저 시-비 리- 경 지 2.0 한민

는 아래 조건 르는 경 에 한하여 게

l 저 물 복제, 포, 전송, 전시, 공연 송할 수 습니다.

다 과 같 조건 라야 합니다:

l 하는, 저 물 나 포 경 , 저 물에 적 된 허락조건 명확하게 나타내어야 합니다.

l 저 터 허가를 면 러한 조건들 적 되지 않습니다.

저 에 른 리는 내 에 하여 향 지 않습니다.

것 허락규약(Legal Code) 해하 쉽게 약한 것 니다.

Disclaimer

저 시. 하는 원저 를 시하여야 합니다.

비 리. 하는 저 물 리 목적 할 수 없습니다.

경 지. 하는 저 물 개 , 형 또는 가공할 수 없습니다.

이학박사학위논문

통증 조절을 위한 다각적 접근:

국소 진통제, 약침 요법 및 시간 제한 섭식에

관한 연구

Multimodal approach for pain management:

Local analgesics, Pharmacopuncture and Time-

restricted feeding

2020 년 2 월

서울대학교 대학원

자연과학대학 뇌인지과학과 전공

이 정 윤

1

ABSTRACT

Multimodal approach for pain management:

Local analgesics, Pharmacopuncture and Time-

restricted feeding

Jeong-yun Lee

Brain Cognitive Neuroscience

The Graduate School

Seoul National University

Pain is defined as an unpleasant sensory and emotional experience

associated with actual or potential tissue damage by International Association for

the Study of Pain (IASP). Since pain has both sensory-discriminative and

affective-motivational dimensions, it is difficult to effectively modulate pain with

a conventional pharmacological approach (NSAIDs and opioids) alone. In an

effort to explore multimodal approaches to pain management, I have focused on

the therapeutic effect of local analgesics (sinomenine), pharmacopuncture

(acupoint stimulation with drug, Scolopendra subspinipes) and time-restricted

feeding (fasting/refeeding) on animal pain models as below.

In the first chapter, I revealed that sinomenine can produce peripheral

analgesic effect by reducing cellular excitability of nociceptive neurons. The

intraperitoneal injection of sinomenine suppressed formalin-induced pain

behavior and c-Fos expression. Sinomenine inhibited voltage-gated sodium

2

current in nociceptive neurons. Furthermore, the intraplantar injection of

sinomenine also suppressed pain behavior. Therefore, sinomenine could be a

potential pharmacological therapeutic agent for local anesthesia and analgesia.

In the second chapter, I demonstrated that acupoint stimulation with

Scolopendra subspinipes (pharmacopuncture with Scolopendra subspinipes, SSP)

produces an analgesic effect via alpha2-adrenoreceptors in the spinal cord. SSP

into Zusanli acupoint had a significant analgesic effect on oxaliplatin-induced

neuropathic pain model. The intrathecal injection of yohimbine (alpha2-

adrenoeceptor antagonist) reversed SSP-induced analgesic effect. Therefore, SSP

produces an analgesic effect by activating descending pain inhibitory system,

which suggests a possibility for pain management.

In the third chapter, I focused on the analgesic effect of fasting and

refeeding. Both 24h fasting and 2h refeeding produce analgesic effect but via

different mechanisms, suggesting that endocannabinoid and opioid system is

only involved in fasting-induced analgesia, whereas refeeding-induced analgesia

is associated with eating behavior and calorie recovery effect. Therefore, the

regulation of feeding (time-restricted feeding) may have abundant benefits to

modulate pain and might provide a novel strategy for the management of pain.

Key Words: pain; sinomenine; voltage-gated sodium channel; Scolopendra

subspinipes; pharmacopuncture; alpha2-adrenoceptors; fasting; time-restricted

feeding

Student Number: 2015-22669

3

CONTENTS

Contents ............................................................................................................... 3

List of figures ....................................................................................................... 5

Background .......................................................................................................... 7

Purpose ............................................................................................................... 22

CHAPTER 1:The analgesic effect of sinomenine via inhibition of voltage-

gated sodium channel ...................................................................................... 23

Abstract ...................................................................................................... 24

Introduction ................................................................................................ 25

Materials and Methods ............................................................................... 26

Results ........................................................................................................ 31

Discussion .................................................................................................. 44

CHAPTER 2: Pharmacopuncture with scolopendra subspinipes suppresses

mechanical allodynia in oxaliplatin-induced neuropathy: Mediation by

spinal alpha2-adrenoceptor ............................................................................. 48

Abstract ...................................................................................................... 49

Introduction ................................................................................................ 50

Materials and Methods ............................................................................... 51

Results ........................................................................................................ 55

Discussion: ................................................................................................. 65

CHAPTER 3:Both acute fasting and refeeding alleviate inflammatory pain

but via different pathways ............................................................................... 68

Abstract ...................................................................................................... 69

Introduction ................................................................................................ 70

4

Materials and Methods ............................................................................... 72

Results ........................................................................................................ 75

Discussion .................................................................................................. 88

Conclusion ......................................................................................................... 91

References .......................................................................................................... 92

국문초록 ............................................................................................................ 98

5

LIST OF FIGURES

Background

Figure 1. Pain pathway ...................................................................................... 12

Figure 2. Approaches to pain management ....................................................... 14

Figure 3. Local analgesics ................................................................................. 16

Figure 4. The mechanism of ascending nociceptive control ............................. 18

Figure 5. Benefits of time-restricted feeding .................................................... 20

Chapter 1

Figure 6. Effects of sinomenine on formalin-induced pain ............................... 34

Figure 7. Effects of sinomenine on action potentials in small sized dorsal root

ganglion neurons .................................................................................. 36

Figure 8. Effects of sinomenine on voltage-gated sodium channel in small sized

dorsal root ganglion neurons ................................................................ 38

Figure 9. Intraplantar administration of sinomenine on formalin-induced pain

behavior ................................................................................................ 40

Figure 10. Effects of sinomenine in rota-rod test .............................................. 42

Chapter 2

Figure 11. Effect of Scolopendra subspinipes pharmacopuncture (SSP) on

formalin-induced acute inflammatory pain model ............................... 57

Figure 12. Effect of Scolopendra subspinipes pharmacopuncture (SSP) on

oxaliplatin (OXA)-induced neuropathic pain model ............................ 59

6

Figure 13. Peripheral nerve activation of the specific acupoint is associated

with Scolopendra subspinipes pharmacopuncture (SSP)-induced anti-

allodynia ............................................................................................... 61

Figure 14. Effects of pretreatment of intrathecal injection of yohimbine,

naloxone, or methysergide on Scolopendra subspinipes

pharmacopuncture (SSP)-induced anti-allodynia................................. 63

Chapter 3

Figure 15. The effect of fasting and refeeding on acute pain behavior ............. 78

Figure 16. Changes in body weight, blood glucose level and food intake by

refeeding in naïve mice ........................................................................ 80

Figure 17. The analgesic effect of refeeding with non-calorie agar on

inflammatory pain behavior ................................................................. 82

Figure 18. The analgesic effect of intraperitoneal administration of D-glucose

on inflammatory pain behavior ............................................................ 84

Figure 19. The effect of opioid receptor and cannabinoid receptor (CB1)

antagonist on fasting and refeeding-induced analgesia ...................... 86

7

BACKGROUND

1. Pain pathway

Pain is somatic and emotional sensations1. The sensory-discriminative aspect

of pain (nociception) provides sensory information such as location, quality, and

intensity. Emotional pain consists of hedonic aspect (pleasantness/unpleasantness)

and affective-motivation aspect which creates behavior to escape from pain as a

secondary pain effect. Pain pathways are largely divided into three categories:

two ascending pain pathways (the lateral pain pathway for nociception and the

medial pain pathway for emotional pain) and one descending pain pathway (Fig.

1 A)2. In the lateral pain pathway, noxious stimuli activate nociceptive primary

afferent neurons (1st order neuron) which transmit signals from the periphery to

the dorsal horn of the spinal cord (2nd order neurons). The thalamocortical

neurons (3rd order) project from the ventral posterior nucleus of the thalamus to

the primary somatosensory cortex, thus contributing to nociception (Fig.1 B and

C). On the other hand, the medial pain pathway projects from the medial thalamic

nuclei to brain regions including the prefrontal, the anterior cingulate cortex

(ACC) and the insular cortex which are critical for regulation of emotions and

susceptible to various cognitive factors (Fig.1 C)3. While the ascending pain

pathway conveys pain signal from periphery to central cortex, the descending

pain pathway including the ACC, the periaqueductal gray (PAG), and the

rostroventral medial medulla (RVM) modulates pain signal from central cortex

to the spinal cord (Fig.1 D)4. The main neurotransmitters involved in descending

inhibitory pain pathway, are known as serotonin, noradrenalin, and opioid.

8

2. Approaches to pain management

The World Health Organization (WHO) proposed the analgesic ladder

according to pain intensity (Fig. 2A)5,6. The guidelines from the WHO ladder

suggest non-opioid drugs, especially nonsteroidal anti-inflammatory drugs

(NSAIDs) for the first steps in pain therapy and also recommend the adjuvant

treatments (anticonvulsants, antidepressants, alpha2-adrenoceptor agonists, N-

Methyl-D-aspartate receptor antagonists, etc.) for neuropathic pain. In the case

of pain that is not controlled with non-opioid drugs, the use of opioid-based

analgesics is recommended. Although this therapeutic guideline is widely

available for the administration of analgesics, conventional analgesics limited

in pain treatment and many patients still experience pain7. Furthermore, pain is

a multifaceted phenomenon with various etiologies and symptoms. Recently,

integrated pain management is being recognized as an effective care for pain

management (Fig. 2B)8. For example, multimodal analgesia combines various

groups of medications such as local anesthetics, NSAIDs, gabapentin, tricyclic

antidepressants, opioids etc. to produce a synergistic effect. Physical (sensory)

interventions including massage, positioning, hot/cold, acupuncture, and

transcutaneous electrical nerve stimulation (TENS) are also well known to

relive pain9. Furthermore, behavioral intervention and improvement of lifestyle

manage pain better through psychotherapy, sleep, nutrition, mood, and exercise

(active self-care therapies).

9

3. Local analgesics

The most commonly used systemic analgesics have various side effects (Fig.

3A)10, while local analgesics have the advantage of minimizing the side effects

by treating drugs only in actual or potential tissue damage sites11. The mechanism

of local analgesics is largely within the peripheral nervous system. Since the

activity of nociceptive neurons (Aδ and C-fiber) is regulated by ion channels, ion

channels expressed on nociceptive neurons are the key molecular target for

diverse analgesics (Fig. 3B)12. For example, eugenol, the main component in

clove plant, is well known for having local anesthetic actions in periphery by

regulating various ion channels (TRPV1, P2X, voltage-gated sodium channels,

voltage-gated potassium channels, voltage-gated calcium channels, etc.), thereby

providing a pharmacological mode of action for their wide use in dental clinic to

alleviate dental pain13. Especially, voltage-gated sodium channels (VGSCs) are

the main ion channels for generating action potentials, thereby responsible for

transmitting pain signals in the nociceptive neurons14-16. Local analgesics

targeting VGSCs, such as procaine, lidocaine, and mepivacaine, are the most

widely used17. It is also possible to selectively block pain signals by blocking

VGSCs with the permanently charged lidocaine derivative QX-314 that enters

through large-pore ion channels selectively expressed in nociceptive neurons18-20

10

4. Pharmacopuncture

Peripheral stimulation by noxious stimuli is well known to produce an

analgesic effect by activating descending pain pathway (Fig. 4)21,22. Acupoint is

anatomically defined areas on the body surface with unique features such as high

electrical conductance, hypersensitivity, needling sensation, therapeutic effect

and connection with internal organ23,24. Interestingly, recent studies have shown

that these features of acupoint are closely related to Aδ and C-fiber. The high

electrical conductance or hypersensitivity at acupoint is associated with

neurogenic substance P (SP) and calcitonin gene-related peptide (CGRP) from

Aδ and C-fiber24. Primary afferent neurons, especially Aδ and C-fiber, are

essential for mediating the therapeutic effect of acupoint stimulation25,26. It is well

known that the low-frequency electrical stimulation into acupoint produced

analgesic effect by modulating mu-opioid receptor, serotonin receptor, and

alpha2-adrenoceptor in spinal cord. Drug-induced acupoint stimulation, termed

pharmacopuncture, is a method of safely stimulating acupoint for a long time.

Bee venom is the most commonly used drug in pharmacopuncture. In clinical

study as well as various animal pain models, pharmacopuncture with bee venom

(BVP) is known to cause pain but has been shown to have a long-lasting analgesic

effect via serotoninergic and adrenergic system, but not opioidergic system27,28.

11

5. Time-restricted feeding

Hunger is well known as a powerful driving force to change cognition.

Several clinical evidences have shown that therapeutic fasting is effective for

pain relief and mood enhancement regardless of body weight change in chronic

pain patients. In various animal pain models, fasting or calorie restriction also

has an analgesic effect. However, it is recently shown that Time-Restricted

Feeding (TRF) may be more successful than Calorie-Restricted Feeding (CRF)

for not only on healthy lifespan but also on neural circuits related to cognition

and emotion29. Many studies have shown that TRF with high fat diet did not cause

metabolic disease and obesity30 (Fig. 5A). As compared with TRF, Ad libitum

feeding interfere with circadian rhythm resulting in metabolic dysfunction and

metabolic disease. Furthermore, TRF is accompanied by intermittent metabolic

switching such as negative energy balance (fasting and/or exercise) followed by

positive energy balance (eating, resting, and sleeping) (Fig. 5B), which is known

to alter cell signaling in neurons and affect neuroplasticity31. The sensation of

hunger and satiety is closely related to the mesolimbic reward system involving

the regulation of food intake32. Since the unpleasantness of pain as well as the

relief of pain is encoded by mesolimbic circuitry, TRF may have many benefits

to modulate pain perception.

12

Figure 1.

13

Figure 1. Pain pathway

(A) Two ascending pain pathway and one descending pain inhibitory pathway.

(B) Ascending pain pathway consists of three neuron pathways (1st, 2nd, and 3rd

order neurons) (C) Ascending pain pathway is divided into lateral and medial

pathway. Lateral pathway is responsible for detecting painful sensation and for

identifying the location of painful stimulus. Medial pathway is responsible for

the emotional aspect of pain. (D) Descending pain inhibitory pathway suppresses

pain signal in the spinal cord by modulating monoaminergic inputs.

Fig. 1A modified and extended from De Ridder et al. 2016 (2)

Fig. 1B modified and extended from Pearson Education, Inc. 2011

Fig. 1C modified and extended from Bushnell et al 2013 (3)

Fig. 1D modified and extended from Benarroch et al 2008 (4)

14

Figure 2.

15

Figure 2. Approaches to pain management

(A) The WHO Pain Treatment 3 Step Ladder. The stages are divided according

to pain intensity and the use of opioids. (B) Integrated care for pain; non-opioid

and non-pharmacologic treatment. The use of non-opioid medication as medical

interventions is useful for multimodal analgesia which combines various groups

of medications (local anesthetics, NSAIDs, opioids, NMDA agonist, alpha-2

agonists, etc.). Physical (sensory) interventions include massage, positioning,

hot/cold, acupuncture and Transcutaneous Electrical Nerve Stimulation (TENS).

Behavioral intervention and improvement of lifestyle manage pain relief through

psychotherapy, sleep, nutrition, mood, and exercise (active self-care therapies).

Fig. 2A modified and extended from bpacnz pain management (5)

Fig. 2B modified and extended from Conschafter, A 2015(8)

16

Figure 3.

17

Figure 3. Local analgesics

(A) Multimodal analgesic sites of action in the central and peripheral nervous

systems and adverse effects of analgesics commonly used. (B) Ion channels

targeting for local analgesics in nociceptive neurons. Ion channels detecting

noxious thermal, chemical, and mechanical stimuli are expressed in nociceptive

nerve ending. Voltage-gated sodium channels (Nav1.7, Nav1.8 and Nav1.9)

mainly are expressed in nociceptive neuron and play an important role in

generating action potential.

Fig. 3A modified and extended from Pitchon et al. 2018 (10)

Fig. 3B modified and extended from Juárez-Contreras R et al. 2018 (12)

18

Figure 4.

19

Figure 4. The mechanism of ascending nociceptive

control

Peripheral stimulations by noxious stimuli such as electrical, chemical or

acupuncture alter the peripheral nerve activity, which induces the activation of

descending pain inhibitory system. In persistent pain condition, adequate

peripheral stimulation effectively suppresses pain signaling.

Fig. 4 modified and extended from BMJ 2014;348:f7656

20

Figure 5.

21

Figure 5. Benefits of time-restricted feeding

(A) Time-Restricted Feeding (TRF) improves circadian clock and metabolism

rhythms. TRF increase resistance to disease caused by high fat diets. (B)

Metabolic switching enhances neuroplasticity and optimize brain function.

Fig. 5A modified and extended from Hatori, M. et al. 2012 (30)

Fig. 5B modified and extended from Mattson, M. P. et al. 2018 (31)

22

PURPOSE

The standard of care (NSAIDs and opioids) for pain currently has many

limitations. In an ongoing attempt to control pain through a multimodal approach

based on pain mechanisms, I have focused on the therapeutic effect of local

analgesics (sinomenine), pharmacopuncture (acupoint stimulation with drug,

Scolopendra subspinipes), and time-restricted feeding (fasting/refeeding) as

below.

To confirm whether sinomenine produces an analgesic effect on

animal pain behavior and if so, to investigate the mechanism of

sinomenine as a local analgesic.

To investigate the analgesic mechanism of pharmacopuncture with

Scolopendra subspinipes.

To examine whether animal pain behavior is affected by fasting and

refeeding, with a focus on calorie state, and if so, to investigate their

potential mechanism.

23

CHAPTER 1:

The analgesic effect of sinomenine

via inhibition of voltage-gated

sodium channel

*This chapter has been largely reproduced from articles published by

Jeong-Yun Lee, Seo-Yeon Yoon, Jonghwa Won, Han-Byul Kim, Youngnam Kang, Seog

Bae Oh. Sinomenine produces peripheral analgesic effects via inhibition of voltage-

gated sodium currents. Neuroscience, 2017, Sep;358:28-36. doi:10.1016/

24

ABSTRACT

Sinomenium acutum has been used in traditional medicine to treat a

painful disease such as rheumatic arthritis and neuralgia. Sinomenine, which is a

main bioactive ingredient in Sinomenium acutum, has been reported to have an

analgesic effect in diverse pain animal models. However little is known about the

detailed mechanisms underlying peripheral analgesic effect of sinomenine. In the

present study, I aimed to elucidate its cellular mechanism by using formalin-

induced acute inflammatory pain model in mice. I found that intraperitoneal (i.p.)

administration of sinomenine (50 mg/kg) suppressed formalin-induced paw

licking behavior in both the first and the second phase. Formalin-induced c-Fos

protein expression was also suppressed by sinomenine (50 mg/kg, i.p.) in the

superficial dorsal horn of spinal cord. Whole cell patch clamp recordings from

small-sized dorsal root ganglion (DRG) neurons revealed that sinomenine

reversibly increased the spike threshold and the threshold current intensity for

evoking a single spike and decreased firing frequency of action potentials evoked

in response to a long current pulse. Voltage-gated sodium currents (INa) were also

significantly reduced by sinomenine in a dose-dependent manner (IC50 = 2.3 ±

0.2 mM). Finally, I confirmed that intraplantar application of sinomenine

suppressed formalin-induced pain behavior only in the first phase, but not the

second phase. Taken together, these results suggest that sinomenine has a

peripheral analgesic effect by inhibiting INa.

25

INTRODUCTION

Sinomenium acutum has been traditionally used as a herbal medicine to

treat rheumatic arthritis and neuralgia33. Sinomenine is its main bioactive

ingredient in Sinomenium acutum and is well known to have immunosuppressive

and anti-inflammatory effects34. Interestingly, recent studies have also

demonstrated analgesic effects of sinomenine in rodents with nociceptive,

inflammatory and neuropathic pain35. Sinomenine-induced analgesia is reversed

by a GABAA receptor antagonist in postoperative pain and neuropathic pain

models36. Apart from the pathological animal pain models, tail flick test shows

that sinomenine has analgesic effects by activating opioid μ-receptor37. While

these studies have found potential cellular targets for sinomenine in the central

nervous system (CNS)38, little is known about the cellular mechanism or

sinomenine-induced peripheral analgesia.

VGSCs are the main ion channels for transmitting pain signals in the

nociceptive neurons14-16. Thus, VGSCs in primary afferent neurons is the key

molecular target for diverse analgesics including local anesthetics such as

lidocaine17. In chapter 1, I sought to explore the analgesic effect of sinomenine

by using formalin-induced acute pain model and further focused its peripheral

mechanisms by examining its effects on the excitability of nociceptive neurons.

I found that sinomenine can produce peripheral analgesic effect by reducing

cellular excitability of nociceptive neurons and the inhibition of VGSC is likely

to contribute to its action.

26

MATERIALS AND METHODS

Animals

Male C57BL/6 mice weighing 20-28 g were used for the experiment.

They were housed 4-6 per cage at a temperature-controlled room (23 ± 1 , 12

h/12 h light/dark cycle) and maintained with pellet diet and tap water ad libitum.

All surgical and experimental procedures were reviewed and approved by the

Institutional Animal Care and Use Committee (IACUC) at Seoul National

University.

Formalin test

Mice were acclimated in cage at least for a week and then adapted in an

acrylic observation chamber (size ranges 12×12×12 cm) before the experiment

at least three times. A mirror was located at 45° angle below the chamber to

observe the paws. Formalin test was performed by intraplantar (i.pl.) injection of

1% formalin. 20 μl of 1% formalin was injected subcutaneously into the plantar

surface of the right hind paw with a 31-gauge needle of 0.3 ml insulin syringe.

Following formalin injection, the animals were immediately placed in a test

chamber and recorded using a video camera for a period of 40 minutes. The time

mice spent licking or biting was measured during each 5 minutes by an observer

who was blinded to the treatment. Formalin-induced pain behaviors during 0-10

minutes after formalin injection represented the first phase and during 10-40

minutes after formalin injection represented the second phase.

c-Fos immunohistochemistry

Animals were sacrificed 2 h after formalin injection for analysis of c-Fos

27

proteins. Animals were anaesthetized by intraperitoneal (i.p.) injection of

pentobarbital (60 mg/kg) and transcardially perfused with 0.01 M phosphate-

buffered saline (PBS) including 500 U/L heparin followed by 4%

paraformaldehyde (PFA) in 0.01 M PBS. The spinal cord was post-fixed by 4%

PFA overnight and transferred to 30% sucrose in 0.01 M PBS for 2 - 3 weeks.

Frozen specimen was transversely sectioned into 30 μm with cryotome and

sections were stored in cryoprotectant at -20 . Free floating sections were

washed with 0.01 M PBS and incubated in 0.3% H2O2 (in distilled water) for 30

minute at room temperature (RT). After elimination of endogenous peroxidase,

sections were washed with 0.01 M PBS and Pre blocked with 5% normal goat

serum (NGS) in PBS with 0.3% triton x (PBST) for 1 h at RT. Sections were

incubated in 1st antibody (PC38, Calbiochem, USA; 1:1000 in 1% NGS (in 0.3%

PBST)) for 48 h at 4 , washed with 0.01 M PBS to remove 1st antibody and

incubated in biotinylated goat anti rabbit (BA1000, Vector laboratories, USA;

1:400 in 0.01 M PBS) for 2 h at RT. Sections were processed with ABC kit

(PK-6100, Vectastain ABC kit, Vector laboratories, USA), visualized with DAB

kit (DAB substrate kit for peroxidase, Vector laboratories, USA) and then

mounted on slide glass. After air drying, all sections were cover slipped with

mountant (H-1000, Vectashield mounting medium, Vector laboratories, USA).

Cell counting and image analysis

For the quantification of c-Fos expression, I confirmed that most c-Fos

expression induced by injection of 1% formalin (i.pl.) localized in Lumbar (L) 4-

5 segment. Four sections with highest expression of c-Fos per animal were

chosen and superficial dorsal horn (lamina I-II) of L4-5 segments were selected

for analysis. The number of c-Fos positive neurons was counted blindly and the

28

mean value was used as representative counts. Using image J, the image of

selected sections was converted to grey scale, background subtracted, enhanced

and sharpened. Intensity threshold was adjusted and then analyzed.

Dorsal root ganglion (DRG) culture

Mice were sacrificed by exposure to isoflurane and decapitated. DRG

were isolated in cold HBSS (Welgene, Korea) and incubated in 3 ml HBSS

containing 1 mg/ml collagenase (Roche, USA) / 2.4 U/ml dispase II (Roche, USA)

for 1 h at 37 . Then DRG were digested by 0.25% trypsin in PBS for 7 min at

37 , inactivated by 2 ml 0.25% trypsin inhibitor (Sigma, USA) and 2 ml

DMEM containing 10% fetal bovine serum (Gibco, USA) / 1% pen strep (Gibco,

USA) and washed by 4 ml DMEM (FBS / pen strep). DRG neurons were

triturated using fire-polished pasteur pipette to separate cells. The cells were

resuspended in neurobasal medium (Gibco, USA) containing B-27 supplement

(Gibco, USA), 1% pen strep, L-glutamine (1 mM), placed on cover slips coated

with poly-D-lysine (Sigma, USA). After 1h cells were fed with fresh neurobasal

medium (B-27 supplement / pen strep / L-glutamine) and maintained in a 5%

CO2 - 95% O2 incubator at 37 °C.

Electrophysiological recordings

Whole-cell current- and voltage-clamp recordings were performed in

small-sized DRG neurons (<20 μm diameter) at RT with an Axopatch 200B

amplifier to record action potentials (APs) and INa. Data were sampled at 10 kHz.

The patch pipettes were pulled from borosilicate capillaries. When filled with

following pipette solutions, their resistances were 2-6 MΩ. A -10 mV liquid

junction potential was corrected. The extracellular solution was driven by gravity

29

and continuously perfused (1-2 ml/min). The pipette solution for current clamp

contained (in mM): K-gluconate 140, CaCl2 1, MgCl2 2, EGTA 10, K2ATP 5,

HEPES 10, adjusted to pH 7.4 with KOH, osmolality 300 mOsm. Extracellular

solution for current clamp contained (in mM): NaCl 140, HEPES 10, CaCl2 2,

MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH, osmolality 300

mOsm. Single APs were evoked by 5 ms depolarizing current pulses. The spike

threshold was measured at the onset of the positive deflection of the first

differentiation of the spike potential. A series of APs were evoked by 500 ms

depolarizing current pulses. The pipette solution for voltage clamp contained (in

mM): CsCl 100, sodium L glutamic acid 5, TEACl 30, CaCl2 0.1, MgCl2 2, EGTA

11, HEPES 10 adjusted to pH 7.4 with CsOH osmolality 300 mOsm.

Extracellular solution for voltage clamp contained (in mM): NaCl 140, HEPES

10, CaCl2 2, MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH,

osmolality 300 mOsm. The INa was evoked by a test pulse stepped to -10 mV

from a holding potential of -90 mV every 20 s.

Rota-rod test

Mice were placed on the horizontal bar rotating at a speed of 4 r.p.m

using rota-rod apparatus (DJ-4009, Dae-Jong Engineering &Clean Technology,

Seoul, Korea). Twenty-four hours before the actual rota-rod test all mice were

tested and those that were able to remain on the rod for at least 120 s were

included in the study. Performance time on the bar (in sec) and number of falls

over a 2 minutes were measured. The scores were then compared and analyzed 5

minutes (PRE) before and 30 minutes (POST) after treatment with vehicle or

sinomenine. The test was repeated 3 times and the mean value for each animal

was recorded.

30

Administration of sinomenine

Sinomenine hydrochloride was purchased from TCI chemicals (Tokyo,

Japan). In the formalin test, to examine the systemic effects of sinomenine,

sinomenine was diluted in normal saline and intraperitoneally injected at a dose

of 25 mg/kg, 50 mg/kg or 75 mg/kg in a volume of 10 ml/kg body weight 30

minutes prior to the formalin injection. Control animals received (i.p.) an

equivalent volume of vehicle. To evaluate the peripheral effects of sinomenine,

formalin test was performed after i.pl. injection of sinomenine (500 μg per mouse)

which was diluted in 1% formalin (20 μl/saline). For electrophysiological

recordings, sinomenine was dissolved in the extracellular solutions to their

concentration. The solution was driven to cell by gravity and continuously

perfused (1-2 ml/min).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 6.0

(GraphPad Software, USA). Comparison between two groups was made using

the unpaired or paired Student's t-test to analyze the expression of c-Fos,

electrophysiological recordings of APs, formalin test and rota-rod test. For

multiple comparisons, data were analyzed using the one-way ANOVA followed

by Tukey’s test to determine formalin test and electrophysiological recordings of

INa. Data are presented as mean ± SEM. Differences with p<0.05 were considered

significant.

31

RESULTS

The effect of sinomenine on formalin-induced pain behavior

I investigated the analgesic effect of sinomenine using formalin model as

it is a reliable model for testing the analgesic effect and mechanism of a drug39-

41. Injection of 1% formalin (i.pl.) elicited typical biphasic pain behavior (Fig.

6Aa). As compared with the vehicle group, 25 mg/kg (i.p.) sinomenine did not

induce statistically significant decrease in formalin-induced pain behavior (Fig.

6Ab). However, sinomenine treated group (i.p.) had a significant analgesic effect

in both the first phase and second phase, at the dose of 50 mg/kg and 75 mg/kg

(Fig. 6Ab). In the first phase, formalin-induced pain behavior was reduced by 45

± 9.8% at the dose of 50 mg/kg (*p<0.05) and 81.4 ± 5.1% at the dose of 75

mg/kg, respectively (***p<0.001). In the second phase, formalin-induced pain

behavior was reduced by 38.5 ± 6.8% at the dose of 50 mg/kg (**p<0.01) and

96.3 ± 2% at the dose of 75 mg/kg, respectively (***p<0.001).

The expression of c-Fos protein is a measure of acute nociception

following noxious stimulation42. Thus, it is commonly used to show the analgesic

property of the drug43. Injection of 1% formalin (i.pl.) caused high c-Fos

expression in superficial dorsal horn innervated by L4-5 DRG neurons (Fig. 6B).

As compared with the vehicle group, sinomenine (50 mg/kg i.p.) reduced the

number of c-Fos positive neurons in superficial dorsal horn by 32.1 ± 10.1%

(*p=0.0292) (Fig. 6B).

Collectively, these results showed that sinomenine produces an analgesic

effect in acute inflammatory pain, suggesting that peripheral nociceptive neurons

might be targeted by sinomenine.

The effect of sinomenine on APs and VGSCs in small-sized DRG neurons

32

I next investigated the effects of sinomenine on the excitability of small-

sized DRG neurons (<20 μm diameter). 1mM sinomenine increased the spike

threshold from –31 ± 1.6 mV to –26.6 ± 2.1 mV (**p=0.0023) and the current

threshold by 21.5 ± 5% (**p=0.0032) (Fig 6A), and also significantly decreased

AP amplitude by 18.1 ± 3.2% (**p=0.0013) and AP overshoot level by 28.1 ±

4.9% (**p=0.0021) (Fig. 7A). Concomitantly, half- width of AP was increased

by 89.3 ± 29.9% (**p=0.0057) (Fig. 7A). When I examined the effect of

sinomenine on a series of APs, I found that AP firing frequency was also

decreased by 3 mM sinomenine and restored partially after washout (Fig. 7B).

Compared with the control, 3 mM sinomenine reduced AP firing frequency by

39.5 ± 6% (**p=0.007).

Because the VGSCs are responsible for the generation of the APs, I tested

the effect of sinomenine on INa in small-sized DRG neurons by a command pulse

stepped from -90 to -10 mV. As compared with the control, 3 mM sinomenine

significantly inhibited INa in small-sized DRG neurons by 63.3 ± 3.2%

(***p<0.001) (Fig. 8A). INa was decreased by sinomenine in a dose-dependent

manner with 0.1 mM by 7.7 ± 1%, 0.3 mM by 13 ± 2%, 1 mM by 29.9 ± 2.3%,

3 mM by 63.3 ± 3.2% and 10 mM by 83.3 ± 3.2%. (Fig. 8B, IC50 = 2.3 ± 0.2

mM).

These results suggest peripheral action of sinomenine on VGSCs in

nociceptive neurons, which might be associated with analgesic effect of

sinomenine on acute inflammatory pain.

Intraplantar application of sinomenine on formalin-induced pain behavior

To clarify the peripheral action of sinomenine, I then examined whether

peripheral injection of sinomenine indeed produces analgesic effects. I found that

33

Injection of 500 μg sinomenine (i.pl.), co-administrated with 1% formalin, only

reduced the first phase of formalin-induced pain behavior by 38.2 ± 5.9%

(**p=0.0057), whereas the second phase of formalin-induced pain behavior was

not affected by sinomenine (Fig. 9).

The effect of sinomenine on motor function in rota-rod test

Because drug-induced motor function impairment may result in false

positive results in nociceptive pain behavior, I carried out rota-rod test to examine

the effect of sinomenine on motor function. I did not observe motor activity

impairment at the dose of 25 mg/kg and 50 mg/kg, whereas there was a

significant motor dysfunction at the dose of 75 mg/kg (Fig. 10). 75 mg/kg

sinomenine (i.p.) significantly increased number of falls (*p=0.0267) and

decreased performance time (***p=0.0002) in rota-rod test.

34

Figure 6.

35

Figure 6. Effects of sinomenine on formalin-induced

pain

(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced

pain behavior (a). Formalin-induced pain behavior was divided into two phases

and analyzed (b). The bar graph represented first phase (0-10 min) and second

phase (10-40 min) of formalin-induced pain behavior. Intraperitoneal (i.p.)

injection of sinomenine inhibited formalin-induced pain behavior in both first

phase and second phase at the dose of 50 mg/kg or 75 mg/kg. *p<0.05, **p<0.01

and ***p<0.001 (one-way ANOVA followed by Tukey’s test) (B) Intraplantar

injection of 1% formalin induced c-Fos expression in superficial dorsal horn of

Lumbar 4-5 (L4-5) segments (a). Sinomenine (SIN, 50 mg/kg, i.p.) decreased

formalin-induced c-Fos expression in superficial dorsal horn of L4-5 (b). The

quantification of c-Fos expression (c). 50 mg/kg sinomenine suppressed

formalin-induced c-Fos expression in superficial dorsal horn of L4-5 as

compared with vehicle. *p<0.05 (unpaired t test, two-tailed), Scale bar = 200 μm,

SDH; superficial dorsal horn

36

Figure 7.

37

Figure 7. Effects of sinomenine on action potentials in

small sized dorsal root ganglion neurons

(A) The inhibitory effect of sinomenine on action potentials (APs). A

representative APs trace (a). 1 mM sinomenine (SIN) increased the spike

threshold (b) and the current threshold (c), and inhibited AP amplitude (d) and

peak (e). After application of 1 mM sinomenine, half-width of AP was increased

(f). (B) sinomenine decreased APs firing frequency. Series of APs were recorded

during 500 ms depolarizing current step. 3 mM sinomenine inhibited APs firing

frequency (n=4). Series of APs returned partially after washout. **p<0.01 (paired

t test, two-tailed)

38

Figure 8.

39

Figure 8. Effects of sinomenine on voltage-gated sodium

channel in small sized dorsal root ganglion neurons

(A) 3 mM sinomenine (SIN) inhibited voltage-gated sodium current (INa). Time

course of effects of 3 mM sinomenine on INa (1) before, 2) during, 3) after the

application of 3 mM sinomenine) (a). INa trace at the points indicated (b).

Normalized INa. As compared with control 3 mM sinomenine significantly

decreased INa (n=6) (c). ***p<0.001 (repeated measures ANOVA followed by

Tukey’s test) (B) INa was decreased by sinomenine in a dose dependent manner

with IC50 = 2.3 ± 0.2 mM. (0.1 mM n=4, 0.3 mM n=5, 1 mM n=8, 3 mM n=6,

10 mM n=5)

40

Figure 9.

41

Figure 9. Intraplantar administration of sinomenine on

formalin-induced pain behavior

(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced

pain behavior. (B) Formalin-induced pain behavior was divided into two phases

and analyzed. The bar graph represented first phase (0–10 min) and second phase

(10–40 min) of formalin-induced pain behavior. Intraplantar injection of

sinomenine (500 μg) inhibited formalin-induced pain behavior only in the first

phase. **p<0.01 (unpaired t test, two-tailed)

42

Figure 10.

43

Figure 10. Effects of sinomenine in rota-rod test

The performance time on rota-rod (A) and the number of falls (B) was measured

5 minutes (PRE) before and 30 minutes (POST) after intraperitoneal (i.p.)

injection with vehicle or sinomenine. Sinomenine had no effect on rota-rod test

at the dose of 25 mg/kg (n=6) or 50 mg/kg (n=7). 75 mg/kg sinomenine showed

significant difference in rota-rod score (n=6). *p<0.05 and ***p<0.0001 (paired

t test, two-tailed)

44

DISCUSSION

In chapter 1, I show peripherally mediated-analgesic action of sinomenine.

I found that not only i.p application, but also i.pl. application, of sinomenine

produces analgesic effect on formalin-induced pain model. Sinomenine inhibited

formalin-induced c-Fos expression in the superficial dorsal horn of spinal cord.

Using whole-cell patch clamp recordings, I also found that sinomenine reduces

cellular excitability by inhibiting INa in small-sized DRG neurons.

The formalin-induced pain model in rodents typically show biphasic pain

behavior, each caused by different mechanisms40,41,44. The first phase occurs due

to direct stimulation of nociceptor in C-fiber and the second phase is a pain

response caused by peripheral inflammation and functional changes in central

processing44. In the present study, i.p. injection of sinomenine significantly

decreased formalin-induced pain behavior in both first phase and second phase.

However, i.pl. injection of sinomenine decreased formalin-induced pain behavior

only in the first phase (Fig. 6 and Fig. 9). These results suggest that sinomenine

has peripheral analgesic mechanism.

The analgesic effects of sinomenine via i.p. route on the second phase

might result from its central mechanisms. Recent studies showed that analgesic

effects of sinomenine are related to a GABAA receptor and opioid μ-receptor,

both of them are cellular targets for sinomenine in the CNS. Furthermore, in a

previous study, systemic administration (i.p.) of VGSC blockers attenuates

formalin-induced pain behavior in the second phase45. However, peripherally

administered VGSC blocker such as lidocaine suppresses formalin-induced pain

behavior only in the first phase17. Thus, it seems that sinomenine has peripheral

action, in addition to its central actions previously demonstrated by other groups.

45

The gene c-Fos encodes nuclear protein Fos in response to neuronal

activation and has been used as a marker for neuronal activity42. Following the

noxious stimulation, primary afferent nociceptive neurons (Aδ and C-fiber)

transmit pain signal to second order neurons in the superficial dorsal horn of

spinal cord. Thus, c-Fos are expressed in the superficial dorsal horn of spinal cord

after noxious stimulation46 and the reduction of c-Fos expression is commonly

used to show peripheral analgesic effects43. In this study, I found that sinomenine

(50 mg/kg i.p.) decreased formalin-induced c-Fos expression in the superficial

dorsal horn. This result further supports a peripheral action of sinomenine (Fig.

6).

The activity of primary nociceptive neurons (Aδ and C-fiber) is regulated

by ion channels and is important for pain pathway16. Because pain threshold and

intensity are directly correlated with AP threshold and AP frequency, respectively,

in such afferent, I examined whether sinomenine modulate the excitability of

small-sized DRG neurons. Using patch clamp recordings, I first showed that

sinomenine increased AP threshold and decreased AP firing frequency (Fig. 7).

This strongly suggests that sinomenine has an analgesic effect. Consistent with

an involvement of VGSCs in the generation of AP, sinomenine affected VGSCs.

Furthermore, it is well known that VGSCs contribute to AP overshoot phase47

and, sinomenine inhibited AP overshoot phase (Fig. 8). So, the cause of widening

of the AP width could be the reduction of potassium channel activation due to the

sinomenine-induced decrease in VGSCs activation. In the present study, INa was

decreased by sinomenine in a dose dependent manner. Sinomenine has been

demonstrated to inhibit L-type calcium channel and ASIC channel in cortical

neurons48. In addition, vasodilatory action of sinomenine is involved with

inhibition of calcium channels49. These studies show the possibility of ion

46

channel regulation by sinomenine in various cells. On the other hand, present

study proposes that sinomenine regulate VGSCs in primary sensory neurons,

which can produce peripheral analgesic effect by reducing cellular excitability of

nociceptive neurons.

Sinomenine has pharmacological actions on diverse ion channels at a

wide range of concentrations. Sinomenine inhibits voltage-gated calcium

currents in cultured rat cortical neurons at concentration of 0.05–1 μM48. 10 μM

sinomenine inhibited ATP-activated current in HEK293 cells expressing the

P2X3 receptor50. In this study, I observed that sinomenine was effective at a

concentration range of ~ mM on the cellular excitability and VGSCs in small-

sized DRG neurons. Thus, VGSCs may exhibit less sensitivity to sinomenine

compared to VGCCs and P2X3 channels. Likewise, previous studies showed that

diverse ion channels expressed in sensory neurons are targeted by eugenol with

different sensitivity51-53. Given VGCCs and P2X3 channels are also expressed

in DRG neurons, it remains to be determined whether VGCCs and P2X3 channels

in nociceptive neurons are indeed targeted by sinomenine.

The effective analgesic dose of sinomenine (50 mg/kg i.p.) does not elicit

motor activity impairment. When I investigated the effect of sinomenine on

motor function in rota-rod test, the dose of 50 mg/kg (i.p.) were analgesic without

motor activity impairment (Fig. 10). However, I observed motor dysfunction and

some sedative effect at high dose of sinomenine (75 mg/kg i.p.). Because

systemic administration of sinomenine has a side effect such as sedation, gastric

intestine and kidney damage at high dose, it is important to use sinomenine in

low concentrations. In many analgesics as well as sinomenine, the side effects of

systemic administration are an important issue. Thus, various drug administration

methods have been applied to reduce drug concentration and side effects. For

47

example, patch form of lidocaine was used to reduce systemic adverse effects and

effective in neuropathic patients in clinical practice54. Sinomenine also showed

anti-inflammatory effects when topically administrated via patch and spray

formulations55. In present study, I found that i.pl administration of sinomenine

show analgesic effect at the low concentration that did not show analgesic effects

when administered systemically (20 ~25 mg/kg i.pl. versus 25 mg/kg i.p.), imply

its potential therapeutic use as a local analgesic in the periphery.

Based on these findings, I propose a peripheral analgesic mechanism for

sinomenine other than its well-known anti-inflammatory actions, which is

reduction of cellular excitability via the inhibition of INa in small-sized DRG

neurons. Therefore, sinomenine could be a potential pharmacological therapeutic

agent as local anesthetic and analgesics.

48

CHAPTER 2:

Pharmacopuncture with

Scolopendra subspinipes suppresses

mechanical allodynia in oxaliplatin-

induced neuropathy: Mediation by

spinal alpha2-adrenoceptor

*This chapter includes reproductions from an article published by Seo-Yeon Yoon,

Jeong-Yun Lee, Dae-Hyun Roh, Seog Bae Oh. 2018. Pharmacopuncture With

Scolopendra subspinipes Suppresses Mechanical Allodynia in Oxaliplatin-Induced

Neuropathic Mice and Potentiates Clonidine-induced Anti-allodynia Without

Hypotension or Motor Impairment. JOURNAL OF PAIN, 2018, 19, 10, 1157-1168. doi:

10.1016/j.jpain.2018.04.015

49

ABSTRACT

Pharmacopuncture is a new form of acupuncture therapy that combines

acupuncture with herbal medicine by injection of the herbal extract into the

acupoint and has been reported to be more effective than acupuncture alone.

Scolopendra subspinipes (SS) has been used in traditional medicine to treat

chronic neuronal diseases. In this study, I examined the effect of

pharmacopuncture with Scolopendra subspinipes (SSP) into the ST36 acupoint

by using formalin-induced acute inflammatory pain model and oxaliplatin-

induced neuropathic pain model. Subcutaneous (s.c.) treatment with SSP (0.5 %,

20 μl, ST36) did not affect formalin-induced paw licking behavior in both the

first and second phase. On the other hand, SSP (0.5 %, 20 μl, ST36, s.c.)

significantly decreased oxaliplatin-induced mechanical allodynia, which was

completely prevented by acupoint pretreatment of lidocaine. SS injection into a

non-acupoint site had no effect. Intrathecal (i.t.) treatment with yohimbine (25

μg, 5 μl), an alpha2-adrenoceptor antagonist, prevented the anti-allodynic effect

of SSP. In contrast, SSP treatment still produced significant anti-allodynic effect

after treatment with naloxone (20 μg, 5 μl, i.t.), an opioid receptor antagonist, or

methysergide (20 μg, 5 μl, i.t.), a serotonin receptor antagonist. Collectively,

these findings demonstrate that SSP produces an analgesic effect in oxaliplatin-

induced neuropathic pain via neuronal conduction at the acupoint and activation

of spinal alpha2-adrenoceptors.

50

INTRODUCTION

For relieving pain, pharmacological approaches are not the only option.

While medications are targeting the somatic sensation of the pain, non-

pharmacological therapies aim to improve mood states, the affective component

of pain, by modulating the endogenous pain control system9. Acupuncture has

been known to have a great therapeutic potential for relieving pain as a non-

pharmacological therapy56. Especially, Zusanli acupoint (ST36) located below

the knee, on the tibialis anterior muscle has been widely used to elucidate the

analgesic mechanism of acupuncture in animal experiments. Electrical

stimulation at acupoint (EA) is a suitable method to study the analgesic

mechanism of acupuncture even in anesthetized animals57. However, EA has

limitations of short duration and is difficult to apply in non-anesthetized animals.

Pharmacopuncture is a new form of acupuncture therapy that combines

acupuncture with herbal medicine related to the diseases, which is known as a

method of safely stimulating acupoint for a long time in non-anesthetized animals.

Scolopendra subspinipes (SS), a venomous arthropod, has been used in

China and Korea for several hundred years to treat neuronal disorders including

stroke, epilepsy, tetanus, joint problems, neoplasm, and Alzheimer’s disease58,59.

It has been reported that crude extract of SS venom glands is composed of many

ingredients, including histamine, 5-hydroxytryptamine, lipids, polysaccharides,

enzymes, and amino acids 60. Recently, peptides isolated from SS venom were

shown to produce an analgesic effect in animal pain models61,62. However, little

is known about the effect of pharmacopuncture with SS (SSP) on pain. In chapter

2, I examined the effect of SSP into ST36 on animal pain model and further

focused its underlying mechanisms.

51

MATERIALS AND METHODS

Animals

All experimental procedures were reviewed and approved by the

Institutional Animal Care and Use Committee (IACUC) at Seoul National

University. Male C57BL/6 mice weighing 20-28 g and adult male 129S4/SvJae

x C57BL/6J F1 hybrid mice (25- 30 g) were used for the experiment. They were

housed in a temperature- and light-controlled room (22°C ± 2°C, 12-hour

light/dark cycle with lights on at 07:00). Food and water were available as desired.

All animals were habituated to the mouse house 1 week before the start of the

study.

Pharmacopuncture with SS

SS extract (0.5% in saline solution) was obtained from Korean

Pharmacopuncture Institute (Seoul, Korea), and the following is the description

about the extraction method of SS extract. Briefly, dried centipedes (112 mm x 7

g) were used after the head and legs were removed, and 128.6 g of dried

centipedes were immersed in distilled water (DW, 1 L) and stirred at room

temperature for 3 hours. The crude extract was stored in refrigerator overnight to

precipitate, and the supernatant was collected (1´ DW extraction). The DW

extraction using the precipitate was repeated twice (2´ and 3´ DW extraction). All

supernatants of the 1´, 2´, and 3´ DW extracts were passed twice through a filter

paper (Whatman, 0.8 mm) and concentrated under reduced pressure at 70°C for

3 hours using a rotary vacuum evaporator (EYELA, Tokyo, Japan). The viscous

extract was concentrated to an ethanol series (90%, 80%, and 70%) using a rotary

vacuum evaporator. The extract was filtered through Whatman paper in the order

52

of 8.00, 0.45 and 0.10 mm, and lyophilized using a freeze dryer (IlshinBioBase,

Gyeonggido, Korea) for 200 hours. The final product powder to be tested was

dissolved in saline solution at a concentration of 0.005 g/mL (0.5%; pH 11.7) and

stored at 4°C. Saline solution was used as the vehicle for 0.250% or 0.125% SS.

Pharmacopuncture was performed by subcutaneous (s.c.) injection of 20

μl of SS solution at bilateral ST36 acupoints under isoflurane anesthesia. The

ST36 acupoint of mouse was identified based on the human acupoint landmark

and a mouse anatomical reference. It is located in the lateral stifle joint, 5 mm

lateral to the anterior tubercle of the tibia. Vehicle control mice were injected with

the saline into the ST36. Non-acupoint located on the back and buttocks were

selected. 40 μl of SS was injected into a non-acupoint (arbitrary position on the

back) or 20 μl of SS was injected into bilateral non-acupoints (midway between

coccyx and hip joint).

Formalin-induced acute pain model

Formalin test was performed as previously described in chapter 1. 10

minutes after SSP, 20 μl of 1% formalin was injected subcutaneously into the

plantar surface of the right hind paw. Following formalin injection, the animals

were immediately placed in a test chamber and recorded using a video camera

for a period of 40 minutes.

Oxaliplatin-induced neuropathic pain model

Oxaliplatin was purchased from Tocris Bioscience (Bristol, United

Kingdom). A single dose of oxaliplatin (10 mg/kg) was injected i.p. into the

mouse. Vehicle control mice were administrated with an equivalent volume of 5%

dextrose. Oxaliplatin-induced mechanical allodynia was measured using an

53

ascending series of von Frey filaments (North Coast Medical, Morgan Hill, CA).

Mice were acclimated to an acrylic cylinder (6.5 cm diameter, 17 cm height) on

the metal mesh grid for 30 minutes before mechanical allodynia test. Each

monofilament was applied 6 times in ascending order (range of bending force:

0.008, 0.020, 0.040, 0.070, 0.160, 0.400, and 0.600 g) to the midplantar region of

each mouse hind paw. The force of monofilament that elicited paw withdrawal,

licking, or flinching in 3 of the 6 applications was defined as the mechanical Paw

Withdrawal Threshold (PWT).

Drugs

To examine whether peripheral neuronal activation in the acupoint was

related to the effect of SSP, regional nerve block was performed by injection of

lidocaine (0.5% in 20% ethanol; Sigma, St. Louis, MO, 20 μl, s.c.) into the ST36

acupoint 10 minutes before SSP or saline injection. To evaluate the role of the

major descending inhibitory pathway including adrenergic, opioidergic, and

serotonergic neurotransmitter systems in SSP-induced analgesia, yohimbine

(alpha2-adrenoceptor antagonist, 20 μg, 5 μl), naloxone (opioid receptor

antagonist, 20 μg, 5 μl), or methysergide (serotonin receptor antagonist, 20 μg, 5

μl) was administered 5 minutes before SS injection. Using a 50 μl Hamilton

syringe, intrathecal (i.t.) injections were performed by insertion of a 30 G needle

(0.5inch length) into the subarachnoidal space between lumbar vertebrae L5 and

L6. A reflexive flick of the tail indicated a successful entry of the needle in the

subarachnoidal space. All drugs were purchased from Sigma and diluted in saline

except yohimbine, which was diluted in DW.

Statistical Analysis

54

All of the data are presented as mean ± SEM. Statistical analysis was

performed using graphpad prism version 5.1 (Graphpad software). Comparison

between two groups was made using the unpaired student's t test to determine

formalin test. For multiple comparisons, data were analyzed using the one-way

ANOVA followed by Tukey’s test to determine formalin test. Data were analyzed

using two-way ANOVA followed by the Tukey post hoc test for multiple

comparison in mechanical allodynia. Differences with p < 0.05 were considered

significant.

55

RESULTS

SSP do not affect formalin-induced acute inflammatory pain behavior

To investigate the effect of SSP on acute inflammatory pain, I measured

spontaneous pain behavior in the formalin-induced acute inflammatory pain

model (Fig. 11). 1% formalin was injected in right hind paw 10 minutes after

treatment of SSP (0.5%, 20 μl, ST36). Formalin-induced spontaneous pain

behavior was not affected by unilateral (ipsi-ST36) treatment of SSP, while

pharmacopuncture with bee venom (BVP) suppressed formalin-induced

spontaneous pain with a significant analgesic effect in the second phase (Fig.

11A). Again, bilateral treatment of SSP also had no significant effect in formalin-

induced spontaneous pain behavior (Fig. 11B).

SSP suppress Oxaliplatin-induced mechanical allodynia

I investigated whether SSP affected the oxaliplatin-induced neuropathic

pain (Fig. 12). Animals underwent oxaliplatin injection (10 mg/kg i.p.) showed a

significant decrease in mechanical PWT in both hind limbs at 14 days after the

injection (POST) compared with the mean values before the injection (PRE),

indicating induction of mechanical allodynia by oxaliplatin chemotherapy (Fig.

12A and B). Treatment of SSP (20 μl, ST36) in oxaliplatin-treated mice produced

a significant dose-dependent increase in PWT at 30 minutes and 1 hour after SS

injection compared with injection of vehicle (0.9% saline) (Fig. 12B). In contrast,

injection of vehicle (5% dextrose i.p.), instead of oxaliplatin, did not show the

change in PWT at 14 days after injection (Fig. 12C). SSP also had no effect on

PWT in those control mice not treated with oxaliplatin (Fig. 12C). These results

indicate that SS venom extract (0.5%, but not lower concentrations) injected into

56

ST36 location temporarily decreased allodynia (as measured by PWT), and the

effect is over within 2 hours after injection.

Peripheral nerve activation of the specific acupoint is essential for the

analgesic effect of SSP in oxaliplatin-induced mechanical allodynia

Next, I tested whether peripheral nerve activation in the acupoint region

was involved in the SSP-induced anti-allodynic effect. The effect of SSP was

blocked by pretreatment with lidocaine (0.5%, 20 μl) into the ST36 acupoint

(lidocaine + SSP) compared with pretreatment with vehicle only (20% ethanol,

20 μl, vehicle + SSP) (Fig. 13A). Lidocaine without SSP (lidocaine + saline) had

no effect on oxaliplatin-induced mechanical allodynia (Fig. 13A). Furthermore,

I compared effects between acupoint and non-acupoint stimulation (Fig. 13B).

Based on previous studies, the locations of non-acupoint were selected on back

or buttocks. SS injection into non-acupoint sites had no effect on oxaliplatin-

induced decrease in PWT (Fig. 13B).

Spinal alpha2-adrenoceptors mediate the analgesic effect of SSP

I then examined the involvement of the descending endogenous pain

inhibitory system associated with spinal alpha2-adrenergic, opioidergic, and/or

serotonergic system using an i.t. injection of an antagonist of each receptor before

SSP treatment. Yohimbine (alpha2-adrenoceptor antagonist) significantly

decreased the SSP-induced anti-allodynic effect (Fig. 14A), suggesting that SSP-

induced analgesia is mediated by spinal alpha2-adrenoceptors. In contrast,

pretreatment with naloxone (opioid receptor antagonist) or methysergide

(serotonin receptor antagonist) did not affect the SSP anti-allodynic effect (Fig.

14B).

57

Figure 11.

58

Figure 11. Effect of Scolopendra subspinipes

pharmacopuncture (SSP) on formalin-induced acute

inflammatory pain model

(A) Effects of SSP (unilateral injection) on the time course curves of formalin-

induced pain behavior (a). Formalin-induced pain behavior was divided into two

phases and analyzed (b). The bar graph represented the first phase (0-10 min) and

second phase (10-40 min) of formalin-induced pain behavior. Unilateral injection

of SSP did not affect formalin-induced pain behavior in both the first phase and

second phase. Unilateral injection of bee venom pharmacopuncture (BVP)

inhibited formalin-induced pain behavior only in the second phase. *p<0.05,

(one-way ANOVA followed by Tukey’s test) (B) Effects of SSP (bilateral

injection) on the time course curves of formalin-induced pain behavior (a).

Formalin-induced pain behavior was divided into two phases and analyzed (b).

Bilateral injection of SSP did not affect formalin-induced pain behavior in both

first phase and second phase. (unpaired t test, two-tailed)

59

Figure 12.

60

Figure 12. Effect of Scolopendra subspinipes

pharmacopuncture (SSP) on oxaliplatin (OXA)-induced

neuropathic pain model

(A) The experimental design is shown in the time line diagram (B) Intraperitoneal

(i.p.) treatment with OXA (10 mg/kg) showed a significant decrease in

mechanical paw withdrawal threshold (PWT) in both hind limbs after 14 days

(POST) compared with the mean PWT before OXA injection (PRE). SSP

injection into bilateral ST36 locations (0.5%, 0.25%, or 0.125%, 20 μl) dose-

dependently reversed OXA-induced mechanical allodynia. (C) Treatment of

vehicle (5% dextrose) instead of OXA did not affect PWT, and injection of SS or

saline into bilateral ST36 acupoints also did not affect PWT in those vehicle-

treated mice (C). ***P < 0.001, *p<0.05 (two-way ANOVA followed by Tukey’s

test)

61

Figure 13.

62

Figure 13. Peripheral nerve activation of the specific

acupoint is associated with Scolopendra subspinipes

pharmacopuncture (SSP)-induced anti-allodynia

(A) Effect of subcutaneous lidocaine pretreatment on pharmacopuncture with

Scolopendra subspinipes (SSP)-induced anti-allodynic effect in oxaliplatin

(OXA)-treated mice. By comparison with subcutaneous (s.c.) injection of the

vehicle (20% ethanol) with SSP (vehicle + SSP), which induced anti-allodynia,

lidocaine (0.5%/20 μl, s.c.) pretreatment into the ST36 acupoint (lidocaine + SSP)

10 minutes before SSP reversed SSP-induced anti-allodynia. Lidocaine alone

(lidocaine + saline) did not affect OXA-induced mechanical allodynia (vehicle +

saline). (B) SSP reversed oxaliplatin-induced mechanical allodynia with the

bilateral ST36 acupoint injection but did not affect PWT when SSP at non-

acupoint location (back or buttocks). ***p<0.001, *p<0.05 versus vehicle +

saline group (two-way ANOVA followed by Tukey’s test)

63

Figure 14.

64

Figure 14. Effects of pretreatment of intrathecal

injection of yohimbine, naloxone, or methysergide on

Scolopendra subspinipes pharmacopuncture (SSP)-

induced anti-allodynia

Intrathecal treatment of yohimbine (an alpha2-adrenoceptor antagonist),

naloxone (an opioid receptor antagonist) or methysergide (a serotonin receptor

antagonist) was performed 5 minutes before SSP. (A) Yohimbine (25 μg, 5 μl),

prevented the anti-allodynic effect of SSP. (B) In contrast, SSP treatment still

significantly produced anti-allodynic effect after treatment with naloxone or

methysergide. ***p<0.001 versus vehicle + SSP in each group (two-way ANOVA

followed by Tukey’s test)

65

DISCUSSION

In chapter 2, I found out that SSP into bilateral ST36 acupoint in adult

mice relieved mechanical allodynia associated with oxaliplatin chemotherapy-

induced neuropathy. The SSP-induced anti-allodynia was mediated via peripheral

neuronal activation at the SSP acupoint and via activation of spinal alpha2-

adrenoceptors.

Compared to conventional acupuncture combined with oral medication,

the benefits of pharmacopuncture are synergistic effects of acupuncture and

pharmacological medicine. Pharmacopuncture has more immediate effects

compared to oral drug administration because the pharmacological materials are

absorbed directly, without passing through the gastrointestinal tract. In non

acupoint experiment, a total 40 μl of SS, the same amount with analgesic effect

when treated into ST36 (20 μl on each ST36 acupoint), was injected into an

arbitrary non-acupoint location but there was no effect (Fig. 13B). Therefore, the

analgesic effect of SSP may not be due to the systemic effect of SS.

Since peptides isolated from SS venom produce an analgesic effect by

inhibiting TRPV1 and Nav1.761,62, there is a possibility that the analgesic effect

of SS injection into ST36 could be mediated by inhibiting TRPV1 and Nav1.7 at

ST36. However, lidocaine into ST36 point itself did not affect the pain sensation

in both oxaliplatin and vehicle-treated mice (Fig. 13A). Furthermore,

anatomically, ST36 is quite close to the deep peroneal nerve and the tested plantar

surface of the paw in this study is mainly innervated by the tibial nerve (partially

the sural and saphenous nerves) rather than peroneal nerve63. Therefore, it is

plausible that the analgesia induced by SSP at ST36 is not just mediated by the

simple pharmacologic effect of nerve agents especially into a peroneal nerve.

66

Peripheral nerve block by pretreatment of lidocaine at ST36 completely

reversed the SSP-induced analgesia (Fig. 13A). These findings indicate that the

analgesic effect of SSP on oxaliplatin-induced neuropathy might be related to

peripheral neuronal activation in the ST36 acupoint. Acupoint stimulation

activate peripheral nociceptive neurons and induce a sensation of heaviness,

soreness or numbness (deqi, a composite of unique sensations), which has been

shown to be essential for the analgesic effects of acupuncture56 and induces

neuronal signals that transmit to the spinal cord and then to the brain, resulting in

activation of the descending endogenous pain inhibitory system57. With this in

mind, I evaluated whether the anti-allodynic effect of SSP is mediated by the

descending endogenous pain inhibitory system associated with spinal alpha2-

adrenergic, opioidergic, and/or serotonergic system. I found that intrathecal

pretreatment with yohimbine blocked the SSP-induced anti-allodynia in

oxaliplatin-induced neuropathic mice, whereas pretreatment with naloxone or

methysergide did not (Fig. 14). This result indicates that spinal alpha2-

adrenoceptors, but not opioid or serotonin receptors, are involved in the analgesic

effect of SSP.

SSP-induced neuronal signaling might activate the descending

noradrenergic system and spinal a2-adrenoceptors, leading to the analgesic effect

on oxaliplatin chemotherapy-induced neuropathic pain. There are several articles

demonstrating that the physiological mechanisms underlying pharmacopuncture

with bee venom, which is another useful venom for oriental medicine, induced

anti-nociceptive and anti-inflammatory effect in various rodent models27,64. BVP

into the ST36 acupoint potently activates catecholaminergic neurons in locus

coeruleus, ultimately leading to the activation of descending noradrenergic

pathway into the spinal cord65. Moreover, this increased activity of spinal

67

noradrenergic neurons mediates an alpha2-adrenoceptor, which is mainly

involved in pharmacopuncture-induced effect in acute and neuropathic pain

models. However, it is unclear how the SSP-induced activation of descending

noradrenergic pathway leads to significant analgesic effects in chemotherapy-

induced neuropathic pain. Further studies need to be performed to delineate these

concerns.

This study demonstrates that SSP suppresses oxaliplatin chemotherapy-

induced mechanical allodynia via peripheral nerve activation in the injected

acupoint and activation of spinal alpha2-adrenoceptors. Although this study

shows a potential therapeutic effect of SSP in established oxaliplatin-induced

pain, the prevention of chemotherapy-induced pain is also important clinically.

Further study is needed to evaluate the effect of SSP on the development of

chemotherapy-induced neuropathic pain.

68

CHAPTER 3:

Both acute fasting and refeeding

alleviate inflammatory pain but via

different pathways

*This chapter has been largely reproduced from articles published by

Jeong-Yun Lee, Grace J Lee, Pa Reum Lee, Chan Hee Won, Doyun Kim, Youngnam Kang,

Seog Bae Oh, The analgesic effect of refeeding on acute and chronic inflammatory pain.

Scientific Reports, 2019, 14;9(1):16873. doi: 10.1038/s41598-019-53149-7

69

ABSTRACT

Pain is susceptible to various cognitive factors. Suppression of pain by

hunger is well known, but the effect of food intake after fasting (i.e. refeeding)

on pain remains unknown. In the present study, I examined whether inflammatory

pain behavior is affected by 24h fasting and 2h refeeding. In formalin-induced

acute inflammatory pain model, fasting suppressed pain behavior only in the

second phase and the analgesic effect was also observed after refeeding.

Refeeding with non-calorie agar produced an additional analgesic effect

compared with fasting. Besides, administration of glucose (i.p.) after fasting,

which mimics calorie recovery following refeeding, induced a greater analgesic

effect than vehicle group. Administration of opioid receptor antagonist (naloxone,

i.p.) and cannabinoid receptor antagonist (SR 141716, i.p.) reversed fasting-

induced analgesia, but did not affect refeeding-induced analgesia in acute

inflammatory pain model. Taken together, these results show that fasting and

refeeding produce analgesia through different mechanisms, suggesting that

endocannabinoid and opioid system is only involved in fasting-induced analgesia,

whereas refeeding-induced analgesia is associated with eating behavior and

calorie effect.

70

INTRODUCTION

Pain perception is a multifaceted experience, largely divided into sensory

and affective dimension1,66. The sensory dimension of pain provides sensory-

discriminative information such as location, quality, and intensity. Affective pain

consists of hedonic aspect (pleasantness/unpleasantness) and affective-

motivation aspect which creates behavior to escape from pain as a secondary pain

effect67. Since pain is both a sensation and the emotion which is an unpleasant

state motivating an organism to react in favor of its survival, it is susceptible to

modulation by cognitive factors3,68.

Hunger is well known as a powerful driving force to change cognition.

Several clinical evidences have shown that mood states, such as confusion, anger

and tension can be improved by limiting food intake69. Furthermore, therapeutic

fasting is also effective for pain relief and mood enhancement regardless of body

weight change in chronic pain patient70,71. In various animal pain models, it is

also well established that fasting or calorie restriction also has an analgesic

effect72-75. Interestingly, recent studies have revealed that a part of the

parabrachial nucleus (PBN) is involved in the suppression of pain response by

fasting76,77. Endogenous opioid and endocannabinoid system not only play a

critical role in both homeostatic and hedonic aspects of feeding but also are

involved in the endogenous pain inhibitory system78,79. Therefore, activation of

opioid and endocannabinoid system after fasting is likely to modulate pain.

However, the relationship between the analgesic effect after fasting and these

endogenous pain control systems have not been elucidated.

While several studies have found the fasting-induced analgesia, little is

known about the effect of food intake after fasting on pain. Previous study has

71

revealed that hedonic drinking induces analgesic effect in fasted animal80.

Moreover, food ingestion after fasting reduced pain perception in healthy

volunteers81. In animal study, thermal pain threshold also increased at returning

to free feeding after calorie-restriction82. Thus these studies strongly suggest the

possibility that food intake after fasting can suppress pain. Nevertheless, the ph

enomenon or the mechanism for it is still unknown.

In this study, I thus sought to explore the change of pain behavior

following fasting and refeeding using inflammatory pain conditions with the

formalin-induced acute pain model. I found that fasting-induced analgesia was

mediated by opioid and endocannabinoid system. On the other hand, refeeding-

induced analgesia is mediated by eating behavior and calorie recovery.

72

MATERIALS AND METHODS

Animals

Male C57BL/6 mice weighing 18-25 g were purchased from DooYeol

Biotech (Korea) and maintained with standard lab chow (pellet diet) and water

ad libitum except when food was removed for deprivation experiments. All

experimental procedures were reviewed and approved by the Institutional Animal

Care and Use Committee (IACUC) at Seoul National University (SNU-170705-

1, SNU-180518-1). All experiments were performed in accordance with relevant

guidelines and regulations that was confirmed by IACUC.

Formalin-induced pain model

Formalin test was performed as previously described in chapter 1. The

time mice spent licking was measured during each 5 minutes by an observer who

was blinded to the treatment. Formalin-induced licking behaviors during 0-10min

after formalin injection represented the first phase and during 10-40min after

formalin injection represented the second phase. The total sum of the licking

times for each phase was statistically analyzed.

von Frey test in naïve mice

To assess mechanically evoked pain, both the 50% paw withdrawal

threshold and paw withdrawal frequency was measured using von Frey filaments

(North Coast Medical, Morgan Hill, CA, USA). Mice were acclimated in cage at

least for a week and then adapted in an acrylic cylinder (6.5 cm diameter, 17 cm

height) on the metal mesh floor before the experiment at least three times. All

animals left to an acrylic cylinder on the metal mesh floor for 30min-1h prior to

73

mechanical test. The 50% paw withdrawal threshold was determined based on

the up-down method with an ascending series of von Frey filaments (0.16 g, 0.4

g, 0.6 g, 1 g, and 2 g). The response frequency was used to assess the paw

withdrawal frequency to the repeated application of a single von Frey filament

(0.16 g or 0.6 g, 10 times each).

Hargreaves test in naïve mice

To assess heat-evoked thermal pain, paw withdrawal latency was

measured using Hargreaves method (IITC Life Science Plantar Test, Victory Blvd

Woodland Hills, CA, USA) with a glass surface, heated to 30 °C. Thermal latency

was measured 4 to 7 times and averaged.

Administration of drug

Naloxone (Tocris) was diluted in 0.9% saline. SR 141716 (Tocris) was

diluted in 0.9% saline with 10% DMSO and 1% tween 80. These drugs were

intraperitoneally (i.p.) injected at a dose of 10 mg/kg in a volume of 10 ml/kg

body weight.

Feeding schedule for pain behavior test

Food deprivation began between the hours of 09.00-13.00. For refeeding

with the non-calorie agar pellet experiment, additive-free agar powder was

melted in tap water (4%, in a microwave oven and then cooled in a refrigerator)

and cut into about 1.5 x 1 x 0.5 cm. After 24h food deprivation, mice had free

access to 4% agar for 2h. Water was freely accessible in all experiments.

74

Measurement of body weight, blood glucose level and food intake

Glucose levels were detected in blood samples collected from the tail

vein using an ACURA PLUS (automated glucometer, Korea). Body weight was

measured before and after 24h food deprivation and 2h and 24 h of refeeding.

And then, fasting and refeeding body weight was normalized to body weight

before fasting. The weight of the diet was measured per cage by weighing food

before and after ingestion and divided by the number of mice per cage.

Administration of D-glucose

D-glucose was diluted in 0.9% saline and injected at a dose of 1 mg/kg

(i.p.) in a volume of 10 ml/kg body weight. In the formalin test, after 15min of

D-glucose injection, formalin was injected in 24h fasted mice.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 5.0

(GraphPad Software, USA). Comparison between two groups was made using

the unpaired or paired Student's t-test. For multiple comparisons, data were

analyzed using the one-way ANOVA, repeated ANOVA followed by the post hoc

Bonferroni test. Detailed statistics for each experiment were shown in the figure

legend. Data are presented as mean ± SEM. Differences with p<0.05 were

considered significant.

75

RESULTS

Both fasting and refeeding suppress acute inflammatory pain behavior

To investigate the effect of fasting and refeeding on acute pain behavior,

I measured spontaneous pain behavior in the formalin-induced acute

inflammatory pain model and mechanical/thermal pain threshold in naïve mice

(Fig. 15). Consistent with previous studies74,77,83, 24h acute fasting suppressed

formalin-induced spontaneous pain behavior with a significant analgesic effect

only in the second phase (Fig. 15A). As compared with the free fed group, 2h

refed group had a significant analgesic effect only in the second phase, and this

analgesic effect was disappeared at 24h refeeding (Fig. 15A). In naïve mice, both

paw withdrawal threshold for mechanical stimuli and paw withdrawal latency for

thermal stimuli were not affected by 24h fasting and remained unchanged after

2h/24h refeeding (Fig. 15B).

To make satiety condition, mice were fasted for 24h and then allowed to

free access to normal chow for 2h. Induction of satiety was confirmed by body

weight, blood glucose level, food intake and stomach size (Fig. 16). After 24h

fasting, body weight and blood glucose level decreased, which was recovered

after 2h refeeding (Fig. 16 A and B). 2h refeeding increased food intake and

stomach was significantly expanded compared to the free fed mice (Fig. 16C and

D).

These results suggest that both fasting and refeeding produces an

analgesic effect in acute inflammatory pain but did not affect non-inflammatory

pain (nociception).

76

Refeeding of non-calorie agar pellet induces analgesic effect

Next, I investigated whether eating behavior or calorie recovery is

involved in refeeding-induced analgesia. To eliminate the effect of calorie

recovery, I used non-calorie agar pellet.

In the formalin-induced acute pain model, both refeeding of non-calorie

agar and normal lab chow had an analgesic effect only in the second phase (Fig.

17A and B). Interestingly, the agar-refeeding group had a greater analgesic effect

than fasting or normal chow-refeeding group (Fig. 17C), whereas there was no

difference in the analgesic effect between high-calorie chow (Oreo) and normal

chow (Fig. 17C). As compared to refeeding with normal chow, refeeding of non-

calorie agar for 2h did not increase stomach size, body weight, and blood glucose

level (Fig. 17D). Thus, agar-refeeding engaged eating behavior but did not induce

satiety signal by calorie recovery or stomach expansion.

These results indicate that eating behavior during refeeding contributes to

refeeding-induced analgesia, regardless of calorie.

Intraperitoneal administration of glucose induces analgesic effect

An additional analgesic effect by refeeding with non-calorie agar

suggests that satiety signal that consists of calorie recovery and stomach

expansion may have interfered with refeeding-induced analgesia. So I next

investigated the effect of calorie recovery on refeeding-induced analgesia. To

determine the effect of calorie recovery on pain without engaging eating behavior

and stomach expansion, I injected D-glucose (1 g/kg, i.p.).

Formalin was injected 15min after D-glucose injection in fasted mice (Fig.

18Aa). Compared with the vehicle (0.9% saline, i.p.) treated group, D-glucose

treated group had no significant difference in the sum of formalin-induced

77

spontaneous pain behavior from 10min to 40min (Fig. 18Ab and c). However,

only when comparing formalin-induced pain behavior from 10min to 30min, pain

behavior was significantly decreased in D-glucose treated group (Fig. 18Ab and

d). As compared to the vehicle group, administration of D-glucose increased

blood glucose level over time, peaked after 15 minutes and recovered after 1h

(Fig. 18B).

Taken together, these findings suggest that calorie recovery after fasting

may serve as an additional factor for refeeding-induced analgesic effect.

Opioid and endocannabinoid system contribute to fasting-induced analgesia,

but not refeeding-induced analgesia

To determine the involvement of opioid and endocannabinoids system in

fasting and refeeding-induced analgesia, I used naloxone (opioid receptor

antagonist) and SR 141716 (cannabinoid receptor (CB1) antagonist). In the

previous study, it was confirmed that SR 141716 significantly inhibit food

intake after fasting at dose of 10 mg/kg but not at 3 mg/kg84,85. I also confirmed

that 3mg/kg of naloxone (i.p.) had no effect on fasting-induced analgesia (data

not shown). Naloxone (10 mg/kg, i.p.) and SR 141716 (10 mg/kg, i.p.) was

administrated 30min before formalin injection (Fig. 19A). In formalin-induced

acute inflammatory pain model, both naloxone and SR 141716 inhibited fasting-

induced analgesia but did not affect refeeding-induced analgesia (Fig. 19B-E).

These results suggest that endogenous opioid and endocannabinoid

system mediate fasting-induced analgesia, but not refeeding-induced analgesia.

Refeeding might recruit distinctive analgesic factors from fasting-induced

analgesia.

78

Figure 15.

79

Figure 15. The effect of fasting and refeeding on acute

pain behavior

(A) Experimental design and schedule for formalin test (a). Time course of

spontaneous pain behavior following intraplantar injection of formalin (b).

Formalin-induced pain behavior was divided into two phases and the total sum

of the licking times for each phase was statistically analyzed; Free fed (n=8),

Fasted (n=9), 2h refed (n=8), 24h refed (n=7) (c). (B) The effect of 24h fasting

and 2h refeeding on paw withdrawal threshold and frequency for mechanical

stimuli (n=6) and paw withdrawal latency for thermal stimuli (n=5). Data are

presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (A one-way ANOVA

followed by Bonferroni test, B repeated measures ANOVA)

80

Figure 16.

81

Figure 16. Changes in body weight, blood glucose level

and food intake by refeeding in naïve mice

(A, B) Change in body weight and blood glucose level after refeeding in naïve

mice. Refeeding for 2h after 24h fasting caused weight gain; (n=5). The decrease

in blood glucose level due to fasting was also increased after 2h refeeding; (n=7).

(C) Stomach expansion induced by refeeding and food inside stomach (D)

Changes in food intake after refeeding. Compared with free fed group, the

amount of food intake for 2h was significantly increased in 24h fasted group. The

difference in food intake between two groups decreased after 2h refeeding. Data

are presented as mean ± SEM. *p,0.05, ***p<0.001 (unpaired t test, two-tailed)

82

Figure 17.

83

Figure 17. The analgesic effect of refeeding with non-

calorie agar on inflammatory pain behavior

(A, B) Effect of 2h refeeding non-calorie agar or normal chow on formalin-

induced pain behavior; Free fed (n=9), Agar-refed (n=12), Free fed (n=12),

Normal chow-refed (n=12). (C) Comparison of 2nd phase according to

experimental groups; Free fed (n=19), Fasted (n=12), Agar-refed (n=12), Normal

chow-refed (n=12), Oreo-refed (n=4). (D) Change in stomach size, body weight,

blood glucose level and food intake after agar refeeding in naïve mice. Data are

presented as mean ± SEM. **p<0.01, ***p<0.001 (A, B unpaired t-test, two-

tailed, C one-way ANOVA followed by Bonferroni test)

84

Figure 18.

85

Figure 18. The analgesic effect of intraperitoneal

administration of D-glucose on inflammatory pain

behavior

(A) Experimental design and schedule for formalin test (a). Time course of

spontaneous pain behavior following intraplantar injection of formalin after D-

glucose (1g/kg) administration (b); Free fed (n=11), Fasted (n=14), D-glucose

treated (n=8). 2nd phase of formalin test was divided into 10 to 40min and 10 to

30min and analyzed (c, d). (B) Blood glucose level after intraperitoneal

administration of D-glucose (1g/kg); D-glucose treated (n=4), Vehicle treated

(n=3). Data are presented as mean ± SEM. **p<0.01, ***p<0.001 (A one-way

ANOVA followed by Bonferroni test)

86

Figure 19.

87

Figure 19. The effect of opioid receptor and cannabinoid

receptor (CB1) antagonist on fasting and refeeding-

induced analgesia

(A) Experimental design and schedule for formalin test. Naloxone (opioid

receptor antagonist, 10 mg/kg) and SR 141716 (CB1 receptor antagonist, 10

mg/kg) were intraperitoneally (i.p.) administered 30 min before the formalin

injection. (B, C) Time course of spontaneous pain behavior following intraplantar

injection of formalin; Free fed-vehicle (n=9), Free fed-naloxone (n=7), 24h

fasted-vehicle (n=13), 24h fasted-naloxone (n=11), 2h refed-vehicle (n=12), 2h

refed-naloxone (n=10) / Free fed-vehicle (n=11), Free fed-SR 141716 (n=11),

24h fasted-vehicle (n=9), 24h fasted-SR 141716 (n=9), 2h refed-vehicle (n=10),

2h refed-SR 141716 (n=9). (D, E) Formalin-induced pain behavior was divided

into two phase and the total sum of the licking times for each phase was

statistically analyzed. Data are presented as mean ± SEM. **p<0.01, ***p<0.001

(D, E one-way ANOVA followed by Bonferroni test)

88

DISCUSSION

In chapter 3, I compared pain behaviors between fasting and refeeding

mice and discovered that refeeding only alleviates pathological pain induced by

inflammation. In the formalin-induced acute inflammatory pain model, refeeding

suppressed only spontaneous pain behavior especially in the second phase which

represents inflammatory pain, whereas the first phase of the formalin test, the von

Frey test and Hargreaves test were not affected by both fasting and refeeding. I

confirmed that fasting and refeeding produces analgesic effect through different

mechanisms where fasting produces analgesic effect via the opioid and

endocannabinoid system, but these systems are not involved in refeeding-induced

analgesia. Both refeeding of non-calorie agar and the calorie recovery by D-

glucose injection (i.p.) after fasting had an additional analgesic effect, compared

to fasting-induced analgesia.

Feeding behavior is crucial for maintaining homeostasis and it is well

investigated that pain perception is changed in eating disorder patients86,87. Since

opioid and endocannabinoid system are critical modulators of pain as well as

feeding78,79, these systems are important factors in determining the relationship

between feeding and pain. Diurnal fluctuations in pain sensitivity were not

caused by circadian rhythm but food deprivation, which was related to opioid

system72. Intermittent fasting produced analgesic effect via kappa-opioid system

in spinal cord88. It is also well known that leptin-deficient (ob/ob) obese mice

have increased pain threshold by activating endocannabinoid system in

descending pain pathway89. In the present study, I found that acute fasting leads

to analgesic effects in acute inflammatory pain through opioid and cannabinoid

system, whereas the analgesic effect of refeeding was not associated with these

89

systems (Fig. 19). Thus refeeding might recruit distinctive factors which are

different from fasting-induced analgesia.

Eating behavior and satiety signal such as calorie recovery and stomach

expansion may result from refeeding after fasting. I determined which factors

play critical roles in refeeding-induced analgesia. First, I identified the effect of

eating behavior on pain. Several pre-clinical studies have suggested that pain

signals are suppressed during drinking or eating regardless of calorie80,90,91.

Moreover, previous study also showed that not only drinking a sucrose solution

but also water drinking increased pain thresholds in the Hargreaves test of

thermal sensitivity80. However, these studies are not suitable to investigate the

effects of refeeding on pain because eating and pain signals exist at the same time.

In the present study, I performed a pain behavior test after eating behavior was

completed, and found that even eating non-calorie food produces a greater

analgesic effect than fasting-induced analgesia, indicating that eating behavior

itself can suppress pain perception, regardless of calorie (Fig. 17).

Next, I examined the effect of calorie recovery after fasting on pain.

Glucose administration after fasting is known to enhance neuroplasticity and

cognitive function31,92, so it is possible that pain perception is also affected by

glucose administration92. In the present study, it was found that glucose

administration (i.p.) had a greater analgesic effect compared to fasting only while

blood glucose level remained elevated (Fig. 18), suggesting that refeeding-

induced analgesia is also associated with a calorie recovery. Therefore, refeeding-

induced analgesia is multiple phenomena occurring simultaneously, which are

clearly distinctive from simple fasting-induced analgesia.

Finally, stomach expansion might affect pain perception. Physiologically,

slight distention of the stomach does not cause a significant increase in gastric

90

pressure, but severe stomach distension may activate the affective pain system93.

Hence it is possible that the affective component of pain from significant stomach

expansion may interfere with refeeding-induced analgesia. Furthermore, PBN

receives satiety signal such as ingestion, gut distention and satiety hormones

directly from nucleus of the solitary tract (NTS)94 and is activated by not only

meal-related satiety but also noxious stimuli95. Therefore, fullness due to stomach

expansion could interfere with the analgesic effect of refeeding in formalin test

by activating PBN (Fig. 17C and D).

I have not yet determined the brain circuits that mediate refeeding-

induced analgesia. Painful stimuli, such as formalin or electrical shock, is known

to induce c-Fos (neural activity marker) expression in PBN77,96. A previous study

reveals that hunger suppress inflammatory pain behavior by inhibiting PBN via

the agouti-related protein (AgRP)-expressing neuron77. However, it is well

known that the expression of c-Fos in PBN is increased by 2h refeeding after 20-

40h acute fasting97-99 which is involved in determination of meal size and meal

termination during refeeding100. Hence, I suggest that 2h refeeding-induced

analgesia is less likely mediated by PBN.

Feeding and fasting drive oscillation of energy metabolism. These

oscillations are known to have positive effects not only on healthy lifespan but

also on neural circuits related to cognition and emotion29. Thus, alternation of

fasting-refeeding may have many benefits to modulate pain. Based on these

findings, I propose that both fasting and refeeding modulate pathological pain

more effectively through different mechanisms.

91

CONCLUSION

Pain is a highly individualized phenomenon with the mismatch between

pain complaint and pathological condition. Therefore, integrated multimodal care

is required for pain management. In this thesis, I have explored various

therapeutic approaches to pain based on pain pathways (the lateral pain pathway,

the medial pain pathway, and the descending pain pathway).

In the first chapter, I tried to reduce pain by regulating the lateral pain

pathway. I showed that sinomenine can produce peripheral analgesic effect by

inhibiting INa in nociceptive afferent neurons. In the second chapter, I wanted to

find a form of pain treatment by enhancing the descending pain pathway. I found

that acupoint stimulation with Scolopendra subspinipes activated the descending

pain inhibitory pathway associated with the spinal alpha2-adrenergic system. In

the third chapter, I sought to investigate whether the medial pain pathway could

be affected by modulation of feeding patterns. Both the fasting and refeeding

produced analgesic effect only in pathological pain. The nociception that mainly

related to the lateral pain pathway, was not affected by fasting and refeeding.

This thesis shows that the regulation of each pain pathway has potential

therapeutic effects. However, it cannot be confirmed whether these approaches

control pain in the patients who are not managed by conventional pain treatment.

Thus, further research is needed regarding combination therapy for clinical

applications.

92

REFERENCES

1 Price, D. D., Harkins, S. W. & Baker, C. Sensory-affective relationships among different

types of clinical and experimental pain. Pain 28, 297-307 (1987).

2 De Ridder, D. & Vanneste, S. Burst and Tonic Spinal Cord Stimulation: Different and

Common Brain Mechanisms. Neuromodulation 19, 47-59, doi:10.1111/ner.12368

(2016).

3 Bushnell, M. C., Ceko, M. & Low, L. A. Cognitive and emotional control of pain and its

disruption in chronic pain. Nat Rev Neurosci 14, 502-511, doi:10.1038/nrn3516 (2013).

4 Benarroch, E. E. Descending monoaminergic pain modulation: bidirectional control and

clinical relevance. Neurology 71, 217-221, doi:10.1212/01.wnl.0000318225.51122.63

(2008).

5 The principles of managing acute pain in primary care, <https://bpac.org.nz/2018/acute-

pain.aspx> (2018).

6 Vargas-Schaffer, G. Is the WHO analgesic ladder still valid? Twenty-four years of

experience. Can Fam Physician 56, 514-517, e202-515 (2010).

7 Pain Treatment Guidelines,

<http://www.oregonpainguidance.org/app/content/uploads/2016/05/OPG_Guidelines_

2016.pdf> (2016).

8 Conschafter, A. IfPA Policy Brief Identifies Barriers to Integrated Pain Care,

<https://allianceforpatientaccess.org/ifpa-policy-brief-identifies-barriers-integrated-

pain-care-2/> (2015).

9 Ahmed El Geziry, Y. T., Fathi Al Kadhi, Muhammad Pervaiz and Mohammad Al Nobani.

Non-Pharmacological Pain Management,

<http://dx.doi.org/10.5772/intechopen.79689> (2018).

10 Pitchon, D. N., Dayan, A. C., Schwenk, E. S., Baratta, J. L. & Viscusi, E. R. Updates on

Multimodal Analgesia for Orthopedic Surgery. Anesthesiol Clin 36, 361-373,

doi:10.1016/j.anclin.2018.05.001 (2018).

11 Derry, S. et al. Topical analgesics for acute and chronic pain in adults - an overview of

Cochrane Reviews. Cochrane Database Syst Rev 5, CD008609,

doi:10.1002/14651858.CD008609.pub2 (2017).

12 Juarez-Contreras, R., Rosenbaum, T. & Morales-Lazaro, S. L. Lysophosphatidic Acid

and Ion Channels as Molecular Mediators of Pain. Front Mol Neurosci 11, 462,

doi:10.3389/fnmol.2018.00462 (2018).

13 Chung G., O. S. B. in Natural Products. (ed Mérillon JM. (eds) In: Ramawat K.)

(2013).

14 McEntire, D. M. et al. Pain transduction: a pharmacologic perspective. Expert Rev Clin

Pharmacol 9, 1069-1080, doi:10.1080/17512433.2016.1183481 (2016).

15 Mathie, A. Ion channels as novel therapeutic targets in the treatment of pain. J Pharm

Pharmacol 62, 1089-1095, doi:10.1111/j.2042-7158.2010.01131.x (2010).

16 Waxman, S. G. & Zamponi, G. W. Regulating excitability of peripheral afferents:

emerging ion channel targets. Nat Neurosci 17, 153-163, doi:10.1038/nn.3602 (2014).

17 Inan, S., Dun, N. J. & Cowan, A. Inhibitory effect of lidocaine on pain and itch using

formalin-induced nociception and 5'-guanidinonaltrindole-induced scratching models in

mice: behavioral and neuroanatomical evidence. Eur J Pharmacol 616, 141-146,

doi:10.1016/j.ejphar.2009.06.026 (2009).

93

18 Puopolo, M. et al. Permeation and block of TRPV1 channels by the cationic lidocaine

derivative QX-314. J Neurophysiol 109, 1704-1712, doi:10.1152/jn.00012.2013 (2013).

19 Binshtok, A. M. et al. Coapplication of Lidocaine and the Permanently Charged Sodium

Channel locker QX-314 Produces a Long-lasting Nociceptive Blockade in Rodents.

Anesthesiology 111, 127–137 (2009).

20 Kim, H. Y. et al. Selectively targeting pain in the trigeminal system. Pain 150, 29-40,

doi:10.1016/j.pain.2010.02.016 (2010).

21 Tobaldini, G., Sardi, N. F., Guilhen, V. A. & Fischer, L. Pain Inhibits Pain: an Ascending-

Descending Pain Modulation Pathway Linking Mesolimbic and Classical Descending

Mechanisms. Mol Neurobiol 56, 1000-1013, doi:10.1007/s12035-018-1116-7 (2019).

22 Gear, R. W., Aley, K. O. & Levine, J. D. Pain-induced analgesia mediated by mesolimbic

reward circuits. J Neurosci 19, 7175-7181 (1999).

23 Kim, D. H. et al. Acupuncture points can be identified as cutaneous neurogenic

inflammatory spots. Sci Rep 7, 15214, doi:10.1038/s41598-017-14359-z (2017).

24 Fan, Y. et al. Neuropeptides SP and CGRP Underlie the Electrical Properties of

Acupoints. Front Neurosci 12, 907, doi:10.3389/fnins.2018.00907 (2018).

25 Tobaldini, G., Aisengart, B., Lima, M. M., Tambeli, C. H. & Fischer, L. Ascending

nociceptive control contributes to the antinociceptive effect of acupuncture in a rat

model of acute pain. J Pain 15, 422-434, doi:10.1016/j.jpain.2013.12.008 (2014).

26 Chang, S. et al. Peripheral Sensory Nerve Tissue but Not Connective Tissue Is Involved

in the Action of Acupuncture. Front Neurosci 13, 110, doi:10.3389/fnins.2019.00110

(2019).

27 Yoon, S. Y. et al. Diluted bee venom injection reduces ipsilateral mechanical allodynia

in oxaliplatin-induced neuropathic mice. Biol Pharm Bull 36, 1787-1793 (2013).

28 Park, J. W. et al. Effects of sweet bee venom pharmacopuncture treatment for

chemotherapy-induced peripheral neuropathy: a case series. Integr Cancer Ther 11, 166-

171, doi:10.1177/1534735411413265 (2012).

29 Longo, V. D. & Panda, S. Fasting, Circadian Rhythms, and Time-Restricted Feeding in

Healthy Lifespan. Cell Metab 23, 1048-1059, doi:10.1016/j.cmet.2016.06.001 (2016).

30 Hatori, M. et al. Time-restricted feeding without reducing caloric intake prevents

metabolic diseases in mice fed a high-fat diet. Cell Metab 15, 848-860,

doi:10.1016/j.cmet.2012.04.019 (2012).

31 Mattson, M. P., Moehl, K., Ghena, N., Schmaedick, M. & Cheng, A. Intermittent

metabolic switching, neuroplasticity and brain health. Nat Rev Neurosci 19, 63-80,

doi:10.1038/nrn.2017.156 (2018).

32 Fetissov, S. O. [Hunger and satiety factors in the regulation of pleasure associated with

feeding behavior]. Biol Aujourdhui 210, 259-268, doi:10.1051/jbio/2016025 (2016).

33 Zhao, X. X., Peng, C., Zhang, H. & Qin, L. P. Sinomenium acutum: a review of

chemistry, pharmacology, pharmacokinetics, and clinical use. Pharm Biol 50, 1053-

1061, doi:10.3109/13880209.2012.656847 (2012).

34 Wang, Q. & Li, X. K. Immunosuppressive and anti-inflammatory activities of

sinomenine. Int Immunopharmacol 11, 373-376, doi:10.1016/j.intimp.2010.11.018

(2011).

35 Gao, T., Hao, J., Wiesenfeld-Hallin, Z., Wang, D. Q. & Xu, X. J. Analgesic effect of

sinomenine in rodents after inflammation and nerve injury. Eur J Pharmacol 721, 5-11,

doi:10.1016/j.ejphar.2013.09.062 (2013).

36 Zhu, Q. et al. Antinociceptive effects of sinomenine in a rat model of neuropathic pain.

94

Sci Rep 4, 7270, doi:10.1038/srep07270 (2014).

37 Wang, M. H. et al. Activation of opioid mu-receptor by sinomenine in cell and mice.

Neurosci Lett 443, 209-212, doi:10.1016/j.neulet.2008.07.088 (2008).

38 Zhu, Q. et al. Antinociceptive effects of sinomenine in a rat model of postoperative pain.

Br J Pharmacol 173, 1693-1702, doi:10.1111/bph.13470 (2016).

39 Cho, I. H. et al. Systemic administration of minocycline inhibits formalin-induced

inflammatory pain in rat. Brain Res 1072, 208-214, doi:10.1016/j.brainres.2005.12.039

(2006).

40 Hunskaar, S. & Hole, K. The formalin test in mice dissociation between non

inflammatory inflammatory pain. Pain 30 103-114 (1987).

41 Tjolsen, A., Berge, O. G., Hunskaar, S., Rosland, J. H. & Hole, K. The formalin test: an

evaluation of the method. Pain 51, 5-17 (1992).

42 Harris, J. A. Using c-fos as a neural marker of pain. Brain Res Bull 45, 1-8 (1998).

43 Badral, B. et al. Pain fiber anesthetic reduces brainstem Fos after tooth extraction. J Dent

Res 92, 1005-1010, doi:10.1177/0022034513505620 (2013).

44 Shibata, M., Ohkubo, T., Takahashi, H. & Inoki, R. Modified formalin test characteristic

biphasic pain response. Pain 38, 347-352 (1989).

45 Blackburn-Munro, G., Ibsen, N. & Erichsen, H. K. A comparison of the anti-nociceptive

effects of voltage-activated Na+ channel blockers in the formalin test. Eur J Pharmacol

445, 231-238 (2002).

46 Catherine, A., K., T. B., Alex, P. M. & I., B. A. Differential contribution of the two phases

of the formalin test to the pattern of c-fos expression in the rat spinal cord: studies with

remifentanil and lidocaine. pain 69, 101-110 (1997).

47 Renganathan, M., Cummins, T. R. & Waxman, S. G. Contribution of Nav1.8 Sodium

channels to Action Potential Electrogenesis in DRG Neurons. J Neurophysiol 86 (2001).

48 Wu, W. N. et al. Sinomenine protects against ischaemic brain injury: involvement of co-

inhibition of acid-sensing ion channel 1a and L-type calcium channels. Br J Pharmacol

164, 1445-1459, doi:10.1111/j.1476-5381.2011.01487.x (2011).

49 Nishida, S. & Satoh, H. In vitro pharmacological actions of sinomenine on the smooth

muscle and the endothelial cell activity in rat aorta. Life Sci 79, 1203-1206,

doi:10.1016/j.lfs.2006.03.024 (2006).

50 Rao, S. et al. The effect of sinomenine in diabetic neuropathic pain mediated by the

P2X3 receptor in dorsal root ganglia. Purinergic Signal, doi:10.1007/s11302-016-9554-

z (2017).

51 Lee, M. H. et al. Eugenol inhibits calcium currents in dental afferent neurons. J Dent

Res 84, 848-851, doi:10.1177/154405910508400913 (2005).

52 Park, C. K. et al. Eugenol inhibits sodium currents in dental afferent neurons. J Dent Res

85, 900-904, doi:10.1177/154405910608501005 (2006).

53 Yeon, K. Y. et al. Eugenol reverses mechanical allodynia after peripheral nerve injury

by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Pain

152, 2108-2116, doi:10.1016/j.pain.2011.05.018 (2011).

54 Gammaitoni, A. R., Alvarez, N. A. & Galer, B. S. Safety and tolerability of the lidocaine

patch 5%, a targeted peripheral analgesic: a review of the literature. J Clin Pharmacol

43, 111-117 (2003).

55 Li, X. et al. Development of patch and spray formulations for enhancing topical delivery

of sinomenine hydrochloride. J Pharm Sci 99, 1790-1799, doi:10.1002/jps.21947 (2010).

56 Lin, J. G. & Chen, W. L. Acupuncture analgesia: a review of its mechanisms of actions.

95

Am J Chin Med 36, 635-645, doi:10.1142/S0192415X08006107 (2008).

57 Kim, W., Kim, S. K. & Min, B. I. Mechanisms of electroacupuncture-induced analgesia

on neuropathic pain in animal model. Evid Based Complement Alternat Med 2013,

436913, doi:10.1155/2013/436913 (2013).

58 Li, C. et al. Inhibitory Effects of Scolopendra Pharmacopuncture on the Development

and Maintenance of Neuropathic Pain in Rats: Possible Involvement of Spinal Glial

Cells. J Acupunct Meridian Stud 8, 236-244, doi:10.1016/j.jams.2015.01.001 (2015).

59 Lee, K. H., Cho, Y. Y., Kim, S. & Sun, S. H. History of Research on Pharmacopuncture

in Korea. J Pharmacopuncture 19, 101-108, doi:10.3831/KPI.2016.19.010 (2016).

60 Gomes, A., Datta, A., Sarangi, B., Kar, P. K. & Lahiri, S. C. Isolation, purification &

pharmacodynamics of a toxin from the venom of the centipede Scolopendra subspinipes

dehaani Brandt. Indian J Exp Biol 21, 203-207 (1983).

61 Yang, S. et al. A pain-inducing centipede toxin targets the heat activation machinery of

nociceptor TRPV1. Nat Commun 6, 8297, doi:10.1038/ncomms9297 (2015).

62 Yang, S. et al. Discovery of a selective NaV1.7 inhibitor from centipede venom with

analgesic efficacy exceeding morphine in rodent pain models. Proc Natl Acad Sci U S A

110, 17534-17539, doi:10.1073/pnas.1306285110 (2013).

63 Lu, G. W. Characteristics of afferent fiber innervation on acupuncture points zusanli. Am

J Physiol 245, R606-612, doi:10.1152/ajpregu.1983.245.4.R606 (1983).

64 Yoon, J. et al. Sweet bee venom pharmacopuncture for chemotherapy-induced

peripheral neuropathy. J Acupunct Meridian Stud 5, 156-165,

doi:10.1016/j.jams.2012.05.003 (2012).

65 Kang, S. Y. et al. Repetitive treatment with diluted bee venom reduces neuropathic pain

via potentiation of locus coeruleus noradrenergic neuronal activity and modulation of

spinal NR1 phosphorylation in rats. J Pain 13, 155-166, doi:10.1016/j.jpain.2011.10.012

(2012).

66 Rainville, P., Carrier, B., Hofbauer, R. K., Bushnell, M. C. & Duncan, G. H. Dissociation

of sensory and affective dimensions of pain using hypnotic modulation. Pain 82, 159-

171 (1999).

67 Price, D. D. Psychological and neural mechanisms of the affective dimension of pain.

Science 288, 1769-1772 (2000).

68 Craig, A. D. A new view of pain as a homeostatic emotion. Trends Neurosci 26, 303-307

(2003).

69 Hussin, N. M., Shahar, S., Teng, N. I., Ngah, W. Z. & Das, S. K. Efficacy of fasting and

calorie restriction (FCR) on mood and depression among ageing men. J Nutr Health

Aging 17, 674-680, doi:10.1007/s12603-013-0344-9 (2013).

70 Michalsen, A. et al. Prolonged fasting in patients with chronic pain syndromes leads to

late mood-enhancement not related to weight loss and fasting-induced leptin depletion.

Nutr Neurosci 9, 195-200, doi:10.1080/10284150600929656 (2006).

71 Fond, G., Macgregor, A., Leboyer, M. & Michalsen, A. Fasting in mood disorders:

neurobiology and effectiveness. A review of the literature. Psychiatry Res 209, 253-258,

doi:10.1016/j.psychres.2012.12.018 (2013).

72 McGivern, R. F. & Berntson, G. G. Mediation of diurnal fluctuations in pain sensitivity

in the rat by food intake patterns: reversal by naloxone. Science 210, 210-211 (1980).

73 Liu, Y. et al. Antinociceptive effects of caloric restriction on post-incisional pain in

nonobese rats. Sci Rep 7, 1805, doi:10.1038/s41598-017-01909-8 (2017).

74 Jang, S. P. et al. Characterization of changes of pain behavior and signal transduction

96

system in food-deprived mice. Anim Cells Syst (Seoul) 22, 227-233,

doi:10.1080/19768354.2018.1490348 (2018).

75 Hargraves, W. A. & Hentall, I. D. Analgesic effects of dietary caloric restriction in adult

mice. Pain 114, 455-461, doi:10.1016/j.pain.2005.01.010 (2005).

76 Campos, C. A., Bowen, A. J., Roman, C. W. & Palmiter, R. D. Encoding of danger by

parabrachial CGRP neurons. Nature 555, 617-622, doi:10.1038/nature25511 (2018).

77 Alhadeff, A. L. et al. A Neural Circuit for the Suppression of Pain by a Competing Need

State. Cell 173, 140-152 e115, doi:10.1016/j.cell.2018.02.057 (2018).

78 Roques, B. P., Fournie-Zaluski, M. C. & Wurm, M. Inhibiting the breakdown of

endogenous opioids and cannabinoids to alleviate pain. Nat Rev Drug Discov 11, 292-

310, doi:10.1038/nrd3673 (2012).

79 Cota, D., Tschop, M. H., Horvath, T. L. & Levine, A. S. Cannabinoids, opioids and eating

behavior: the molecular face of hedonism? Brain Res Rev 51, 85-107,

doi:10.1016/j.brainresrev.2005.10.004 (2006).

80 Davies, A. J. et al. Hedonic drinking engages a supra-spinal inhibition of thermal

nociception in adult rats. Pain, doi:10.1097/j.pain.0000000000001482 (2019).

81 Zmarzty, S. A., Wells, A. S. & Read, N. W. The influence of food on pain perception in

healthy human volunteers. Physiol Behav 62, 185-191 (1997).

82 Hamm, R. J. & Lyeth, B. G. Nociceptive thresholds following food restriction and return

to free-feeding. Physiol Behav 33, 499-501 (1984).

83 Gheibi, N., Saroukhani, M. & Azhdari-Zarmehri, H. The effect of food deprivation on

nociception in formalin test and plasma levels of noradrenaline and corticosterone in

rats. Basic Clin Neurosci 4, 341-347 (2013).

84 Son, M. H. et al. Peripherally acting CB1-receptor antagonist: the relative importance

of central and peripheral CB1 receptors in adiposity control. Int J Obes (Lond) 34, 547-

556, doi:10.1038/ijo.2009.253 (2010).

85 Tam, J. et al. Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic

risk in mouse models of obesity. J Clin Invest 120, 2953-2966, doi:10.1172/JCI42551

(2010).

86 Lautenbacher, S., Pauls, A. M., Strian, F., Pirke, K. M. & Krieg, J. C. Pain perception in

patients with eating disorders. Psychosom Med 52, 673-682 (1990).

87 de Zwaan, M., Biener, D., Bach, M., Wiesnagrotzki, S. & Stacher, G. Pain sensitivity,

alexithymia, and depression in patients with eating disorders: are they related? J

Psychosom Res 41, 65-70 (1996).

88 de los Santos-Arteaga, M., Sierra-Dominguez, S. A., Fontanella, G. H., Delgado-Garcia,

J. M. & Carrion, A. M. Analgesia induced by dietary restriction is mediated by the

kappa-opioid system. J Neurosci 23, 11120-11126 (2003).

89 Cristino, L. et al. Orexin-A and Endocannabinoid Activation of the Descending

Antinociceptive Pathway Underlies Altered Pain Perception in Leptin Signaling

Deficiency. Neuropsychopharmacology 41, 508-520, doi:10.1038/npp.2015.173 (2016).

90 Foo, H. & Mason, P. Analgesia accompanying food consumption requires ingestion of

hedonic foods. J Neurosci 29, 13053-13062, doi:10.1523/JNEUROSCI.3514-09.2009

(2009).

91 Foo, H. & Mason, P. Sensory suppression during feeding. Proc Natl Acad Sci U S A 102,

16865-16869, doi:10.1073/pnas.0506226102 (2005).

92 Sibille, K. T., Bartsch, F., Reddy, D., Fillingim, R. B. & Keil, A. Increasing

Neuroplasticity to Bolster Chronic Pain Treatment: A Role for Intermittent Fasting and

97

Glucose Administration? J Pain 17, 275-281, doi:10.1016/j.jpain.2015.11.002 (2016).

93 Vandenbergh, J. et al. Regional brain activation during proximal stomach distention in

humans: A positron emission tomography study. Gastroenterology 128, 564-573 (2005).

94 Roman, C. W., Derkach, V. A. & Palmiter, R. D. Genetically and functionally defined

NTS to PBN brain circuits mediating anorexia. Nat Commun 7, 11905,

doi:10.1038/ncomms11905 (2016).

95 Palmiter, R. D. The Parabrachial Nucleus: CGRP Neurons Function as a General Alarm.

Trends Neurosci 41, 280-293, doi:10.1016/j.tins.2018.03.007 (2018).

96 Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an

Affective Pain Circuit that Creates a Threat Memory. Cell 162, 363-374,

doi:10.1016/j.cell.2015.05.057 (2015).

97 Zseli, G. et al. Elucidation of the anatomy of a satiety network: Focus on connectivity

of the parabrachial nucleus in the adult rat. J Comp Neurol 524, 2803-2827,

doi:10.1002/cne.23992 (2016).

98 Wu, Q. et al. The temporal pattern of cfos activation in hypothalamic, cortical, and

brainstem nuclei in response to fasting and refeeding in male mice. Endocrinology 155,

840-853, doi:10.1210/en.2013-1831 (2014).

99 Pinol, R. A. et al. Brs3 neurons in the mouse dorsomedial hypothalamus regulate body

temperature, energy expenditure, and heart rate, but not food intake. Nat Neurosci 21,

1530-1540, doi:10.1038/s41593-018-0249-3 (2018).

100 Campos, C. A., Bowen, A. J., Schwartz, M. W. & Palmiter, R. D. Parabrachial CGRP

Neurons Control Meal Termination. Cell Metab 23, 811-820,

doi:10.1016/j.cmet.2016.04.006 (2016).

98

국문초록

통증 조절을 위한 다각적 접근:

국소 진통제, 약침 요법 및 시간 제한 섭식에

관한 연구

통증은 국제 통증 연구 협회 (IASP)에 의해 실질적/잠재적

조직 손상과 관련된 불쾌한 감각 또는 정서적 경험으로 정의된다.

통증은 감각 및 정서적 요소를 모두 가지고 있기 때문에, 기존의

약리학적 접근법 (NSAID 및 오피오이드) 만으로는 통증을

효과적으로 조절하는 것이 어렵다. 따라서 본 학위 논문은 통증

관리에 대한 다각적인 접근 방식을 모색하기 위해 국소 진통제

(sinomenine), 약침 요법 (Scolopendra subspinipes, 오공 약침) 및 시간

제한 섭식 (금식 / 금식 후 재 섭식)의 통증 조절 가능성을 주요 연구

대상으로 하였다.

첫 번째 장에서는 sinomenine이 통각 수용 감각 신경의 흥분을

감소시킴으로써 말초에서 진통 효과를 유도 할 수 있음을 밝혔다.

Sinomenine의 복강 내 투여는 포르말린으로 유도된 통증 행동 및 c-

Fos 발현을 감소 시켰으며, sinomenine이 전압에 따라 개폐 되는 소디

99

움 채널을 억제함을 통각 수용 감각신경에서 확인하였다. 또한

sinomenine을 하지 말단에 국소 투여하였을 때도 통증 행동 감소가 관

찰되었다. 이러한 결과는 sinomenine이 국소 마취제 및 진통제로서 적

용 가능함을 제시한다.

두 번째 장에서는 오공 약침을 이용한 경혈 자극이 척수에 존

재하고 있는 alpha2 아드레날린 수용체를 통해 진통 효과를 유도한

다는 것을 밝혔다. 오공약침을 족삼리 영역에 피하 투여 시 옥살리플

라틴 처치로 인해 유발된 신경 병증성 통증을 감소시킴을 확인하였으

며, alpha2 아드레날린 수용체의 길항제로 알려진 yohimbine을 척수 내

에 주입하였을 때 오공 약침으로 유도된 진통 효과가 억제되었다. 따

라서, 오공 약침에 의한 경혈 자극은 하행성 통증 억제 시스템을 활

성화시켜 진통 효과를 나타내며, 이는 오공 약침을 이용한 통증 관리

의 가능성을 시사한다.

세 번째 장에서는 금식과 재 섭식 시 보이는 진통 효과에

중점을 두었다. 자율 섭식과 비교하였을 때, 24시간 금식과 2 시간 재

섭식은 모두 진통 효과를 보였으나 진통 효과를 유도하는 기전이

다름을 확인하였다. 24 시간 금식 시 유도된 통증 억제 효과는

엔도카나비노이드와 오피오이드 시스템이 관여하는 반면, 2 시간 재

섭식으로 유도된 진통 반응에는 섭식을 하는 행동 자체와 칼로리

회복이 관여 하였다. 따라서 섭식 조절 (시간 제한 섭식)은 통증

관리를 위한 새로운 치료 전략이 될 수 있을 것이라 기대된다.

100

주요어: 통증; sinomenine; 소디움 채널; 오공 약침; alpha2 아드레날린

수용체; 금식; 시간 제한 섭식

학번: 2015-22669