Role of extracellular matrix in acupoint region in acupuncture effect

ZHANG Zhi-ying, et al. Role of Extracellular Matrix in Acupoint Region in Acupuncture Effect

2006 Shanghai Research Institute of Acupuncture and Meridian︱

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325

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Basic Study

Role of Extracellular Matrix in Acupoint Region in

Acupuncture Effect

ZHANG Zhi-ying (张志英)

,DANG Rui-shan (党瑞山)

,JIN Jian-hua (金建华)

,HUANG Xuan(黄轩)

REN Cong-li(任丛莉)

,LI Liang(李亮)

,CHEN Er-yu(陈尔瑜)

1. Teaching and Research Section of Anatomy, NO.2 Military Medical University, Shanghai 200433, P. R. China
2. Teaching and Research Section of Anatomy, Wenzhou Medical College, P.R.China;
3. Clinical Medical Student Enrolled in 2003, NO.2 Military Medical University, Shanghai 200433, P.R .China

摘要

目的：探讨穴区细胞外基质在针刺信号转导中的作用机制。方法：SD大鼠 40 只，

雌雄各半，随机分为

4 组，每组 10 只，以大鼠热辐射甩尾实验的温度变化为观察指标。

实验组大鼠

"后三里"区注射人工合成的五肽甘氨酰－精氨酰－甘氨酰－天冬酰－酪氨酸

(Gly-Arg-Gly-Asp-Tyr, GRGDY)30 min后再针刺，以大鼠基础痛阈组、"后三里"区注射
0.9%NaCl组及针刺后三里组为对照。结果:GRGDY注射后 30 min再针刺，大鼠甩尾的温
度与针刺组相比未见明显改变。结论：

GRGDY未能阻断针刺效应，针刺效应的初始机制

可能与细胞外基质与整合素特异性的结合位点

RGD无关。

关键词

穴，足三里；RGD；细胞外基质；整合素

Abstract Objective: To investigate the action mechanism of extracellular matrix in acupoint
region in acupuncture signal transmission. Methods: Forty SD rats, half male and half female,
were randomly allocated to the experiment group, basic pain threshold group, normal saline
group and acupuncture group, with 10 rats in each group, the tail temperature at which rat flicks
its tail in heat radiation tail flick test was taken as an observation index. In experiment group, the
rats were given acupuncture treatment 30 min after injection of synthetic pentapeptide GRGDY
(glycyl-arginyl-glycyl-aspartyl-tyrosine, Gly-Arg-Aly-Asp-Tyr) into "Housanli" area, basal pain
threshold group, normal saline group which was given injection of 0.9% NaCl into "Housanli"
area and acupuncture group which was given acupuncture at "Housanli" were used as controls.
Results: As compared with acupuncture group, the temperature at which rat flicked its tail did
not significantly changes following acupuncture 30 min after injection of GRGDY. Conclusion:
GRGDY could not prevent the acupuncture effect, and the initial mechanism of acupuncture
effect might have no relation to the structural site that extracellular matrix combines with
integrin specially
Key Words Point, ST 36; RGD; Extracellular matrix; Integrin
CLC Number: R2.03
Document Code: A

It is still unclear that how mechanical signal

produced by acupuncture in acupoint region changes
into the effective biological signal to regulate the

Author: ZHANG Zhi-ying(1963-), female, associate professor,
Ph,D, mainly engaged in study on mechanism of acupuncture
effect, E-mail: zhiying1963@163.com
Foundation project: Financially supported by Shanghai
development fund of science and technology ( 04DZ19839 ) and
national key basic research development plan (2005CB523306 )

corresponding functions of viscera and organ.
Langevin et al

[1]

believed that in the process of

twirling, rotating, lifting and thrusting needle, the
collagenous fibers and elastic fibers in acupoint
region were twined and tightened up to cause the
deformation of extracellular matrix and activation of
fibroblast. The experiment in vitro proved that
structural site of extracellular matrix protein could
bind with integrin belonging to the cell surface
receptor family to form the focal adhesion plaques

Journal of Acupuncture and Tuina Science, Volume 4, Number 6, 2006 December

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326

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(FAP), then to regulate intracellular Ca

concentration and activate phospholipase, protein
kinae and lipid kinase, further to result in a series of
signal transmissions

[2,3]

. In order to discover whether

the mechanical coupling of acupuncture and
extracellular matrix can trigger the combination of
structural site of extracellular matrix protein and
integrin, so as to bring about a series of acupuncture
effects, the authors injected synthetic pentapeptide
GRGDY (Gly-Arg-Aly-Asp-Tyr) into "Housanli" area
of rat to bind with integrin on cell membrane and then
performed acupuncture at "Housanli" to observe the
change of acupuncture effect, so as to find out the
initial mechanism of acupuncture signal transmission.

Materials and Methods

1. Experimental animals

SD rats, weighting between 200 g and 300 g, half

male and half female, with the rats that were specially
sensitive to pain or not specially sensitive to pain
sifted out through the heat radiation tail flick test,
were divided randomly into basal pain threshold
group(Group A), acupuncture group(Group B),
normal saline group(Group C) and experiment group
(Group D), with 10 rats in each group. For Group A,
only rat's basal pain thresholds was measured; For
Group B, acupuncture was performed at "Housanli"
of rat; For Group C, 20 µL of 0.9% NaCl was injected
into "Housanli" area of rat; For Group D, 20 µL of
0.1% GRGDY(molecular weight 792.88, Shanghai
Bio-engineering Ltd., Co.) was injected into rat's
"Housanli" area and then acupuncture was performed
at "Housanli".

Heat radiation tail flick test

The Model IITC 336 Paw/Tail Stimulator

Analgesia Meter of IITC Life Science Company of
U.S.A. was adopted to measure rat's pain threshold.
The rat's tail was placed over the tail flick panel, and
the site 1.5 cm above each rat's tail tip was set up as
the radiated spot, tail temperature at which the test is
to be started (starting temperature) was set at

degrees Celsius, when the rat was quiet, pressed the
heat button to heat to a certain degrees, and after the
rat flicked its tail, heating was automatically stopped,
and the tail temperature was automatically presented
on a digital display. For basal pain threshold group,
each rat was tested for 3 times, and the average was

taken as basal pain threshold.

3. Acupuncture methods

"Housanli" was located at 0.84 mm below and 3.35

mm anterior to the head of the fibula, after routine
disinfection, the filiform needle 13 mm in length was
obliquely inserted into "Housanli" at an angle of 45
degrees, when the tense and distending feeling
appeared under the needle (arrival of qi), the needle
was twirled, rotated, lifted and thrust for many times,
and then retained. The pain threshold at 10 min, 20
min and 30 min after acupuncture was measured.

4. "Housanli" injection method

20 µL of 0.1% GRGDY or 20 µL of 0.9% NaCl

was injected into rat's "Housanli" area with 20 µL-
micro-injector, then the syringe needle was retained
for 1min, during which the injected local region was
slightly pressed to make the GRGDY or NaCl
distribute evenly.

5. Statistical method

The data were analyzed with one-factor analysis of

variance by SPSS 11.0 statistic software, and then
compared between any two groups.

Results

Acupuncture could enhance the pain threshold, and

the acupuncture analgesic effect was most obvious at
20 min after acupuncture (Table 1). After injection of
20 µL of 0.9% NaCl into "Housanli" area, the rat's
pain threshold was enhanced, which lasted for 30 min,
then it gradually returned to the basal pain threshold
(Table 1). After injection of 20 µL of 0.1% GRGDY
into rat's "Housanli" area, the rat was given
acupuncture at "Housanli" area, the results showed
that there was significant difference in the rat's pain
threshold as compared with normal saline group, and
no significant difference was found as compared with
acupuncture group (Table 1).

Table 1. Effect of different managements on the rat's tail

temperature in heat radiation tail flick test (x¯ ±s， )

℃

Groups N

10 min

20 min

30 min

A 10

35.96±2.49

B 10

59.47±4.81

62.70±4.15

47.84±5.48

C 10

40.44±3.82

39.48±2.89

39.81±2.55

D 10

61.49±4.23

1)2)

65.23±5.44

1)2)

57.83±4.03

1)2)

Notes: in comparison with basal pain threshold group, 1)

P<0.05; in comparison with normal saline group, 2) P<0.05

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Discussion

The rat's "Housanli" area corresponds to the

acupoint Zusanli (ST 36) in human being,
acupuncture at acupoint Zusanli (ST 36) has a
remarkable analgesic effect, the present study was to
further prove the analgesic effect of acupuncture at
rat's "Housanli" area by the heat radiation tail flick
test, and the acupuncture analgesic effect was the
most obvious at 20 min after acupuncture, indicating
that the duration of acupuncture treatment should not
exceed 20 min in clinic, otherwise, the stimulation-
induced adaptation phenomenon may appear, and the
acupuncture effect will reduce.

After injection of 0.9% NaCl into "Housanli" area,

the rat's pain threshold was enhanced, which lasted for
30 min, suggesting that single needling stimulation
and the injected liquid also had the acupuncture-like
effect. Acupoint-injection therapy is to combine the
pharmacological action of drug and the physical
action of liquid. In present study, the normal saline
group was used as control to preclude the actions of
syringe needle and injected liquid.

In the acupuncture treatment, the most important

characteristic reaction is "arrival of qi" or "needling
sensation". The arrival of qi refers to soreness,
numbness, distending or heaviness feeling around the
acupoint after the needle is inserted into a certain
depth, or their transmission upward and downward
along the meridians; at the same time, the operator
may feel tenseness or unevenness around the needle,
which seems "a fish bites on fishing pulling the line
downward". But how this local signal of "arrival of
qi" or "needling sensation" changes into the effective
biological signal to transmit? The research from
Langevin et al showed that in the process of twirling,
rotating, lifting and thrusting needle, the collagenous
fibers and elastic fibers in acupoint region were
twined and tightened up, then the needle and tissues
formed the mechanical coupling

[1,5]

. The twined

collagenous fibers may lead to the cell adhesion,
consequently, the glucoprotein of extracellular matrix
combined with integrin to link with cytoskeleton in
cell, triggering a series of phosphorylations of protein
complex, so as to further activate the intracellular and
extracellular signal molecules to transmit the effective

biological signal. On the structure of extracellular
matrix protein molecule, some different structural
sites that can combine with integrin have been
identified, the glucoprotein structure of extracellular
matrix contains RGD tripeptide sequential structure
which is the main structural site that extracellular
matrix adheres to cell. In the present study, the
synthetic pentapeptide GRGDY was injected into the
rat's "Housanli" area, 30 min later, "Housanli" area
was given acupuncture, the results showed that there
was no significant difference in acupuncture analgesic
effect between injection of GRGDY plus acupuncture
group and acupuncture group, indicating that
GRGDY could not prevent the acupuncture effect in
the acupoint region. We could not achieve the
expected effect, because: The mechanical signal
produced by acupuncture in acupoint region might not
transmit via combination of glucoprotein RGD
sequence of extracellular matrix and integrin; and Due
to the complexity of acupoint structure in vivo, the
synthetic GRGDY can not simply combine with
integrin to prevent acupuncture effect. The present
results showed that acupuncture analgesic effect was
slightly better in injection of GRGDY plus
acupuncture group (experiment group) than in
acupuncture group, and lasted for a longer time, this
might be related to the double action of syringe needle
and pentapeptide GRGDY liquid.

References

[1] Langevin HM, Churchill DL, Wu J, et al. Evidence of

Connective Tissue Involvement in Acupuncture. FASEB J,
2002, 16(8): 872-874.

[2] Chiquet M, Renedo AS, Huber F, et al. How do Fibroblasts

Translate Mechanical Signals into Changes in Extracellular
Matrix Production? Matrix Biol, 2003, 22(1): 73-80.

[3] DeMali KA, Wennerberg K, Burridge K. Integrin Signaling

to the Actin Cytoskeleton. Curr Opin Cell Biol, 2003,15(5):
572-582.

[4] LIN Wen-zhu. Science of Acupuncture and Moxibustion.

Shanghai: Shanghai Science and Technology Publishing
House, 1994:327.

[5] Langevin HM, Churchill DL, Cipolla MJ. Mechanical

Signaling Through Connective Tissue: a Mechanism for the
Therapeutic Effect of Acupuncture. FASEB J, 2001,15(12):
2275-2282.

Translator: ZHU Zhong-chun (朱忠春)

Received Date: February. 20, 2006