30-2 06 [63-78]jkom.org/upload/30-2 06 [63-78].pdf · ‘BY-500’, was treated with cBSA (9 / i.p) and BYMSB extract (500 / , p.o). After cBSA and BYMSB extract treatment for 4 weeks,

63

서 론

(membranous nephropathy, MN)

, ,

, 1-4).

補陽膜性方

임범수, 조충식, 김철중

대전대학교 한의과대학 신계내과학 교실

Original Article

접수：2008년 10월 2일 수정：2009년 1월 17일 채택：2009년 2월 4일

교신저자：김철중(Cheol-Jung Kim)

대전광역시 중구 대흥동 대전대학교 부속대전한방병원 신계내과

Tel：+82-42-229-6880, Fax：+82-42-254-3403, E-mail：[email protected]

Effects of Boyangmaksung-bang (BYMSB) on the Membranous Nephropathy Induced by Cationic Bovine Serum Albumin in Mouse Model

Bum-Soo Lim, Chung-Sik Cho, Cheol-Jung Kim

Department of Internal Medicine, College of Oriental Medicine, Daejeon University, Daejon, Korea.

Objective: The purpose of experimental study was to prove the effects of Boyangmakseong-bang (BYMSB) treatment on cBSA-induced in a MN mouse model.Methods: We divided mice into 4 groups. The Normal group had no treatment. We used cBSA and induced MN mouse model to the other 3 groups. The Control group was treated with cBSA (9 / i.p) only. The second group, named ‘BY-250’, was treated with cBSA (9 / i.p) and BYMSB extract (250 / , p.o). The third group, named ‘BY-500’, was treated with cBSA (9 / i.p) and BYMSB extract (500 / , p.o). After cBSA and BYMSB extract treatment for 4 weeks, the increase in percentage of body weight, proteinuria, serum albumin, total cholesterol, creatinine and BUN of all groups were measured. The CD3+, CD19+, CD4+, CD8+ cell levels of spleen of all groups were analyzed. IgA, IgG, IgM, IL-1β, TNF-α, IL-6 and IFN-γ levels of all groups were gauged. H&E staining, immunofluorescence staining and electron microscopy of kidney were observed.Results: BYMSB showed significant decrease in the 24hrs proteinuria, serum total cholesterol, serum IgG levels and BUN levels, and showed significant increase in the serum albumin levels compared with the control group. BYMSB showed increase in the increasing percentage of body weight and IFN-γ levels compared with the control. BYMSB showed decrease in the CD3+ T cells, CD4+ Th cells, IL-1β, TNF-α and IL-6 levels, but did not show significant change compared with the control. BYMSB showed considerable decrease in the thickening of the GBM on H&E staining, deposition of IgG on immunofluorescence staining and deposition of electron-density on electron microscopy of kidney compared with the control.Conclusions: According to the above results, it is suggested that BYMSB decreases the symptoms of MN induced by cBSA in a mouse model. Therefore BYMSB seems to be applicable to MN in clinical practice.

Key Words : Boyangmakseong-bang (BuYangMoXing-fang), membranous nephropathy (MN), cationic bovine serum albumin (cBSA)

30 2 (2009 3 )J Korean Oriental Med 2009;30(2):63-78

30 2 (2009 3 )

64

, IgG

(glomerular basement

membrane, GBM) ,

5-10).

, IgG C3

, 5,11-15).

MN 20~40%

,

steroid 3~6

,

14,16-22).

MN ‘ ’, ‘ ’, ‘ ’, ‘

’ 2,3,23,24), , , 25,26), , ,

, , ,

, , , 2,23,24).

MN 24,27) , ,

, 28)

. 29,30)

29), 30)

MN

.28) 31)

, ,

(BYMSB)

, cationic bovine serum albumin

(cBSA) MN mouse , 24

,

.

실 험

1. 재료

1)

mouse ( ) 8

ICR female 1

.

,

22.1% , 8.0% ,

5.0% , 8.0% , 0.6% ,

Herbs Scientific Name Galenical Name Amount(g)

黨蔘 Codonopsis Pilosulae Radix Codonopsis pilosula(FR.) NANNF. 30.0白花蛇舌草 Oldenlandiae Diffusae Herba Oldenlandia Diffusa(WILLD.) ROXB. 30.0薏苡仁 Coicis Semen Coix lachryma-jobi var. mayuen(ROMAN.) STAPF 30.0黃 Astragali Radix Astragalus membranaceus BUNGE 60.0當歸 Angelicae Gigantis Radix Angelica gigas NAKAI 20.0淫羊藿 Epimedii Herba Epimedium koreanum NAKAI 15.0巴戟天 Morindae Officinalis Radix Morinda officinalis HOW. 15.0破古紙 Psoraleae Fructus Psoralea corylifolia L. 12.0鹿茸 Cervi Pantotrichum Cornu Cervus nippon TEMMINCK 9.0

total amount 221.0

Table 1.

(236)

2 : Cationic Bovine Serum Albumin Mouse Membranous Nephropathy

65

0.4% , (

Co.) . 22±2

1 12 200-300 Lux

, 12 conventional

system .

2)

,

(Table 1).

3)

(1)

bovine serum albumin(BSA),

anhydrous ethylenediamine(EDA), 1-ethyl-3{(3-dim-

ethylaminopropyl)-carbodiimide hydrochloride}(EDC),

HCl, complete Freund's adjuvant, chloroform, gluta-

ldehyde, glycine, NP-40, ethanol, hematoxylin, eosin,

paraformaldehyde, triton X-100, RPMI-1640 media,

phosphate buffered saline(PBS), RBC lysis buffer

, 10% buffered formalin Sigma(U.S.A.)

, fetal bovine serum(FBS), horse serum,

Hoechst Hyclone(U.S.A.) .

(2)

BÜCHI

rotavapor R-200 BÜCHI heating bath B-

490 (Flawil, Switzerland) . cBSA

isoelectric point(pI) Bio-

Rad(U.S.A.) ,

cBSA Bio-Rad(U.S.A.)

5% SDS-PAGE

. (proteinuria) Bayer(Germany)

Albustix , cytometer FACSCalibur(Becton

Dikinson, U.S.A), ELISA Molecular Devices

(Minnesota, U.S.A.) .

Serotec(U.S.A.) cooling

microtome(Washington DC, U.S.A.), metabolic cage

Techniplast(Varese, Italy) .

2. 방법

1)

2,000

3

(rotary vacum evaporator)

, (freeze dryer) 26g

( : 12%)

.

2) Antigen, cationic BSA

Cationic BSA(cBSA, Sigma Chemical Co., USA)

Border BSA

. 20g BSA 12

, 20 2.68

EDA HCl pH 4.75 anhydrous

EDA 25 . EDA solution

BSA solution ,

1-ethyl-3-{(3-dimethylaminopropyl)-carbodi-

imide hydrochloride} (EDC) 150 4

. 1 EconoPac

High S cationic ion-exchange column(Bio-Rad, CA)

pH 6.8 phosphate buffer(40mM) washing

BSA pH

10.6 phosphate buffer(40mM) elution . 5%

thin-layer polyacrylamide gel cBSA

control BSA loading (Bio-

Rad, USA) cBSA isoelectric point(pI)

. BCATM protein assay

kit (Pierce, USA) spectrophotometer 540nm

OD .

3) MN model

8 female ICR mouse (Normal),

(Control), BY-250 (BY-250), BY-500 (BY

-500) 4 3

complete Freund's adjuvant 0.2 cBSA

(intra-peritoneal injection) 2

(immunization) . antigen saline

(237)

30 2 (2009 3 )

66

.

2 4 9 / cBSA

3

MN BY-250

250 / , BY-500 500 /

MN

. 4 meta-

bolic cage 24

.

blood, kidney, spleen sample

.

4)

.

5)

Mouse urine metabolic cage

-20 Albustic(Bayer, Germany)

TBPB

albumin .

6)

ethyl ether

1

3,000rpm 10

-80 . creatinine, blood urea

nitrogen(BUN), albumin, total cholesterol sero-

logical analysis ELISA .

7)

frozen tissue embedding solution freezing

10% buffered formalin ,

glutaldehyde fixing .

8) Spleen lymphocytes

Mouse spleen

cell cell strainer(Falcon, USA)

, red blood

cell(RBC) lysis buffer(8.3g NH4Cl, 1g KHCO3,

0.1mM EDTA in 1L of demineralized water)

5 RBC PBS trypan

blue . Splenocyte

1×106 PE FITC conjugated

anti-mouse CD3+, CD4+, CD8+, CD19+ antibody

0.5% BSA in PBS washing flow

cytometry study . cell quest

program .

9) Enzyme-linked immuno-sorbent assay

(immunoglobulin, Ig) cytokines

IgA, IgM, IgG, IL-1β, IL-6,

IFN-γ, TNF-α ELISA .

antibody goat anti-mouse IgA, IgM, IgG, rat anti-

mouse IL-1β, IL-6, IFN-γ, rabbit anti-mouse TNF-α

, antibody coating 96-well plate

50 2

washing solution 3 washing strepvidin-

RP antibody TMB substrate

ELISA reader 450nm optical density

.

10) Hematoxylin & Eosin staining

Embedding solution freezing

rotary microtome 20 dissection

-80 .

rehydration hema-

toxylin 1 nuclear

washing . Eosin 1

cytoplasm

washing . 70%,

95%, 100% ethanol series

dehydration , xylen 1

mounting solution cover glass

.

(238)


67

11) Immunofluorescence staining

(immunofluorescence staining)

frozen section tissue

, slide glass 4% para-formald-

ehyde 1 fixing 3 5 washing

NP-40 45

, BSA blocking rat anti-mouse IgG,

anti-mouse CD54(ICAM-1), anti-mouse CD11a(LFA-1),

anti-mouse complement component C3 monoclonal

antibody 4 washing

solution 10 3 washing fluorescen

isothiocyanate(FITC) conjugated secondary anti

-body 4 . Washing solution 10

1 washing Hoechst solution nuclear

washing cover slip

.

12) Electron microscopy

3% glutaldehyde fixing kidney tissue Carson-

Milonig's solution washing 1% aqueous

osmium tetroxide 2 alcohol series

dehydration , epon resing

diamond knives ultrathin section uranyl

acetate lead citrate double staining

. tissue transmission electron microscope

.

13)

mean±standard deviation

Student's T-test

. (two-tail p value)

p

30 2 (2009 3 )

68

2. 24시간 단백뇨에 미치는 영향

(index of proteinuria) 0.5±0.2, 2.0

±0.6 , BY-250 1.0

±0.1(p


69

4) BUN

BUN 18.2±2.2( / )

, 21.4±1.1( / )

, BY-250 17.6±0.9( / )

(p

30 2 (2009 3 )

70

±0.5(%) , BY-250

14.7±2.7(%), BY-500 13.5±2.1(%)

(Fig. 8).

5. 혈청 IgA, IgM, IgG에 미치는 영향

1) IgA IgM

IgA 308.5±61.7( / )

, 463.1± 66.6( / )

, BY-250 379.6±82.4

( / ), BY-500 367.1±81.3( / )

.

IgM 115.0±21.1( / )

, 349.5±34.8( / )

, BY-250 282.2±70.3( /

), BY-500 239.2±32.7( / )

(Fig. 9).

2) IgG

IgG 308.5±28.4(ng/ )

, 454.6±21.3(ng/ )

, BY-250 428.0±21.1

(ng/ )(p


71

6. 혈청 IL-1β, TNF-α, IL-6, IFN-γ에

미치는 영향

IL-1β 112.6±15.0(pg/

) , 306.6±7.1(pg/ )

, BY-250 241.6±9.2(pg

/ ), BY-500 242.6±8.5(pg/ )

.

TNF-α 45.8±4.1 (pg/ ) ,

102.3±9.3(pg/ )

, BY-250 81.4±3.9(pg/ ), BY-

500 71.7± 8.8(pg/ )

(Fig. 11).

IL-6 36.9±5.4(pg/ )

, 55.0± 3.4(pg/ )

, BY-250 26.6±9.3(pg/

), BY-500 24.3±19.0(pg/ )

.

IFN-γ 271.8±17.5(pg/ ) ,

AAAA BBBB

CCCC DDDD

Fig. 13.

AAAA BBBB

CCCC DDDD

Fig. 14.

300

0

Normal Control BY-250 BY-500

200

IL-6

INF-r

150

250

50

Serum

cytokin

elevel (p

g/m

l) 100

Fig. 12. γ

(243)

30 2 (2009 3 )

72

201.0±22.4(pg/ )

, BY-250 236.0±15.1(pg/ ),

BY-500 216.8±33.0(pg/ )

(Fig. 12).

7. Morphology에 미치는 영향

1) H&E staining analysis

, cBSA

, BY-250

BY-500

(Fig. 13).

2) Immunofluorescence staining analysis

IgG ,

IgG , BY-250

BY-500

(Fig. 14).

3) Electron microscopy analysis

,

, BY- 250 BY-500

(Fig. 15).

고 찰

MN

1,19,20).

MN

,

penicillamine, gold, captopril drugs cancer,

hepatitis B C virus infections, mercury,

formaldehyde, gasoline , 1,14,15).

,

1,6,7,14,15).

- ,

,

, ,

cytokines reactive

oxygen species ,

10). MN CD4+ /CD8+

, T cell B cell ,

T cell Th2 cytokines

, B cell IgG32).

MN ,

AAAA BBBB

CCCC DDDD

CBM

CBM

CBM

CBM

Fig. 15.

(244)


73

,

‘spike’

.

.

IgG

C3 .

11,13,15,21).

MN steroid

, steroid

,

. steroid

,

.

20~40% 14,16-22).

MN ‘ ’, ‘ ’, ‘ ’ 2,3,23,24),

, , , , , 2,23,24).

, , 28)

, , , 29),

, 30)

. 31) , ,

.

MN

12,33).

, , cationic bovine serum albumin

(cBSA)

27,34).

pH

4.5-5.1 bovine serum albumin(BSA)

(cationic) pH 9.5 cBSA35,36), cBSA

cBSA

MN .

MN 37), cBSA

mouse MN 38).

cBSA MN

.

cBSA MN

cBSA

,

. ,

.

charge-selective barrier

size-selective barrier .

sialoglycoprotein

,

heparan sulfate proteoglycan

, pH 7.4

.

, ,

. charge-

selective barrier size- selective barrier

,

.

(245)

30 2 (2009 3 )

74

2,3,14,39-41).

24

.

.

.

,

.

.

2,11,13,14,41,42).

albumin

.

.

,

very low density lipoprotein

(VLDL)

, lipoprotein

lipoprotein lipase(LPL)

VLDL

2,14,42,43).

total cholesterol

, BY

-250 .

.

.

, ,

BUN, creatinine 2,14).

creatinine

. BUN

. MN creatinine

BUN , 20).

MN ,

Th2 type27,32,38). MN

T B-lymphocyte population34). CD3+ T cell

MN

.

. MN

CD3+ T cell

. CD19+ B

cell

.

T cell CD3

, ,

- . CD4+

Th cell

, Th1 cytokines

, Th2 cytokines

,

.

CD8+ Tc cell

,

. T cell

,

(246)


75

Th cell Tc cell MN9,38,44).

CD4+ Th cell

.

cBSA MN CD4+ Th

cell

CD4+ Th cell

. CD8+ Tc cell

.

IgA Ig 10~15%

, , , ,

Ig . IgM

Ig 5~10%

B cell Ig44-46). IgA IgM

. cBSA

IgA

IgM .

IgG Ig

75% ,

1 231,44,46).

IgG

. cBSA

MN IgG

IgG

.

IL-1β

, ,

. TNF-α

. IL-1β TNF-α44).

IL-6 B

cytokines ,

B cell

. , IFN-γ

T NK cell , MHC

class B cell , IL-2 IL-4

cytokines44).

IL-1β

.

proinflammatory cytokines TNF-α

. IL-1β TNF-

α

cytokines32).

B-cell differentiational factor IL-6

. IFN-γ

. IL-6

cytokines .

IFN-γ

IL-4

.

MN

cBSA

.

IgG

, 5,8,10,14,47,48).

,

.

.

(247)

30 2 (2009 3 )

76

cBSA

MN mouse , albumin

, total cholesterol , BUN

, IL-1β,

TNF-α IL-6 cytokines

, IFN-γ , IgG ,

MN

, MN

,

.

결 론

, CD3+ T cell CD4+ Th cell

IL-1β, TNF-α, IL-6 , IFN-γ

. 24

, total cholesterol, BUN IgG

,

albumin .

, IgG

, .

cBSA

mouse MN

. MN

,

.

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Documents

30-2 06 [63-78]jkom.org/upload/30-2 06 [63-78].pdf · ‘BY-500’, was treated with cBSA (9 / i.p) and BYMSB extract (500 / , p.o). After cBSA and BYMSB extract treatment for 4 weeks,