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1 26 1(2005 3) J Korean Oriental Med 2005;26(1): The Protective Effects of Seokchangpowonji-san on H2O2-Mediated Cell Death of Neuro 2A as an Alzheimer Model System Jun-Mo Yim 1), Min-Goo Lee 1),2), Jong-Min Yun 1),2), In Lee 2), Byung-Soon Moon 1),2) Professional Graduate School of Oriental Medicine, Wonkwang University 1) Department of Internal Medicine, College of Oriental Medicine, Wonkwang University 2) Objective : The water extract of Seokchangpowonji-san (SWS) has traditionally been used for treatment of dementia in oriental medicine. However, little is known about the mechanism by which the water extract of SWS rescues cells from neurodegenerative disease such as Alzheimer's disease. Methods & Results : This study was designed to investigate the protective mechanisms of SWS on -amyloid or H2O2- induced cytotoxicity in neuro 2A cells. H2O2 markedly decreased the viability of neuro2a cells, which was characterized by apparent apoptotic features such as membrane blebbing as well as fragmentation of genomic DNA and nuclei. However, the water extract of SWS significantly reduced H2O2-induced cell death and apoptotic characteristics through reduction of intracellular peroxide generation. Also, the extract prevented the mitochondrial dysfunction including the disruption of mitochondria membrane permeability transition (MPT) and the modulation in expression of Bcl-2 family proteins in H2O2- treated neuro 2A cells. Furthermore, pretreatment with SWS inhibited the activation of caspase-3, and in turn, degradation of ICAD/DFF45 was completely abolished in H2O2-treated cells. Conclusion : Taken together, the data suggest that the protective effects of the water extract of SWS against -amyloid induced oxidative injuries may be achieved through modulation of mitochondrial dysfunction. Key Words: Seokchangpowonji-san, Alzheimer, neuro 2A cells : : : :, (Tel : , Fax , mbs@wonkwang.ac.kr ) * (03-PJ9- PG6-SO ). 1) ().,, 161

2 (162) 26 1(2005 3), 2).,,,,,,, 3).,,,,,, 4,5), Alzheimer, 6). 7), (neuritic plaques) (neurofibrillary tangles), 8). 2,,,,, Ca 2+,, free radical.. (neurotransmitter;, ).. 9) CMS Morris, 10) pct105, 11), Neuro 2A. (-amyloid peptide) Neuro 2A,,. 1) Rat Neuro 2A ATCC (American Type Culture Collection) 10% Fetal bovine serum (FBS) dulbeccos modified eagles medium (DMEM) 5% CO2, 95% 37. 2), (Table1). 3) Dubleccos modified Eagles medium(dmem),, trypsin (fetal Table 1. The Prescription of Seokchangpowonji-san (g) Rhizoma Acori Graminei 50 Radix Polygalae 50 Total amount

3 (163) bovine serum, FBS) GIBCO BRL(Grand Island, NY, USA), (24-well plate, 10 cm dish) Falcon(Becton Dickinson, San Jose, CA, USA). methylthizol-2- yl-2,5-diphenyl, tetrazolium bromide(mtt), crystal violet Sigma(St. Louis, Missouri, USA). Bcl-XL/S, ICAD/DFF45, HO-1 Santa Cruz(San Diego, CA, USA), anti-rabbit IgG conjugated horse-radish peroxidase Enhanced chemiluminescence kit(elc kit) Amersham(Buckinghamshine, England). 1) 100g rpm 20 (Rotary evaporater) -70(Deep Freezer) 12 freeze dryer 21.2g. 2) Neuro 2A Rat Neuro 2A(ATCC, CRL1446) CO2 (37, 5% CO2) 10% DMEM % trypsine-edta, 12. 3) (24-well plate) ( cells/ml) 1 ml 12 CO2, 1/10 MTT (5 mg/ml in PBS) 4. formazan 10% sodium-dodesyl sulfate(sds) 0.01N HCl 100 /well (ELISA reader, Molecular Devices Co., Sunnyvale, CA, USA) 570 nm. 4) Neuro 2A phosphate buffered sailne(pbs, ph 7.4) 2. (3.7%) 0.5% crystal violet 5 PBS (Phase contrast microscope, Nicon, TE300, Japan). 5) H2O2 Neuro 2A 50M 2, 7-dichlorofluorescin diacetate(dcf-da) 30 PBS (Leica MPS 60, Germany) H2O2. DCF-DA H2O2 deacetylation dichlorofluorescin. 6) Western blot analysis, Hanks balanced salt solution(hbss, ph7.4) 2. (50 mm HEPES ph 7.4, 150 mm NaCl, 1% deoxy-cholate, 1 mm EDTA, 1 mm PMSF, 1 / aprotinin) ,000 rpm 20 BCA. ( : 200 g) 2ample buffer % SDS-PAGE. gel semi-dry 0.8mA 2 nitrocellulose membrane. Nitrocellulose membrane blocking buffer(5% skim milk) 1. Bcl-XL/S, ICAD/DFF45 HO %(v/v) Tween-20 3% skim milk/tbs 1:

4 (164) 26 1(2005 3) (anti-rabbit IgG conjugated horseradish peroxidase) 1. Nitrocellulose membrane TBS 3 ECL kit ECL. 7) (membrane potential) JC-1 Rhodamine 123. PBS 2. JC-1 (10/ml) Rhodamine 123(5 /ml) DMEM 20 CO2 PBS (Leica MPS 60, Germany). 8) 3 students t-test, p-value 0.05(p0.05). -amyloid Neuro 2A H2O2 2, 7-dichlorofluorescin diacetate (DCF-DA) H2O2. H2O2 DCF-DA 150 M H2O2 2. (Fig. 1A B), 1 M -amyloid 4 DCF-DA 6 (Fig. 1C). H2O2 Neuro 2A H2O2 12 MTT. H2O2 100 M H2O2 71%, 400 M 23% (Fig. 2) H2O2 Neuro 2A. -amyloid H2O2, Neuro 2A M H2O2 12 MTT. H2O2 49% 75 g/ml 58%, 150 g/ml 63%, 300 g/ml 68%, 600 g/ml 75%, 1200 g/ml 87% (Fig. 3). H2O2 Neuro 2A crystal violet. Neuro 2A H2O2(300 M) 6 (Fig. 4B b), (1200 g/ml) 30 (Fig. 4D d)., (Fig. 4A a) (Fig. 4C c). 164

5 (165) A B C Fig. 1. Reduction of intracellular peroxide by -amyloid-treated Neuro 2A cells. Cells were treated with -amyloid in Neuro 2A cells for 6 hr. Then, cells were incubated with 2, 7-dichlorofluorescin diacetate (DCF-DA, 50 M) and fluorescence intensity of cells was visulalized under a fluorescent microscope. The data were one of three independent experiments. (A) control, (B) H2O2-treated, (C) -amyloid treated cells. Fig. 2. Effects of H2O2 on viability of Neuro 2A cells in a dose dependent manner. Cells were treated with various concentrations of H2O2 for 12 hr Cell viability was measured by MTT assay. Results were expressed as means.d. of quadruplicates. * p<0.001 by students t-test, compared with control group. Fig. 3. Seokchangpowonji-san(SWS) prevented the cytotoxicity by H2O2 in Neuro 2A cells in a dose-dependent manner. Cells were pretreated with various conentrations of SWS for 30 min and followed by the addition of 300 M H2O2 for 12 hr. Cell viability was measured by MTT assay. Results were expressed as means.d. of quadruplicates. * p<0.01 by students t-test. 165

(A, a), control cells. (B, b), cells treated with H2O2. (C, c), cells treated with SWS and (D, d), cells pretreated with SWS before H2O2. A B C D Fig. 5.

6 (166) 26 1(2005 3) A B C D a b c d Fig. 4. Pretreatment of Neuro 2A cells with SWS prevented the cytotoxicity and morphological change by H2O2. Cells were treated with H2O2 (300 M) alone, SWS (1200 g/ml) alone and SWS with H2O2 for 6 hr. Then, cells stained with crystal violet and observed under phase contrast microscopy. (A, a), control cells. (B, b), cells treated with H2O2. (C, c), cells treated with SWS and (D, d), cells pretreated with SWS before H2O2. A B C D Fig. 5. Pretreatment of Neuro 2A cells with SWS prevented the morphological change by H2O2. Cells were treated with H2O2 (300 M) alone, SWS (1200 g/ml) alone and SWS with H2O2 for 6 hr. Then, cells stained with Hoechst and observed under fluorecense microscopy. (A), control cells. (B), cells treated with H2O2. (C), cells treated with quercetin and (D), cells pretreated with quercetin before H2O2. H2O2 Neuro 2A DNA Hoechst. H2O2 Neuro 2A (Fig. 5B), (Fig. 5D). (Fig. 5C), H2O2 Neuro 2A. HO- 1 Western blot. Neuro 2A HO M H2O2 6. H2O2 6 HO g/ml

(167) 600 1200 H2O2(300M) SWS(/ml) HO-1(31kDa) -actin(43da) Fig. 6. SWS suppressed the HO-1 expression induced by H2O2 in Neuro 2A cells.

Cell lysates were used to measure the HO-1 and -actin expression by Western blot analysis with anti-ho-1 and -actin antibodies.

SWS inhibited the decrease in Bcl-XL expression as well as increase in Bcl-XS expression and SWS inhibited the degradation ICAD/DFF45 in H2O2-treated Neuro 2A cells.

7 (167) H2O2(300M) SWS(/ml) HO-1(31kDa) -actin(43da) Fig. 6. SWS suppressed the HO-1 expression induced by H2O2 in Neuro 2A cells. Cells were treated with 300 M H2O2 in the absence and presence of SWS for 6 hr. Cell lysates were used to measure the HO-1 and -actin expression by Western blot analysis with anti-ho-1 and -actin antibodies H2O2(300M) SWS(/ml) Bcl-XL(29kDa) Bcl-XS(23kDa) ICAD/DFF45(45kDa) -actin(43da) Fig. 7. SWS inhibited the decrease in Bcl-XL expression as well as increase in Bcl-XS expression and SWS inhibited the degradation ICAD/DFF45 in H2O2-treated Neuro 2A cells. Cells were treated with 300 M H2O2 in the absence and presence of SWS for 8 hr. Cell lysates were used to measure the expression of their patterns by Western blotting with anti-bcl-xl/s(a), ICAD(B) and -actin (C) antibodies. A B C D a b c d Fig SWS inhibited the disruption of mitochondrial membrane potential transition in H2O2-treated Neuro 2A cells. Cells were treated with 300 M H2O2 in the absence and presence of 1200 g/ml SWS for 8 hr. Cells were stained with 10 g/ml of JC-1 (A, B, C and D, 50), 5 g/ml of Rhodamine 123 (a, b, c and d, 50) and visualized under a fluorescent microscope. The data were one of three independent experiments. Control cells (A, a), 300 M H2O2 (B, b), 1200 g/ml SWS only (C, c) and 300 M H2O2 with 1200 g/ml SWS treated Neuro 2A cells. 167

8 (168) 26 1(2005 3). -actin (Fig. 6) caspase-3 Bcl-2 family, Bcl-XL Bcl-XS. Neuro 2A 600, 1200 g/ml M H2O2 Western blot. Neuro 2A anti-apoptotic Bcl- XL 300 M H2O (Fig. 7A). H2O2 pro-apoptotic Bcl- XS.(Fig. 7A). effector caspase-3 ICAD/DFF45. H2O2(300 M) 8 ICAD/DFF g/ml H2O2 ICAD/DEF45.(Fig. 7B) H2O2 Neuro 2A caspase-3. -actin (Fig. 7C). 300 M H2O2 Neuro 2A caspase-3, Bcl-2 family. (membrane potential transition; MPT) JC-1 Rhodamine M H2O2 (Fig. 8B), 1200 g/ml (Fig. 8A) (Fig. 8D). Rhodamine 123 H2O2 diffusedpattern, 1200 g/ml punctuated-pattern, 1200 g/ml (Fig. 8).,,,,,,,,, 12)., 6), ), 65, 85 50%, Evans % 14). -amyloid, estroge, apolipoprotein E(Apo E), presenilin, oxidants (hydrogen, superoxide, hydroxyl radicals),,,, 168

9 (169) 15), -amyloid (neuritic plaques) (Tau) (neurofibrillary tangles, NFTs) neuritic plaques NFTs 16). Neuritic plaques (senile plaque) (amyloid plaque), -amyloid, (glial cell). Neuritic plaques plaques 17). amyloid plaque. Amyloid 40 -amyloid, amyloid -estrogen -secretase amyloid precursor protein (APP) amyloid APP amyloid plaque 18).., amyloid, St. George-Hyslop 19) 21 APP, -amyloid., Presenilin, presenilin 1 presenilin , 70% 20)., apolipoprotein E4 65, Apo E 19, Apo E4 Apo E2 17). APP pathway 21), -secretase -amyloid domail, amino terminal. sapp (secreted form of APP) cyclic GMP level 22)., alternative processing pathway -secretase -amyloid N terminal, -amyloid sequence (14-22KD) C anchor 23). C -amyloid sequence, secretase -amyloid, C.,,,,,, 24). 25),,,,,,,,,, 26). 27). 26) 28),. 27),,,, 29),,,,,. 169

10 (170) 26 1(2005 3),, 30).,,,,,,,,,,,, 31).,.,.,,,,,,,,, 2).,,,,,,,,,,,,,,, 32).,,,,,,,,,,,,, 2).,,,,,, 33)., 2). Neuro 2A H2O2, H2O2. Nuero 2A, H2O2. Neuro 2A. H2O2(300 M).. endotoxin,,,. heme heme (biliverdin, bilirubin) (Fe 2+ ) Heme Oxygenase(HO) 34). Heme Oxygenase isozyme(ho-1 HO-2). HO-2, HO-1. H2O2 Neuro 2A HO-1 HO-1. Bcl-2 (human follicular lymphoma) 170

11 (171) 35,36). Bcl-XL., Bcl-XS Bcl-XL 37). Bcl-2 family c-terminal. Bcl-2 Bcl-XL (MPT) cytochrome c 38). Bax Bcl-XS cytochrome c, 39). H2O2 Neuro 2A Bcl-XS Bcl-XL. apoptogenic Bcl-XS antiapoptotic Bcl-XL H2O2 Neuro 2A. Bcl- XL Bcl-XS H2O2. intermembrane space respiratory chain 40) cytochrome c Apaf-1, datp, procaspase-9 caspase-9. cytochrome c Bcl-2 Bcl-XL Bax 38). caspase- 9 caspase-3. H2O2 caspase-3 ICAD/DFF45,. Bcl-2 caspase Neuro 2A. Neuro 2A H2O2, caspase-3 ICAD/DFF45, Bcl-X L, Bcl-XS. Neuro 2A. 2-D DNA microarray. H2O2 Neuro 2A,, HO-1, Bcl-X L, Bcl-XS,. 1. H2O2 Neuro 2A. 2. H2O2. 3. H2O2 HO proapoptotic Bcl-XS, antiapoptotic Bcl-X L,. 5. H2O2 ICAD/DFF45 171

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical