133

서 론

,

1).

,

, , 2),

,

, ,

1,3).

4),

, , , ,

, 5).

Improved Axonal Regeneration Responses in the Injured Sciatic Nerve of Rats by Danggui Treatment

Hong Soon-Sung, Oh Min-Seok

Dept. of Oriental Rehabilitation Medicine, College of Korean Medicine, Daejeon University, Korea.

Objective: This study was performed to examine Danggui (DG, Angelica gigas Nakai)’s potential activity for promoting axonal regeneration in the injured peripheral nerve.

Methods: Using the sciatic nerve in the rats, DG extract 5 (10 / in 0.5% saline) was dripped into the injury

site of the nerve.

Results: DG treatment facilitated axonal elongation responses in the distal portion to the injury site. GAP-43 protein

levels were upregulated by DG treatment in the injured nerve and also in the DRG, suggesting the induction of

GAP-43 expression at gene expression level after nerve injury. Phospho-Erk1/2 protein levels were upregulated in the injured nerve area and also in the DRG, suggesting retrograde transport of phospho-Erk1/2 protein from the injury

area to the cell body. Cdc2 protein levels were slightly upregulated by DG treatment. DG treatment increased the

number of non-neuronal cells in the distal portion to the injury site.

Conclusions: The present data suggest that DG is effective for enhanced axonal regrowth after sciatic nerve injury.

Key Words : Danggui (DG, Angelica gigas Nakai), sciatic nerve, axonal regeneration

2008 3 14 2008 5 9

: , 2 1136

(Tel:+82-42-470-9136, Fax:+82-42-470-9008,

E-mail:ohmin@dju.ac.kr)

홍순성, 오민석

Original Article

29 2 (2008 5 )J Korean Oriental Med 2008;29(2):133-150

29 2 (2008 5 )

134

6)

, rat

,

,

.

재료 및 방법

1. 실험재료

1)

7

Sprague-Dawley(SD, , Korea)

30 . 1

.

, 22 2

4 , 50±10% ,

(12

/ ) .

2)

(Angelica gigas Naka)

.

3)

PVDF membrane

(Pall Corporation, USA), anti-rabbit IgG(Santa

Cruz Biotech, USA), anti-GAP-43 antibody(H-100,

rabbit polyclonal, Santa Cruz Biotech, USA),

anti-Cdc2 antibody(Santa Cruz Biotech, USA),

anti-β -tubulin antibody(Tuj-1, Covarice, USA),

anti-p-Erk antibody(Santa Cruz Biotech, USA),

anti-neurofilament200 antibody(NF-200, N52, Sigma,

USA), anti-actin antibody(MP Biomedicals, USA),

rho-damin-goat anti-rabbit antibody(Molecular Probes,

USA), carbocyanine dye l, l'-dioctodecyl-3 ,3 ,3'

,3' tetramethylindocarbo-cyanine perchlorate(DiI;

dimethylsulfixide 3%, Sigma, USA), poly-L-ornithine

(0.1 / , Sigma, USA), laminin(0.02 / ,

Collaborate Research, USA), DMEM medium

(Gibco, USA), type XI collagenase(2500 U/ ,

Sigma, USA) .

( , Korea), rotary

vaccum evaporator(Büchi B-480, Switzerland),

freeze dryer(EYELA FDU-540, Japan), CO2

incubator(Forma Scientific Co., USA), clean

bench(Vision Scientific Co., Korea), autoclave

(Sanyo, Japan), micro-pipet(Gilson, France), water

bath(Vision Scientific Co., Korea), vortex mixer

(Vision Scientific Co., Korea), blood glucose

meter(Lifescan, USA), spectro-photometer(Shimazue,

Japan), centrifuge(Sigma, USA), deep-freezer(Sanyo,

Japan), thermocycler system(MWG Biotech, Ger-

many), ice-maker(Vision Scientific Co., Korea),

homogenizer(OMNI, USA), plate shaker (Lab-Line,

USA), i-solution software(Image & Microscope

Technology Goleta, Canada), Kodak scientific

imaging film(Eastman Kodak Co., USA),

(Nikon E-600, Kawasaki, Japan)

.

2. 방 법

1)

54 g 2,000

3

6 g

, (-84 )

.

(356)

1 : rat

135

2)

3

.

(Control),

( ),

(

) .

(10 / in 0.5% ) 5

pipette

,

. 0.5% 5

.

3) 7)

. ketamine(80 / )

rompun(5 / )

(intraperitoneal injection) ,

(30×20 )

.

30 1

, 30 .

,

heating pad

36 37 .

4) Western blot analysis

137 mM NaCl, 2.7 mM KCl,

10mM Na2PO4, 2 mM KH2PO4(pH 7.4)

PBS 50 200 triton lysis

buffer(20 mM tris, pH 7.4, 137 mM NaCl, 25

mM β-blycerophosphate, pH 7.14, 2 mM sodium

pyrophosphate, 2 mM EDTA, 1 mM Na3VO4,

1% triron X-100, 10% glycerol, 5 / leupeptin,

5 / aprotinin, 3 µM benzamidine, 0.5 mM

DTT, 1 mM phenylmethylsulfonyl fluoride)

. sample

, 10

western blot analysis .

anti-glu 4 antibody .

Membrane proteins 12% SDS-PAGE(1.5 M

trizma base, 10% sodium dodecyl sulfate, 30%

acrylamide, 10% ammonium sulfate, TEMED)

PVDF membrane

. antibody

3% BSA, 0.1% tween 20

TBS buffer membrane 1

4 16 .

membrane wash-ing rat glut

4 C-terminal polyclonal antibody

blocking buffer(1× TBS buffer, 3% BSA, 0.1%

tween-20) 1:1000

30 . membrane

anti-rabbit IgG horser-

adish peroxidase 1:1000

30

. western blotting detection

system membrane

Kodak scientific imaging film

.

5) Hoechst

-20 cryostat

20

. (double immunofluo-

rescence staining) , 4% parafo-

rmaldehyde, 4% sucrose PBS

45 .

(357)

29 2 (2008 5 )

136

blocking buffer 4

1 . 1

anti-neurofilament200 antibody(NF-200),

anti-GAP-43 antibody, anti-Cdc2 antibody, anti-β

-tubulin antibody(Tuj-1), anti-p-Erk antibody

. 2.5% BSA, 2.5% horse

serum blocking buffer 1:600

4 16

. 1

PBST , 2.5% BSA, 2.5%

horse serum blocking buffer

fluorescein-goat anti-mouse rhodamin-goat anti-

rabbit antibody 1:100 1

30 2 . 2

3 PBST(phosphate-buffered

saline with 0.1% triton ×100) . Hoechst

2 0.25%

Hoechst 33258 PBST

PBST .

sample ,

Adobe photoshop

(version 5.5) green red

.

photoshop program layer blending mode options

.

3. 통계 처리

data student's t-test8)

. p 0.05

.

성 적

1. GAP-43 단백질 유도 생성의 변화

1) 3 western

blot analysis

3

, GAP-43

,

.

actin

(Fig. 1).

CTL SAL DG

GAP-43

Actin

Fig. 1.

(358)

1 : rat

137

2) 3

3

NF-200 ,

, 1

, 3

, 1

3

.

NF-200

GAP-43

, 1 3

GAP-43

(Fig. 2).

3) 7

7

NF-200 ,

7 ,

10

, GAP-43

CTL

SAL

1: Injury site

2: 1mm distal

3: 3mm distal

1: Injury site

2: 1mm distal

3: 3mm distal

DG

Fig. 2.

(359)

29 2 (2008 5 )

138

10

.

4) 3

3

, GAP-43

,

.

Tuj-1

(Fig. 4).

DG

SAL

Fig. 3.

CTL

DG

SAL

Fig. 4.

(360)

1 : rat

139

5) 7

7

, GAP-43

,

(Fig. 5).

2. p-Erk1/2 단백질 유도 생성의 변화

1) 3 western

blot analysis

3

, p-Erk1/2

,

. Actin

(Fig. 6).

2) 3

3

p-Erk1/2 ,

,

3

,

SAL DG

Fig. 5.

CTL SAL DG

p-Erk 1/2

Erk 1/2

Actin

Fig. 6.

(361)

29 2 (2008 5 )

140

3

(Fig. 7).

3) 7

7

p-Erk1/2 ,

3

,

3

(Fig. 8).

CTL

SAL

DG

Injury site 3 mm distal

Fig. 7.

SAL

DG

Injury site 3 mm distal

Fig. 8.

(362)

1 : rat

141

4) 3, 7

3

p-Erk1/2 ,

,

(Fig. 9A).

7

p-Erk1/2 ,

,

(merged image)

(Fig. 9B).

3 7

p-Erk1/2 7 3

.

3. Cdc2 단백질 유도 생성의 변화

1) 7 western

blot analysis

SAL

DG

SAL

DG

A B

Fig. 9.

CTL

Cdc2

SAL DG

Actin

Fig. 10.

(363)

29 2 (2008 5 )

142

7

, Cdc2 ,

. Actin

(Fig. 10).

2) 3, 7

3

Cdc2 ,

3

,

3

,

.

7

Cdc2 ,

3

(Fig. 11).

4. 비신경 세포 증식 효과

1) 3 Hoechst

3

Hoechst

, 0.1

450±83 , 1, 3

0.1

758±67 ,

780±79

(p<0.05).

CTL

SAL

DG

Injury site 3mm distal

3 days post crushInjury site 3mm distal

7 days post crush

Fig. 11.

(364)

1 : rat

143

,

,

,

,

(Fig. 12).

2) 7 Hoechst

7

Hoechst

CTL

SAL

DG

proximal Injury site 1 mm distal 3 mm distal

A

1200

1000

800

600

400

200

0

The number of nuclei

DG

SAL

Distance from the injury site (mm)

-1 0 1 3

B

Fig. 12.

SAL

DG

Injury site 3 mm distal 7 mm distal 10 mm distal

A

1200

1000

800

600

400

200

0

The number of nuclei

DG

SAL

Distance from the injury site (mm)

0 3 7 10

B

Fig. 13.

(365)

29 2 (2008 5 )

144

, 0.1

862±96 , 852

±102

(p<0.01).

3, 7, 10

,

(Fig. 13).

고 찰

,

1),

7).

,

9).

4),

, ,

, ,

, ,

, ,

,

, ,

, , ,

, 5).

·10)

“

”

,

,

, , , 11)

, ·12)

“ ”, ·12)

“ ”

11).

,

13),

14)

.

, , , ,

, , ,

6).

15),

16) 17),

18),

19)

, 20)

, 21,22)

,

.

, rat

, ,

.

(366)

1 : rat

145

GAP-43, p-Erk1/2, Cdc2

.

GAP-43 43 kDa axonal growth-

associated protein 1980

23), rat

,

24).

GAP-43 ,

. GAP-43

(presynaptic terminal)

protein kinase C

CaM kinase kinase25)

.

, GAP-43

26).

,

GAP-43

24).

3 7

GAP-43 ,

,

. GAP-

43

. GAP-43

.

.

rat GAP

-43

GAP-43

,

.

Erk1/2 MAP kinase

, 27)

.

MAP kinase c-Jun N-terminal

kinase(JNK) p38

28).

MAP kinase

, kinase

. , Erk1/2 kinase MEK1/2

, JNK kinase

MEK 4, 627)

. JNK

Erk1/2

.

MEK kinase

, Erk1/2 JNK

29). Erk1/2

.

p-Erk1/2 3

7

.

,

(367)

29 2 (2008 5 )

146

.

p-Erk1/2

p-Erk1/2

MEK 1/2 p-Erk1/2

.

p-Erk1/2

30).

p-Erk1/2

Erk1/2

, Erk1/2

Rsk CREB(cAMP responsiveness element

binding protein)27)

.

p-Erk1/2

.

Cdc2 cycle G2 phase M

phase

cyclin B 31)

. , Cdc2

G2 phase

cyclin B1 B2

,

32).

Cdc2 cyclin

, Cdc2

33). Cdc2

.

Cdc2

,

Cdk

(apoptosis) ,

(proapoptotic protein) Bad

34).

Cdc2

Cdc2

35).

Cdc2 7

western blot analysis .

3 7

Cdc2

,

.

Cdc2

.

.

,

37). ,

,

,

1).

Cdc2

,

(368)

1 : rat

147

,

. 3 3

,

. ,

7

,

(0 10 )

.

.

.

5 DiI

. 3

,

21.8%

. 7

3

,

69.7%

(p<0.01),

DiI

.

3

(T11-12) ventral horn

,

62.5%

(p<0.01),

7 ventral horn 3

,

50%

(p<0.05).

, 3

, DiI

5

..

3

.

.

DiI

10

20% .

.

.

.

(preconditioning)

3 10

,

in vivo

(369)

29 2 (2008 5 )

148

36,37).

(lesion signal)

,

.

37).

,

.

. 1

, 2

.

GAP-43 ,

. in vivo

.

.

,

(multiple effects)

.

, GAP-43, p-Erk1/2

,

.

,

.

,

.

결 론

.

1. NF-200

.

2. GAP-43

.

3. p-Erk1/2

.

4. Cdc2

.

5.

.

.

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