 ;
IL-1β -Fw: 50 -AAACAGATGAAGTGCTCCTTCCAGG-30 ; IL-1β-Rv: 50 -TGGAGAACACCACTTGTT
GCTCCA-30 ; Caspase-1-Fw: 50 -GAATGTCAAGCTTTGCTCCCTAGA-30 ; Caspase-1-Rv:50 -AAGACGTG
TGCGGCTTGACT-30 ; ASC-Fw: 50 -ATCCAGGCCCCTCCTCAGT-30 ; ASC-Rv: 50 -GTTTGTGACCCTC
CGCGATAAG-30 ; P2X7 receptor-Fw: 50 - TGTGCCTACAGGTGCTACGCC-30 ; P2X7 receptor-Rv: 50 -
GCCCTTCACTCTTCGGAAACTC-30 ; β-actin-Fw: 50 -GAGACCTTCAACACCCCAGCC-30 ; β-actin-Rv:
50 -GGATCTTCATGAGGTAGTCAG-30 .

4.6. Measurement of Active Caspase-1


Active caspase-1 was measured with a covalently bound fluorescent inhibitor, FAM-YVAD-fmk
(Immunochemistry Technologies, Bloomington, MN, USA). Briefly, the LPS-primed cells were
sequentially treated with ginger phytochemicals and ATP for 1 h, FAM-YVAD-fmk were added
and incubated for another 1 h. After which, cells were further incubated with fresh medium for 1 h to
remove the unbound inhibitors. The fluorescence intensity was measured using a multimode reader
with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

4.7. Statistical Analysis


Data are expressed as the mean ± SD of triplicate independent experiments. Statistical comparisons
among different treatments were analyzed by the one way ANOVA followed by Duncan’s test.
Differences were considered to be statistically significant for p values < 0.05. Statistical analyses
were performed with the SPSS 22.0 software (IBM, Armonk, NY, USA).

5. Conclusions
Our results showed that 10-gingerol/shogaols, especially 6-shogaol, had a potent capacity to
attenuate canonical NLRP3 inflammasome-mediated IL-1β secretion in THP-1 macrophages by using
LPS and ATP as the priming and activation stimulation. They exerted dual inhibiting actions, targeting
both the expression of pro-IL-1β and NLRP3 governed by NF-κB as well as the activation of caspase-1
triggered by assembly of NLRP3 inflammasome. Although it needs more efforts to explore the detailed
molecular mechanism and its effectiveness in clinical studies, ginger phytochemicals, particularly
6-shogaol, have great potentials to play a vital role as a novel canonical IL-1β inhibitor and may be
applied in the future to prevent sterile inflammation-associated diseases.

Acknowledgments: This study was supported by the Ministry of Science and Technology, Taiwan, ROC, under the
grant number, MOST 104-2320-B-264-001-MY3.
Molecules 2018, 23, 466 11 of 12

Author Contributions: S.-C.H. conceived and designed the experiments; S.-C.H. performed the experiments;
S.-C.H. and Y.-H.C. analyzed the data; S.-C.H. and Y.-H.C. wrote the paper.
Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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