Journal of Sasang Constitutional Medicine

(1)

사상체질의학회지

J. ^of ^Sasang ^Canst. ^M^뼈.

Vol. 13. No 1. 2001

Studies on the Cytokine Production Regulation in Human

Astrocytes by Yuldahansotang

Choi Ji-sook* . ^Kim ^Kyoung-yo* . ^Kim Hyoung-min- . ^Ju ^‘^Jong-chon*

| ^{초 혹} l

A 間腦星狀細뼈에서 熱多寒少漫에 의한 細뼈活性物質生빼 調節에 關한 iff ^究

최지숙,* . 김경요* . _{김형민 **} . ^주종전

원광대학교한의과대학 사상체질과

.. 원광대학교 한의과대학

사상의학적 견지에서 太陰A의 중풍 , 치매와 같은 신경계질환에 다용되고 있는 熱多寒少陽은 최근에 그 엄

상적 효과를 뒷받청할 다각적인 연구들이 이루어지고 있음에도 불구하고 그 정확한 약리학적 기전에 대해서 는 밝혀지지 않고 있다.

본 연구에서는 인간성상세포를 이용하여 熱多寒少楊이 substance P (SP) 와 lipopolysaccharide αPS) 에 의해 유

도되는 다양한 세포활성물질의 분비량의 조절올 검토함으로써 熱多寒少陽의 약리기전을 연역학적 측면에서

보다 세밀하게 살펴보고자 하였다 .

熱多寒少楊 수침액은 인간 뇌 성상세포로 부터 LPS 와 SP 의 동시자극에 의해 생성되는 세포활성물질중

interleukin (IL)-I, IL-4, IL-6 및 tumor necrosis faccor-a (TNF- 미의 분비를 농도의존적으로 억제했다 . 그러나

interferon- r (lFN- r) 및 IL-2 의 분비 조절에는 영향을 미치지 않았다

그리고 항 IL-I P 항체에 의해 SP 유도성 TNF-a 분비의 증가가 억제되기 때문에 IL-I 은 TNF-a 증가를 매개 하는 역할을 하는 것으로 사료된다.

이상의 결과는 熱多寒少陽에 의한 급성기 중풍환자 치료 효과가 뇌 성상세포로부터 분비되는 세포활성물

질의 조절과 밀접한 관련성이 있다는 것올 암시하고 있다.

Keywords: Yulda-Hanso-Tang, Ascrocytes, Tumor necrosis faccor-a, Substance P, Lipopolysaccharide, Interleukin-I

I. INTRODUCTION constitutions inco four cy야s, according co the strengths

and weaknesses in funccions of the internal organs. They

are Taeyangin, Tae 띠run Soyangin 뼈d Soumin. Sasang

The Sasang Constitutional medicine class 퍼g

야ople

’s

. ^Dept. ^of ^Sasang ConstitUtional m뼈마Ie, College of Oriental M 때icine, Wonkwang Univ.

.. ^College ^of ^Oriental ^M벼icine, Wonkwang Univ

교신저자 최지숙 주소) 광주광역시 북구 일곡용49 천지인한의원 천화)062-575-4333 E-mail)ANAlOOO@ch 빼^n.net

(2)

- ^{사상책짙의학회지 찌}13권 쩌 1 호때1 -

Constitutional philosophy forms the basis of treatment

by correcting the imbalance of the internal organs

caused by the constitutional properties in each body

type. For example, Taeumin is thought to have a higher

rate of stroke, hypertension 뻐d hyperlipidemia than the

other types because he or she has a large liver and

small lungs. Thus, a pe πon of this body type has a

weak dispersing qi and excessive gathering qi I.²⁾

From the viewp 이 nt of the Sasang Constitutional

Medicine, Y띠dahansotang αH-Tang) is a prescription

which onen has been 따ed clinically for neurological

diseases, such as Taeumin's stroke and dementia

Recently, many researches support the clinical effect of

3-^’). ^I _I'

YH-TangJ'''. Among them, Choj"' has reported that the

serum level of cytokine was regulated by YH-Tang in

an acute cerebral infarction (0) of Taeumin.

Astrocytes, one type of the neurological cells, have an

important role in maintaining central ne πous system

(CNS) homeostasis. To carty out homeostatic function,

astrocytes synthesize various immune-mediated cytokines

and interact with those substancel-IO). It is known that

cycokines are involved in various neuropathological

diseases, such as Alzheimer ^’s disease, multiple sclerosis,

and acquired immunodeficiency syndrome (AIDS) ^11-13)

Also the change of the s야cific cytokine level was

reported in an acute 0 patientI4). Astrocytes were

induced to secrete cytokines after interacting with

lipopolysaccharide αPS) or virus.

Substance P (SP) is a neurotransmitter and nerve-

originating chemical that mediates neurogenic infl 입n-

’. ¹⁶⁾

mation u. ,u'. In addition, SP stimulaces che production

of inflammatoty cycokines, such as cumor necrosis

factor-Q (TNF-Q), interleukin (IL)-117. ¹⁸⁾ and IL-617), and

affects che number of SP receptors when che CNS ^잉

injured IW"'. SP 얀 widely distribuced in che CNS and is

believed co scimulate cytokines to influence che

pachological process in the CNS I

In chis study, the reg 띠 acing effect of YH-Tang on

cycokine secretion induced by LPS and SP in astr εyces

was invescigated. An experiment on che regulating effect

of YH Tang on cycokine secretion in astrocytes was

conduCted co determine how the reg 띠 acing mechanism

providechebasisforcheclinicalcherapeuciceffeCton

YH-Tanginacuce0 patients

II. 써ATER 뻐A 해D METHODS

1. Materials 1) Reagents

Fetal bovine serum (FBS), SP, LPS,

penicillin/screpcomycin, 때d cween-20 were obcained

from Sigma Chemical Co. (Chicago, IL). Cycokines, such

as IFN-r, 1L-1, 1L-2, ι,-4, and 1L-6 were obcained

from R & D systems (Minneapolis, MN). Human

recombinanc TNF- a (rTNF- a), polyclonal anti-mouse

止-I a and anci-human TNF- a anci 야dy were obcained

from Genzyme (Cambridge, MA). Dulbecco ^’s m 여ified

Eagle ^’s me φum (DMEM) was obtained from Life

Technologies (Grand Island, NY). Enzyme-linked

immunosorbent assay (EUSA) places were obcained from

Nunc (Baperville, IL).

2) Cell line

Human 싫trocytes, CCF-STIG 1 cells were used

3) Preparation of YH- Tang

The plant sample was obtained from che Kwangju

Oriencal Medicine Hospital of Wonkwang University.

A prescripcion of YH-Tang weighs 48g, consiscing of

16 g of Puerariae Radix (Ge Gen), 8g of Scucellariae

R찌Dc (Huang Qin), 8g of Ligustici Tenuissimae Radix

(Gao Ben), 4g of Raphani Semen (Lai Fu Zi), 4g of

Angelicae Dahuricae Radix (Bai Zhi), 4g of Cimicifugae

Rhizoma (Sheng Ma), and 4g of Platycodi Radix Oil'

Geng). A water excract of YH-Tang was prepared by

de α>cting che prescripcion of dried herbs in discilled hot

wacer. The excract was mtered through a 0.45 _띠nfih«

and freeze-dried.

The YH -Tang was preserved ac 4"C ^and ^used ⁱⁿ ^this

study. The yield of che extracc was abouc 10% (w/w).

(3)

- 최지숙 외 3: A뻐g

훌狀뼈뼈에서 多흉少홉에 의한JlllII 홉↑

*^빼^it ^호 ^해^II^{애 뻐한} ^6]11,: ^-

2. Methods

1) Astrocytes were cultured at 4 X 105 per well

and were grown for 3 days with DMEM containing

10% FBS in C02 inαbator.

2) SP preparation:

Special care was taken with SP to avoid possible iPS

contamination. Fiπt, peptide SP was dissolved in 0.01 %

acetic acid. Acetic acid was made of 1/1ááoo glacial

acetic acid and f1ltered through 2.2 _띠n f1lter. SP stock

solution was kept in a refrigerator at -20'C after being

diluted in iPS non-contaminated dist 피ed water

immediately before use.

3) Measurement of cytokines :

Assays of cycokines were performed under modified

EUSA according to the procedure outlined by Scuderi et

al20) ηlat is, anti-cytokine monoclonal antibody was ^‘

treated with a coating buffer solution [0.02% s여ium

azide contained phosphate buffered saline (PBS) pH = 7.

2) on flat-bottomed 9ιwell plate (Coming, Rochester,

NY) at a final concentration of 6.25 ng in each well,

and coated at 4'C ^for ¹² ^h. ^After ^coating, ^to ^reduce

non-spec 퍼c binding, blocking buffered solution, PBS

containing 2% BSA, was added and coated at 37

'C ^for

2 h. Each well was added to 100 μ of recombinant

cytokine standard solution and su 야matant of each

specimen, and after washing 4 띠les with wash buffer

(PBS containing 0.05% Tween 20), were subsequently

incubated at 37

'C ^for ^{2 h.}

The wells were then washed 4 times again with PBS

containing 0.05% rween 20. The wells were treated

with anti-cytokine antibody which had been diluted by

PBS containing 0.05% rween 20, and again incubated

at 37

'C ^for ^{2 h.} ^After ^washing ^repeatedly ^{7 times} ^with

a wash buffer, each well was treated with 100 ng/mL of

phosphatase-bound anti IgG antibody (Sigma Co.) and

incubated at 37.C for 2 h, then washed 7 t띠les agam.

After the last washing step, 100 때 of p-nirro phenyl

phosphate was added to each well, which was 버ssolved

in buffer solution of the mixture of 0.05M N 상K03

and 0.05mM MgCI2. Ten min after the color changed,

the absorbance of each cycokine was measured at 405

run wave-length in the EUSA reader. Appropriate

specificity controls were included.

3. Statistical analysis

D염^{a are} ^given ^as ^means ^:!:: ^S.E. Statistical 뻐혀ys 잉

was performed with Student ^’s t-test. Results with

P<0.05 were considered statistically significant.

삐 . RESULTS

1. The effect of YH-Tang on LPS and SP

induced in’erferon- r (lFN- r) sec-

retion in as ’rocytes

To evaluate the effect of YH-Tang on IFN- r

secretion in 잃trαγtes, the amount of IFN- r ^secretion

was measured after 18 h of incubation of astrocyres

which was added by iPS and SP at ^’ various

concentration. As shown in Figure I, YH-Tang did not

have an effea on iPS and SP induced IFN- r ^secretion

m 싫trocytes.

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사캠 h ^버니

l

o 10 100 1000

YH-Tang (

μg/ml)

Fig. 1. Effect 이 YH- Tang on LPS and SP induced IFN-r

secretion in astr< α;ytes. The cells (4 x 105 cel 때ml)

were incubated for 24 h in medium ∞ntaining LPS

(1 _μwml) plus SP (2 μ잉ml) with various con-

centrations of YH- Tang and the supernatants were

∞lIected and frozen at -8(re until assayed for IFN-

r. Each datum value indicates the mean :t S.E.

of six separated experiments.

*: statistically significant differences from the control values at P <

0.05

(4)

- 사상채짙의학회지 제13권 제 1 효 2X)\ -

2. The effect of YH- Tang on ^LPS and SP

induced IL -1 secretion in astrocytes

The analysis of the effeer of YH-Tang on U엄 and

SP induced IL-l secretion in astrocytes showed that

YH-Tang decreased IL-l concentration dependently, as

shown in Figur ξ 2. The εffeer of YH-Tang was

significant at a concentration of 100-1000 μpjrnLe

0.55

0.50

“”찌

%

nununu

(-FISt)

ζ。

;@」

Q@m

‘-

닐 0.30

0.25

o ¹ 10 100 1000

YH-Tang (

따잉ml)

Fig. 2. Effect 이 YH- Tang on LPS and SP induced IL-1

않cretion in astrl α:ytes. The ∞lis (4 x 105 ∞II 의ml)

(1 뼈ml) plus SP (2 빼ml) with various ∞ncen-

trations of YH- Tang and the supematants were

∞lIected and frozen at -BOt until assayed for

IL -1. Each datum value indicates the mean :.t S.E.

of' six separated experiments

*; sratisrically significanc differences from rhe concrol values at P <

0.05.

3. The effect of YH-Tang on ^LPS and SP

induced IL-2 secretion in astrocytes

A5 shown in Figure 3. 1L-2 secretion was not

increased significantly and the regulatory effeer of

YH- Tang on 1L-2 secretion was not significant.

0.50

“”애

%%

nunununu

=EBI)c。

;eu잉”

N」-

t-Y

0.25

o 10 100 1000

YH-Tang (vg/ml)

Fig. 3. Effect 이 YH- Tang on LPS and SP induced IL-2

secretion In astr ∞ytes. The ∞lis (4 x 105 ∞II 잉ml)

(1 μ잉ml) plus SP (2 μ밍'ml) with various ∞n ∞n-

!rations 이 YH- Tang and the supematants were

∞lIected and froze.n at -BOt until assayed for

IL-2. Each datum value indicates the mean :.t S.E.

이 six separated experiments.

*; ^sta 뼈에Iy 맹^1ificanr diffcrcnccs from the conad 때‘JfS at P < 0.05.

induced IL-4 secra ’ion in astrocytes

The effect of YH-Tang on LPS and SP i뼈따ed 1L-4

secretion 띠 astrocytes was evaluated. As shown in Figure 4,

YH-Tang decreased 1L-4 concentration dependently.

0.46

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u!unu

”

u

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u

”

u

=E‘

@C)=。

;p8$*」

-

0.30

o 10 100 1000

YH-Tang (

μg/ml)

Fig. 4. Effect 이 YH- Tang on LPS and SP indu α퍼 IL-4

secretion in astr, ∞ytes. The ∞|_잉 (4 x 105 ∞II 잉ml)

(1 _μml) plus SP (2 μ밍ml) with various ∞ncen-

!rations of YH- Tang and the supematants were

IL-4. Ea 어1 datum value indicates the mean :.t S.E.

이 SIX se 며rated experiments.

*; ^stat ^‘^srically sisnificanc differences from rhe concrol v꾀ucs at

P < 0.05.

(5)

- 최지숙 외 3: A뻐뼈훌狀뼈뼈애서 !t 少훌에 의한IIII 효양. ^호 여WI 에 뻐한 iiI'9.: -

0.55 0.60

0.50

0.45

0.40

0^‘35

-E~aZ)t。

;밑

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induced IL-6 secretion in astrocytes

The effect of YH-Tang on LPS and SP induced IL-6

secretion in ascrocytes was evaluated. As shown in

Figure 5, YH-Tang decreased 1L-6

dependently.

concentration

0.55

0.50 0.30

1000 100

o 10 0.45

YH-Tang (ng/ml)

F밍. 6. Effect 이 YH- Tang on LPS and SP induced TNF- a

똥crehon in astr α:;ytes. The ∞lis (4 x 105 cell 의mO

were inωbated for 24 h in medium ∞ntaining LPS (1 μ밍ml) plus SP (2 _μwml) 에th various ∞ncen-

TNF- a. Each datum value indicates the mean ::t

S.E. of six separated experiments.

1000 100 1 10

o

0.40

0.35

0.30

0.25

(-ESZ)t。

;@』

Q%

얘」

-

0.05

7. The effect of anti IL-1 β antibody on

LPS and SP induced TNF- a secretion

in astrocytes

To ev외uate whether YH- Tang ^’s inhibirory effect on

TNF- a secretion was mediated via IL-I, the effect of

anti IL-I /3 antilx 삐y was as양ssed. After LPS( I_μglmL)

and SPwere introduced ro an 양trocyte incubating

solution, anti IL-l /3 antibody was added and the

amount of TNF- a secretion was me 싫따ed after 24 h.

As shown in Figure 7, TNF- a concentration decreased

dependently in the anti IL-I /3 anti φdy treated group.

The inhibitory effect of the anti IL-I /3 antibody was

significant at a concentration of 1-100 뼈/mL

Fig. 5. Effect 이 YH- Tang on LPS and SP indu ∞d IL-경

secretion in astr ∞ytes. The ∞lis (4 x 105 ∞II 잉ml)

(1 μ밍ml) plus SP (2 μ잉ml) with various ∞ncen-

∞lIected and frozen at -BOt until assayed for IL용. Each datum value indicates the mean ::t S.E.

of six separated experiments.

YH-Tang (_μg/ml)

0.05.

induced TNF- a secretion in astro-

cytes

The effect of YH-Tang on LPS and SP induced

TNF- a secretion in astrocytes was evaluated. TNF- a

is an important inflammatory cyrokine. TNF- a sec-

retion by ascrocytes is induced by LPS and SP. As

shown in Figure 6, YH-Tang decreased TNF- a concen-

tration dependently

(6)

- 사상쩨짙의학회지 째13권 채’효 ::m1 -

0.60

%%

“””

%%

n“

nunununu (-E~@C)

ζ。

;@』

U@maι

ZLF

0.30

o 0.1 10 100

IL-1 II antibody (ng/ml)

Fig. 7. EHect of anti IL-1 β anti α잉y on LPS and SP

induced TNF-a 똥cretion in astr αγtes. The ∞lis

(4 x 105 cell 의ml) were incubated for 24 h in

medium ∞ntainlng LPS (1 μ잉ml) plus SP (2 μg/ml)

with various ∞ncentrations 이anti IL-1 β antitx: 찌.

The supematants were ∞lIected and frozen at -8

O.C until assayed for TNF-a. Each datum value

indicates the mean :t S.E. 이 four separated

experiments.

*: statistically significant differences from the concrol values at P <

0.05.

N. DISCUSSION

In this study, the regulatory effect of YH-Tang on

LPS and SP induced cytokine secretion in astrocytes was

investigated. It has been reported that asrrocytes in

CNS pathophysiologically are likely to involve in

cytokines, both as stimulators and mediatots of asrrocyre

10)

function ,u', with which CNS homeostasis is maintianed.

LPS controls many biological properties of gtam-

negative bacteria. A serological spec 퍼ciry exists in the

part of varied polysaccaride and this part belongs to

O-antigen of gram-negative bacteria. O-antigen displays

a specific receptor of bacteriophage. LPS controls

endotoxic acciviry. If ba αeria loses its ability to

synthesize LPS, the pathogenicity of bacteria is often

lose. The properties of endotoxin include pyrogenicity,

leth 외ity, complement activity, B-cell mitogen activity in

the mouse, adjuvant acitivity, anti-rumor activity, etc2l).

SP has been reported to induce inflammatory

cytokine production in human neuroglial cells and

peripher 쇠 lymphoid cells as well 22)

It was noted thar YH-Tang inhibited the secretion of

IL-I, ι4, IL-6, and TNF- a significantly among the

various cyrokines produced by simultaneous stimulation

of LPS and SP in asrrocyres. The effect of YH-Tang

w^’as concentration-dependem. It was derermined that

TNF- a and IL-I secretion in asrrocyres needed LPS

stimulation, and LPS stimulation was augmented by SP.

Because anti-IL-I p antibody inhibited the secretion of

SP-induced TNF- a, IL-l was considered as a mediator

of TNF- a incre 앓. These results are the evidence thar

SP, a neurotransmitter produced in the ne πe of the

CNS, is an important molecule involved in i바la-

mmarion. IL-l is an endogenous pyrogen, the activating

factor of lymph αyre products. It is made in many cells,

especially macrophage cells, where it is made most. IL-l

activates T-cell 뻐d B-cell, 뼈d causes the inflammatory

response. In the brain, IL-l causes heat and increases

the secretion of corticosteroid 23)

According 【o T ouzani et aI^’s recenrrepor(24), IL-I has

an important role in the development of brain damage

following cerebral ischemia. The expression of IL-l in

the brain is dramatically increased during the early and

chronic stages of infarction. The most direct evidence

that IL-I conrributes significantly to ischemic injury is

that (I) cenrral administration of IL-I p exacerbates

brain damage, and (2) injection or over- expr δsion of

IL-l receptor antagonist, and blockade of IL-I P

converting enzyme activity dramatically reduce infarction

and improve behavioral deficit. The mechanisms

underlying IL-I actions in srroke have not been clearly

리 ucidated, and it seems likely that its effects are

mediated through stimulation and inhibition of a wide

range of pathophysiological processes.

2))

According to Yamasaki et aI^’s report"', the conrribution

of cytokines in an inflarrunatory cascade on cerebral

reperfusion injury are characterized as consisting of

typical phases; leukocytes invasion, microglial activation

and remodeling. Within 1-2 days, IL-I and TNF- a

induce the expression of adhesion molecules that cause

leukocytes to adhere to endothelial cells.

Journal of Sasang Constitutional Medicine

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사캠 h ^버니