Transcript Cancer Pain: Role of formaldehyde and TRPV1

Tumor cell-derived endogenous formaldehyde
activates TRPV1 in cancer pain
You Wan (万有), MD, Ph.D.
Neuroscience Research Institute, Peking University
E-mail: [email protected]
2015/7/18
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Cancer pain epidemiology
 Populations ageing and unhealthy lifestyles
increase cancer patients.
 Each year
11 million people develop malignant tumours
7 million die from the disease
2020: sharp increase from 10 million to 15 million
 More cancer patients develop into advanced
stage
IASP/WHO: 50 % of cancer patients develop pain
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Multiple nociceptive and non-nociceptive
mechanisms contributing to cancer pain
 Mechanisms
Nociceptive pain or inflammatory pain in the relatively
early stage after metastasis
Neuropathic pain in the relatively late stage when
nerves were injured
 According to the locations
Bone pain
Visceral pain
……
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Mechanisms of cancer pain: largely
unknown, but peripheral factors is important
ET-1
IL-1
TNF
I
II
III
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Two facts about formalin
 In pain research field: Formalin test
 In clinical medicine: formalin is one of the
indoor-contaminants, and possibly a cause of
cancer (i.g. leukemia)
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Formalin test
(5% formaldehyde, 1662 mM)
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Formaldehyde is present in all cells and
tissues
Formaldehyde is a ubiquitous compound, which is present in all cells (Tyihak, et al. 1998)
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Formaldehyde metabolisms
Synthesis
Degradation
Cancer-linked DNA hypomethylation and
hypermethylation
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Huba Kalász. 2003
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Endogenous formaldehyde level increased in
cancer patients
 It is known that prostate cancer, bladder
cancer, breast cancer patients
frequently has bone cancer pain
 Formaldehyde level elevated in these
patients (Ghilardi JR, et al. 2005)
 Formaldehyde could be a valuable
indicator of the presence of early-stage
tumors in the body
 Presence of formaldehyde in the breath
of some breast cancer patients
 Formaldehyde is emitted from cultured
tumor cells (Spanel, et al. 1999)
Formaldehyde as a marker
or a cause of tumor
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TRPV1 receptor is an integrator
The structure of capsaicin
The ligands of TRPV1
Capsaicin
Heat （>43°C）
Protons （H+）
Lipids-anandamide (AEA)
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The structure of TRPV1
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TRPV1 plays a role in cancer pain
 Cancer pain model mice appeared heat and
mechanical hyperalgesia (Nagamine K, et al.
2006; Asai H, et al. 2005)
 Selective blockade of TRPV1 attenuated bone
cancer pain (Menendez L, et al. 2006)
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Expression of TRPV1 correlated with cancer
pain in patients
TRPV1 expression in normal human pancreas, chronic
pancreatitis, and pancreatic adenocarcinoma – RT-PCR.
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Correlation of different pain levels with TRPV1
expression in patients with pancreatic cancer
Hartel M, et al. Vanilloids in pancreatic cancer: potential for chemotherapy and pain
management. Gut 2006; 55(4): 519-28.
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Expression of the TRPV1 correlated with
cancer pain in patients
Quantification of TRPV1-immunoreactive, intra-epidermal fibres and breast pain. Scatter grams show
TRPV1- mmunoreactivity in (A) intra-epidermal fibres and (B) intra-epidermal fibre "clusters" in patients with and
without breast pain.
Gopinath P, et al. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in
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keratinocytes in human breast pain. BMC Women’s Health 2005; 5: 2.
tumor
H+
formaldehyde
Hypothesis
 Formaldehyde increases in tumor and activates
peripheral TRPV1 in cancer pain especially in
the acidic environment
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
4. Formaldehyde at low concentration level induces
discharges of peripheral C-fibers
5. TRPV1 antagonism or formaldehyde scavenger inhibits
cancer pain in cancer pain rats
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
4. Formaldehyde at low concentration level induces
discharges of peripheral C-fibers
5. TRPV1 antagonism or formaldehyde scavenger inhibits
cancer pain in cancer pain rats
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Formaldehyde increased in the lung cancer
patients’ tissues
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Formaldehyde increased in the breast
cancer patients’ tissues in vivo
Patients`
Number
Concentration of
Formaldehyde (mM)
Patients'
Number
Concentration of Formaldehyde (mM)
16
0.398676
0.235319
17
0.301124
0.261872
0.694605
18
0.846995
0.001539
4
0.519895
19
0.600516
0.264758
5
0.826407
M+S.E.M
0.5368±0.1207
0.1909±0.0635
6
0.20511
7
0.599169
8
0.361348
9
0.620911
10
2.345686
11
0.303625
12
0.310359
13
1.115793
14
0.697491
15
1.447703
2
0.449088
3
M+S.E.M
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0.8088±0.1533
1.0
n=19
.8
.6
##
.4
n=4
.2
0.0
All Specimens
Cancer-adjacent
Concentrations of Formaldehyde (mM)
Cancer-adjacent Tissues
1.635304
Concentrations of Formaldehyde (mM)
Cancer Tissues
1
.7
n=4
Breast cancer
.6
.5
.4
.3
**
.2
.1
0.0
Cancer Tissues Cancer-adjacent Tissues
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Formaldehyde increased in the ascetics
cancer model in vivo
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n=6
MRMT-1 cancer cell lines
.030
.025
.020
.015
.010
.005
0.000
Concentrations of Formaldehyde (mM)
1 day
.025
n=6
2 day
##
**
.020
.015
.010
.005
0.000
2 day
.14
3 day
n=6
Melanoma cancer cell lines
.12
.10
.08
.06
.04
.02
**
0.00
3 day
H 1299 cancer cell lines
1 day
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Concentration of Formaldehyde (mM)
.035
1 day
Concentrations of Formaldehyde (mM)
Concentrations of Formaldehyde (mM)
Formaldehyde increased in cultured cancer
cell lines
.035
n=6
2 day
3 day
PC-3 cancer cell lines
.030
.025
.020
.015
.010
.005
0.000
0 day
2 day
3 day
20
Cancer pain model:
MRMT-1 breast cancer cells injected into tibea bone
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Formaldehyde concentration increased in
tibia bone in bone-cancer pain rats
 Just finished
 Up to 2 mM
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
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TRPV1 increased in DRG in cancer pain rats
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
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Formaldehyde at low pathological concentrations
activated or sensitized TRPV1: Calcium influx
TRPV1-transfected CHO cell
100
TRPV1-CHO cell
TRPV1-CHO cell
100
Fluorescence Intensity
Fluorescence Intensity
120
80
CAP
60
40
20
80
60
10mM FA
40
20
0.1mM CPZ+10mM FA
CPZ+CAP
0
0
0
50
100
150
200
0
250
50
80
60
40
20
TRPV1-CHO cell
80
10 mM
70
100 mM
FA
60
1 mM
50
40
30
0.1 mM
0 mM
20
0
0
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1mM
10mM
Formaldehyde
100mM
200
250
CHO cell
40
10 mM
FA
30
100 mM
20
0 mM
1 mM
0.1 mM
10
0
10
0.1mM
FLuorescence Intensity
FLuorescence Intensity
Increase Fluorescence Intensity
50
90
150
Time (Sec)
Time (Sec)
100
100
50
100
Time (Sec)
150
200
250
0
50
100
Time (Sec)
150
200
26
250
Formaldehyde at low pathological concentration
activated or sensitized TRPV1
40
10mM FA
CPZ+10mM FA
Increase
Fluorescence Intensity
Fluorescence Intensity
40
35
30
25
20
FA
15
10
5
0
CPZ+10mMFA
0
50
100
150
200
35
30
25
20
15
10
5
0.1mM
250
1mM
TRPV1- COS-7
100
90
10 mM
FA
80
100 mM
70
1 mM
0.1 mM
1000 mM
0 Mm
60
50
Fluorescence Intensity
110
10mM
100mM
Formaldehyde
90
COS-7 Cell
1000 mM
80
70
FA
60
100 mM
10 mM
50
40
0 mM
30
1 mM
0.1 mM
20
10
0
40
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TRPV1-COS-7 Cell
Time (Sec)
Fluorescence Intensity
Calcium imaging
TRPV1-transfected COS-7 cell
0
50
100
150
Time (Sec)
200
250
50
100
150
Time (Sec)
200
250
27
Formaldehyde at low pathological concentrations
activated or sensitized TRPV1
120
FLuorescence Intensity
FLuorescence Intensity
DRG neurons
CAP
CPZ+CAP
170
CAP
160
150
140
80
50
30mM
50mM
10mM
FA
60
0.1mM
0mM
40
CPZ+CAP
0
20
100
150
200
250
0
300
50
100
Increase Fluorescence Intensity
130
FA
CPZ+FA
110
FA
100
90
80
70
CPZ+FA
60
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0
50
100
150
Time (Sec)
150
200
250
300
Time (Sec)
Time (Sec)
120
100mM
100
130
Fluorescence Intensity
Calcium imaging
180
200
250
300
35
DRG neurons
30
25
20
15
10
5
0
0.1mM
10mM
30mM
50mM
Formaldehyde
100mM
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Formaldehyde-induced TRPV1 current was
enhanced by low pH
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
4. Formaldehyde at low concentration level induces
discharges of peripheral C-fibers
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Formaldehyde induced fiber discharges
Conduction velocity:
1.25 m/s
FA (1 mM)
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
4. Formaldehyde at low concentration level induces
discharges of peripheral C-fibers
5. TRPV1 antagonism or formaldehyde scavenger inhibits
cancer pain in cancer pain rats
2015/7/18
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Cancer pain model:
MRMT-1 breast cancer cells injected into tibea bone
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Behavior in cancer pain rats
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Formaldehyde activated TRPV1 receptors in
vivo
n = 10
Vehicle
FM
FM + CPZ (50)
FM + CPZ (100)
140
120
100
**
80
*
60
**
40
20
n=10
160
Mean time of licking(sec)
Mean time of licking (sec)
160
**
0
Vehicle
FM
FM + MT (1mg)
FM + MT (5mg)
140
120
*
100
80
60
**
**
40
20
0
-20
0
10
20
30
40
50
60
-20
70
0
Time (min)
Mean time of licking (Sec)
FM
FM + Res (0.2 mg)
FM + Res (0.4 mg)
140
120
*
100
80
**
60
*
**
40
20
**
0
0
10
20
** **
30
40
20
30
40
50
60
70
Time (min)
50
60
160
Mean time of licking (Sec)
n = 10
160
10
n = 10
FM
FM + GSH (6.25mg)
FM + GSH (25mg)
140
120
100
*
80
60
**
40
*
20
0
70
0
Time (min)
10
20
30
40
50
60
70
Time (min)
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5%FM=1662 mM
35
Mean time of nociceptive response
Formaldehyde at low concentration induced
nociceptive responses
70
n=10
50
**
40
30
**
20
**
10
*
**
1mM
5mM
0
CON 0.1mM
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**
60
10mM 30mM 50mM 100mM
36
Endogenous formaldehyde and TRPV1 in
MRMT-1 bone cancer pain rats
PBS
Mechanical Threshold (g)
16
Killed
MRMT-1
CPZ
Melatonin
Glutathione
Resveratrol
14
12
10
8
6
#
** * *
**
**
4
2
0
0 Day
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3 Day
7 Day
11 Day
37
TRPV1 antagonist or formaldehyde scavenger
inhibited formaldehyde-induced pain
Mean number of flinches
14
n=10
##
12
10
8
6
**
4
2
**
**
**
0
CON
FA
CPZ
MT
MK-801 APV
Formaldehyde (5mM)
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Evidence include the followings:
1. Formaldehyde concentration increase to pathological
level
2. TRPV1 was up-regulated in the tibia bone in MRMT-1
cancer pain model rats
3. Formaldehyde at low concentration level activates
TRPV1 especially under acidic environment (low pH)
4. Formaldehyde at low concentration level induces
discharges of peripheral C-fibers
5. TRPV1 antagonism or formaldehyde scavenger inhibits
cancer pain in cancer pain rats
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Conclusions
 Formaldehyde increased in the cancer cell lines
and in the tissues of cancer patients
 Formaldehyde under acidic environment
induced C-fibers excitation by activating and/or
sensitizing peripheral TRPV1, resulting in cancer
pain
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Summary: formaldehyde action on
peripheral TRPV1
FA
H+
TRPV1
FA
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FA
Formaldehyde and TRPV1:
more evidence needed
?
FA
?
FA
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Antonio Ferrer-Montiel, et al. Molecular architecture of the vanilloid receptor: nsights for drug design. Eur. J. Biochem. 271, 1820–1826 (2004)
42
Acknowledgements
 Zhi-Qian TONG
 Yan LI
 Prof. Wen-hong LUO
 Prof. Hong-mei ZHAO
 Prof. Jun WANG
 Prof. Yan-qing WANG
 Prof. Huang-ying TAN
 Prof. David JULIUS
 Prof. Zhuan ZHOU
 Prof. Tian-le XU
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Thank you for your attention!
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FA activated and/or sensitized TRPV1
DRG neurons
CAP
CPZ
CPZ
FA
FA
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Formaldehyde activated and/or sensitized
TRPV1
TRPV1-transfected CHO cell
TRPV1-transfected COS-7 cell
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Formaldehyde activates current of DRG
neurons and TRPV1-transfected 293 HEK
cellneurons
DRG
TRPV1-transfected 293 HEK cell
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Our viewpoint II.
Endogenous formaldehyde and TRPV1
participates in bone cancer pain
 Effects of formaldehyde capturer and
antagonists of TRPV1 on formaldehyde level
and bone cancer pain responses
 Formaldehyde may activate TRPV1 and/or
NMDA receptor in vitro and in vivo
 Formaldehyde activates and/or sensitizes
TRPV1
 1 mM formaldehyde can induce current and
calcium influx of DRG neurons
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Question II.
2. Which receptors does FA activate, TRPV1
? FA
or NMDA receptor?
?
FA
?
FA
2015/7/18 121 (2003) 667–680
Neuroscience
49
2.2. MT may be an antagonist of TRPV1
80
18
##
n=10
**
**
**
16
60
**
40
**
20
Hot Plate Latecency (S)
Mean time of licking (sec)
n=10
14
12
10
8
6
4
2
0
0
Vehicle
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CAP
MT(0.5mg) MT(1mg) MT(5mg)
CAP
Base
CON
MT (0.5mg) MT (1mg) MT (5mg)
50
Summary
II
Formaldehyde
and TRPV1 participates in
bone cancer painC-fibers
 Formaldehyde level
increased in the spinal cord
 Decreases formaldehyde
level in spinal cord
attenuated bone cancer
pain
 Antagonists of TRPV1
attenuated bone cancer
pain by antagonizing
TRPV1
 Formaldehyde may activate
TRPV1 and/or NMDA
receptor
 1 mM formaldehyde can
induce current and calcium
influx.
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1 mM
FA 1
SP
FA
51
2
Discussion
How does FA induce pain responses in bone cancer pain model?
H+
H+
Acidic microenvironment created by osteoclasts causes bone pain associated with tumor colonization
Nagae M. et al. J Bone Miner Metab 2007
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1. Cancer cell and formaldehyde
Production of FA:
① Genetic factors: Cancer-linked DNA hypomethylation and hypermethylation;
② Environmental factors: Xenobiotics and some antitumor drug metabolism;
③ Physiological factors: Abnormal SSAO metabolism;
④ Food
Metabolisms of formaldehyde
Huba Kalász. 2003
Cancer DNA
demethylation
FA metabolic detoxification enzyme systems
Shirley Teng. et al. 2001
gene-transcriptions
Localization of ADH and ALDH
in the human spinal cord and brain
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formaldehyde
protein
Motavkin PA, et al. 1990
4. How dose formaldehyde activate TRPV1?
TRPV1
TM6
O
formaldehyde
H
TRPA1
C
H
O
acrolein
H C
C
C
H
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Nature 2007;445(7127):541-5.
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Cell 124, 1269–1282, March 24, 2006
5. Endogenous formaldehyde may act as an pain signing molecule
CCI model
N-acetyl-L-cysteine(NAC)
SNL model
Bone cancer pain model
GSH
?
trigeminal neuralgia
(neuropathic pain)
FA
multiple sclerosis
diabetic pain
glycine
Evolution
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microorganism, plants, animal model, human
Infection, temperature
wound
pain
55
MRMT-1 bone cancer pain
25
14
$$
Hot Latency (Sec)
#
*
12
$$
15
$$
10
Mechanical Threshold (g)
#
*
20
**
10
##
8
&
6
4
$
5
2
0
0
L R L R L R L R L R L R L R L R L R
Con
Con Model Con Model Con Model Con Model
0 day
12 day
3 day
6 day
9 day
Days after surgery
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L R L R L R L R L R L R L R L R L R
Con
Con Model Con Model Con Model Con Model
0 day
6 day
3 day
9 day
12 day
Days after surgery
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Function of endogenous formaldehyde in vivo
cell proliferation----- tumor and activating glia
cell
nervous
aging
lung
FA
sensory function -------pain
respiratory function --------asthma
heart
brain
cardiovascular function ---------stroke
learning function------ neurodegenaration
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FA
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FA
a.
FA
c.
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FA
b.
d.
FA
59
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Bone
Nerve fiber
FA
TRPV1
Inflammatory
Cell
ASICs
Conduction
Up-regulation
FA
TRPV1
H+
Aδ-fiber
DRG Neurons
H+
FA
Tumor
Cell
FA
ASICs
C-fiber
H+
FA
H+
Spinal cord
TRPV1
FA
H+
H+
Cancer pain
FA
Macrophage
Bone
destruction
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Brain
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2. Mechanism of formaldehyde induced-sensitization of peripheral and central nervous.
Sensitization of peripheral nervous
Sensitization of central nervous
FA
H+
TRPV1
FA
I
II
III
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FA