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Ru-Rong Ji, PhD
Pain Research Division
Department of Anesthesiology
Department of Neurobiology
Duke University Medical Center
July 9, 2015, Ion Channel Retreat, Vancouver
Ion Channel and Pain Targets
Modulation of TRP channels by
resolvins in mouse and human
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Omega-3 polyunsaturated fatty acids
Ji et al., Trends Neurosci, 2011
Powerful metabolic products
(Resolvins)
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Chronic pain is a major heath problem
• Over 100 million Americans have chronic pain
• The total financial cost of is near 600 billion
a year in the US.
• Annual US expenditure on pain (medical costs
and lost wages) is higher than that for cancer,
heart disease, and diabetes combined.
• Co-morbidity: Chronic pain is associated with depression, anxiety, insomnia, etc.
• Prevention and treatment of this epidemic are equally important.
Pizzo & Clark, N Engl J Med., 2012
American Pain Society
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Peripheral mechanisms of pain
Ji et al., Nat Rev Drug Disco., 2014
Nociceptor
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Glial
mediators
p38-P
ERK-P
Injury
Microglia
Activation
Astroglia
activation JNK-P
Glutamate
AMPA NMDA TrkB
Primary afferents
Spinal cord dorsal horn neurons
BDNF Glu
GABA-A-R Gly-R
DRG
Inhibition
Excitation
Persistent pain
TNF-a
IL-1b
BDNF
CCL2
IL-1b
Tissue injury
&
Nerve injury
Central mechanisms of pain
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Serhan et al., 2008; Nature Reviews Immunology
(RvE1)
(RvD1)
Charles Serhan
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DHA
17R-Resolvin D Series
Acetylated COX-2
Aspirin:COX-2
LOX
17S-Resolvin D Series
Lipoxygenase mechanism
LOX
EPA
Aspirin:COX-2
P450
microbial
18R-H(p)EPE
LOX
RvD2
RvE1
RvD1
Biosynthesis of resolvins from PUFA Precursors
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Resolvins are potent inhibitors of inflammation
in various animal models
Ji et al., Trends Neurosci, 2011
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Vehicle
RvD1, 20 ng (60 pmol)
RvE1, 20 ng (60 pmol)
0
2
4
6
8
10
12
BL 2 h 4 h 24 h
Time after CRG
PW
L (
s)
* * *
Resolvins prevent CRG-induced heat hyperalgesia
Xu et al., Nat Med, 2010
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0
20
40
60
Naive CRG CRG+
(RvE1, 20ng)
M
PO
activity
(U m
g1 p
rote
in)
* *
0
20
40
60
80
Pa
w v
olu
me
in
cre
ase
(% o
f B
L)
CRG CRG+
(RvE1, 20ng)
*
Paw edema
RvE1 inhibits CRG-induced inflammation
Neutrophil infiltration
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0
5
10
15
20
25
30
Fo
ld o
f n
aïv
e c
on
tro
l
TNF-a
IL-1b
IL-6
MCP-1
CRG CRG+
(RvE1, 20ng)
MIP-1a * * * * *
# # # # #
Naive
CRG, 2 h
RvE1 + CRG, 2 h
3
1
2
4 5
P
P
P
3
1
2
4 5
P
P
P
3
1
2
4 5
P
P
P
a
b
c
Cytokine array
RvE1 inhibits cytokine/chemokine expression
after inflammation
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No effect of RvE1 on basal pain
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Broad actions of resolvins
Ji et al., Trends Neurosci, 2011
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ChemR23 expression in macrophages and microglia
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ChemR23 expression in neurons in the PNS and CNS
Ji et al., Trends Neurosci, 2011
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ng/ml0.1 1 10Inhib
itio
n r
atio
of
ca
psa
icin
0.0
0.2
0.4
0.6
0.8
1.0
1.2
sec0 400 800
340/3
80
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 IC50 = 3.2 ng/ml
RvE1
300 nM Capsaicin
10 ng/ml RvE1
50 mM KCl
Control Capsaicin RvE1 + Capsaicin
RvE1 blocks capsaicin-induced signaling in mouse DRG neurons
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0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 10
Time (min)
Capsaicin
(500 ng, i.t)
Capsaicin +
RvE1 (10 ng, i.t.)
Du
ratio
n o
f n
ocife
nsiv
e
b
eh
avio
r (s
) * *
*
* *
Patch clamp
recording
6 s
Control
Capsaicin (100 nM)
RvE1 (1 ng ml) + Capsaicin
20
pA
RvE1 blocks TRPV1-mediated
spinal synaptic transmission & pain
0
0.5
1
1.5
2
2.5
3
Frequency
Amplitude
+ Capsaicin (100 nM)
Ra
tio
of
sE
PS
C
*
#
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Resolvins are potent endogenous inhibitors of TRPV1/A1
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Capsaicin and mustard oil-induced pain
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Resolvin E1 (RvE1) blocks TRPV1 current in mouse DRG neurons
GAPDH
TRPV1
1 2 3
ChemR23
4 5 6 7 8 9 10 N
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TRPV1-expressing HEK cells
TRPV1 and ChemR23-coexpressing HEK cells
Capsaicin (100 nM)
RvE1 (10 ng/ml)
ChemR23 is sufficient to mediate RvE1’s effect in HEK 293 cells
100 pA
1 min
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RvE1 fails to inhibit TRPV1 signaling in b-arrestin 2 KO mice
WT b-arrestin 2 KO
100 pA
1 min
CAP (100 mM) RvE1 (10 ng/ml)
Control RvE1 (10 ng/ml), 15 min
barr2 barr2
20 mm
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Minutes after capsaicin injection
0
4
8
12
16
20
0 1 2 3 4 5 6 7 8 9 10
CKO
WT
Nu
mb
er
of
flic
kin
g/l
ick
ing
/bit
ing
1 2 3 4 5 N M
WT barr2 CKO
GAPDH
Nav1.8
barr2
barr1
1 2 3 4 5 N M
TRPV1 signaling is potentiated in b-arrestin 2 CKO mice
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TLR7
1 2 3 4 5 6 7 8 9 10 neg
TRPV1
GAPDH
TRPA1
Single-cell PCR
let-7b (30 µM) let-7b (30 µM)
HC030031(100 µM)
10 s
10
0 p
A
30 mm
Ion channel research on Primary sensory neurons of human DRGs
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TRPV1 / ChemR23
100 mm
Co-expression of TRPV1 and ChemR23 in human DRG neurons
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25 µm 1 n
A
20 s
CAP
RvE1 (100 ng/ml)
Capsaicin. Cap (100 nM)
RVE1
10 n
g/ml
100
ng/ml
100
ng/ml
0
20
40
60
80
100
120
n = 12 / 17
n = 5 / 17n = 3 / 3
% o
f 1
st C
AP
resp
on
se
CAP RvE1
RvE1 inhibits TRPV1 currents in human DRG neurons
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• Anti-inflammatory actions
• Pro-resolution actions
• Block TNF-a synthesis and signaling
• Modulate synaptic plasticity
• Powerful endogenous inhibitors of TRPV1/A1
• No effects on basal pain
• Unknown side effects
Limitations
• Unstable
• Difficult to measure
• Expensive to produce
Multiple beneficial effects of resolvins
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DHA
17R-Resolvin D Series
Acetylated COX-2
Aspirin:COX-2
LOX
17S-Resolvin D Series
Lipoxygenase mechanism
Epoxidation
NPD1 Protectins
Neuroprotectins
LOX
EPA
Aspirin:COX-2
P450
microbial
18R-H(p)EPE
LOX
RvD2
RvE1
RvD1
Biosynthesis of Protectins from PUFA Precursors
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Acknowledgements Temugin Berta
Supported by
NIH grants
R01: NS67686
R01: DE17794
R01: NS54932
R01: DE22743
R01: NS89479
Pfizer/Neusentis/Icagen
Zhen-Zhong Xu Ling Zhang Chul-Kyu Park Ning Lu
Charles Serhan Seok-Yong Lee