The Effects of Hirudo on Streptozotocin-induced Diabetic Nephropathic Rats

서 론

,

1) , ,

5 20% .

水蛭

糖尿病性 腎症 影響

서수환, 김용성, 한양희 동신대학교 목포한방병원 신계내과 교실

Original Article

The Effects of Hirudo on Streptozotocin-induced Diabetic Nephropathic Rats

Su-Hwan Seo, Young-Seong Kim, Yang-Hee Han

Dept. of Internal Medicine. College of Oriental Medicine, Dongshin University

Objectives: The aim of present study was to investigate recovery effects of Hirudo, which has been used clinically in diabetes therapy.

Methods: We established three groups: normal, control, Hirudo, and assigned 6 rats to each group. The normal group was not treated by any process and fed by normal saline. The control & Hirudo groups were administered streptozotocin (STZ) to induce diabetes. Hirudo extract was orally administered to the Hirudo group for 10 days. After 8 weeks, the rats were sacrificed and their body weight, 24hrs urinary protein excretion, glucose, albumin, BUN, creatinine, total-cholesterol, LDL-cholesterol, triglyceride in blood, and level of glycation end-product (AGE) and transforming growth factor (TGF-β1) in serum were measured. Morphological profiles and morphometric studies of the kidney cortex, renal transforming growth factor (TGF-β1) expression, and renal receptor for advanced glycation end-products (RAGE) expression were studied.

Results: The following results were obtained. The protein amount in urine per 24hrs of the Hirudo-treated group as compared to the control group was significantly reduced. The BUN and creatinine level in serum of the Hirudo-treated group as compared to the control group was significantly inhibited. The construction change in kidney of the Hirudo-treated group as compared to the control group was significantly inhibited. The factor of the Hirudo-treated group as compared to the control group was significantly inhibited, which induced the construction change in kidney.

Conclusions: The above results suggest that Hirudo partially improved the function of the kidney.

Key Words : Hirudo, streptozotocin, diabetic nephropathy

접수：2008년 7월 11일 수정：2008년 8월 9일 채택：2008년 8월 11일

교신저자 : 김용성(Young-Seong Kim)

전남 목포시 상동 834번지 동신대목포한방병원 2내과

Tel : +82-61-280-7700, Fax : +82-61-280-7788, E-mail : [email protected]

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streptozotocin(STZ) rat

.

재료 및 방법

1. 재료 1)

(Hirudo nipponica Whitman)

. 2)

280-320g Sprague-Dawley rat (

, Korea) cage 40-70%

12 1 .

( ) ,

. . 3)

Sigma Chemical Co.(St. Lousi,

MO, USA) ,

Falcon Lavware(Becton-Dickinson, Franklin Lakes,

NJ, USA) .

Rotary evaporator

(Model NE-1, , Japan),

(Model FD-1, , Japan),

(SureStep, LIFESCAN INC. USA),

(Olympus BX-50, Olympus Optical, Tokyo, Japan), MetaVue (Molecular devices, USA), Vectastain ABC ELITE kit <rat IgG> (Vector Laboratories,

Inc) .

2. 방법 1)

400g 1,500 4

, Rotary

evaporator ,

. 1 1g 10

95 2

, 14,000rpm 20

. 0.2

,

18% . -7

0 .

2)

pH4.5 citrate buffer(2.5 /

) streptozotocin(STZ, Sigma, USA) rat

45 / 1 .

3)

6 (Normal group), (Control group),

(Hirudo group) 3 .

STZ citrate buffer ,

. STZ 3

300 / rat STZ 4

5 / 8 .

STZ 3 300

/ rat STZ

4

500 / zonde 1 1

8 .

4) 24

24 4

8 rat metabolic cage 24

albumin . STZ 4

1 9

. 5)

rat

glucose, albumin, (BUN) creatinine, total cholesterol, low density lipoprotein(LDL), cholesterol,

triglyceride .

6)

rat 0.05M phosphate buffered saline(PBS) 4% paraformaldehyde(PFA) perfusion

pump ,

.

10% , 70%,

80%, 95%, 100%

, .

microtome 4

gelatin coated slide .

xylene , 100%, 95%, 80%,

70% , rehydration

. Periodic acid-Schif(PAS) high-resolution camera

.

MetaVue(Molecular devices, USA)

. rat 10

pink RGB

.

7) Macrophage/monocyte antigen(ED-1), transforming growth factor(TGF-β1), type IV collagen

.

,

15) , (Indirect

method) (unlabelled antibody method) ABC(avidinbiotin complex) ¹⁶⁾ .

4 gelatin coated slide

xylene , 100%,

95%, 80%, 70% , rehydration

1% H 2 O 2 / methanol 15 ,

Protein Blocking Agent

1 . slide

primary antibody

overnight . 50mM PBS 3

Secondary antibody 1

. 50mM PBS 3 Vectastain ABC ELITE kit (rat IgG) 1

. Diaminobenzidine(DAB) hematoxylin

, 70%, 80%, 95%, 100% ,

xylene dehydration

(permount) high-resolution

camera

.

MetaVue

. rat 10

ED-1, TGF-β1, type IV

collagen RGB

.

Protein Blocking Agent 1% Bovine serum albumin(BSA, Sigma-Aldrich) 10% Normal horse serum (Vector Laboratories, Inc)

. primary antibody ED-1 mouse anti-rat monocyte/ macrophage antigen(Chemicon International Inc. Temecula, USA) , TGF-ß1 polyclonal rabbit anti-rat TGF-ß1 antibodies(Santa Cruz Biotechnology, Santa Cruz, USA) , type IV collagen Goat Anti- Type IV Collagen (Southern Biotech) . Secondary antibody Biotinylated Anti-Mouse/

Rabbit/Goat IgG <H+L>, made in horse(Vector

Laboratories, Inc) .

8) Receptor for advanced glycation end-product(RAGE)

dehydration,

protein blocking RAGE primary

antibody overnight ,

secondary antibody

1 .

.

MetaVue . rat

10

RAGE RGB

.

Primary antibody goat polyclonal antibody(Santa Cruz Biotechnology, Santa Cruz, USA)

, secondary antibody donkey anti-goat IgG-FITC:

(Santa Cruz Biotechnology, Santa Cruz, USA) .

9) TGF-β1 AGE ELISA

(ELISA17) : Enzyme-Linked Imm- unosorbent Assay)

,

17) . TGF-β1 TGF-β1

Emax ImmunoAssay System (Promega, USA)

. rat

,

TGF-β1 200 recom-

binant TGF-β1 sRII coating microplates , 200 TGF-β1 conjugate, 200 substrate solution, 50 stop solution

450nm ELISA reader

TGF-β1 pg/ .

AGE Indirect ELISA

. 96 well plate glutaraldehyde(100 /10

PBS) 200 37 1 incubation

, washing buffer 3 .

PBS 90 rat 10 well

plate shaker 2 shaking , washing

buffer 3 . blocking buffer

200 shaker 1 shaking

washing buffer 3 primary antibody

dilution buffer 10 shaker 1 shaking

, washing buffer 3 . secondary antibody dilution buffer 10 shaker 1

shaking washing buffer 3 .

substrate A B 1:1 200

15 incubation , 1M H2SO4 50 450nm ELISA reader AGE optical density (O.D.)

.

Primary antibody Rabbit polyclonal AGE antibody(Novus Biologicals, Inc) 4 , secondary antibody goat anti-rabbit IgG conju- gated to horseradish peroxidase(Abcam) 4

. 10)

GraphPad PRISM statistical package(ver 2.00, Graphpad software inc. San Diego, USA) .

± (mean ± S.D.) ,

one-way analysis of variance(ANOVA)

Tuckey’s post-hoc test ,

(Two-tailed p value) p 0.05 .

실험결과

1. 24 시간 요단백 배설량과 체중 측정

24 8

24.26±3.49 11.12±4.45

(P<0.001) .

8 18.57±4.07

(P<0.05) (Table 1, Fig. 2).

8 341.33±44.64 g

463.17±45.74 g

(P<0.001) .

(Table 1, Fig. 1).

2. 혈청 중 각종 성분의 변화에 미치는 영향

glucose, BUN, creatinine (P<0.001, P<

Normal (n=6) Control (n=6) Hirudo (n=6)

Body weight (g) - 4 weeks 389.00±25.67 335.17±35.18^* 335.67±54.78

Body weight (g) - 8 weeks 463.17±45.74 341.33±44.64^†*** 339.25±14.93

Urinary protein excretion (㎎/㎏/day) - 4weeks 11.35±3.75 14.69±9.74 24.01±5.47 Urinary protein excretion (㎎/㎏/day) - 8weeks 11.12±4.45 24.26±3.49^*** 18.57±4.07^‡#

* Significantly different from the normal group (P<0.05)

† Significantly different from the normal group (P<0.001)

‡ Significantly different from the control group (P<0.05) Table 1.

B o d y W eig ht ( g )

500

400

300

200

Normal Contol Hirudo

0 1 2 3 4 5 6 7 8 9

Weeks after STZ injection

Fig. 1.

0.05, P<0.05) , albumin

(P<0.05) ,

(Table 2).

3. 신장의 조직 병리학적 변화에 미치는 영향

(Fig. 3, A), (diffuse)

,

(capillary lumen) (mesangial cell)

. ,

(mesangial matrix) (extracellular matrix: ECM) , (segmental)

(crescent) (sclerosis) (Fig. 3, B).

U rin ar y p ro te in e xc re tio n ( m g /d ay )

30

25

20

15

10

5

0

Normal Control Hirudo

4weeks 8weeks

Fig. 2.

Normal

(n=6) Control (n=6) Hirudo (n=6) Glucose (㎎/㎗) 78.66±8.98 393.16±105.12^* 485.4±32.65 Albumin (㎎/㎗) 2.28±0.19 1.77±0.35^† 1.80±0.21

BUN (㎎/㎗) 19.56±2.04 33.64±4.97^* 46.05±13.77 Creatinine (㎎/㎗) 0.53±0.03 0.65±0.04^* 0.62±0.09

Total-cholesterol

(㎎/㎗) 73.16±9.66 59.00±18.83 56.00±6.48 LDL-cholesterol

(㎎/㎗) 11.83±3.43 14.40±2.51 14.25±4.19 Triglyceride

(㎎/㎗) 67.50±31.48 54.40±20.86 88.5±35.84

* Significantly different from the normal group (P<0.05)

† Significantly different from the normal group (P<0.001) Table 2.

A A A

A BBBB CCCC

M es an g ia l m at rix a re as (% o f g lo m er ulu s)

25 20 15

5 0 10

D D D D

Normal Control Hirudo

Fig. 3.

,

. ,

, (crescent)

(sclerosis) (Fig. 3, C).

18.74±3.36% 8.13±3.35%

(P<0.001) , 9.46±3.91 %

(P<0.001) .(Fig. 3, D).

4. Macrophage/monocyte antigen (ED-1), TGF - β 1 과 type IV collagen 의 면역조직화학염색 의 변화에 미치는 영향

ED-1

ED-1 10.48±2.99% 0.55

±0.27% (P<0.001) ,

3.21±2.37%

(P<0.001) . (Fig. 4).

TGF-β1

TGF-β1 12.46±1.98% 1.68

±1.38% (P<0.001) ,

6.73±3.01%

(P<0.001) . (Fig. 5).

A A A

A BBBB CCCC

TG F - B 1 p o sit iv e ce lls (% o f g lo m er ulu s)

16 14

6

DDD D

Normal Control Hirudo 12

10 8

4 2 0

Fig. 5. β

β AA

AA BBBB CCCC

E D - 1 p o sit iv e ce lls (% o f g lo m er ulu s)

14

0

D D D D

Normal Control Hirudo 12

10 8 6 4 2

Fig. 4.

type IV collagen

type IV collagen 10.81±2.52%

2.93±2.63% (P<0.001)

, 5.91±2.17%

(P<0.001) . (Fig. 6).

5. Receptor for advanced glycation end-product (RAGE) 의 면역조직형광염색의 변화에 미치는 영향

RAGE RAGE

(Renal cortex)

(tubule) (interstitium) ,

.

RAGE

0.57±0.54% 0.01±0.01

% (P<0.01) ,

0.13±0.00%

(P<0.05) . (Fig. 7)

6. 혈청 advanced glycation end-product(AGE) 와 transforming growth factor(TGF- β 1) 농 도에 미치는 영향

A AA

A BBBB CCCC

Ty p e IV c o lla g en p o sit iv e ce lls (% o f g lo m er ulu s)

14

Normal Control Hirudo 12

10 8 6 4 2 0

Fig. 6.

A A A

A B B B B C C C C

R A G E p o sit iv e ce lls (% o f g lo m er ulu s)

1.2

Normal Control Hirudo 1.0

0.8 0.6 0.4 0.2 0.0

Fig. 7.

AGE 3.94±0.06

(O.D.) 3.77±0.13(O.D.)

(P<0.05) ,

(Table 3, Fig. 8).

TGF-β1 62.10±

8.10pg/ 37.91±5.68 pg/

(P<0.01) ,

38.89±13.18 pg/

(P<0.05) . (Table 3, Fig. 8).

고 찰

,

1,2) .

, ,

40% ,

18,19)

.

, ,

1) , , Albumin , Albumin

Albumin

20) .

, (extracellular matrix, ECM)

, ( ,

angiotensin , endothelin, AGEs) , (

, )

( , ,

)

1,3,21)

.

.

angiotensin

, ,

,

1,19)

.

AGE (O.D.) 3.77 ± 0.13 3.94±0.06^* 3.86±0.07 TGF-β1 (pg/㎖) 37.91±5.68 62.10±8.10^† 38.89±13.18^‡

* Significantly different from the normal group( P<0.05 )

† Significantly different from the normal group( P<0.01 )

‡ Significantly different from the control group(# P<0.05) Table 3.

β

TG F - B 1 (p g /m l)

80 70 60 50 40 30 5 4 3 2 1 0 A G E ( O

D

)

AGE TGF-β 1

Normal Control Hirudo

Fig. 8.

β

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“ ” “ ” ⁶⁾ ,

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7) . , ²⁶⁾

. (hidudinidae)

/ (Whitmania pigra Whitman) (Hirudo nipponica Whitman) / (Whitmania acranulata Whitman) ⁸⁾ .

,

. Hirudo

medicinalis , 5

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hirudin fibrinogen trombin

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streptozotocin(STZ)

,

STZ 1963

. STZ β-

insulin

40) . STZ

rat

.

STZ

3 rat

300 / rat

.

24 4

, . 8

(P<0.001)

.

(Fig. 2.).

,

glucose (P<0.001)

albumin (P<0.05)

BUN creatinine

(P<0.05) .

(Table 2). BUN

glucose ,

. ,

, ,

, 12

.

,

, ^2,3) ,

PAS ,

,

, ,

,

,

, (crescent) (sclerosis)

(Fig. 3. A, B, C).

(P<0.001) (Fig. 3, D). AGE

, , ,

2,19)

. AGE ELISA (P<0.05) ,

(Table 3, Fig. 8). AGE RAGE

RAGE

(P<0.01) ,

(P<0.05) (Fig. 7).

TGF-β1 ,

, , AGE

angiotensin

,

3,19,21)

. TGF-β1 ELISA

(P<0.01) ,

(P<0.05) (Table 3, Fig. 8). TGF-β1

TGF-β1

(P<0.001) ,

(P<0.001) (Fig. 5).

AGE

,

2,19)

. ,

(P<0.001) ,

(P<0.001) (Fig. 4).

type IV collagen

angiotensin

1,19)

. type IV collagen

(P<0.001) ,

(P<0.001) (Fig. 5).

AGE

AGE RAGE, ED-1

TGF-β1, type IV collagen ,

, ,

.

결 론

Sprague-Dawley rat streptozotocin ,

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.

1. 24

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3.

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참고문헌

1. Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., FAuci, A.S., & Kasper, D.L.

HARRION’S Principles of Internal Medicine.

:MIP. 2003,2188-91.

2. . . : . 1999:

1006, 1127-33.

3. . . : .

2001:321-38.

4. .

. . 1998; 19

(2):137-51.

5. , , .

. . 1998;14(3 4):

245-56.

6. , . .

: . 1991:527-30.

7. , .

. . 2005;

11(2):1-13.

8. , , , .

. : . 2004:2566.

9. , , . 3

, . 1994;34(3):343-5.

10. , . . .

1993;1(2):55-66.

11. , , , .

. . 2002

;23(2):151-63.

12. .

. . 1994;16(2):5-42.

13. . , ,

. . 1990;11(2):92-101.

14. , , , , , ,

, , .

. .

2008;29(2):81-95.

15. Shi, S-R., Key, M.E., & Kara, K.L. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunoh- istochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytoc- hem. 1991;39(6):742-8.

16. Hsu, S.M., Raine, L., & Fanger, H. The use of avidin-biotin-peroxidase complex (ABC) in imm- unoperoxidase techniques. A comparison between ABC and unlabeled antibody PAP procedures. J Histochem Cytochem. 1981;29(4):577-80.

17. , , , , .

Smad 2 3 . .

2006;44(3):304-308

18. . . : . 1999:113.

19. . . : . 1998:

471-5.

20. Futoshi Shintani. Steps to Internal medicine.

: . 2002:155-6, 167-76.

21. . .

: . 2002:218-22.

22. . . : . 1986:57,

384-5.

23. . . : . 1975:294.

24. . .. : . 1992:1064,

1078.

25. . ( ). : .

1995:502.

26. . . :

. 2003:152.

27. . - . :

. 1995:19, 41, 89.

28. . . :

. 199:118.

29. . . : . 1994:708.

30. . . : . 1993:234.

31. . . : . 1982:

2285-8.

32. . . : . 2006:

2542.

33. . .

: . 1992:.431.

34. . . :

. 1998:522-4.

35. . . . 1993;34(4):197-9.

36. , , . Alloxan

mouse

. . 2000;5(1):17-25.

37. , , . 1

Streptozotocin

. .

2005;22(5):1-10.

38. , . Alloxan

mouse .

. 2005;26(4):741-52.

39. , . , ,

Streptozotocin

. . 2005;26

(4);767-75.

40. , . Streptozotocin

.

. 2004;19(1):111-9.