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Acacetin inhibits inflammation by
blocking MAPK/NF-κB pathways
and NLRP3 inflammasome
activation
Juan Bu
1
, Yeledan Mahan
1
, Shengnan Zhang
1
, Xuanxia Wu
1
,
Xiaoling Zhang
1
, Ling Zhou
1
* and Yanmin Zhang
2
*
1
Medical and Translational Research Center, People’s Hospital of Xinjiang Uygur Autonomous Region,
Urumqi, China,
2
Scientific Research and Education Center, People’s Hospital of Xinjiang Uygur
Autonomous Region, Urumqi, China
Objective: Our preliminary research indicates that acacetin modulates the
nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain
containing 3 (NLRP3) inflammasome, providing protection against Alzheimer’s
Disease (AD) and cerebral ischemic reperfusion injury. The mechanisms of
acacetin to inhibit the activation of the NLRP3 inflammasome remain fully
elucidated. This study aims to investigate the effects and potential
mechanisms of acacetin on various agonists induced NLRP3 inflammasome
activation.
Methods: A model for the NLRP3 inflammasome activation was established in
mouse bone marrow-derived macrophages (BMDMs) using Monosodium Urate
(MSU), Nigericin, Adenosine Triphosphate (ATP), and Pam3CSK4, separately.
Western blot analysis (WB) was employed to detect Pro-caspase-1, Pro-
Interleukin-1β(Pro-IL-1β) in cell lysates, and caspase-1, IL-1βin supernatants.
Enzyme-Linked Immunosorbent Assay (ELISA) was used to measured the release
of IL-1β, IL-18, and Tumor Necrosis Factor-alpha (TNF-α) in cell supernatants to
assess the impact of acacetin on NLRP3 inflammasome activation. The lactate
dehydrogenase (LDH) release was also assessed. The Nuclear Factor Kappa B
(NF-κB) and Mitogen-Activated Protein Kinase (MAPK) signaling pathways related
proteins were evaluated by WB, and NF-κB nuclear translocation was observed
via laser scanning confocal microscopy (LSCM). Disuccinimidyl Suberate (DSS)
cross-linking was employed to detect oligomerization of Apoptosis-associated
Speck-like protein containing a Caspase Recruitment Domain (ASC), and LSCM
was also used to observe Reactive Oxygen Species (ROS) production. Inductively
Coupled Plasma (ICP) and N-(6-methoxyquinolyl) acetoethyl ester (MQAE) assays
were utilized to determined the effects of acacetin on the efflux of potassium (K+)
and chloride (Cl-) ions.
Results: Acacetin inhibited NLRP3 inflammasome activation induced by various
agonists, reducing the release of TNF-α, IL-1β, IL-18, and LDH. It suppressed the
expression of Lipopolysaccharides (LPS)-activated Phosphorylated ERK (p-ERK),
p-JNK, and p-p38, inhibited NF-κB p65 phosphorylation and nuclear
translocation. Acacetin also reduced ROS production and inhibited ASC
aggregation, thus suppressing NLRP3 inflammasome activation. Notably,
acacetin did not affect K+ and Cl-ions efflux during the activation process.
OPEN ACCESS
EDITED BY
David M. Pereira,
University of Porto, Portugal
REVIEWED BY
Junping Zhang,
First Teaching Hospital of Tianjin University of
Traditional Chinese Medicine, China
Muhammad Furqan Akhtar,
Riphah International University, Pakistan
*CORRESPONDENCE
Ling Zhou,
zlrmyy@sina.com
Yanmin Zhang,
1518486455@qq.com
RECEIVED 31 August 2023
ACCEPTED 26 January 2024
PUBLISHED 08 February 2024
CITATION
Bu J, Mahan Y, Zhang S, Wu X, Zhang X, Zhou L
and Zhang Y (2024), Acacetin inhibits
inflammation by blocking MAPK/NF-κB
pathways and NLRP3 inflammasome activation.
Front. Pharmacol. 15:1286546.
doi: 10.3389/fphar.2024.1286546
COPYRIGHT
© 2024 Bu, Mahan, Zhang, Wu, Zhang, Zhou
and Zhang. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
Frontiers in Pharmacology frontiersin.org01
TYPE Original Research
PUBLISHED 08 February 2024
DOI 10.3389/fphar.2024.1286546
Conclusion: Acacetin shows inhibitory effects on both the priming and assembly
processes of the NLRP3 inflammasome, positioning it as a promising new candidate
for the treatment of NLRP3 inflammasome-related diseases.
KEYWORDS
inflammasome, NLRP3, acacetin, MAPK pathway, NF-κB pathway
1 Introduction
Inflammation represents a biological paradox. While moderate
inflammatory responses can confer benefits to the host, aiding in the
combat against foreign pathogen invasions, excessive or chronic
inflammation may lead to tissue damage and consequential
pathological changes. The nucleotide-binding oligomerization
domain (NOD)-like receptor pyrin domain containing 3
(NLRP3) inflammasome plays a critical role in these
inflammatory responses. Comprised of Apoptosis-associated
Speck-like protein containing a Caspase Recruitment Domain
(ASC), NLRP3, and pro-caspase-1, the NLRP3 inflammasome,
upon activation, catalyzes the activation of caspase-1, which
mediates the discharge of inflammatory cytokines Interleukin-1β
(IL-1β) and Interleukin-18 (IL-18), thereby leading to inflammation
and pyroptosis (Xu Y. et al., 2023;Ma, 2023). Studies support the
implication of mitogen-activated protein kinase (MAPK) and
Nuclear Factor Kappa-B (NF-κB) pathways in modulating
NLRP3 inflammasome activation, thereby participating in the
inflammatory response (He et al., 2013;Robblee et al., 2016). The
activation of NLRP3 inflammasome correlates with a variety of
diseases, including peritonitis (Qin and Zhao, 2023), arthritis (Wang
et al., 2023), Alzheimer’s disease (AD) (Lv et al., 2023), and ischemic
stroke (Han and Le, 2023), etc. Hence, the therapeutic efficacy of
NLRP3 inflammasome inhibitors, which have manifested significant
prognostic improvements in animal models of these diseases,
underscores the imperative for their discovery.
At present, identified inhibitors of NLRP3 inflammasome
encompass agents such as sulforaphane (Kiser et al., 2021),
oridonin (He et al., 2018), BAY11-7082 (Irrera et al., 2017),
INF39 (Shi et al., 2021), MCC950 (Luo et al., 2019), CY-09
(Wang et al., 2022), INF-200 (Gastaldi et al., 2023), SB-222200
(Zhou et al., 2023), Alantolactone (Li W. et al., 2023), and
Tabersonine (Xu H.-W. et al., 2023), among others. Nevertheless,
these therapeutic agents encounter drawbacks including limited
specificity, short half-lives, suboptimal bioavailability, and an
uncertain clinical application outlook. Acacetin, also referred to
as robinin, is a flavonoid compound extracted from various plants,
including chrysanthemums, Robinia pseudoacacia, and Saussurea
involucrata. This compound, characterized by its small molecular
weight, ability to penetrate the blood-brain barrier, minimal toxicity,
and extensive pharmacological activities (Singh et al., 2020). Our
previous study (Bu et al., 2019) revealed that acacetin mitigated
ischemic stroke in mouse models by inhibiting microglial
overactivation, modulating the NF-κB/NLRP3 pathway, and
downregulating inflammatory cytokines such as TNF-α, IL-1β,
and IL-6. In addition, acacetin showed significant neuroprotective
properties by diminishing infarct volume and ameliorating
neurological scores in these models. In vitro study, acacetin
increased the survival rate of microglial cells following the
oxygen-glucose deprivation and reoxygenation (OGD/R) injury,
decreased lactate dehydrogenase (LDH) release, stimulated
autophagy, and inhibited NLRP3 inflammasome activation (Bu
et al., 2023). Furthermore, acacetin has demonstrated an ability
to impede NLRP3 inflammasome activation, reduce inflammatory
cytokine release, attenuate senile plaques development in AD mice,
improve cognitive and exploratory abilities in AD mice, and thereby
impart a protective role against AD (Bu et al., 2022). Given the
critical role that NLRP3 inflammasome plays in the onset and
progression of inflammatory diseases and the significant
protective effects of acacetin on these diseases, questions arise
regarding the potential relationship between acacetin’s protective
role and its capacity to inhibit NLRP3 inflammasome activation.
Herein, we initially isolated macrophages from mouse bone
marrow to explore the impact of acacetin on the activation of
NLRP3 inflammasome induced by both canonical (i.e., by
activators monosodium urate (MSU), Nigericin, and adenosine
triphosphate (ATP)) and non-canonical pathways. The aim was
to clarify whether acacetin could suppress NLRP3 inflammasome
activation, thus exerting anti-inflammatory effects. Subsequently, we
investigated whether acacetin impacts the NF-κB and MAPK
pathways, sequentially inhibiting the priming of
NLRP3 inflammasome. Following this, the study employed
Disuccinimidyl Suberate (DSS) cross-linking to detect ASC
oligomerization and laser scanning confocal microscopy (LSCM)
to monitor the production of Reactive Oxygen Species (ROS). The
impact of acacetin on potassium (K+) and chloride (Cl-) ions efflux
was quantified using Inductively Coupled Plasma (ICP) and N-(6-
methoxyquinolyl) acetoethyl ester (MQAE) assays, respectively.
These methods facilitated a precise understanding of acacetin’s
role in the assembly and upstream processes of the
NLRP3 inflammasome.
2 Materials and methods
2.1 Cell preparation, stimulation,
and grouping
Bone marrow-derived macrophages (BMDMs) were isolated
from 6–8 weeks old C57BL/6J mice supplied by Xinjiang Medical
University (License Number: SCXK (Xin) 2018–0002). Following
euthanization and sterilization, both femurs and tibias were excised
and the bone marrow was flushed into DMEM (C11965500BT,
Gibco, United States) medium using sterile phosphate buffer saline
(PBS, ZLI-9062, ZSGB-Bio, China). The obtained cell suspension
was centrifuged at 110 gfor 10 min at room temperature, after which
the supernatant was discarded. The resultant cell pellet was
resuspended in 2 mL of red blood cell lysis buffer and
maintained on ice for 5 min. Subsequently, 8 mL of DMEM
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supplemented with 10% fetal bovine serum (FBS, FND500, Excell
Bio, China) and 1% penicillin-streptomycin (15070–063, Gibco,
United States) was added. After mixing, the solution was
centrifuged at 220 g(with a centrifuge radius of 10 cm) for 5 min
at room temperature. The supernatant was discarded, and the cells
were then resuspended in DMEM containing 10 ng/mL of
Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF,
P00184, Solarbio, Beijing, China), 10% FBS, and 1% penicillin-
streptomycin. Ensuring a homogenous suspension through
repeated pipetting, the cells were cultured in a 37°C incubator.
After 3 days in culture, a complete medium replacement was
conducted, followed by a half medium change on day 5.
Microscopic examination revealed that the adherent cells had
satisfactory attachment, extending pseudopodia, and adopted a
spindle shape.
When cell confluency reached 80%, BMDMs cells (5 × 10
5
) were
seeded to a 12-well plate and then treated with LPS (50 ng/mL,
L2630, Sigma, United States) for 3 h. Afterwards, the cells were
treated with Acacetin (00017, Sigma, United States) for 0.5 h and
respectively stimulated with MSU (150 μg/mL, tlrl-msu/MSU-
41–07, InvivoGen, France) for 4 h, Nigericin (10 μM, tlrl-nig,
InvivoGen, France) for 0.5 h, and ATP (2.5 mM, A2383-5G/
SLCD1216, Sigma, United States) for 0.5 h. For the activation of
non-canonical pathways, cells were pretreated with Pam3CSK4
(400 ng/mL, Tlrl-pms/PMS-41-03, InvivoGen, France) for 3 h,
then treated with Acacetin for 0.5 h, and then transfected with
LPS (500 ng/mL) using Lipofectamine 3000 (L3000-008/2218558,
ThermoFisher, United States of America) for 16 h. Both the
supernatant and cell pellets were collected for subsequent analyses.
Groups for assessing acacetin’s impact on NF-κB and MAPK
signaling pathways included: a control group, groups treated with
LPS for varying durations (0 min, 10 min, 30 min, 60 min), and
groups first treated with LPS for these durations followed by the
addition of acacetin (10 μM). For evaluating acacetin’s effect on
NLRP3 inflammasome activation, the study encompassed a control
group, an LPS treated group, an LPS + Nigericin group, and groups
treated with LPS + Nigericin followed by different concentrations of
acacetin (2.5 μM, 5 μM, and 10 μM). Further details are available in
the figure legends.
2.2 Cell samples collection and proteins
extraction
After the above treatments, supernatants from each group were
collected and combined with an equal volume of methanol and a
quarter volume of chloroform. This mixture was then vortexed
thoroughly and centrifuged at 14,000 × g at room temperature for
5 min. Following centrifugation, the resulting tri-layered solution
had proteins localized in the intermediate layer. The top layer was
carefully discarded, and 500 μL of methanol was added to the
remaining solution. This was followed by another centrifugation
at 16,000 × g for 5 min, after which the protein precipitate settled at
the bottom. The supernatant was removed, and the resultant pellet
was air-dried for about 5 min to get rid of residual methanol. The
dried protein pellet was then reconstituted in sample buffer and
subjected to heating at 101°C in a metal bath for 10 min. The
prepared protein sample was deemed fit either for immediate
Western Blotting assays or storage at −20°C.
For the cellular proteins, cells were enzymatically digested
using 0.25% Trypsin-EDTA (25200–056, Gibco, United States)
and gathered. These cells were then resuspended in 100 μLof
radioimmunoprecipitation assay (RIPA) buffer (AR0105, Boster
Bio, China), ensuring a homogenous mixture. The cell-lysis
mixture was allowed to incubate at 4°Cfor60min.A
centrifugation step at 14,000 × g for 15 min at 4°C allowed us
to collect the supernatant, which was subsequently mixed with
an appropriate volume of 5 × SDS-PAGE loading buffer infused
with β-mercaptoethanol. The denaturation of proteins was
achieved by placing this mixture in a 100°C-water bath for
5 min. After this, a final centrifugation at 14,000 × g for
5 min was done to obtain a clear supernatant. Lastly, the
protein content was quantified using the standard BCA
method (Easy II Protein Quantitative Kit, DQ111-01,
TransGen Biotech, China), from which the volume equivalent
to 30 μg of protein was calculated, setting the stage for
downstream experimental endeavors.
2.3 Western blot
Electrophoresis conditions: the stacking gel was set at 80 V and
the separating gel at 100 V. Following electrophoresis, proteins from
the gel were transferred onto PVDF membranes (ISEQ00010/
IPVH00010, Millipore, United States). Then the membrane was
blocked with 5% bovine serum albumin (BSA) in TBST at room
temperature for 2 h. It was then incubated with primary antibodies
overnight at 4°C(Table 1). After washing thrice with TBST for
10 min each, the membrane was incubated with corresponding
secondary antibodies (diluted 1:5,000) at room temperature for
2 h. Another three 10-min TBST washes were done, followed by
an application of chemiluminescent reagent (mixed 1:1 of Solution
A and B, 2 mL each) on the membrane. Protein bands were detected
and captured using a chemiluminescence imaging system
(Chemiscope 3000, Clinx, China). The relative expression level
was calculated based on the absorbance ratio of the target gene
band to the β-actin band.
2.4 Enzyme-linked immunosorbent assay
Cytokine levels of IL-1β, IL-18, and TNF-αin collected cell
supernatants were measured using ELISA kits (EK201B/3-48,
EK218-48 and EK282/3-48, LiankeBio, China) following the
manufacturer’s instructions. After adding a stop solution to
terminate the reaction, the color change was measured
spectrophotometrically at a dual wavelength of 450 nm
(maximum absorption wavelength) and 570 nm (reference
wavelength) using a microplate reader (xMarkTM, Bio-Rad,
United States). The optical density (OD) at 570 nm was
subtracted from the OD at 450 nm to correct for optical
imperfections. The corrected OD values were used to determine
the concentrations of the cytokines in the supernatants by
comparing them to the standard curve.
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2.5 LDH release assay
To quantify the level of cellular cytotoxicity and necrosis, a LDH
release assay was performed following the manufacturer’s
instructions. LDH is a stable enzyme, released upon cell lysis,
and it serves as a marker of cellular damage or death.
Supernatants from each group were collected and assayed for
LDH activity. The LDH assay was conducted using the LDH
Cytotoxicity Detection Kit (A020-2, NJJCBio, China). This kit
utilizes the conversion of lactate to pyruvate by LDH, with
simultaneous conversion of a tetrazolium salt (INT) into a red
formazan product. The amount of formazan product, which is
directly proportional to the LDH activity in the sample, can be
quantified by measuring the absorbance at 450 nm using a
microplate reader. The calculation formula of LDH activity is:
LDH Activity U/L
()
Absorbance of sample −Absorbance of blank
Absorbance of standard −Absorbance of standard blank
× Standard concentration × 1000
2.6 NF-κB nuclear translocation
The nuclear translocation of NF-κB was visualized using laser
scanning confocal microscopy (LSCM). After the above treatments,
the coverslips were thoroughly washed with three 2-min wash cycles
using phosphate-buffered saline (PBS). Subsequent steps were
fixation with 4% paraformaldehyde for 20 min, permeabilization
with 0.5% Triton X-100 for 5 min, and blocking with 1% BSA
for 30 min, each followed by identical wash cycles. Following
blocking, cells were incubated at 37°C with primary NF-κB
p65 antibody (2 μg/mL, ab76302, Abcam, United Kingdom) for
2 h. A post-incubation wash cycle was performed before the cells
were exposed to a goat anti-rabbit IgG H&L secondary antibody
(2 μg/mL, ab205718, United Kingdom) for 1 h at 37°C in the dark.
Another wash cycle preceded DAPI counterstaining (1 μg/mL,
C0065, Solarbio, Beijing, China) for 5 min at room temperature.
After a final wash cycle, cells were mounted with 50% glycerol and
imaged using a laser scanning confocal microscope (LSM700, Carl
Zeiss AG, Germany).
2.7 K + ions concentration
Prepared cell suspensions were centrifuged at 110 gfor 10 min,
discarding the supernatant and retaining the cellular pellet. Cells were
then washed twice with PBS, followed by centrifugation at 110 gfor
10 min, retaining the cellular pellet. The cellular pellet was resuspended
in 0.2 mL of deionized water and homogenized in an ice bath.
Homogenateswerethencentrifugedat450gfor 5 min, and 20 μL
of the supernatant was mixed with 180 μL of protein precipitant. This
was further centrifuged at 1370 gfor 5 min, and 50 μLofthe
supernatant was collected for potassium measurement. Protein
concentration in the samples was also determined to normalize the
K+ ions concentration. The concentration of K+ ions was measured
using a potassium ion assay kit (C001-2-1, NJJCBio, China), following
the manufacturer’s instructions. The calculation formula of the K+ ions
concentration in mmol/g of protein (mmol/gprot) is:
Potassium ions concentration mmolgprot
Absorbance of sample –Absorbance of blank
Absorbance of standard –Absorbance of blank
× standard concentration× sample dilution factor
÷ protein concentration in sample
TABLE 1 Primary antibodies used in Western blot analysis.
Protein Cat no. Dilution Transfer time (min) Company Info
NLRP3 ab263899 1:1000 120 abcam, United Kingdom
Caspase-1 ab179515 1:1000 60 abcam, United Kingdom
IL-1βab9722 1:500 60 abcam, United Kingdom
NF-κB p65 ab32536 1:800 60 abcam, United Kingdom
Phospho-NF-κB p65 ab76302 1:800 60 abcam, United Kingdom
IκBαab32518 1:1000 60 abcam, United Kingdom
Phospho-IκBα5209s 1:800 60 CST, United States
ERK ab184699 1:800 60 abcam, United Kingdom
Phospho-ERK ab201015 1:800 60 abcam, United Kingdom
JNK ab179461 1:1000 60 abcam, United Kingdom
Phospho-JNK ab76572 1:800 60 abcam, United Kingdom
p38 ab170099 1:1000 60 abcam, United Kingdom
Phospho-p38 ab195049 1:800 60 abcam, United Kingdom
β-Actin 100166-MM10 1:1000 60 Sino Biological, China
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2.8 Cl- ions concentration
The concentration of Cl-ions was determined using MQAE
fluorescent indicator. After the previous preparation, the
supernatant was discarded. The cells were then lysed with
ultrapure water and incubated at 37°C for 15 min. The lysate was
transferred to a new 1.5 mL EP tube and placed at −80°C for 30 min.
After centrifuging at 6200 × g for 5 min, the supernatant was
transferred to a fresh 1.5 mL EP tube. An equal volume of 50 μL
of MQAE (HY-D0090, MedChemExpress, United States) at a
concentration of 10 μM was added to the supernatant, and the
mixture was vortexed to ensure uniform mixing. Then, 80 μL of the
mixed solution was dispensed into a 96-well plate. Fluorescence
intensity was measured using a fluorometer (VLB000D2,
ThermoFisher, United States), with the excitation wavelength set
at 355 nm and the emission wavelength at 460 nm.
2.9 Mitochondrial reactive oxygen
species levels
The levels of mitochondrial ROS were evaluated using LSCM.
Cells were adjusted to a concentration of 1 × 10
5
cells/mL and plated
onto adhesive glass coverslips (200 μL per coverslip). Following the
intervention, cells were incubated with 100 μL MitoSOX Red
(M36008, ThermoFisher, United States) mitochondrial
superoxide indicator at a final concentration of 5 μM for 5 min at
37°C. Then, cells were washed with a three 2-min wash cycle using
PBS. Subsequently, cells were fixed at room temperature for 20 min
using 4% paraformaldehyde, followed by another wash cycle. The
nuclei were counterstained with DAPI (1 μg/mL) in a dark room for
5 min at room temperature, followed by a final wash cycle.
Coverslips were finally mounted using 50% glycerol and cells
were visualized using a laser scanning confocal microscope.
Quantification of ROS levels was performed using ImageJ
software (National Institutes of Health, United States).
2.10 ASC oligomerization assay
Cells were collected and lysed in 100 µL of RIPA buffer
(supplemented with protease and phosphatase inhibitors). The
lysate was incubated in an ice bath for 60 min before
centrifugation at 14000 g,4
°C for 15 min, with the supernatant
collected for further processing. The cell lysate was then treated for
oligomerized protein processing. The cells were digested with
trypsin (25200–056, Gibco, United States) and resuspended in
0.5 mL of ice-cold buffer A. The lysate was centrifuged at 1800 g
at 4°C for 8 min to remove the precipitate. A 30 μL aliquot of lysate
was reserved for Western Blot of ASC as input controls. The
remaining supernatant was diluted in a 1:1 ratio with buffer A
and centrifuged at 2000 gat 4°C for 5 min. The supernatant was
collected and diluted with an equal volume of CHAPS buffer before
a centrifugation at 5000 gfor 8 min to precipitate ASC oligomers.
The supernatant was discarded and the pellet was resuspended in
50 µL of CHAPS buffer containing 4 mM of DSS (S1885, Sigma,
United States) and incubated at room temperature for 30 min for
protein cross-linking. The mixture was then centrifuged again at
5000 × g for 8 min at 4°C. The supernatant was discarded, and the
pellet was resuspended in 30 µL of 2× protein loading buffer and
denatured at 90°C for 2 min. Subsequent steps were conducted
according to standard Western Blot procedures with anti-ASC
antibody (sc-514414, Santa Cruz, United States).
2.11 Statistical analysis
Data were analyzed using GraphPad Prism 9 software
(GraphPad Software Inc., United States). The Shapiro-Wilk test
was used for normality, while the Brown-Forsythe test was applied
to test for homogeneity of variances. For multiple comparisons, one-
way analysis of variance (ANOVA) was used, and post hoc pairwise
comparisons were conducted using the Least Significant Difference
(LSD) method. Data are presented as means ± standard error of the
mean (SEM). A p-value of less than 0.05 was considered statistically
significant.
3 Results
3.1 Effect of acacetin on
NLRP3 inflammasome activation induced
by Nigericin
To investigate whether Acacetin could inhibit the activation of the
NLRP3 inflammasome, several methods were conducted. Western blot
analysis was used to quantify the expression levels of pro-caspase-1, pro-
IL-1β, and NLRP3 in the cell lysates, as well as caspase-1 and IL-1βin the
cell supernatant (Figure 1A). ELISA was utilized to measure the
expression of TNF-α, IL-1β, and IL-18 in the supernatant (Figures
1B–D), and the LDH assay was conducted to evaluate cell death
(Figure 1E). The results revealed that, compared to the control group,
Nigericin induced the activation of NLRP3 inflammasome, leading to a
significant increase in the expression of caspase-1, IL-1β,andNLRP3in
the supernatant and cell lysates. Intervention with Acacetin effectively
reduced the expression of caspase-1, IL-1β, and NLRP3. In comparison to
the control group, the expression of inflammatory cytokines TNF-α,IL-
1β, IL-18, and LDH release was significantly elevated, while acacetin
inhibited the expression of them. It indicates acacetin’s inhibition of
Nigericin-induced NLRP3 inflammation activation. Among the tested
concentrations, the inhibitory effect was most prominent at a dose of
10 μM. Consequently, this dose was selected for subsequent studies.
3.2 Effect of acacetin on
NLRP3 inflammasome activation induced by
multiple agonists
We further investigated the effects of Acacetin on
NLRP3 inflammasome activation induced by the canonical
activators MSU, ATP, and Nigericin. The results revealed that
compared to the control group, all three activators—MSU, ATP,
and Nigericin—were able to induce the activation of
NLRP3 inflammasome, leading to an increase in the expression
of caspase-1 and IL-1β(Figure 2A). There was also an elevation in the
release of TNF-α,IL-1β,IL-18,andLDHactivity(Figures 2B–E).
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FIGURE 1
Effect of Acacetin on NLRP3 inflammasome activation induced by Nigericin. After pretreatment with LPS (50 ng/mL) for 3 h, BMDMs were treated
with different concentrations of Acacetin (2.5 μM, 5 μM, and 10 μM) for 0.5 h, and then Nigericin (10 μM) was added for stimulation for 0.5 h. The culture
supernatant and cell lysate were collected. (A) Western blot detected IL-1βand caspase-1 in the supernatant, and pro-IL-1β, pro-caspase-1, and NLR P3 in
the cell lysate. (B–D) ELISA detected levels of IL-1β(B), IL-18 (C), and TNF-α(D) in the supernatant. (E) LDH activity was assessed. The data is
presented as mean ± SEM (n= 3) and was analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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FIGURE 2
Effect of Acacetin on NLRP3 inflammasome activation induced by multiple agonists. BMDMs cells were pretreated with LPS (50 ng/mL) for 3 h and
then treated with Acacetin (10 μM) for 0.5 h. After that, the cells were stimulated with MSU (150 μg/mL for 4 h), ATP (2.5 mM for 0.5 h), and Nigericin
(10 μM for 0.5 h). The culture supernatant and cell lysate were collected. (A) Western blot detected IL-1βand caspase-1 in the supernatant, and pro-IL-1β
and pro-caspase-1 in the cell lysate. (B–D) ELISA detected the levels of IL-1β(B), IL-18 (C) and TNF-α(D) in the supernatant. (E) LDH activity was
assessed. The data is presented as mean ± SEM (n= 3) and was analyzed with ANOVA followed by LSD *p<0.05, **p<0.01, ***p<0.001.
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FIGURE 3
Effect of Acacetin on non-canonical NLRP3 inflammasome activation. After pretreatment with Pam3CSK4 (400 ng/mL) for 3 h, BMDMs were
treated with acacetin of different concentrations (2.5 μM, 5 μM, and 10 μM) for 0.5 h. Subsequently, the cells were transfected with LPS (500 ng/mL) using
Lipofectamine 3000 for 16 h. The culture supernatant and cell lysate were collected. (A) Western blot detected IL-1βand caspase-1 in the supernatant,
and pro-IL-1βand pro-caspase-1 in the cell lysate. (B–D) ELISA detected the levels of IL-1β(B), IL-18 (C) and TNF-α(D) in the supernatant. (E) LDH
activity was also assessed. The data is presented as mean ± SEM (n= 3) and was analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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FIGURE 4
Effect of Acacetin on the NF-kB pathway. BMDMs were primed with LPS for different durations (0, 10 min, 30 min, and 60 min) and then treated with
acacetin (10 μM). (A) Western blot was used to detect the relative expression levels of NF-κB p65 (B),p-NF-κB p65 (C),IκBα(D), and p-IκBα(E) in the cells.
The data is presented as mean ± SEM (n = 3) and was analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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Remarkably, acacetin demonstrated an inhibitory effect on
NLRP3 inflammasome activation by these canonical activators and
decreased the secretion of TNF-α,IL-1β, IL-18, and LDH release.
3.3 Effect of acacetin on non-canonical
NLRP3 inflammasome activation
To explore the potential effects of acacetin on non-canonical
NLRP3 inflammasome activation, BMDMs were pre-treated with
Pam3CSK4 and then stimulated with LPS. Our findings
demonstrated that acacetin effectively interrupted the cleavage of
caspase-1 during non-canonical NLRP3 inflammasome activation.
This interruption led to a subsequent inhibition of the secretion of
IL-1βand IL-18, along with the release of LDH. Interestingly, the
expression of TNF-αremained unaffected by acacetin under these
conditions. Figure 3 summarizes these results.
3.4 Effect of acacetin on the NF-κB pathway
Western Blot was employed to detect the protein expression
levels of phosphorylated p65 (p-p65), p65, phosphorylated IκBα
(p-IκBα), and IκBα. As shown in Figure 4, the protein expression
levels of NF-κB p65 and IκBαdid not differ in all groups. Compared
to the control group, the expression levels of p-65 and p-IκBαwere
upregulated with the progression of LPS stimulation time. Upon
treatment with acacetin, the expression of both p-65 and p-IκBαwas
markedly reduced.
3.5 Effect of acacetin on nuclear
translocation of NF-κB p65
The results from LSCM revealed that in the blank control group,
NF-κB p65 (green) was broadly distributed within the cytoplasm,
FIGURE 5
Effect of Acacetin on nuclear localization of NF-κB p65. BMDMs were primed with LPS for different durations (0, 10 min, 30 min, and 60 min) and
then treated with acacetin (10 μM). (A) Immunofluorescence staining of NF-κB p65 was performed. NF-κB p65 is depicted in gre en, and the cell nuclei are
shown in blue. Scale bar: 50 μm. (B) Quantification of relative fluorescence intensity of NF-κB p65. The data is presented as mean ± SEM (n= 3) and was
analyzed with ANOVA followed by LSD. ***p<0.001.
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with no observable localization within the cell nucleus (blue). Upon
LPS stimulation, a substantial amount of NF-κB p65 was observed to
localize to the cell nucleus, indicating that LPS promotes the
activation and nuclear translocation of NF-κB p65. Following
treatment with acacetin, the levels of LPS-mediated NF-κB
p65 localized to the nucleus were significantly reduced, indicating
FIGURE 6
Effect of Acacetin on the MAPK pathway. BMDMs were primed with LPS for different durations (0, 10 min, 30 min, and 60 min), then treated with
acacetin (10 μM). (A) Western blot was conducted to detect the relative expressions of ERK (B), p-ERK (C),JNK(D),p-JNK(E), p38 (F), and p-p38 (G) in the
cells. The data is presented as mean ± SEM (n = 3) and was analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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that acacetin significantly inhibits LPS-mediated NF-κB p65 nuclear
translocation. A visual overview of the results is given in Figure 5.
3.6 Effect of acacetin on the mitogen-
activated protein kinase pathway
The MAPK family signaling pathways include p38, ERK, and
JNK. To assess their activity, Western blot analysis was conducted to
measure the expression levels of phosphorylated ERK (p-ERK),
ERK, phosphorylated JNK (p-JNK), JNK, phosphorylated p38
(p-p38), and p38. In the blank control group, the expression
levels of p-ERK, p-JNK, and p-p38 were found to be at a low
level. Upon LPS stimulation, there was an increase in the expression
of these phosphorylated proteins. Acacetin effectively inhibited the
LPS-induced expression of p-ERK, p-JNK, and p-p38, underscoring
its modulatory effect on the MAPK pathway. The results are shown
in Figure 6.
3.7 Effect of acacetin on K+ and Cl- ions
efflux during NLRP3 inflammasome
activation
Many studies have highlighted the important role of K+ and
Cl-ions in the activation process of the NLRP3 inflammasome. In
our investigation, compared to the blank control group, the
expression of intracellular K+ and Cl-ions decreased in the
LPS + Nigericin group. The intervention of acacetin did not
reverse the reduction of K+ and Cl-ions within the cells,
suggesting that acacetin does not affect the efflux of K+ and
Cl-ions induced by Nigericin. Therefore, acacetin does not affect
NLRP3 inflammasome activation through the efflux of K+ and
Cl-ions. See Figure 7 for the results.
3.8 Effect of acacetin on mitochondrial
damage and reactive oxygen species during
NLRP3 inflammasome activation
ROS are key upstream signals in the NLRP3 inflammasome
activation. We utilized MitoSOX to label ROS and observed their
expression levels in cells using a laser confocal microscope. The
study found compared to the control group, ROS production
increased following Nigericin induction. Intervention with acacetin
subsequently led to reduced ROS production in a dose-dependent
manner. Thus, acacetin may influence NLRP3 inflammasome
activation by modulating ROS production (Figure 8).
3.9 Effect of acacetin on ASC aggregation
during NLRP3 inflammasome activation
ASC aggregation is an important hallmark of
NLRP3 inflammasome activation. In comparison with the control
group, Nigericin was observed to induce ASC oligomerization.
FIGURE 7
Effect of Acacetin on K+ and Cl- Ions Efflux During NLRP3 Inflammasome Activation. BMDMs were primed with LPS (50 ng/mL) for 3 h, followed by
treatment with different concentrations of acacetin (2.5 μM, 5 μM, and 10 μM) for 0.5 h, and then stimulated with Nigericin (10 μM) for 0.5 h. The cell
suspension was collected and processed. (A) K+ ions concentration was measured using a potassium ion assay kit (mmol/gprot). (B) Cl-ions
concentration was determined using MQAE fluorescent indicator. The data is presented as mean ± SEM (n= 3) and was analyzed with ANOVA
followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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Treatment with Acacetin led to a dose-dependent reduction in ASC
oligomerization. The outcome is illustrated in Figure 9.
4 Discussion
Acacetin has been recognized for its pharmacological properties,
including antioxidant and anti-inflammatory effects, and has
demonstrated therapeutic potential against various diseases in
cellular or animal studies. Multiple studies have found that
acacetin can exert protective effects on cardiovascular diseases by
regulating pathways such as TGF-β/Smad3, MAPK, and PI3K/Akt,
inhibiting the expression of inflammatory cytokines, and promoting
the secretion of anti-inflammatory factors (Liu et al., 2016;Wu W.-
Y. et al., 2018;Li Z.-Y. et al., 2023). Wu D. et al., 2018 found in
cellular experiments that acacetin can inhibit the production of ROS,
increase the activity of HO-1 and Nrf2, and suppress the expression
of inflammatory factors TNF-αand IL-1β, thereby alleviating LPS-
induced lung injury. In vitro studies by Ha et al. (Ha et al., 2012)
showed that acacetin could suppress the expression of NO, iNOS,
FIGURE 8
Effect of Acacetin on Mitochondrial Damage and ROS During NLRP3 Inflammasome Activation. BMDMs were primed with LPS (50 ng/mL) for 3 h,
followed by treatment with different concentrations of acacetin (2.5 μM, 5 μM, and 10 μM) for 0.5 h, and then stimulated with Nigericin (10 μM) for 0.5 h.
The cells were then incubated with MitoSOX Red Indicator (5 μM) to detect the levels of mitochondrial ROS. The images (A) were acquired using laser
scanning confocal microscopy and the bar plot (B) was quantitated by fluorescen ce intensity. Scale bar: 20 μm. The data is presented as mean ± SEM
(n= 3) and was analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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PGE2, COX2, TNF-α, and IL-1βin LPS-stimulated BV2 cells.
Moreover, acacetin has been found to inhibit 6-OHDA-induced
neuronal death and ROS-mediated apoptotic cascades, thereby
exerting protective effects against Parkinson’s Disease (PD) (Kim
et al., 2017). Wang et al. (Wang et al., 2015) discovered that acacetin
could mediate the transcriptional regulation of APP and BACE-1,
downregulate their protein expression, reduce Aβformation, and
also inhibit APP synthesis, thus lessening the formation of senile
plaques and providing protection against Alzheimer’s Disease (AD).
Although these studies confirm the anti-inflammatory effect of
acacetin, the specific mechanisms remain to be further elucidated,
and it is still unclear whether acacetin could become a candidate
drug for inhibiting the NLRP3 inflammasome. In this study, we
found that acacetin inhibits the activation of the
NLRP3 inflammasome induced by canonical activators such as
MSU, ATP, and Nigericin. Acacetin is able to suppress caspase-1
cleavage, inhibit the secretion of IL-1βand IL-18, reduce LDH
activity, and furthermore, it can also inhibit non-classical
NLRP3 inflammasome activation.
The activation of the NLRP3 inflammasome involves two
steps—priming step and activation step. The priming step mainly
activates the NF-κB pathway, thereby upregulating the expression of
NLRP3 and Pro-IL-1β. The activation step primarily triggers the
assembly of the NLRP3 inflammasome, leading to the secretion of
IL-1βand IL-18 mediated by caspase-1, as well as pyroptosis
(Bauernfeind et al., 2009;Swanson et al., 2019). LPS, a major
outer membrane component of Gram-negative bacteria, binds to
toll-like receptor 4 (TLR4), activates the MAPK pathway, and
FIGURE 9
Effect of Acacetin on ASC Aggregation During NLRP3 Inflammasome Activation. BMDMs were primed with LPS (50 ng/mL) for 3 h, followed by
treatment with different concentrations of acacetin (2.5 μM, 5 μM, and 10 μM) for 0.5 h, and then stimulated with Nigericin (10 μM) for 0.5 h. The cells
were lysed with pellets crosslinked with DSS to detect ASC aggregation. (A) Western blot was performed to detect the relative expression of Polymers (B),
Dimers (C), and ASC in pellets (D) in the cells, with ASC in INPUT serving as the loading control. The data is presented as mean ± SEM (n = 3) and was
analyzed with ANOVA followed by LSD. *p<0.05, **p<0.01, ***p<0.001.
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induces an NF-κB-dependent inflammatory cascade, resulting in the
overproduction of pro-inflammatory mediators such as nitric oxide
(NO) and prostaglandin E2 (PGE2) as well as cytokines like TNF-α
and IL-6 (Zeng et al., 2017).
Here, we explored the role of acacetin during the
NLRP3 inflammasome priming step by isolating and inducing
mouse BMDMs with LPS. We found that LPS increased TNF-α
secretion and NLRP3 expression in BMDMs. While acacetin could
inhibit TNF-αsecretion and reduce NLRP3 expression, indicating its
influence on the priming of NLRP3 inflammasome activation. Several
NLRP3 inflammasome inhibitors can regulate the priming process.
Compounds such as curcumin (Hasanzadeh et al., 2020), resveratrol
(Liu et al., 2019), green tea polyphenols (Wang et al., 2019), BAY11-
7082 (Irrera et al., 2017), and INF-39 (Shi et al., 2021) have all shown
inhibitory effects on the NF-κB pathway. Chloroquine (Chen et al.,
2017), Sinigrin (Lee et al., 2017), and ECGG (Luo et al., 2021)can
inhibit both NF-κB and MAPK pathways, thereby regulating the
transcriptional levels of NLRP3 protein components and modulating
NLRP3 inflammasome activation. Whether acacetin plays an
inhibitory role during the priming is not yet clear, and whether it
inhibits NLRP3 inflammasome activation by suppressing the NF-κB
and MAPK pathways remains unreported. In our study, we
discovered that acacetin can inhibit the expression of p-65 and
p-IκBα, suppress nuclear translocation of NF-κB, and inhibit the
expression of p-ERK, p-JNK, and p-P38, thus modulating the
priming process.
Following the priming step, the activation step of the
NLRP3 inflammasome is marked by pivotal roles of ASC
aggregation, ROS production, K+ and Cl-ions efflux. Various
inhibitors of NLRP3, such as MNS (He et al., 2014), NBC6
(Baldwin et al., 2017), and BOT-4-one (Shim et al., 2017), have
been shown to prevent the formation of ASC specks during the
activation of the NLRP3 inflammasome. Youm et al. (He et al.,
2018) demonstrated that β-hydroxybutyrate (BHB) could
specifically inhibit NLRP3 inflammasome activation by
suppressing K+ ions efflux. Similarly, MCC950 was found to
attenuate Cl-ions efflux during NLRP3 inflammasome activation
(Jiang et al., 2017). Yang et al., 2016 pinpointed that sulforaphane
can impede NLRP3 inflammasome activation via inhibiting
unsaturated fatty acid-induced ROS production through the
AMP-activated protein kinase (AMPK) autophagy pathway. In
ourpreviousstudies(Bu et al., 2019;2022;2023), we identified that
acacetin could diminish ROS level in microglial cells post-oxygen
glucose deprivation (OGD) injury. However, whether acacetin
affects ASC aggregation, as well as the efflux of K+ and Cl-ions,
thereby modulating NLRP3 inflammasome activation, remains
unreported. In this study, we observed that acacetin inhibited
ASC aggregation and ROS production following
NLRP3 inflammasome activation but did not alter the efflux of
K+ and Cl-ions during the activation step.
In summary, this study has unveiled that acacetin can inhibit the
activation of the NLRP3 inflammasome. Specifically, acacetin
modulates both the priming and assembly of NLRP3 inflammasome
activation by suppressing the NF-κBandMAPKpathways,
subsequently hindering ASC aggregation and ROS production.
While acacetin displays inhibitory effects on NLRP3 inflammasome
activation, further studies are warranted to determine its protective role
against diseases mediated by the NLRP3 inflammasome.
Data availability statement
The original contributions presented in the study are included in
the article/Supplementary material, further inquiries can be directed
to the corresponding authors.
Ethics statement
The animal study was approved by the Ethics Committee of
People’s Hospital of Xinjiang Uygur Autonomous Region. The study
was conducted in accordance with the local legislation and
institutional requirements.
Author contributions
JB: Conceptualization, Data curation, Funding acquisition,
Investigation, Project administration, Writing–original draft,
Writing–review and editing. YM: Data curation, Formal Analysis,
Funding acquisition, Writing–review and editing, Writing–original
draft. SZ: Formal Analysis, Investigation, Writing–review and
editing. XW: Formal Analysis, Investigation, Writing–review and
editing. XZ: Formal Analysis, Investigation, Writing–review and
editing. LZ: Conceptualization, Data curation, Project
administration, Supervision, Writing–review and editing. YZ:
Supervision, Writing–review and editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This research
was funded by grants from “Tianshan Youth Project”of Program for
Fostering Excellent Young Talents in Science and Technology of
Xinjiang Uygur Autonomous Region (Grant No. 2020Q046),
Natural Science Foundation of Xinjiang Uygur Autonomous
Region (Grant No. 2020D01C095), and Program for Fostering
Excellent Young Talents in Science and Technology of Urumqi,
China to JB and Foundation of People’s Hospital of Xinjiang Uygur
Autonomous Region (Grant No. 20210110) to YM
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Frontiers in Pharmacology frontiersin.org15
Bu et al. 10.3389/fphar.2024.1286546
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