Naringenin attenuates the release of pro-inflammatory mediators from lipopolysaccharide-stimulated BV2 microglia by inactivating nuclear factor-κB and inhibiting mitogen-activated protein kinases.
ABSTRACT Naringenin, one of the most abundant flavonoids in citrus fruits and grapefruits, has been reported to exhibit anti-inflammatory and antitumor activities. However, the cellular and molecular mechanisms underlying the naringenin anti-inflammatory activity are poorly understood. In this study, we conducted an investigation of the inhibitory effects of naringenin on the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators in BV2 microglial cells. We found that pre-treatment with naringenin prior to treatment with LPS significantly inhibited excessive production of nitric oxide (NO) and prostaglandin E2 (PGE2) in a dose-dependent manner. The inhibition was associated with downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Naringenin also attenuated the production of pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) by suppressing expression of mRNAs for these proteins. In addition, the molecular mechanism underlying naringenin-mediated attenuation in BV2 cells has a close relationship to suppressing translocation of the nuclear factor-κB (NF-κB) p65 subunit into the nucleus and the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs). These findings suggest that naringenin may provide neuroprotection through suppression of pro-inflammatory pathways in activated BV2 microglial cells.
- [show abstract] [hide abstract]
ABSTRACT: Inflammatory events in the CNS are associated with injuries as well as with well-known chronic degenerative diseases, such as Multiple Sclerosis, Parkinson's, or Alzheimer's disease. Compared to inflammation in peripheral tissues, inflammation in brain appears to follow distinct pathways and time-courses, which likely has to do with a relatively strong immunosuppression in that organ. For this reason, it is of great importance to get insights into the molecular mechanism governing immune reactions in brain tissue. This task is hard to achieve in vivo, but can be approached by studying the major cell type responsible for brain inflammation, the microglia, in culture. Since these cells are the only professional antigen-presenting cells resident in brain parenchyma, molecular mechanisms of antigen presentation are being discussed first. After covering the expression and regulation of anti- and proinflammatory cytokines, induction and regulation of two key enzymes and their products-COX-2 and iNOS-are summarized. Possibly, pivotal molecular targets for drug therapies of brain disorders will be discovered in intracellular signaling pathways leading to activation of transcription factors. Finally, the impact of growth factors, of neurotrophins in particular, is highlighted. It is concluded that the presently available data on the molecular level is far from being statisfying, but that only from better insights into molecular events will we obtain the information required for more specific therapies.Microscopy Research and Technique 08/2001; 54(1):47-58. · 1.59 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Neuroinflammatory processes play a significant role in the pathogenesis of Parkinson’s disease (PD). Epidemiologic, animal, human, and therapeutic studies all support the presence of a neuroinflammatory cascade in disease. This is highlighted by the neurotoxic potential of microglia. In steady-state, microglia serve to protect the nervous system by acting as debris scavengers, killers of microbial pathogens, and regulators of innate and adaptive immune responses. In neurodegenerative diseases, activated microglia affect neuronal injury and death through production of glutamate, pro-inflammatory factors, reactive oxygen species, quinolinic acid among others and by mobilization of adaptive immune responses and cell chemotaxis leading to transendothelial migration of immunocytes across the blood–brain barrier and perpetuation of neural damage. As disease progresses, inflammatory secretions engage neighboring glial cells, including astrocytes and endothelial cells, resulting in a vicious cycle of autocrine and paracrine amplification of inflammation perpetuating tissue injury. Such pathogenic processes contribute to neurodegeneration in PD. Research from others and our own laboratories seek to harness such inflammatory processes with the singular goal of developing therapeutic interventions that positively affect the tempo and progression of human disease.Clinical Neuroscience Research 01/2007; · 0.80 Impact Factor
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ABSTRACT: Microglia-derived inflammatory neurotoxins play a principal role in the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and HIV-associated dementia; chief among these is reactive oxygen species. The detrimental effects of oxidative stress in the brain and nervous system are primarily a result of the diminished capacity of the central nervous system to prevent ongoing oxidative damage. A spectrum of environmental cues, mitochondrial dysfunction, accumulation of aberrant misfolded proteins, inflammation, and defects in protein clearance are known to evolve and form as a result of disease progression. These factors likely affect glial function serving to accelerate the tempo of disease. Understanding the relationships between disease progression, free radical formation, neuroinflammation, and neurotoxicity is critical to elucidating disease mechanisms and the development of therapeutic modalities to combat disease processes. In an era where populations continue to age, the prevalence and incidence of age-related neurodegenerative diseases are on the rise; therefore, the need for novel therapeutic strategies that attenuate neuroinflammation and protect neurons against oxidative stress is ever more immediate.International Review of Neurobiology 02/2007; 82:297-325. · 1.65 Impact Factor
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30: 204-210, 2012
Abstract. Naringenin, one of the most abundant flavonoids
in citrus fruits and grapefruits, has been reported to exhibit
anti-inflammatory and antitumor activities. However, the
cellular and molecular mechanisms underlying the naringenin
anti-inflammatory activity are poorly understood. In this
study, we conducted an investigation of the inhibitory effects
of naringenin on the production of lipopolysaccharide (LPS)-
induced pro-inflammatory mediators in BV2 microglial cells.
We found that pre-treatment with naringenin prior to treatment
with LPS significantly inhibited excessive production of nitric
oxide (NO) and prostaglandin E2 (PGE2) in a dose-dependent
manner. The inhibition was associated with downregulation of
inducible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) expression. Naringenin also attenuated the production
of pro-inflammatory cytokines and chemokines, including
interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and
monocyte chemoattractant protein-1 (MCP-1) by suppressing
expression of mRNAs for these proteins. In addition, the molec-
ular mechanism underlying naringenin-mediated attenuation in
BV2 cells has a close relationship to suppressing translocation
of the nuclear factor-κB (NF-κB) p65 subunit into the nucleus
and the phosphorylation of Akt and mitogen-activated protein
kinases (MAPKs). These findings suggest that naringenin may
provide neuroprotection through suppression of pro-inflamma-
tory pathways in activated BV2 microglial cells.
Microglia are glial cells that enter the brain early in embryo-
genesis and develop in parallel with the maturation of the
nervous system. Under normal conditions, these microglia
play a major role in host defense and tissue repair in the central
nervous system (CNS) (1-3). However, in response to injury,
infection or inflammation, microglia readily become activated
and secrete neurotoxic and pro-inflammatory mediators,
including nitric oxide (NO), prostaglandin E2 (PGE2), reactive
oxygen species (ROS) and pro-inflammatory cytokines such
as interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α
(TNF-α) (4-6). Therefore, microglial activation appears to
play a pivotal role in the initiation and progression of neuro-
degenerative diseases, including Alzheimer's disease (AD),
Parkinson's disease (PD), cerebral ischemia, multiple sclerosis
and trauma (7-9). Thus, regulating microglial activation and
downregulation of pro-inflammatory mediators in microglia
may have the therapeutic potential to reduce neuronal injury
or death in neurodegenerative diseases.
Flavonoids are a diverse group of plant natural products
synthesized from phenylpropanoid and acetate-derived precur-
sors. They are becoming an important source of novel agents
with pharmaceutical potential and have attracted a great deal
of attention in recent years for their role in the prevention
of chronic diseases (9-13). Among them, naringenin and its
glycoside naringin are abundant in grapefruit and citrus fruits
and juices (14,15). Previous studies have shown that narin-
genin inhibits CYP3A4 activity and exhibits aorta dilatory,
antioxidant, antiestrogenic, antiproliferative and antimeta-
static effects (16-20). Recently, naringenin, but not naringin,
has been reported to induce apoptosis in various human cancer
cells and treatment with a similar dose showed no toxic effect
on normal cells (21-25). Although numerous studies on the
antioxidant and anticancer effects of naringenin have been
reported, the cellular and molecular mechanisms underlying
naringenin-induced anti-inflammatory effects are not clear.
In this study, we investigated the inhibitory effects of
naringenin and the way in which it induces anti-inflammatory
mechanisms in lipopolysaccharide (LPS)-stimulated inflam-
matory mediator production in murine BV2 microglia. As a
result of our findings, we suggest that naringenin may be a
candidate for use in treatment of various neurodegenerative
disorders in the brain.
Naringenin attenuates the release of pro-inflammatory mediators
from lipopolysaccharide-stimulated BV2 microglia by inactivating
nuclear factor-κB and inhibiting mitogen-activated protein kinases
HYE YOUNG PARK1,2, GI-YOUNG KIM3 and YUNG HYUN CHOI1,4
1Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052; 2Department of
Pharmacy, Busan National University, Busan 609-735; 3Department of Marine Life Sciences, Jeju National University,
Jeju 690-756; 4Department of Biomaterial Control (BK21 Program), Graduate School and Blue-Bio Industry
Regional Innovation Center, Dongeui University, Busan 614-714, Republic of Korea
Received January 25, 2012; Accepted March 23, 2012
Correspondence to: Dr Yung Hyun Choi, Department of Bio chemistry,
Dongeui University College of Oriental Medicine, Busan 614-052,
Republic of Korea
Key words: naringenin, anti-inflammation, nuclear factor-κB, Akt,
mitogen-activated protein kinase
PARK et al: ANTI-INFLAMMATORY EFFECTS OF NARINGENIN
Materials and methods
Cell culture. BV2 murine microglial cells were obtained
from Professor I.W. Choi (Inje University, Busan, Republic
of Korea). The cells were cultured in Dulbecco's modified
Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD)
supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin and 100 µg/ml streptomycin and were maintained
in a humidified incubator with 5% CO2. Naringenin (Sigma-
Aldrich, St. Louis, MO) was dissolved in dimethyl sulfoxide
(DMSO) and dilutions were made in DMEM. The final
concentration of DMSO in the medium was <0.05% (vol/vol)
which showed no influence on cell growth. In all experiments,
cells were pre-treated with the indicated concentrations of
naringenin for 1 h before addition of LPS (Escherichia coli
Cell viability assay. Cell viability was measured based on
formation of blue formazan metabolized from colorless
(MTT, Sigma-Aldrich) by mitochondrial dehydrogenases,
which are active only in live cells. BV2 cells were plated into
24-well plates at a density of 2x105 cells/well for 24 h and then
washed. The cells incubated with various concentrations of
naringenin were treated with or without 0.5 µg/ml LPS for 24 h
and then incubated in 0.5 mg/ml MTT solution. Three hours
later, the supernatant was removed and formation of formazan
was measured at 540 nm using a microplate reader (26).
Measurement of NO production. The concentrations of NO in
culture supernatants were determined as nitrite, a major stable
product of NO, using the Griess reagent (Sigma-Aldrich).
After cells (5x105 cells/ml) were stimulated in 24-well plates
for 24 h, 100 µl of each cultured medium was mixed with the
same volume of the Griess reagent [1% sulfanilamide/0.1%
H3PO4]. Nitrite levels were determined using an ELISA plate
reader at 540 nm and nitrite concentrations were calculated by
reference to a standard curve generated by known concentra-
tions of sodium nitrite (27).
Measurement of PGE2 production. BV2 cells were incubated
with naringenin in either the presence or absence of LPS
(0.5 µg/ml) for 24 h. Following the manufacturer's instruc-
tions, a volume of 100 µl of culture-medium supernatant was
collected for determination of PGE2 concentration by ELISA
(Cayman Chemical, Ann Arbor, MI).
Reverse transcriptase-polymerase chain reaction. Total-RNA
was isolated using TRIzol reagent (Invitrogen Life
Technologies, Carlsbad, CA). Total-RNA (1.0 µg) obtained
from cells was primed with random hexamers to synthesize
complementary DNA using M-MLV reverse transcriptase
(Promega, Madison, WI) according to the manufacturer's
instructions. Polymerase chain reaction (PCR) was performed
for amplification of the inducible nitric oxide synthase (iNOS),
cyclooxygenase-2 (COX-2), IL-1β, TNF-α and monocyte
chemoattractant protein-1 (MCP-1) genes from the cDNA and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
used as an internal control. Conditions for the PCR reactions
were 1x (94˚C for 3 min); 35x (94˚C for 45 sec, 58˚C for 45 sec,
and 72˚C for 1 min); and 1x (72˚C for 10 min). Amplification
products obtained by PCR were electrophoretically separated
on 1% agarose gel and visualized by EtBr staining.
Protein extraction and western blot analysis. For western
blot analysis, cells were harvested and washed twice in PBS
at 4˚C. Total cells lysates were lysed in lysis buffer [40 mM
Tris (pH 8.0), 120 mM, NaCl, 0.5% NP-40, 0.1 mM sodium
orthovanadate, 2 µg/ml aprotinin, 2 µg/ml leupeptin and
100 µg/ml phenymethylsulfonyl fluoride]. The supernatants
were collected and protein concentrations were then measured
with protein assay reagents (Pierce Biotechnology, Inc.,
Rockford, IL). Equal amounts of protein extracts were dena-
tured by boiling at 95˚C for 5 min in sample buffer (0.5 M
Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol
blue, 10% β-mercaptoethanol) in a ratio of 1:1, subjected to
8-10% sodium dodecyl sulfate (SDS)-polyacrylamide gels
and transferred to polyvinylidene difluoride membranes
(Schleicher and Schuell Bioscience, Inc., Keene, NH) by
electroblotting. The membranes were blocked with 5% non-fat
dry milk in PBS with Tween-20 buffer (PBS-T) (20 mM Tris,
100 mM NaCl, pH 7.5 and 0.1% Tween-20) for 1 h at room
temperature. Membranes were then incubated overnight at
4˚C with the primary antibodies, probed with enzyme-linked
secondary antibodies and visualized using an enhanced chemi-
luminescence (ECL) kit (Amersham Life Science, Arlington
Heights, IL) according to the manufacturer's instructions. In
a parallel experiment, nuclear proteins were prepared using
the NE-PER nuclear extraction reagent (Pierce Biotechnology,
Inc.) according to the manufacturer's protocol.
Enzyme immunosolvent assay (ELISA). The levels of IL-1β,
TNF-α (R&D Systems, Minneapolis, MN) and MCP-1
(BioLegend, San Diego, CA) were measured by the ELISA kits
according to the manufacturer's instructions. Briefly, BV2 cells
(5x105 cells/ml) were plated in 24-well plates and pre-treated
with the indicated concentrations of naringenin for 1 h before
treatment of 0.5 µg/ml LPS for 24 h. One hundred microliters
of culture-medium supernatants were collected for determina-
tion of IL-1β, TNF-α and MCP-1 concentration by ELISA (28).
Electrophoretic mobility shift assay (EMSA). DNA-protein
binding assays were carried out with nuclear extract. Synthetic
complementary nuclear factor-κB (NF-κB; 5'-AGTTGAGGGG
ACTTTCCCAGGC-3') binding oligonucleotides (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) were 3'-biotinylated using
the biotin 3'-end DNA labeling kit (Pierce Biotechnology, Inc.)
according to the manufacturer's instructions and annealed
for 30 min at room temperature. The reaction mixture was
electrophoretically separated on a 4% polyacrylamide gel in
0.5X Tris-borate buffer and transferred to a nylon membrane.
The transferred DNAs were cross-linked to the membrane at
120 mJ/cm2. Horseradish peroxidase-conjugated streptavidin
was used according to the manufacturer's instructions to detect
the transferred DNA.
NF-κB luciferase assay. A total of 1x106 BV2 cells were
transfected with 2 µg NF-κB-luciferase reporter plasmids (BD
Biosciences, San Jose, CA) using lipofectamine according to the
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30: 204-210, 2012
manufacturer's protocol (Gibco-BRL). After incubating with
DNA-lipofectamine mixtures, the cells were pre-incubated in
the presence or absence of naringenin before being stimulated
with LPS for 6 h. Cells were then washed twice with PBS and
lysed with reporter lysis buffer (Promega). After vortexing and
centrifugation at 12,000 x g for 1 min at 4˚C, the supernatant
was stored at -70˚C for the luciferase assay. After 20 µl of the
cell extract was mixed with 100 µl of the luciferase assay reagent
at room temperature, the mixture was measured a microplate
luminometer LB96V (Perkin-Elmer, Foster City, CA) (29).
Statistical analysis. Data values represent the means ± SD.
Statistical significance was determined using analysis of
variance, followed by Student's t-test. A value of P<0.05 was
accepted as statistically significant.
Naringenin attenuates NO and PGE2 production in
LPS-stimulated BV2 microglia. To determine levels of NO
production, we measured nitrite released into the culture
medium using the Griess reagent. For this study, BV2 cells
were pre-treated with various concentrations of naringenin
for 1 h prior to stimulation with LPS. According to the NO
detection assay, treatment with LPS alone resulted in higher
NO production by cells as compared with that generated by the
control. However, pre-treatment with naringenin significantly
repressed the levels of NO production in LPS-stimulated BV2
cells in a concentration-dependent manner (Fig. 1A). PGE2 is
another important inflammatory mediator. We evaluated the
effects of naringenin on PGE2 production in LPS-stimulated
BV2 cells. As indicated in Fig. 1B, treatment of BV2 cells
with LPS alone resulted in a marked increase in PGE2 release
in comparison to untreated controls. However, naringenin
inhibited LPS-induced PGE2 production in a concentration-
dependent manner. These results suggest that pre-treatment
with naringenin suppresses LPS-mediated expression of pro-
inflammatory mediators. In order to exclude cytotoxic effects
of naringenin in BV2 microglia, we evaluated the viability
of BV2 cells incubated with or without 0.5 µg/ml LPS in
the absence or presence of naringenin using MTT assays.
Concentrations (40-80 µM) used for inhibition of NO and
PGE2 production did not affect cell viability (data not shown),
confirming that inhibition of NO and PGE2 production in
LPS-stimulated BV2 cells was not due to a cytotoxic action
Naringenin decreases expression of LPS-stimulated iNOS
and COX-2 mRNA and protein. We carried out RT-PCR and
western blot analyses to determine whether inhibition of NO
and PGE2 production were associated with decreased levels of
iNOS and COX-2 expression. As shown in Fig. 2, iNOS and
COX-2 mRNA levels were detectable 6 h after LPS treatment,
whereas the protein levels of these enzymes were detected in
whole cell lysates 24 h after LPS treatment. However, narin-
genin markedly decreased both mRNA and protein levels for
iNOS and COX-2. The results suggest that naringenin-induced
reductions in the expression of iNOS and COX-2 were the
cause of the inhibition of NO and PGE2 production.
Figure 1. Inhibition of NO and PGE2 production by naringenin in LPS-stimulated
BV2 microglia. BV2 cells were pre-treated with various concentrations of nar-
ingenin for 1 h prior to incubation with LPS (0.5 µg/ml) for 24 h. (A) Nitrite
content was measured using the Griess reaction. (B) Sample treatment condi-
tions were identical to those described for (A) and a commercially available
ELISA kit was used for measurement of PGE2 in the resulting supernatants.
Each value indicates the mean ± SD and is representative of results obtained
from 3 independent experiments. *P<0.05 indicates a significant difference from
the value obtained for cells treated with LPS in the absence of naringenin.
Figure 2. Inhibition of iNOS and COX-2 expression by naringenin in LPS-
stimulated BV2 microglia. (A) BV2 cells were pre-treated with naringenin
1 h prior to incubation with LPS (0.5 µg/ml) for 24 h. Cell lysates were then
prepared and western blot analysis was performed using antibodies specific
for murine iNOS and COX-2. (B) After LPS treatment for 6 h, total-RNA was
prepared for RT-PCR analysis of iNOS and COX-2 gene expression in LPS-
stimulated BV2 microglia. ERK and GAPDH were used as internal controls
for western blot analysis and RT-PCR assays, respectively. The experiment
was repeated 3 times and similar results were obtained.
PARK et al: ANTI-INFLAMMATORY EFFECTS OF NARINGENIN
Naringenin suppresses induction of inflammatory cytokines in
LPS-stimulated BV2 microglia. We next determined whether
or not naringenin suppresses production of pro-inflammatory
cytokines such as IL-1β and TNF-α. For this study, BV2 cells
were incubated with naringenin in the absence or presence of
LPS for 24 h and cytokine levels were evaluated in the culture
supernatants. As shown in Fig. 3A and B, the levels of IL-1β
and TNF-α were markedly increased in the culture media of
LPS-stimulated BV2 microglia. However, pre-treatment with
naringenin resulted in a significant decrease in the release of
these pro-inflammatory cytokines in a concentration-dependent
manner. In a parallel experiment using RT-PCR, we studied
the effects of naringenin on LPS-induced IL-1β and TNF-α
mRNA expression. As shown in Fig. 3B, IL-1β and TNF-α
mRNA transcription also decreased following naringenin
treatment. These results suggest that naringenin suppresses
pro-inflammatory cytokine production through alteration of the
transcription levels of IL-1β and TNF-α in activated microglia.
Naringenin attenuates MCP-1 protein and mRNA in
LPS-stimulated BV2 microglia. We determined the effects
of naringenin on production of chemokine MCP-1. As shown
in Fig. 4A, naringenin alone had no effect on the production
of MCP-1 in BV2 cells, but the levels of MCP-1 were mark-
edly increased in the culture media of LPS-stimulated BV2
microglia. However, pre-treatment with naringenin resulted in a
concentration-dependent decrease of MCP-1 production, which
was associated with a reduction in LPS-mediated increases in
MCP-1 mRNA levels. These results also indicate that narin-
genin regulates production of MCP-1 at the transcriptional level.
Naringenin blocks NF-κB in LPS-stimulated BV2 microglia.
Because activation of NF-κB is the key event for the induction
of all major pro-inflammatory mediators, we next investigated
whether naringenin modulates the activation of NF-κB in BV2
microglia in response to LPS. As shown in Fig. 5, immuno-
blotting indicated that stimulation of cells with LPS induced
the degradation of IκBα and the translocation of the NF-κB
Figure 4. Effects of naringenin on MCP-1 production in LPS-stimulated BV2
microglia. BV2 cells were pre-treated with the indicated doses of naringenin
for 1 h before LPS treatment (0.5 µg/ml) and total-RNA and supernatants were
isolated at 6 or 24 h after LPS treatment, respectively. (A) Following incuba-
tion for 24 h and centrifugation, supernatants were isolated and the amounts of
MCP-1 were measured by ELISA kits according to the manufacturer's instruc-
tions. Each value indicates the mean ± SD and is representative of results
obtained from 3 independent experiments. *P<0.05 indicates a significant dif-
ference from cells treated with LPS in the absence of naringenin. (B) Following
incubation for 6 h, levels of MCP-1 mRNA were determined by RT-PCR. The
experiment was repeated 3 times and similar results were obtained.
Figure 3. Effects of naringenin on LPS-stimulated IL-1β and TNF-α production and expression in BV2 microglia. (A and B) BV2 cells were pre-treated with
naringenin for 1 h prior to LPS treatment (0.5 µg/ml). After incubation for 24 h, levels of (A) IL-1β and (B) TNF-α present in the supernatants were measured.
Each value indicates the mean ± SD and is representative of results obtained from 3 independent experiments. *P<0.05 indicates a significant difference from
the value obtained for cells treated with LPS in the absence of naringenin. (C) Cells were pre-treated with naringenin for 1 h prior to LPS treatment (0.5 µg/ml)
and total-RNA was isolated 6 h after LPS treatment. RT-PCR was performed for determination of the levels of IL-1β and TNF-α mRNA. The experiment was
repeated 3 times and similar results were obtained.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30: 204-210, 2012
p65 subunit from the cytosol to the nucleus. LPS-induced
IκB degradation was inhibited after 30 min of exposure to
naringenin. Also, naringenin inhibited nuclear translocation
of the NF-κB p65 protein. According to the EMSA assay, LPS
treatment caused a significant increase in the DNA-binding
activity of NF-κB (Fig. 6A). In contrast, treatment with
naringenin significantly suppressed the LPS-induced DNA
binding activity of NF-κB. We next tried to confirm inhibition
of LPS-induced NF-κB activation by naringenin by luciferase
assay. For this study, BV2 cells transfected with NF-κB-
luciferase reporter plasmids were pre-treated with naringenin
for 1 h and stimulated with LPS for 6 h and then luciferase
activity was measured. As shown in Fig. 5B, LPS significantly
enhanced NF-κB activity up to 8-fold over the basal level,
while naringenin significantly inhibited LPS-induced NF-κB
activity. Taken together, the above findings demonstrate
involvement of the NF-κB pathway in the anti-inflammatory
effect of naringenin in LPS-stimulated BV2 cells.
Naringenin reduces LPS-induced phosphorylation of Akt and
MAPKs in LPS-stimulated BV2 microglia. To investigate other
intracellular mechanisms responsible for the inhibitory effect of
naringenin on inflammatory mediators, we examined the effect
of naringenin on Akt and mitogen-activated protein kinases
(MAPKs) signaling pathways. As shown in Fig. 7, phosphoryla-
tion of Akt was increased within 15 min after LPS stimulation
and naringenin pre-treatment resulted in significant blockage
of LPS-induced Akt phosphorylation. Furthermore, stimula-
tion of BV2 cells with LPS led to rapid activation of p38MAPK,
ERK and JNK, with the peak levels of each phospho-MAPK
occurring 15-60 min after addition of LPS. However, narin-
genin pre-treatment significantly inhibited phosphorylation
of MAPKs in LPS-stimulated BV2 microglia (Fig. 7). This
finding suggests that naringenin is capable of disrupting key
signal transduction pathways such as Akt and MAPKs that are
activated by LPS in BV2 microglia; the disruption prevents
production of pro-inflammatory mediators.
In this study, we demonstrated that naringenin in activated
BV2 microglial cells inhibits LPS-induced production of
Figure 5. Effects of naringenin on LPS-induced NF-κB translocation and IκB
degradation in BV2 microglia. Cells were treated with naringenin for 1 h before
LPS treatment (0.5 µg/ml) for the indicated times. Nuclear and cytosolic pro-
teins were subjected to 10% SDS-polyacrylamide gels followed by western blot
analysis using anti-NF-κB p65 and anti-IκB-α antibodies. Results are represen-
tative of those obtained from 3 independent experiments. ERK and lamin B
were used as internal controls for nuclear and cytosolic fractions, respectively.
Figure 6. Effects of naringenin on NF-κB activation in LPS-stimulated BV2
microglia. (A) BV2 cells were pre-incubated with naringenin (80 µM) for 1 h
before stimulation of LPS (0.5 µg/ml) for 30 min. Then, the nuclear extracts were
assayed for NF-κB activity by EMSA as described in Materials and methods.
The experiment was repeated 3 times and similar results were obtained each
time. (B) Transfected BV2 microglia were pre-treated with naringenin for 1 h
and then stimulated with LPS for 6 h. NF-κB activity was expressed as lucif-
erase activities. Each value indicates the mean ± SD and is representative of
results obtained from 3 independent experiments. *P<0.05 indicates a significant
difference from cells treated with LPS in the absence of naringenin.
Figure 7. Effects of naringenin on AKT and MAPKs activation induced by
LPS in BV2 microglia. BV2 cells were treated with the indicated concentra-
tions of naringenin for 1 h prior to LPS (0.5 µg/ml) treatment for the indicated
times. Total protein was subjected to 10% SDS-polyacrylamide gels, fol-
lowed by western blot analysis using the indicated antibodies. Proteins were
visualized using an ECL detection system.
PARK et al: ANTI-INFLAMMATORY EFFECTS OF NARINGENIN
pro-inflammatory mediators such as NO and PGE2, cytokines,
including TNF-α and IL-1β, and the chemokine MCP-1.
These effects were accompanied by downregulation of NF-κB
activity and inactivation of Akt and MAPK signaling pathways.
In theory, downregulators of these inflammatory molecules
have been considered as candidate anti-inflammatory drugs
to alleviate progression of neurodegenerative diseases caused
by activation of microglia (30-32). Therefore, the inhibition
of pro-inflammatory molecules by naringenin shown in this
study could play a beneficial role in the treatment of neurode-
NO, PGE2 and pro-inflammatory cytokines and chemo-
kines, such as TNF-α, IL-6, IL-1β and MCP-1, have been
implicated as important mediators in the process of inflamma-
tion. Several lines of evidence have shown that the activation
of microglia induced by CNS injury or infection is associated
with neurodegenerative disorders and the release of NO and
PGE2, and with subsequent release of pro-inflammatory cyto-
kines and chemokines (4-6). Previously, many studies have
shown that expression of COX-2 and iNOS, key enzymes for
NO and PGE2, are upregulated in activated glial cells. Also,
pro-inflammatory cytokines activate the transcription of
COX-2 and iNOS genes, and anti-inflammatory drugs may
also effectively reduce NO and PGE2 production (1,4-6). In this
study, we demonstrate that naringenin treatment significantly
inhibits NO and PGE2 production in LPS-stimulated BV2
microglia. The inhibitory effects of naringenin attenuated the
expression iNOS and COX-2 mRNA and protein, indicating
that the effect of naringenin occurs at the transcriptional level.
The present data also indicate that naringenin inhibits the
production of pro-inflammatory cytokines and chemokines
such as TNF-α, IL-1β and MCP-1. Thus, the inhibitory actions
of naringenin on the production of inflammatory mediators
occurs at the transcriptional level.
The transcription factor NF-κB is a primary regulator of
genes that are involved in production of pro-inflammatory
cytokines and enzymes involved in the inflammatory process
(33-35). In addition, involvement of the phosphoinositide
3-kinase (PI3K)/Akt pathway in the expression of inflam-
matory mediators in microglia through activation of NF-κB
has been shown (36,37). As a result of its key role in several
pathologic conditions, NF-κB is a major drug target in a
variety of diseases. The blockade of NF-κB transcriptional
activity in microglial is also known to suppress expression of
iNOS, COX-2, pro-inflammatory cytokines and chemokines
including TNF-α, IL-1β and MCP-1 (38-40). Therefore,
many putative anti-inflammatory therapies seek to block
NF-κB activity. We demonstrated that naringenin causes
marked blockage of LPS-induced IκB-α degradation, and of
NF-κB translocation and transcriptional activity. Our findings
suggest that downregulation of pro-inflammatory mediators
by naringenin is due to inhibition of the NF-κB pathway.
Furthermore, naringenin significantly inhibited Akt activation
in LPS-stimulated BV2 microglia, indicating that naringenin
inhibits LPS-induced NF-κB activation via inactivation of the
PI3K/AKT signaling pathway.
Involvement of various intracellular signaling pathways,
such as MAPKs, in inflammatory mediator induction has been
reported (41-44). LPS is also known to activate a series of
MAPKs such as ERK, p38MAPK and JNK in microglial cells
(45). Therefore, experiments were performed to determine
whether naringenin tightly regulates expression of MAPKs
to induce anti-inflammatory effects in LPS-stimulated BV2
microglia. The present study indicates that naringenin is a
potent inhibitor of MAPKs expression induced by LPS stimu-
lation in BV2 microglia. Although, further studies are needed
to validate roles for MAPKs in changes in various inflamma-
tory mediators in microglia, the present results suggest that the
anti-inflammatory effects of naringenin are associated with
inhibition of the MAPKs signaling pathway.
In conclusion, the results presented in this study, demon-
strate that naringenin inhibits LPS-induced NO and PGE2
production by suppressing iNOS and COX-2 mRNA and
protein expression in BV2 microglial cells. Naringenin also
inhibits the production of pro-inflammatory cytokines and
chemokines (TNF-α, IL-1β and MCP-1) by suppressing their
transcriptional activity. The inhibitory action of naringenin
was mediated by prevention of NF-κB activation and by
inhibition of IκB-degradation, which is accompanied by the
blocking of PI3K/Akt and MAPKs pathways. As a result of
the findings presented in this study, we suggest that naringenin
may provide an effective treatment for many inflammatory
and neurodegenerative diseases.
This study was supported by the R&D program of MKE/KEIT
(10040391, Development of functional food materials and device
for prevention of aging-associated muscle function decrease).
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