ª Ÿ (Korean J. Medicinal Crop Sci.) 17(2) : 145 150 (2009) j w s w Magnolol yz w *Á x *Á½ *Á *Á x**á y***á½ * *Á½ *Á½ *Á * * w w, ** w w, *** w w w Magnolol Attenuates Neuronal Cell Death Induced by Kynurenine Metabolite Chang Uk Lee*, Hyun Jung Lee*, Do Hee Kim*, Yeong Mi Jang*, Sang Hyung Lee**, Yoonh wa Jeong***, Dae Jin Kim*, Yoon Hee Chung*, Kyung Yong Kim*, Sung Su Kim*, and Won Bok Lee* *College of Medicine, Chung-Ang University, Seoul 156-756, Korea. **College of Medicine, Seoul National University, Seoul Municipal Borame Hospital, Seoul 156-707, Korea. ***College of Natural Science, Dankook University, Yongin 448-701, Korea. ABSTRACT : This study investigated the protective roles and mechanism of magnolol, from the stem bark of Magnolia officinalis against potential neurotoxin 3-hydroxykynurenine (3-HK)-induced neuronal cell death. For the evaluation of protective role of magnolol, we examined cell viability, apoptotic nuclei, change of mitochondrial membrane potential and caspase activity in human neuroblastoma SH-SY5Y cells. It was found that 3-HK induces neuronal cell death in the human neuroblastoma SH-SY5Y cell line. The reduced cell viability produced characteristic features such as cell shrinkages, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation. The cells treated with 3-HK showed an increase in the concentration of reactive oxygen species (ROS) as well as in caspase activity. In addition, both are involved in the 3- HK-induced apoptosis. Magnolol attenuated the cell viability reduction by 3-HK in both a dose- and time-dependent manner. Optical microscopy showed that magnolol inhibited the cell morphological features in the 3-HK-treated cells. Furthermore, the increase in the ROS concentration and the caspase activities by 3-HK were also attenuated by magnolol. These results showed that magnolol has a protective effect on the 3-HK induced cell death by inhibiting ROS production and caspase activity. Key Words : Magnolol, 3-hydroxykynureinine, Mitochondria, ROS, Caspase-3 3- j (3-Hydroxykynurenine, 3-HK) j, p mq y w. w xql (Pearson and Reynolds, 1992), qk (Ogawa et al., 1992), z v z e (Sardar et al., 1995) nw y ƒ y 3-HK ƒ ƒ ƒ. ƒ 3-HK sww j w (Lapin, 1980; Lapin, 1981). 3-HK w y» y w x w k w š ƒ» š. Nakagami et al. (1996) 3-HK w y» ƒ š šw. 3-HK sü y p s wš, nw y s p w k s w (Okuda et al., 1996; Okuda et al., 1998). ù w w z p w y, w³, w z š ù f w w k (Han et al., 2006; Lee et al., 2005). Magnolol (z ) z ù (Magnolia officinalis) Ë w, y ƒ š (Ikeda and Nagase, 2002; Zhong et al., 2003). w magnolol y, y, w y z, m, t, Corresponding author: (Phone) +82-2-820-5642 (E-mail) whitefox@cau.ac.kr Received March 6, 2009 / 1st Revised April 13, 2009; 2nd Revised April 21, 2009 / Accepted April 22, 2009 145
------------------------------------------------------------------------------------------------------------------ ( ( Á x Á½ Á Á xá yá½ Á Á½ Á½ Á šx, w w w ùkü s x x (Chiu et al., 1999; Lin et al., 2002; Loong et al., 2002). p, magnolol hydroxyl radical ROS scavenger y» w w w y w» š y» ƒ» w ƒ nw y, e w ƒ w ƒ., 3-HK w s s, y» caspase y y w magnolol z w. 3-HK nw y»w w, z ù Ë magnolol 3-HK w s w e ƒ w y w. 1. s s SH-SY5Y 10% fetal bovine serum (Gibco-BRL, CA, USA) Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL) 5% CO 2 ƒ œ» 37æ w. 3- j (3-HK, Sigma) 250 µm w» 2 1% fetal bovine serum DMEM ã. w magnolol w w, 10 µm š w w. 2. s d (alarmablue assay) SH-SY5Y s 96-well plates (Nunc, Slangerup, Denmark) 15,000 cells/well š 24 w. s 3-HK w» 2 1% fetal bovine serum DMEM. z AlarmaBlue (Serotec, Oxford, UK) 10 w z 3 wš ELISA Reader (Molecular Devices, Sunnyvale, CA, USA) w 570 Ÿ d w. Ÿ 600 d w. s œ w (Shimoke and Chiba, 2001). s = ( test sample count ) blank count ) 100 ( untreated control count) blank count) 3. Hoechst 33258 magnolol 3-HK 2 wš, 36 z SH-SY5Y s 4% q s 20 š w, 5 Hoechst dye 33258 8 / k. phosphate buffered saline wš xÿ x w. s ù s xk j p w ƒù w s xk y w. 4. y» d 2', 7'-dichlorofluorescein diacetate (DCF-DA) xÿ w 3-HK w w y» d w (Kim et al., 2008, Lee et al., 2007). d wš w SH-SY5Y s 30 DCF-DA 10 µm k. s PBS w. s š k z s xÿ x w. 5. d (mitochondrial membrane potential, Ψm) y d w» w tetramethylrhodamine ethyl ester (TMRE; Molecular Probes, Eugene, OR, USA) w. Ψm d w» w TMRE 100 nm 15 37æ wš TMRE xÿ fluorescence microscopy (Olympus IX70, Olympus Optical Co. GmbH, Hamburg, Germany) w. 6. caspase-3 y d SH-SY5Y s 10 150 g g š, 50 mm Tris, ph 7.5, 0.03% NP-40, 1 mm dithiothreitol (DTT)ƒ sw lysis 100 s r ww. w s 20 ew z, 15,800 g 5 w. z d w. caspase» w s d 20 caspase-3» Ac-DEVD- AMC 0.25 mm z 1 37æ w. TECAN GENios Fluorescence microplate reader (Tecan, Switzerland) w 380 460 q caspase- 3 y d w. z y txw. 7. m mean ± SEM t w š, x Student's t-test w y w. 1. 3-HK w s w magnolol wz Fig. 1A, 250 µm 3-HK w SH-SY5Y s s w y w 146
에 의한 신경세포 사멸에 대한 3-HK 의 보호효과 Magnolol Effect of magnolol on 3-HK-induced neuronal cell death. (a) SH-SY5Y cells were replaced with 1%FBS/DMEM 2 h prior to the 3-HK treatment. The cells were treated with 3-HK for various time. The cell viability was determined at indicated time points. (b) The cells were pretreated with 10 µm of magnolol for 2 h. 250 µm of 3-HK were then added. The 3-HK was dissolved in DMSO (final conc. < 0.1%). Cell viability was assessed by the alarma blue assay. The values are a mean ± SEM of three separate experiments. Fig. 1. Morphological assessment of apoptosis by phase-contrast and fluorescence microscopy. The SH-SY5Y cells were either not treated (a, d) or treated (b, e) with 250 µm of 3-HK for 36 h. The magnolol pretreated (10 µm of magnolol for 2 h) cells were treated with 250 µm of 3-HK for 36 h (c, f). The figures show the optical microscopic morphology (a, b, c) and Hoechst 33258 stained nuclear morphology over fluorescence microscope (d, e, f). The figures are representative of three different experiments. Fig. 2. 2. 3-HK에 의한 magnolol의 효과 어포토시스형 세포 사멸을 억제하는 3-HK에 의해 나타나는 신경세포 사멸의 형태학적 변화를 관 찰하기 위해 위상차 현미경으로 세포의 외형을 관찰하고 (Fig. 2A-C), 핵 형태의 변화를 확인할 수 있는 Hoechst 33258 염 색을 통해 형광 현미경 하에서 변형된 핵 모양을 관찰하였다 (Fig. 2D-F). 250 µm의 3-HK에 36시간 가량 노출된 SH- 다. 250 µm의 3-HK를 처리한 경우 36시간 내에 세포 생존율 이 50% 정도 감소되었다. 그러나 이때 magnolol을 2시간 전 처리 한 경우 3-HK에 의한 신경세포 사멸을 효과적으로 억제 하였다 (Fig. 1B). 이러한 magnolol의 효과는 3-HK에 의한 세포 사멸이 증가되어도 지속되는 것을 알 수 있었고, 본 연 구에서는 48시간이 지나도 magnolol의 세포 보호효과가 유지 됨을 확인하였다. 147
Á x Á½ Á Á xá yá½ Á Á½ Á½ Á Fig. 3. Determination of ROS generation by 3-HK with 250 µm after pretreatment with magnolol. (a) Levels of ROS generation were measured using the fluorescent probe 2', 7'-dichloroflorescein-diacetate (DCF-DA). The effect of pretreatment of magnolol is presented. For detecting ROS generation, the cells were incubated with 10 µm DCF-DA for 30 min. And excitation at 485 and emission at 530 were measured with fluorometer (Materials and Methods). The difference from the cells treated with 3-HK alone was statistically significant (P < 0.05). SY5Y s s sy, s xk y š, Hoechst 33258 w w xk DNA w y ùkü. ù 3-HK magnolol w s 3-HK w ùk ù sm x s xkw yƒ w š y w (Fig. 2C, F). 3. 3-HK w s magnolol w y z y» s k y p. 250 µm 3-HK w ü w y» ƒ ƒw w (Fig. 3). DCF xÿ y» t y w, 3- HK w 15 ü w y» 30 1 ¾ j ƒwš z w y w. w w magnolol 3-HK w w y» x w û y w (Fig. 3). y» d w» w fluorometer w excitation 485 emission 535 q DCF xÿ w. 4. 3-HK w w magnolol yz y p w» w w Fig. 4. Effect of pre-treatment with magnolol on mitochondrial membrane potential (MMP) in SH-SY5Y cells treated with 3-HK of 250 µm. Upper graph: Level of TMRE fluorescence was quantificated with Tecan Fluorometry in excitation at 550 and emission at 590. Lower images: cells plated on glass coverslips were maintained for 6 h at 37 in either culture medium alone (left) or medium supplemented with 3-HK (medium), 3-HK+ magnolol (right), as indicated. Cells were then loaded with 100 nm TMRE and evaluated by fluorescence microscopy. ƒ š, 3-HK w y» w w, 3-HK w» wƒ y wš, w magnolol w y» w ( Ψm) d w. Ψm d w q w, Ψm d w» w TMRE w. Fig. 4 v 3-HK w 6 z Ψm w. ù magnolol w z 3-HK w 3-HK w Ψm z ww. w xÿx w TMRE w w w w, 3-HK TMRE ù w. ù w magnolol wì w 3-HK w TMRE y w (Fig. 4). 5. 3-HK w s magnolol caspase-3 y y z s w l v l q z wù caspaseƒ 3-HK w s» w pan-caspase w zvad-fmk 148
3-HK w s w Magnolol yz Fig. 5. Effect of magnolol on 3-HK-induced caspase-3 activity. CTL was vehicle-treated SH-SY5Y cells (control). The cells were either not incubated (3-HK) or incubated (magnolol+3- HK) with 10 µm of magnolol for 2 h, which was then followed by treatment with 250 µm of 3-HK for 6 h. Fifty micrograms of the cellular extracts were incubated with 0.5 mm AC-DEVD-AMC in a total volume of 100 at 37 for 1 h. Excitation at 380 and emission at 460 were measured with a fluorescence microplate reader (TECAN, GENios). The enzymatic activity is expressed as arbitrary units of relative values. The values are reported as a mean ± SEM of three separate experiments ( * P < 0.05). w. magnolol caspase y y w z» w caspase-3 xÿ» Ac-DEVD-AMC w caspase-3 y y y w. 3-HKƒ caspase-3 y y 2 ƒ w, magnolol ù pan-caspase w zvad-fmk 3-HK w caspase-3 y y y w û w. š 3-HK xql (Pearson and Reynolds, 1992), qk (Ogawa et al., 1992), z v e (Sardar et al., 1995) nw y x j ƒw. w» n, ý, ƒ x ù ƒ s 3-HK y w q mw (Savvateeva et al., 2000). 3-HK š, w 3-HK k B6 v y» w x (Guilarte and Eastman, 1993). 3-HK sƒ 3-HK s w. w 3-HK w s y» caspase-3 y yƒ ƒw w, sm x s t, 3-HK w ƒw. w 3-HK w s w magnolol w w. magnolol 3-HK wì w s w. 3-HK w s w magnolol yz ƒ y» x wwš, caspase y y ƒ w. y» üv s s ƒ s k sm x s w (Jacobson, 1996). y» ƒ sm x s (Buttke and Sandstrom, 1994), ƒ w y» ƒ s w wš wš. w y p ƒ s w š, w w y ƒ w s ww (Lennon et al., 1991), y» ƒ ƒ w š (Olanow, 1993). 3-HK w s magnolol f y» w w y w w. sm x s, v s ƒ w w. w s t FASù p w, ER p sw. p caspase s w w w l v l family caspase w caspase š y y g s v w w s w w w (Wolf and Green, 1999). w w caspase wùƒ caspase-3 š, s w w z w s w. Caspase-3 w y y ƒ s w w ƒ, s ü caspase-3 y y ƒ w» (Thornberry and Lazebnik, 1998). 3-HKƒ caspase-3 y 2 ƒ š, w ƒ 3-HK w ùkù s ew. w caspase-3» DEVD-AMC mw magnolol w y z r. Magnolol 3-HK wì w caspase-3 y 3-HK 50% ƒ û caspase-3 y y ùkü. w magnolol 3-HK w s w w wš caspase y y wš. y w ƒ nw y ùkù p ùküš, y», s x y 149
Á x Á½ Á Á xá yá½ Á Á½ Á½ Á caspase-3 y y 3-HK w s z ù Ë magnolol ƒ z ww š. w mw l w w w w y e z k w, p magnolol nw y e, wš z w magnolol» w ƒ¾ w w. 2008w w w w w. LITERATURE CITED Buttke TM and Sandstrom PA. (1994). Oxidative stress as a mediator of apoptosis. Immunology Today. 15:7-10. Chiu JH, Wang JC, Lui WY, Wu CW and Hong CY. (1999). Effect of magnolol on in vitro mitochondrial lipid peroxidation and isolated cold-preserved warm-reperfused rat livers. Journal of Surgical Research. 82:11-16. Guilarte TR and Eastman CL. (1993). Is 3-hydroxykynurenine an endogeneous neurotoxin in Huntington's disease? Journal of the Neurological Sciences. 116:227-228. Han SH, Woo NRY, Lee SD and Kang MW. (2006). Antioxidative and antibacterial activities of endemic plants extracts in Korea. Korean Journal of Medicinal Crop Science. 14:49-55. Ikeda K and Kagase H. (2002). Magnolol has the ability to induce apoptosis in tumor cells. Biological & Pharmaceutical Bulletin. 25:1546-1549. Jacobson MD. (1996). Reactive oxygen species and programmed cell death. Trends in Biochemical Sciences. 21:83-86. Kim JY, Ju HS, Ban JY, Song KS and Seong YH. (2008). Mountan cortex extract inhibits amyloids β protein (25-35)- induced neurotoxicity in cultured rat cortical neurons. Korean Journal of Medicinal Crop Science. 16:409-415. Lapin IP. (1980). Taurine selectivity antagonizes L-kynurenineproduced seizures in mice. Journal of Neural Transmission. 48:311-316. Lapin IP. (1981). Kynurenines and seizures. Epilepsia. 22:257-265. Lee SB, Kim JY, Cho SO, Ban JY, Ju HS, Bae KH and Seong YH. (2007). Extract of Cedrela sinensis leaves protects neuronal cell damage induced by hydrogen peroxide in cultured rat neurons. Korean Journal of Medicinal Crop Science. 15:444-450. Lee SY, Hwang EJ, Kim GH, Choi YB, Lim CY and Kim SM. (2005) Antifungal and antioxidant activities of extracts from leaves and flowers of Camellia japonica L. Korean Journal of Medicinal Crop Science. 13:93-100. Lennon SV, Martin SJ and Cotter TG. (1991). Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Proliferation. 24:203-214. Lin SY, Liu JD, Chang HC, Yeh SD, Lin CH and Lee WS. (2002). Magnolol suppresses proliferation of cultured human colon and liver cancers cells by inhibiting DNA synthesis and activating apoptosis. Journal of Cellular Biochemistry. 84:532-544. Loong CC, Chiu JH, Tiao RC, Chiu YY, Wu CW and Lui WY. (2002). Protective effects of magnolol on the small intestinal ischemia-reperfusion injury. Transplantation Proceedings. 34: 2679-2680. Nakagami Y, Saito H and Katsuki H. (1996). 3-Hydroxykynurenine toxicity on the rat striatum in vivo. The Japanese Journal of Pharmacology. 71:183-186. Ogawa T, Matson WR, Beal MF, Myers RH, Bird ED, Milbury P and Saso S. (1992). Kynurenine pathway abnormalities in Parkinson's disease. Neurology. 42:1702-1706. Okuda S, Nishiyama N, Saito H and Katsuki. (1996). Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine. Proceedings of the National Academy of Sciences of the United States of America. 93:12553-12558. Okuda S, Nishiyama N, Saito H and Katsuki H. (1998). 3- Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity. Journal of Neurochemostry. 70:299-307. Olanow CW. (1993). A radical hypothesis for neurodegeneration. Trends in Neurosciences. 16:439-444. Pearson SJ and Reynolds GP. (1992). Increased brain concentrations of a neurotoxin, 3-hydroxykynurenine, in Huntington's disease. Neuroscience Letters. 144:199-201. Sardar AM, Bell JE and Reynolds GP. (1995). Increased concentrations of the neurotoxin 3-hydroxykynurenine in the frontal cortex of HIV-1-positive patients. Journal of Neurochemistry. 64:932-935. Savvateeva E, Popov A, Kamyshev N, Bragina J, Heisenberg M, Senitz D, Kornhuber J and Riederer P. (2000). Agedependent memory loss, synaptic pathology and altered brain plasticity in the Drosophila mutant cardinal accumulating 3- hydroxykynurenine. Journal of Neural Transmission. 107:581-601. Shimoke K and Chiba H. (2001). Nerve growth factor prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced cell death via the Akt pathway by suppressing caspase-3-like activity using PC12 cells: relevance to therapeutical application for Parkinson's disease. Journal of Neuroscience Research. 63:402-409. Thornberry NA and Lazebnik Y. (1998). Caspases: enemies within. Science. 281:1312-1316. Wolf BB and Green DR. (1999). Suicidal tendencies: apoptotic cell death by caspase family proteinases. Journal of Biological Chemistry. 274:20049-20052. Zhong WB, Wang CY, Ho KJ, Lu FJ, Chang TC and Lee WS. (2003). Magnolol induces apoptosis in human leukemia cells via cytochrome C release and caspase activation. Anti-Cancer Drugs. 14:211-217 150