The effects of Jengjengamiyjin-tang on Pancreatitis induced by DBTC in Mouse

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최흥민, 김학재, 한이수, 임성우

The effects of Jengjengamiyjin-tang on Pancreatitis induced by DBTC in Mouse

Heung-min Choi, Hak-jae Kim, Yi-soo Han, Seong-woo Lim

Department of Oriental Medicine, College of Korean Medicine, Dongguk University

Objectives : We examined the effect of Jengjengamiyjin-tang, a traditional herbal formula, on the experimental pancreatitis induced by Dibutyltin Dichloride(DBTC).

Methods : 15 mice were divided into 3 groups; the normal group and the control group which were administered water only after pancreatitis elicitation and the test group which were administered Jengjengamiyjin-tang for 7 days after pancreatitis elicitation.

Results : In the activities of amylase, lipase and alkaline phosphatase in serum, the test group showed significant decrease compared with the control group after 21 days. In the common morphology and histochemical study, various injuries by hemorrhagic erosion were observed in the control group, while they were significantly decreased in the test group. In the immunohistochemical study, the manifestation of COX-1, 2, HSP27, HSP70, MAC387, Ki-67 decreased significantly in the 14th day in the test group, however, they increased in the 21st day more than the control group(p<0.05).

Conclusion : According to the above results, it is supposed that Jengjengamiyjin-tang has anti-inflammatory effect on the experimentally induced pancreatitis and may be applicable to pancreatitis especially in the acute stage.

Key Words : Jengjengamiyjin-tang, pancreatitis, dibutyltin Dichloride (DBTC), COX, HSP, MAC387, Ki-67

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13), Helcobacter pylori ¹⁴⁾ ,

15) ,

DBTC(Dibutyltin Dichloride)

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DBTC

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.

실 험

1. 약재 및 동물

1)

(Jengjeng-

amiyjintang) ⁸⁾

1 (Table 1).

2)

230-250g Sprague-

Dawley 6

,

( 23±2-3°, 50±0.5%, 12

) (DBTC) 7

( , damage )

5 .

Species Scientific name Dose(g)

Crataegi Fructus Cyperi Rhizoma Pinelliae Rhizoma Cnidi Rhizoma

Atractylodis Macrocephalae Rhizoma Atractylodis Rhizoma

Citi Pericarpium

Poria

Massa Medicata Fermentata Amomi Fructus

Hordei Fructus Germiniatus Glycyrrhizae radix Zingiberis Rhizoma Jujubae Fructus

Crataegus pinnatifida BGE.

Cyperus rotundus L.

Pinellia ternata BREIT.

Cnidium officinale MAKINO Atractylodes macrocephala KOIDZ.

Atractylodes japonica KOIDZ.

Citrus unshiu MARKOVICH.

Poria cocos WOLF Massa Medicata Fermentata Amomum xanthioides WALL.

Hordeum vulgare var.hexastichon ASCHERS..

Glycyrrhiza uralensis FISCH.

Zingiberis officinale ROSC.

Zizyphus jujuba MILL.

6.0 4.0 4.0 3.2 3.2 3.2 2.8 2.8 2.8 2.0 2.0 1.2 3.0 2.0

Total amount 42.2

(3)

100 3

. 100ml 100ml/60kg/day 3

. 2) DBTC

100% (2 part) dibutylitin dichloride (DBTC, Sigma) 8mg/kg

glycerol(1 part)

1 .

DBTC (N)

( ,

C) DBTC 1

( , S)

, DBTC 7,

14, 21 , 14 21

. 3)

sodium pentobarbital

syringe ,

3000rpm 10

AU400 (Japan) .

,

12 .

5

. 4)

5 xylene

(rehydration) H&E stain, Luna's stain, Mallory-Azan stain

. 5)

5

xylene ,

proteinase K(20 /ml) 5 prote-

olysis 0.3% H O 5

, 1

(Table 2) 4 humidified

chamber 12 .

0.02M phosphate buffered saline (PBS)

2 biotinylated goat anti-rabbit IgG (1:1,000, DAKO, USA) 10

antibody source company dilution

HSP27 Rabbit Chemicon 1 : 200

HSP70 Human Stressgen 1 : 50

COX1 Rabbit Santa cruz 1 : 100

COX2 Rabbit Santa cruz 1 : 100

MAC387 Rabbit Chemicon 1 : 200

Ki67 Human Santa cruz 1 : 200

(4)

link , steptavidin peroxidase (DAKO, USA)

10 . 0.02M PBS

0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB, Sigma) 0.003% H O

. hematoxylin

. 6) TUNEL

In situ apoptosis detection kit (Apoptag, Intergen, USA) TUNEL (terminal deoxynucleotid transferase-mediated dUTP-biotin nick-end labelling)

.

proteinase K 5 proteolysis ,

quiliation buffer 20 .

strength TdT enzyme 37 1

strength stop/wash buffer 10 . Anti-digoxigeninperoxidase 1

0.05% 3, 3-diaminobenzidine tetrahydrochloride (DAB, Sigma) 0.003%

H2O2

. hematoxylin

(apoptosis) .

7)

SPSS program (ver. 12.0)

, one-way ANOVA p<0.05 .

결 과

1. 혈청분석 결과

Amylase Lipase

21

. ALP 3

(Table. 3).

2. 조직화학 결과

DBTC 7 (vacuole)

,

Parameter (mg/dl) group 7 days 14 days 21 days

Amylase

normal 1456.00±295.43 1502.33±126.89 2215.67±1308.84

control 1619.67±142.86 1547±186.88 1544.67±104.15

sample - 1490.33±253.32 1539.75±256.77

Lipase

normal 14.25±2.22 12.00±1.00 34.67±21.08

control 14.33±2.31 12.67±1.53 19.67±3.51

sample - 14.67±2.08 16.00±4.36

ALP

normal 902.67±244.44 823.67±81.74 959.67±51.64

control 912.00±47.51 819.67±189.21 834.33±65.42

sample - 800.33±65.65 788.5±123.45

Mean±SD; a Significant difference (p<0.01), sample group compared with normal and control groups at the corresponding time. In activities of amylase and ALP, control and sample groups were not different to normal group.

(5)

.

21 14

(Fig.1. B), (mast cell)

,

(Fig.2. B). 21

(Langerhans islet) , Mallory-

Azan DBTC 21

(Fig.3. B, E).

21 (Fig.3. C, F).

3. HSP27 면역조직화학 결과

14.4 ± 4.66 / 10,000 pick cell

(fig.4. A, Table. 4) ,

7

,

, 14 (Fig.4. B). 21

65.2 ±13.62 / 10,000 pick cell .(Fig.4. C, Table 4.).

14

,

. 21

,

A B C

(6)

30.8 ± 7.91 / 10,000 pick cell .(Fig.4. D, Table. 4.).

4. HSP70 면역조직화학 결과

6.4 ± 1.17 / 10,000 pick cell

(fig.5. A. Table 4.).

7

,

(fig.5. D). 14

7 (fig.5. B).

7 . 21

.

74.8 ± 10.07 /10,000 pick

cell (fig.5. C, Table 4.).

14 ,

A B C

D E F

A B

C D

(7)

(fig.5. E). 21

34.4 ± 7.47 / 10,000 pick cell (Fig.5. F, Table. 4).

5. COX-1 면역조직화학 결과

7.6 ± 3.54 / 10,000 pick cell (Fig.6. A, Table 4) 7

(Fig.6. D).

14 7

(Fig.6. B),

7 ,

21

74.4 ± 7.68 / 10,000 pick cell

Antibody

Cell count(mean±S.E)

Normal(N) Control(C) Sample(S)

HSP70 6.4 ± 1.17a 74.8 ± 10.07b 34.4 ± 7.47c*

HSP27 14.4 ± 4.66a 65.2 ±1 3.62b 30.8 ± 7.91c

COX-1 7.6 ± 3.54a 74.4 ± 7.68b 22.8 ± 4.84c

COX-2 4.4 ± 1.94a 88.4 ± 18.08b 31.6 ± 10.36c

MAC387 5.2 ± 2.80a 46.0 ± 5.48b 12.0 ± 6.54c

Ki67 6.4 ± 4.07a 70.4 ± 17.43b 36.8 ± 11.16c**

a-b, a-c Values with the different superscripts are significantly different at p<0.01. a-c* Values with the different superscripts are significantly different at p<0.001. a-c** Values with the different superscripts are significantly different at p<0.05.

Abbreviation : Nomal, No treated mice; Control, water treated mice with pancreatitis by DTBC; Sample, Jengjengamiyjin-tang treated mice with pancreatitis by DTBC

A B C

D E F

(8)

(Fig.6. C, Table 4). 14

(Fig.6. E) 21

22.8 ±4.84 / 10,000 pick cell (Fig.6. F, Table. 4).

6. COX-2 면역조직화학 결과

,

(Fig.7. D). 14 7 ,

(Fig.7. B). 21

A B C

D E F

A B C

D E F

(9)

88.4 ± 18.08 / 10,000 pick cell

(Fig.7. C). 14

(Fig.7. E), 21

31.6 ± 10.36 / 10,000 pick cell

(Fig.7. F, Table. 4).

7. MAC387 면역조직화학 결과

macrophage ,

(Fig.8. D). 14

(Fig.8. B). 21

46.0 ± 5.48 / 10,000 pick cell

(Fig.8. C). 14

7 ,

(Fig.8. E). 21

12.0 ± 6.54 / 10,000 pick cell

8. Ki67 면역조직화학 결과

(Fig.9. D),

6.4 ± 4.07 / 10,000 pick cell

(Fig.9. A. Table 4). 14

.

,

A B C

D E F

(10)

(Fig.9. B). 21 14

,

70.4 ± 17.43 / 10,000 pick cell (Fig.9. C, Table 4). 14

,

(Fig.9. E). 21

36.8 ± 11.16 / 10,000 pick cell

9. Apoptosis 결과 DBTC

21 Apoptosis

, 21

(Fig.10. B, C).

고 찰

. ,

, ,

A B C

D E F

A B C

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1).

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, ¹³⁾

, ¹⁴⁾ H.pylori , ¹⁵⁾

.

100% dibutylitin dichloride (DBTC, Sigma) 8mg/kg

glycerol(1 part)

, ,

.

Sparmann ¹⁷⁾ DBTC (8mg/kg)

24 , 7

(neutrophil), (monocyte) (fibroblast)

(pancreatic acinar cell) , 14

. DBTC

7 (vacuole)

,

. 21

14

. (mast cell)

,

. 21 (Langer-

hans islet) , Mallory- Azan

DBTC 21

A . ,

21 .

DBTC

(12)

.

, HSP(heat shock

protein) HSP family

HSP10, HSP27, HSP60, HSP70 HSP90 ubiquitin

. Joo ¹⁸⁾ HSP27

HSP70

, HSP

, , ,

19). HSP70 HSP27 hormone-

sensitive neoplasm

20). HSP27

,

, 7

14

. 21

,

. 21

. HSP27

HSP27 ,

. DBTC

HSP27

,

.

HSP70 Heat-shock-factor proteins(HSFs)

, HSFs HSP90

HSF , HSFs

HSP70 gene

HSP70 mRNA

21,22)

. endotoxin HSP70

Koh ²³⁾ , Hwang

24) HSP70 cerulein

.

, DBTC 14

21

.

HSP70 ,

7 (DBTC 14 )

, 14 (DBTC

21 )

. DBTC

, 7

(DBTC 14 ) 7

.

, 14 (DBTC 21 )

HSP70 .

. Cyclooxygenase(COX) enzyme

isoenzyme COX-1 COX-2 .

cox enzyme prostaglandins(PGs) , PGs

(13)

, PGs

28). ,

COX-1,2

, 7

, 14

, 7

7 (DBTC 14 )

14 (DBTC

21 ) . DBTC

, COX-1,2

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(macrophage) interleukin-1β (IL-1β) tumor necrosis factor-α (TNF-α) cytokine nitric oxide (NO) prostaglandin (PG)

(inflammatory mediator)

. Macrophage

29,30,31)

. NF-KB

(granulocyte)

(TNF-α, IL-1), , ,

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32).

,

7 7 (DBTC

14 ) , 14

. DBTC

,

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Ki67 G1

S G2

33), Ki67

. S G2

Ki67 . Ki67

[34].

Ki67 ,

, 14

. 21

.

21

,

14 (DBTC

21 )

DBTC

. Apoptosis DBTC

.

(14)

DBTC

,

.

결 론

DBTC

,

, ,

.

1. Amylase Lipase

ALP .

2. vacule,

, ,

. 3. HSP27, 70

21 , HSP70

, 14 .

4. COX-1, 2 21

, 14 .

5. MAC387 21

, 14 .

6. Ki67 21

, 14

.

7. Apoptosis DBTC

.

DBTC

,

.

참고문헌

1. . .

. . 1997:1636-1648

2. . . . . 1995:810

-811

3. .

. . . 1998:308-314

4. 4 .

Trypsin . . 1987;

3(2):268-280

5. 9 . 2

. . 2003;24(2):365-373

6. 3 .

1 . . 2002;23(3):471-478

7. . . , . 1986:69

8. : . . . 1992:131

9. . . . . 1986:131

10. .

.

(1978-1990). . 1992:81

11. .

. . 1997:1-38

12. , , , .

. . 1999;20(2):130-136

13. , , .

HCI-aspirin

. . 1999;39:1168-1179

(15)

-83

15. , , , , ,

. ,

. . 2003;18(2):13-25

16. . .

. 2002(14);149

17. Sparmann G, Merkord J, Jäschke A, Nizze H, Jonas L, Löhr M, Liebe S, Emmrich J.

Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride. Gastroen- terology. 1997;112:1664-1672

18. Joo M, Chi JG, Lee H. Expressions of HSP70 and HSP27 in hepatocellular carcinoma. J.

Korean Med Sci. 2005;20:829-834

19. Park KH, Kim MS, Sun DI, Cho SH, Lee JW.

Study on expression of HSP27 in squamous cell carcinoma of larynx. Korean J. Bron- choesophagology. 2001;7(1):24-28

20. Joo M, Chi JG, Lee H. Expressions of HSP70 and HSP27 in hepatocellular carcinoma. J.

Korean Med. Sci. 2005;20:829-34

21. Fukushima M, Sataka T, Tsuusui K, Arase K, Gomita Y, Asano C. Grcadian pattern of stress response to affective cues of foot shock.

Physiol Behav. 1981;27:915-920

22. Fuchimoto H, Sataka T, Ogawa N, Gomita Y.

Olfactory bulb as aues in the communication of affect between rats. Fukuoka Acta Med.

1973;64:41-46

23. Koh Y, Lim CM, Kim MJ, Shim TS, Lee SD, Kim WS, Kim DS, Kim WD. Heat shock response decrease endotoxin-induced acute lung injury in rats. Respirology. 1999;4(4):

325-330

activation: studies in Hsp70.1 knock-out mice.

The Korean Journal of Gastroenterology.

2002;40:326-333

25. Frungieri MB, Albsequent M, Raemsch R, Mayerhofer A. The action of the mast cell product tryptase on cyclooxygenase-2(COX2) and subsequent fibroblast proliferation involves activation of the extracellular signal- regulated kinase isoforms 1 and 2 (erk1/2). Cellular Signalling. 2005;17:525-533

26. Smith WC, Meade EA, Dewitt DL. Pharmac- ology of prostaglandin endoperoxide synthase isozymes-1 and -2. Ann NY Acad Sci. 1994;

714:136-142

27. Mitchell IA, Larkim S, Williams TJ. Cycloo- xygenase-2 : regulation and relevance in inflammation. Biochem Pharmacol. 1995;50:

1535-1542

28. Kawamori T, Rao CV, Seibert K, Reddy BS.

Chemopreventive activity of elecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res. 1998; 58:

409-412

29. Yeom MJ, Choi BH, Han DO, Lee HJ, Shim I, Kim SH, Hahm DH. In vitro inhibition of pro-inflammatory mediator mRNA expression by nephrite in lipopolysaccharide-induced mouse macrophage cells. Korean J. Oriental Physiology & Pathology. 2004;18(6):1622-1627 30. Isomaki P, Punnonen J. Pro-and antiinfl-

ammatory cytokines in rheumatoid arthritis.

Ann Med. 1997;29(6):499-507

31. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002;105(9):

1135-43

(16)

32. Kim CD. Pancreatitis-Etiology and Pathogenesis.

The Korean Journal of Gastroenterology. 2005;

46:321-332

33. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear

antigen defined by the monoclonal antibody Ki-67. J. Immunol. 1984;133:1710-1715 34. Park IS. Histological study of expreimental

colitis induced by dextran sulfate sodium. The Korean J. Anat. 2006;39(1):27-33