Vinpocetine inhibits NF-kappaB-dependent inflammation via an IKK-dependent but PDE-independent mechanism.

Abstract

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-alpha-induced NF-kappaB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-alpha- or LPS-induced up-regulation of proinflammatory mediators, including TNF-alpha, IL-1beta, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-alpha- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-kappaB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca(2+) regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

Full text

Page 1

Vinpocetine inhibits NF-κB–dependent inflammation
via an IKK-dependent but PDE-independent
mechanism
Kye-Im Jeona,1, Xiangbin Xub,1, Toru Aizawac, Jae Hyang Limb, Hirofumi Jonob, Dong-Seok Kwond, Jun-ichi Abea,
Bradford C. Berka, Jian-Dong Lib,2, and Chen Yana,2
aAab Cardiovascular Research Institute andbDepartment of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry,
Rochester, NY 14642;cTokai University School of Medicine, Kanagawa, 259-1193, Japan; anddGraduate School of Public Health, Seoul National University,
Seoul, 151-742 Korea
Edited* by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved April 8, 2010 (received for review December 18, 2009)
Inflammation is a hallmark of many diseases, such as atherosclerosis,
chronicobstructivepulmonarydisease,arthritis,infectiousdiseases,and
cancer. Although steroids and cyclooxygenase inhibitors are effective
antiinflammatory therapeutical agents, they may cause serious side
effects.Therefore,developinguniqueantiinflammatoryagentswithout
significantadverseeffectsisurgentlyneeded.Vinpocetine,aderivative
of the alkaloid vincamine, has long been used for cerebrovascular dis-
orders and cognitive impairment. Its role in inhibiting inflammation,
however, remains unexplored. Here, we show that vinpocetine acts as
anantiinflammatoryagentinvitroandinvivo.Inparticular,vinpocetine
inhibits TNF-α–induced NF-κB activation and the subsequent induction
of proinflammatory mediators in multiple cell types, including vascular
smooth muscle cells, endothelial cells, macrophages, and epithelial
cells. We also show that vinpocetine inhibits monocyte adhesion and
chemotaxis, which are critical processes during inflammation. More-
over,vinpocetinepotentlyinhibitsTNF-α-orLPS-inducedup-regulation
ofproinflammatorymediators,includingTNF-α,IL-1β,andmacrophage
inflammatory protein-2, and decreases interstitial infiltration of poly-
morphonuclear leukocytes in a mouse model of TNF-α- or LPS-
induced lung inflammation. Interestingly, vinpocetine inhibits NF-
κB–dependent inflammatory responses by directly targeting IKK,
independent of its well-known inhibitory effects on phosphodies-
terase and Ca2+regulation. These studies thus identify vinpocetine
as a unique antiinflammatory agent that may be repositioned for
the treatment of many inflammatory diseases.
vinpocetine|inflammation|NF-κB|IKK
I
obstructive pulmonary disease, asthma) (2), arthritis (3), infectious
diseases, and cancer (4, 5). Steroids and cyclooxygenase inhibitors
have long been used as the main therapeutical antiinflammatory
agents,buttheyarefrequentlyassociatedwithsignificantdetrimental
effects in patients (4, 5). Thus, there is an urgent need for the de-
velopment of unique antiinflammatory agents.
NF-κB is a key transcriptional factor involved in regulating ex-
pression of proinflammatory mediators, including cytokines, che-
mokines, and adhesion molecules, thereby playing a critical role in
mediating inflammatory responses (6). NF-κB is a dimeric tran-
scription factor consisting of homo- or heterodimers of Rel-related
proteins (7). In the inactive state, NF-κB resides in the cytoplasm
and forms a multiprotein complex with an inhibitory subunit, in-
hibitor of NF-κB (IκB). On activation by external stimuli, the in-
flammatory signal converges on and activates a set of IκB kinases
known as the IκB kinase (IKK) complex. The activated IKK com-
plex phosphorylates two conserved N-terminal serine residues of
IκBα, leading to its ubiquitination and degradation by the protea-
some. The liberated NF-κB then enters the nucleus, interacts with
κB elements in the promoter region of a variety of inflammatory re-
sponse genes, and activates their transcription (6). Thus, phosphor-
ylation of IκBα appears to be the central point where diverse stimuli
nflammation is a hallmark of many human diseases, including
atherosclerosis(1),pulmonaryinflammatorydiseases(e.g.,chronic
converge to regulate NF-κB. Two major IKKs, IKK-α (IKK-1) and
IKK-β (IKK-2), have been identified and shown to be key compo-
nentsofthemultiproteinIKKcomplex(8,9).BothIKK-αandIKK-β
are Ser/Thr kinases, and each of them directly phosphorylates
IκB proteins (10). Several other molecules in the IKK complex have
also been identified, such as signal-regulated ERK kinase kinase 1
(MEKK-1), NF-κB–inducing kinase, NF-κB essential modulator/
IKKAP1/IKK-γ,andIKKcomplex-associatedprotein(11–14).These
moleculeshavebeenshowntobeessentialfortransmittingupstream
signals to IKK-α and IKK-β by acting as a kinase, regulatory protein,
or scaffold protein (11–14).
Vinpocetine is produced by slightly altering the vincamine mole-
cule, an alkaloid extracted from the periwinkle plant, Vinca minor.
Vinpocetine was originally discovered and marketed in 1978 under
the trade name Cavinton (Hungary). Since then, vinpocetine has
been widely used in many countries for the prevention of cerebro-
vasculardisordersandcognitiveimpairment,includingstroke,senile
dementia, and memory disturbances (15). For instance, different
types of vinpocetine-containing memory enhancers (Intelectol in
Europe and Memolead in Japan) are currently used as dietary sup-
plements worldwide. Vinpocetine is a cerebral vasodilator that
improves brainbloodflow (16).Vinpocetine hasalsobeenshownto
act as a cerebral metabolic enhancer by enhancing oxygen and glu-
cose uptake from blood and increasing neuronal ATP production
(17). Vinpocetine appears to affect several cellular targets, such as
Ca2+/calmodulin-stimulated cyclical nucleotide phosphodiesterase-
1(PDE-1)andvoltage-dependentNa+channelsandCa2+channels
(18).Todate,therehavebeennoreportsofitssignificantsideeffects,
toxicity, or contraindications at therapeutical doses (19).
PDEs, via regulation of cAMP and/or cGMP, have been known
to regulate inflammatory responses. Because vinpocetine has been
shown to inhibit PDE activity, we hypothesized that vinpocetine,
adrugalreadyavailableintheclinic,mayactasanantiinflammatory
agent. Here, we show that vinpocetine indeed inhibits NF-κB–
dependent inflammatory responses in vitro and in vivo. Unex-
pectedly, the inhibitory effect of vinpocetine on NF-κB signaling is
exertedviadirectinhibitionofIKKbutnotPDE.Thesestudiesthus
identifyvinpocetineasauniqueantiinflammatoryagentthatmaybe
Author contributions: J.A., B.C.B., J.-D.L., and C.Y. designed research; K.-I.J., X.X., T.A.,
J.H.L., and H.J. performed research; D.-S.K. contributed new reagents/analytic tools; K.-I.J.,
X.X., T.A., J.H.L., H.J., J.A., B.C.B., J.-D.L., and C.Y. analyzed data; and B.C.B., J.-D.L., and
C.Y. wrote the paper.
Drs. Li, Yan, and Berk have formed a start-up company, with the hope of licensing the
intellectual property rights from the University of Rochester and commercializing this
technology.
*This Direct Submission article had a prearranged editor.
1K.-I.J. and X.X. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: chen_yan@urmc.rochester.edu or
jian-dong_li@urmc.rochester.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.0914414107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0914414107 PNAS
| May 25, 2010
| vol. 107
| no. 21
| 9795–9800
MEDICAL SCIENCES

Page 2

repositioned as a therapeutical agent for the treatment of various
human inflammatory diseases.
Results
Vinpocetine Inhibits NF-κB Activation in a Variety of Cell Types. Be-
cause NF-κB plays a critical role in regulating inflammatory re-
sponse, we investigated if vinpocetine acts as an antiinflammatory
agent by inhibiting NF-κB. We first evaluated the effects of vinpo-
cetine on NF-κB–dependent transcriptional activity by using a NF-
κB–luciferasereporterplasmidtransientlytransfectedinseveralcell
types. As shown in Fig. 1A, vinpocetine potently inhibited TNF-α–
induced NF-κB–dependent transcriptional activity in vascular
smoothmusclecells(VSMCs)inadose-dependentmanner.Similar
resultswerealsoobservedinhumanumbilicalveinendothelialcells
(HUVECs) (Fig. 1B), human lung epithelial A549 cells (Fig. 1C),
andthemacrophagecell lineRAW264.7(Fig.1D). Dose–response
analysis further revealed that vinpocetine inhibits TNF-α–stimu-
lated NF-κB–dependent transcriptional activity with an approxi-
mate IC50value of 25 μM (Fig. S1A). Cell viability analysis showed
that vinpocetine did not have a significant effect on cell viability
(Fig. S1B).
Vinpocetine Inhibits TNF-α–Induced Proinflammatory Mediators in
Several Cell Types. We next determined if vinpocetine inhibits
TNF-α–induced up-regulation of NF-κB–dependent proinflamma-
tory mediators at the mRNA level by real-time quantitative RT-
PCR. As shown in Fig. 2A and Fig. S2A, vinpocetine potently
inhibitedTNF-α–inducedup-regulationofTNF-α,IL-1β,cytokine-
induced neutrophil chemoattractant (CINC-1; rat form of IL-8),
monocyte chemotactic protein-1 (MCP-1), and vascular cell adhe-
sion molecule-1 (VCAM-1) transcripts in VSMCs. Similarly, vin-
pocetine inhibited TNF-α–induced up-regulation of TNF-α, IL-1β,
IL-8, MCP-1, VCAM-1, and intercellular adhesion molecule-1
(ICAM-1) transcripts in HUVECs (Fig. 2B and Fig. S2B); up-reg-
ulationofTNF-α,IL-1β,andIL-8transcriptsinA549cells(Fig.2C);
and up-regulation of TNF-α, IL-1β, and macrophage inflammatory
protein-2 (MIP-2) transcripts in RAW264.7 (Fig. 2D).
Vinpocetine Inhibits Monocyte Adhesion of HUVECs and Chemotactic
Activity of VSMCs. Toevaluatethephysiologicalconsequencesofthe
inhibitory effect of vinpocetine on induction of proinflammatory
mediators further, we assessed two critical processes during in-
flammation, monocyte adhesion to HUVECs and chemotaxis to
VSMCs, which are known to be dependent on adhesion molecules
(e.g., ICAM-1, VCAM-1) andchemokines (e.g., MCP-1). As shown
in Fig. 3 A and B, TNF-α–induced monocyte adhesion to HUVECs
was significantly inhibited by vinpocetine. Monocyte chemotaxis to
the conditional medium collected from VSMCs was measured by
transwellmigrationusingaBoydenchamber(CorningLifeScience).
As shown in Fig. 3C, monocyte chemotaxis induced by VSMCs
treated with TNF-α was dose-dependently inhibited by vinpocetine.
Vinpocetine Inhibits Lung Inflammatory Response in Vivo. Toconfirm
if vinpocetine also inhibits inflammatory responses in vivo, we
evaluated the effects of vinpocetine on lung inflammation induced
by intratracheal inoculation with LPS or TNF-α for 6 h, a well-
established mouse model of lung inflammation (20). As shown in
Fig.4,i.p.administrationofvinpocetinedose-dependentlyinhibited
induction of TNF-α, IL-1β, and MIP-2 mRNA expression in the
lungs of mice after intratracheal administration of LPS (Fig. 4A).
C
Relative NF- B
Luciferase Activity
0
4
8
12
16
20
*
#
Lung Epithelial Cells
- +
-
-+
- + +
Vinp
*
Macrophages
0
0.5
1
1.5
2
2.5
3
3.5
Relative NF- B
Luciferase Activity
#
- + -
- - + +
+TNF-
Vinp
D
Vinp - - + +
- + - +
TNF-
Vinp - - 50 5 20 50 ( M)
TNF-
TNF-
- + - + + +
0
Relative NF- B
Luciferase Activity
0
1
2
3
4
*
#
HUVECs
B
A
Relative NF- B
Luciferase Activity
2
4
6
8
#
#
*
#
VSMCs
Fig. 1.
moter activity in a variety of cell types. Rat aortic VSMCs (A) or HUVECs (B),
lung epithelial A549 cells (C), and macrophage RAW264.7 cells (D) trans-
fected with NF-κB–luciferase reporter plasmid were pretreated with Vinp for
60 min before treatment with or without TNF-α (10 ng/mL) for 6 h in the
continued presence or absence of various doses of Vinp, as indicated in A, or
50 μM Vinp, as indicated in B, C, and D. Cells were then lysed for luciferase
assay. Data represent the mean ± SD of at least three independent experi-
ments, and each experiment was performed in triplicate. *P < 0.05 vs. con-
trol;#P < 0.05 vs. TNF-α alone.
Vinpocetine (Vinp) inhibits TNF-α–induced NF-κB–dependent pro-
A
Aortic Smooth Muscle Cells
*
TNF-
5
Vinp - - + +
0
Relative mRNA
Levels
1
2
3
4
6
#
TNF-- + - +
- - + +
- - + +
0
*
MCP-1
#
2
4
6
8
10
12
14
- + - +
*
0
1
2
3
4
#
VCAM-1
- + - +
B
Human Umbilical Vein Endothelial Cells
TNF-
*
3
Levels
Vinp - - + +
0.5
1
1.5
2
2.5
3.5
#
- - + +
- - + +
VCAM-1
0
1
2
3
4
5
6
7
*
#
- + - +
ICAM-1
0
20
40
60
80
100
120
140
*
#
- + - +
0
TNF-- + - +
Relative mRNA
TNF-
Vinp - - + +
- + - +
TNF-
0
50
100
150
200
250
300
#
*
IL-1
0
2
4
6
8
10
12
14
#
- + - +
- - + +
*
*
IL-8
#
0
4
8
12
16
20
- + - +
- - + +
C Lung Epithelial Cells
Relative mRNA
Levels
D Macrophages
2.5
Relative mRNA
0
0.5
1
1.5
2
#
TNF-
*
0
1
2
3
4
5
6
7
8
9
*
#
MIP-2
-
- -
+ - +
+ +
*
IL-1
0
1
2
3
4
5
6
#
- +
- -
- +
+ +
TNF-
Vinp -
- + - +
- + +
Levels
Fig. 2.
matory mediators in a variety of cell types. Rat aortic VSMCs (A), HUVECs (B),
lung epithelial A549 cells (C), or macrophage RAW264.7 cells (D) were pre-
treated with 50 μM Vinp for 60 min before treatment with or without TNF-α
(10 ng/mL) for 6 h in the continued presence or absence of Vinp (50 μM).
Expression of TNF-α, IL-1β, IL-8, MCP-1, VCAM-1, ICAM-1, and MIP-1 at mRNA
levels was measured by real-time quantitative RT-PCR. Data represent the
mean ± SD of at least three independent experiments, and each experiment
was performed in triplicate. *P < 0.05 vs. control;#P < 0.05 vs. TNF-α alone.
Vinpocetine (Vinp) inhibits TNF-α–induced expression of proinflam-
9796
| www.pnas.org/cgi/doi/10.1073/pnas.0914414107Jeon et al.

Page 3

Consistent with these results, vinpocetine also significantly inhi-
bited polymorphonuclear neutrophil (PMN) infiltration in bron-
choalveolarlavage(BAL)fluids(Fig.4B)aswellasinperibronchial
and interstitial areas of mouse lungs treated with LPS (Fig. 4C).
Similarly, vinpocetine also inhibited induction of these inflamma-
tory mediators and PMN infiltration in the lungs of mice treated
with TNF-α by intratracheal administration (Fig. S3). Taken to-
gether, these results strongly suggest that vinpocetine is a potent
inhibitor of inflammatory response in vitro and in vivo.
Vinpocetine Inhibits TNF-α–Induced NF-κB Activation by Targeting
IKK. Having identified vinpocetine as an inhibitor for NF-κB–
dependent inflammation, we next determined the molecular target
of vinpocetine. Because IKK-dependent phosphorylation and
degradationofIκBαplayveryimportantrolesinmediatingTNF-α–
induced activation of NF-κB and the subsequent up-regulation of
NF-κB–dependent proinflammatory mediators, we first evaluated
the effects of vinpocetine on IκB phosphorylation and IκB degra-
dation induced by TNF-α in VSMCs. As shown in Fig. 5A, TNF-α
induced phosphorylation and degradation of IκBα in a time-
dependent manner, as measured by Western blot analysis using an
antiphospho-Ser32 IκBα antibody and a nonphosphorylated IκB
antibody. Pretreatment with vinpocetine markedly inhibited TNF-
α–induced IκBα phosphorylation and degradation. These results
suggest that vinpocetine inhibits TNF-α–induced NF-κB activation
bypreventingIκBαphosphorylationanddegradation,thusactingat
or upstream of IκBα.
Because IKK represents the major upstream kinase for IκBα
phosphorylation, we next determined whether vinpocetine inhibits
IKK.IKKkinaseactivitywasmeasuredbyinvitrokinaseassayofthe
IKKimmunocomplex,withGST-IκBα asa substrate inthe presence
of [γ-32P]-ATP. As shown in Fig. 5B, IKK activity was markedly in-
creased in the IKK immunocomplex from cells treated with TNF-α.
Vinpocetine significantly inhibited TNF-α–induced IKK activity,
thereby suggesting that vinpocetine inhibits TNF-α–induced NF-κB
activation at the level (or upstream) of IKK. The dose–response
analysis further revealed that the IC50value of vinpocetine on IKK
inhibitioninthecellis≈26μM(Fig.S4),similartotheIC50valuefor
vinpocetine inhibition of NF-κB–dependent transcriptional ac-
tivity(Fig.S1).Incontrast,wedidnotobservesignificantinhibitory
effects of vinpocetine on TNF-α–induced activation of MAP
kinases ERK1/2, JNK, and p38, thereby demonstrating the spec-
ificity of vinpocetine in inhibiting IKK-NF-κB–dependent in-
flammation (Fig. S5).
Next, we determined whether vinpocetine inhibits TNF-α–
induced NF-κB activation via inhibition of IKK or its major up-
stream kinase, MEKK1 (21). As shown in Fig. 5C, ectopic expres-
sion of constitutively active (CA) MEKK1 alone induced potent
NF-κB–dependent luciferase activity. Pretreatment with vinpoce-
tine significantlyinhibitedCA-MEKK1–inducedNF-κB activation,
indicating that vinpocetine acts at the level (or downstream) of
MEKK1. Furthermore, vinpocetine inhibited NF-κB activation in-
ducedby ectopically expressedCA-IKKα (with serines 176 and 180
mutated to glutamic acid) and CA-IKKβ (with serines 177 and 181
mutated to glutamic acid) (Fig. 5 D and E) but not by expressed
downstream molecule NF-κB p65 subunit (WT) (Fig. 5F). Collec-
tively, these data suggest that vinpocetine inhibits TNF-α–induced
NF-κB activation, likely by targeting IKK.
A
Con
TNF-
Vinp Vinp+TNF-
C
B
Monocyte Adhesion/
Optical Field
0
50
100
150
200
250
300
350
400
*
#
TNF-
Vinp
-
-
+
+
-
-
+
+
0
Migrated Cells (x 104)
10
20
40
30
50
#
*
#
#
- - 5 20 50 M
Vinp
TNF-
- + + + +
Fig. 3.
motactic activity of VSMCs. (A) Microscopic images showing U937 monocytes
adhering to HUVECs as assessed by in vitro adhesion assay. HUVECs were
pretreated with vehicle DMSO (0.5% final concentration) or 50 μM Vinp for
60 min before treatment with TNF-α (10 ng/mL) or vehicle for 6 h in the
continued presence orabsenceof Vinp. U937 monocyte adhesion toTNF-α- or
vehicle–stimulated HUVECs was analyzed. Con, control. (B) Quantitative
monocyte adhesion to HUVECs. (C) Monocyte chemotaxis to VSMCs mea-
sured by transwell migration. Rat aortic VSMCs were treated with or without
TNF-α (10 ng/mL) for 9 h in the presence or absence of various doses of Vinp
(5–50 μM). VSMC-conditional medium was collected and used for monocyte
chemotaxis assays in Boyden chambers. Data represent the mean ± SD of at
leastthree independent experiments,andeach experiment was performed in
triplicate. P < 0.05 vs. control;#P < 0.05 vs. TNF-α alone.
Vinpocetine (Vinp) inhibits monocyte adhesion of HUVECs and che-
Vinp 0 0 2.5 5 100 0 2.5 5 10
A
LPS - + + + +
Relative Quantity
of mRNA Expression
TNF-
0
1
2
3
4
5
6
*
#
#
#
*
IL-1
#
0
2
4
6
8
10
12
- + + + +
MIP-2
0
5
10
15
20
25
*
#
#
- + + + +
0 0 2.5 5 10
B
C
CONLPS
Vinp
Vinp+LPS
CON LPS
VinpVinp+LPS
Fig. 4.
Vinp administered i.p. (at 2.5, 5, and 10 mg/kg of body weight) significantly
inhibited induction of TNF-α, IL-1β, and MIP-2 mRNA in the lungs of mice by
intratracheal administration of LPS (2 μg per mouse). Data represent the
mean ± SD of at least three independent experiments. *P < 0.05 vs. un-
treated group;#P < 0.05 vs. LPS alone. (B) Histological analysis (H&E stain)
showing that Vinp (10 mg/kg of body weight) inhibited PMN infiltration in
BAL fluids from the lungs of mice treated with LPS. Arrows point to PMN.
Con, control. (C) Histological analysis showing that Vinp (10 mg/kg of body
weight) inhibited leukocyte infiltration in peribronchial and interstitial areas
of the lung (H&E stain, magnification ×200).
Vinpocetine (Vinp) inhibits lung inflammatory response in vivo. (A)
Jeon et al. PNAS
| May 25, 2010
| vol. 107
| no. 21
| 9797
MEDICAL SCIENCES

Page 4

To determine whether vinpocetine functions as an inhibitor of
IKK/NF-κB signaling in vivo, we measured phosphorylation and
degradation of IκB, the immediate downstream substrate of
IKK, in lung tissues from LPS-treated mice. As shown in Fig. 5,
vinpocetine significantly inhibited LPS-induced IκB-phosphory-
lation (Fig. 5 G and H) and degradation (Fig. 5 G and I) in the
lung. This result indicates that vinpocetine also inhibits IKK-NF-
κB signaling in vivo.
Vinpocetine Directly Inhibits IKK Kinase Activity. To determine
whethervinpocetine directlytargetsIKK,weexaminedtheeffects
of vinpocetine on IKK kinase activity by directly applying vinpo-
cetinetorecombinantIKKβproteininacell-freesystem.Asshown
in Fig. 6A, vinpocetine dose-dependently inhibited IKKβ kinase
activity, as assessed by in vitro kinase assay using GST-IκBα as
asubstrate.TheIC50valueofvinpocetineondirectIKKinhibition
in the cell-free system is ≈17.17 μM (Fig. 6B).
Inhibitory Effect of Vinpocetine on IKK Kinase Activity Is Independent
of Its Known Actions on PDE Inhibition and Ca2+Regulation. Vin-
pocetine is a well-known PDE-1 inhibitor (18, 22). Therefore, we
determinedwhethertheinhibitoryeffectofvinpocetineonNF-κB
signaling is mediated via inhibition of PDE-1 by examining the
effects of specific PDE-1 inhibitors on NF-κB–dependent tran-
scriptional activity. IC86340, a highly selective PDE-1 inhibitor
thatinhibitsVSMCgrowth(23),didnotinhibitTNF-α–stimulated
NF-κB–luciferase activity (Fig. S6A). Consistently, IC86340 had
minimal inhibitoryeffects on TNF-α–induced IKKkinase activity,
IκBα phosphorylation, and IκBα protein degradation (Fig. 6C).
Because vinpocetine is also known to inhibit Ca2+channels (24,
25)inneurons,wenextdeterminedwhethertheinhibitoryeffectof
vinpocetine on TNF-α–induced IKK activity may involve altered
intracellular Ca2+homeostasis. The effects of nifedipine (Ca2+
channelblocker),EGTA(extracellularCa2+chelator),and1,2-bis
(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic Acid Tetra(aceto-
xymethyl) Ester (BAPTA/AM; an intracellular Ca2+chelator) on
TNF-α–induced NF-κB signaling were examined in VSMCs. As
shown in Fig. 6C, none of these treatments exhibited any signifi-
cant inhibitory effect on TNF-α–induced IKK kinase activity, IκB
phosphorylation, and IκB degradation. It has also been reported
that vinpocetine inhibits neuronal voltage-dependent Na+chan-
nels.WefoundthattheNa+channelinhibitortetrodotoxindidnot
inhibit TNF-α–stimulated NF-κB–dependent transcriptional ac-
tivity in VSMCs (Fig. S6B). Finally, it has been recently reported
that vinpocetine may inhibit PDE-4 activity in vitro (26). Because
PDE-4 is a well-known antiinflammatory target (27), we analyzed
theabilityofvinpocetinetoinhibitPDE-4byPDEassay.Asshown
in Fig. S6, vinpocetine inhibited PDE-1A as expected (Fig. S6C)
but did not effectively inhibit PDE-4D activity compared with the
PDE-4inhibitorrolipram(Fig.S6D),suggestingthattheinhibitory
CA-IKK - + - +
-Actin
A
p-I B
I B
Control
Vinp
0 5 10 15 30 0 5 10 15 30 (min)
GST-I B
IKK
TNF-
TNF-
- + - +
Vinp - - + +
B
Relative NF- B
Luciferase Activity
2
0
4
6
8
10
12
14
Vinp - - + +
CA-MEKK1 - + - +
C
*
#
*
Vinp - - + +
CA-IKK
- + - +
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
#
D
Relative NF- B
Luciferase Activity
Vinp - - + +
F
*
0
2
4
6
8
10
12
Relative NF- B
Luciferase Activity
WT-p65 - + - +
p-I B
I B
LPS - - + + - - + +
Vinp - - - - + + + +
G
-Actin
0
0.2
0.4
0.6
0.8
1.0
1.2
Fold Change
of I B /Actin
Vinp - - + +
LPS - + - +
I
*
0
2
4
6
8
10
12
14
16
Fold Change of
phospho-I B /Actin
Vinp - - + +
LPS - + - +
H
*
#
*
0
2
4
6
8
10
12
14
#
E
Relative NF- B
Luciferase Activity
Vinp - - + +
Fig. 5.
geting IKK. (A) Effects of Vinp on TNF-α–induced IκBα phosphorylation and
degradation. Rat aortic VSMCs were treated with TNF-α (10 ng/mL) for dif-
ferent time periods (0–30 min) as indicated, in the presence or absence of 50
μM Vinp. Western blotting analysis was carried out to evaluate the levels of
phosphorylated IκBα, total IκBα, and β-actin. (B) Vinp inhibits TNF-α–induced
IKK kinase activity in rataortic VSMCs. VSMCs were treated with TNF-α (10ng/
mL) for 10 min in the presence of Vinp (50 μM) or vehicle. IKK kinase activity
was analyzed by an immune complex kinase assay. Effects of Vinp on NF-κB
activation induced by expressing CA-MEKK1 (C), CA-IKKα (D), CA-IKKβ (E), or
WTp65(F)inVSMCsareshown.Datarepresentthemean±SDofatleastthree
independent experiments. *P < 0.05 vs. vector control group;#P < 0.05 vs.
either CA-MEKK1, CA-IKKα, CA-IKKβ, or WT p65 alone. (G) Effects of Vinp on
LPS-induced IκBα phosphorylation and degradation in mouse lungs in vivo.
The lung tissues of mice treated with or without Vinp and LPS were subjected
toWesternblottinganalysesforthelevelsofphospho-IκBαandtotalIκBα.The
relative phospho-IκBα (H) and total IκBα (I) levels were quantified by densi-
tometry and normalized to β-actin. Data represent the mean ± SD of at least
threeanimals.*P<0.05vs.vehiclecontrolgroup;#P<0.05vs.LPS-alonegroup.
Vinpocetine (Vinp) inhibits TNF-α–induced NF-κB activation by tar-
B
01 10 100
0
20
40
60
80
100
IC50=17.17 M
Vinpocetine ( M)
Percentage of
IKK activity
A
GST-I B
IB: IKK
IKK
Activity
IKK
Vinp 0 0 10 25 50 100 M
IKK Recombinant Protein
Vinpocetine
BAPTA/AM
GST-I B
-Actin
p-I B
I B
Vehicle
Nifedipine
EGTA
TNF-- + + + + + +
IC86340
C
Fig. 6.
was analyzed by in vitro kinase assay using recombinant IKKβ. Kinase assays
were conducted using GST-IκBα and [γ-32P]ATP in the presence of various
doses of Vinp for 10 min. (A) Representative autoradiogram showing IKK
kinase activity (Upper) and Western blot analysis showing IKKβ levels
(Lower). (B) Relative IKK activity was indicated. Intensities of the GST-IκBα
bands in the autoradiogram were measured by densitometric scanning.
Results were normalized to the control (Vinp = 0), which is arbitrarily set to
100%. Curve fitting was performed with GraphPad Prism. The IC50values are
defined as the concentrations of Vinp required to produce 50% inhibition.
Data represent the mean ± SD from three independent experiments. *P <
0.05 vs. Vinp = 0. (C) Effects of Ca2+and PDE-1 inhibition on TNF-α–induced
IKK kinase activity, IκBα phosphorylation, and IκBα degradation. Rat aortic
VSMCs were treated with TNF-α (10 ng/mL) for 10 min in the presence of
either 50 μM Vinp, 30 μM nifedipine (Ca2+channel blocker), 15 μM IC86340
(PDE-1 inhibitor), 2 mM EGTA (extracellular Ca2+chelator), or 30 μM BAPTA/
AM (intracellular Ca2+chelator). A representative autoradiogram shows IKK
kinase activity analyzed by an IKK immune complex kinase assay as described
(panel 1). Western blotting analysis was carried out to evaluate the levels of
phosphorylated IκBα (panel 2), total IκBα (panel 3), and β-actin (panel 4).
Data represent at least three independent experiments.
Vinpocetine (Vinp) directly inhibits IKK activity. IKKβ kinase activity
9798
| www.pnas.org/cgi/doi/10.1073/pnas.0914414107 Jeon et al.

Page 5

effectofvinpocetine on NF-κB signalingisunlikely through PDE-
4. Together, we conclude that vinpocetine inhibits TNF-α–in-
ducedIKK-dependentNF-κBactivationindependentofitsknown
actions on PDE, Ca2+, and Na+regulation, thereby revealing
a previously undescribed action of vinpocetine on IKK-NF-κB
signaling.
Discussion
Of particular interest in this study is the identification of vinpo-
cetine as a unique antiinflammatory agent in vitro and in vivo. We
show that vinpocetine inhibited TNF-α–induced NF-κB activation
and the subsequent induction of proinflammatory mediators in
several cell types. Moreover, we show that vinpocetine potently
inhibited LPS- or TNF-α–induced inflammatory responses in
the lungs of mice. Interestingly, vinpocetine inhibited NF-κB–
dependent inflammatory responses by directly targeting IKK (Fig.
7). These findings may lead to the development of previously
undescribed therapeutical repositioning strategies for the treat-
ment of various human inflammatory diseases, including pulmo-
nary inflammatory disease, arthritis, and atherosclerosis (3).
Steroids and cyclooxygenase inhibitors have long been used as
majortherapeuticalantiinflammatoryagents.However,theyoften
cause serious side effects in patients (4, 5). Thus, developing
unique antiinflammatory agents is currently in high demand.
Vinpocetine, a derivative ofthe alkaloid vincamine, has long been
used in the clinic for treatment of cerebrovascular disorders and
cognitive impairment (15). Vinpocetine is well known to enhance
cerebralcirculationandcognitivefunctionandiscurrentlyusedas
adietarysupplementinmanycountriesforpreventativetreatment
of cerebrovascular disorders and related symptoms associated
with aging. Vinpocetine dilates blood vessels and enhances cir-
culationinthebrain.Thisimprovesoxygenutilizationandglucose
uptake from blood, which activate cerebral metabolism and neu-
ronalATPbioenergyproduction(16,17).Inaddition,vinpocetine
elicits neuronal protective effects that increase the resistance of
thebraintohypoxiaandischemic injury(28). Inthepresent study,
weshowthatvinpocetineisanantiinflammatoryagentinvitroand
invivo.Thus,ourfinding,togetherwiththefactthatvinpocetineis
purified from natural products and has already been used in the
clinic for decades, identifies vinpocetine as a highly promising and
attractive antiinflammatory candidate for the treatment of
inflammatory diseases.
Of additional interest is that the inhibitory effect ofvinpocetine
on NF-κB–dependent transcription is independent of its known
actions on PDE, Na+, and Ca2+regulation. The first molecular
target identified for vinpocetine was Ca2+/calmodulin-stimulated
PDE (PDE-1). Vinpocetine inhibits PDE-1 and elevates vascular
cGMP levels at IC50values around 15–30 μM (Table S1). The
positive cerebral vasodilating effect of vinpocetine is at least par-
tially attributable to its effect on PDE-1 inhibition (18). In addi-
tion, vinpocetine inhibits neuronal voltage-dependent Na+
channels and protects neurons against Na+influx in a variety of
cell types (Table S1). Moreover, vinpocetine has been shown to
regulate Ca2+signaling at relatively high concentrations in neu-
rons through interacting with glutamate receptors or inhibiting
voltage-gated Ca2+channels(Table S1).These effects contribute,
atleastpartially, totheneuroprotective effect ofvinpocetine (18).
In the present study, we found that vinpocetine directly inhibits
IKKβkinaseactivityinvitrowithanIC50valueof17.17μM(Fig.6).
Vinpocetine inhibits intracellular IKK kinase activation and NF-
κB–dependent transcriptional activity with an IC50value of about
25μM(Figs.S1andS4).Interestingly,thespecificPDE-1inhibitor
IC86340 exhibited no inhibitory effect on IKK activation, nor did
decreasing intracellular Ca2+concentration by BAPTA/AM, de-
pleting extracellular Ca2+by EGTA, or inhibiting voltage-gated
Ca2+channel by nifedipine (Fig. 6C and Fig. S6). Furthermore,
the voltage-gated sodium channel antagonist tetrodotoxin did not
alter NF-κB–dependent transcription (Fig. S6). Together, these
data suggest that the inhibitory effect of vinpocetine on the NF-
κB–dependent inflammatory response is likely to be independent
of its known actions on PDE-1 as well as on Na+and Ca2+chan-
nels, thereby revealing a previously undescribed action of vinpo-
cetine.Itshouldbenotedthatthepotencyoftheantiinflammatory
effect of vinpocetine is comparable to that of its other well-known
effects (Fig. 6, Figs. S1 and S4, and Table S1).
In conclusion, it is evident that vinpocetine acts as an antiin-
flammatoryagentinvitroandinvivo.VinpocetineinhibitsNF-κB–
dependent inflammatory responses by directly targeting IKK, in-
dependent of its well-known action on PDE-1 activity and Ca2+
regulation. Vinpocetine thus becomes an attractive therapeutical
candidate for treating inflammatory diseases.
Materials and Methods
General Overview. Information on reagents, cell culture and treatment,
Western blot analysis, RNA isolation, real-time RT-PCR assay, dual-luciferase
reporter assay, in vitro IKK kinase assay, monocyte adhesion/chemotaxis
assay, and statistic analysis is provided in SI Materials and Methods.
Mouse Model of Lung Inflammation. C57BL/6 mice at 8 weeks of age were
used, as previously described (20). Under anesthesia, mice were intra-
tracheally inoculated with LPS (Escherichia coli serotype 055:B5, 2 μg per
mouse; Sigma) in 50 μL of PBS vehicle or TNF-α (500 ng per mouse) or with
same volume of saline as control for 6 h. Vinpocetine (10 mg/kg of body
weight) or an equal volume of vehicle control was administered via an i.p.
route 2 h before the intratracheal inoculation of LPS or TNF-α. Lung tissues
were collected and then stored at −80 °C for mRNA expression analysis. For
histological analysis, dissected lung was inflated and fixed with 10% (vol/
vol) buffered formaldehyde, embedded in paraffin, and sectioned at
a thickness of 5 μm. Sections were then stained with H&E to visualize in-
flammatory responses and pathological changes in the lung. For PMN re-
cruitment analysis, BAL was performed by cannulating the trachea with
sterilized PBS, and cells from BAL were stained with Hemacolor (EM Sci-
ence) after cytocentrifugation (Shandon Cytospin4; Thermo Electronic
Co.). Three mice were used for each inoculation group. All animal
experiments were approved by the Institutional Animal Care and Use
Committee at the University of Rochester.
ACKNOWLEDGMENTS. We thank Clint Miller for assistance with curve fitting
using GraphPad Prism (GraphPad Software, Inc., CA) and James Surapisitchat
for critical reading of the manuscript. This work was supported in part by
National Institutes of Health Grant P01 HL077789 (to C.Y., B.C.B., and J.A.);
National Institutes of Health Grant HL088400 (to C.Y.); and National Insti-
tutes of Health Grants DC005843, DC004562, and AI073374 (to J.-D.L.). We
declare that a patent on using vinpocetine for the treatment of inflamma-
tory diseases has been filed by University of Rochester.
TNF-
LPS
MEKK1
IKK /
I B
p50p65
p
TNF- , IL-1 , IL-8, ICAM-1
VCAM-1, MCP-1, MIP-2
Inflammatory Response
Vinpocetine
B sitesTarget Genes
I B
p50 p65
p50p65
Fig. 7.
dependent inflammatory response in vitro and in vivo. As indicated, vin-
pocetine inhibits NF-κB–dependent inflammatory response by directly
targeting IKK, independent of its well-known action on PDE-1, Na+, and
Ca2+regulation.
Schematic diagram depicting how vinpocetine inhibits NF-κB–
Jeon et al. PNAS
| May 25, 2010
| vol. 107
| no. 21
| 9799
MEDICAL SCIENCES