[핸
e J,쁘rnal01 M어i디ne and비e ScienceIsoacteoside induces heme oxygenase-I via activation of
ERK
and factor2
transcription factlDong
Ok
Ko,
Kyoung Ah Kang,
and Jin WOI Departmenl이Biochemistry,Jeju Nati。때UniversitySCh∞
1이MedicirAbstract
Key Words 녕。acteoside,NF-E2 relaled factor 2 (N꺼2). Anlioxidant el
’
ecl5,2009
J
Introduction
Address for correspOndence: Jin Won HyUn
Depar1mentof Bìochemìslry,Jeiu NationalUniversitySchool
。
Medicine.66 Jejudaehakno,690-756,Jeju. KoreaE-mail:jinwonh@jejunu.ac.kr
the modulalion of ARE driven gene expression via Nrf2 activation. ERK and p38 MAPK pathways involvcd the nucleus binding of. Nrf2 to the ARE.13l Isoacteoside was iso!ated from Clerodendron trichotomum Thunb that belongs to Verbenaceae family.14.15) It has pharmacological cffects and 없ü-infl없nmaωryeπ'ects.16. 17)Many researchers have
studied the effect of isoacteoside in a variety of cells However‘HO-l induction of isoacteoside in lung cells has
not been reported unti1 now깨e presenl sludy investigates
the antioxidanl effed via HO-l induction of isoacteoside in V79-4 ceUs
Materials and meth
。
ds 1. ReagentsIsoacleoside was provided by Dr. Sungwook Chae of Korea Jnslitute of Oriental Medicine. Daejeon. Korea Primary rabbit polyclonal phospho ERK. Nrf2 antibodies were purchased from Santa Cruz BiotechnoJo양,Santa Cruz,
CA. USA. Primary mouse monoclonal HO-l antibody was purchas잉d from Str잉ssgen Corp‘Victoria. Canada
2,Cell cullure
Chinese hamster lung fibroblasls (V79-4) ce!is from the American twe culture co!iecüon (Rockvilie,MD,USA) were
maintained at 37
t
in an incubator. with a humidified atmosphere of 5% CÜ2 and cultured in Dulbecco's modified Eagle‘5 medium containing 10% heat-inactivated fetal calf359
Dong Ok Ko,KyoungAh Kang,Jin Won Hyun
seπm. sb .•ptomycin (J()() g/mI) and penicillin (J()() units/mI) Figure 1. Chemi잉1 sσucωre of isoacteoside
0136122
“
8h Figure 3. Effect of isoactcoside on Nrf2 expression Westem blot analysis was performed using Nrf2 specific antibodyFigure 2,Erfect of isoacteoside on HO-l expression Westem blot analysis was performed using HO-1 specific antibody.
관:
,。뿔각
24 48h훌훌
~I
12 6 o HCι1 f\-Actin 3. Western blot analysisThe cells were hroγested. washed twice with PBS‘lySed
。
n ice for 30 min in 100 /L~ of a Iysis buITer [120 mM NaCI.4αnM 까is (pH 8)‘0.1% NP 40] and then cenbifuged at 13‘α)()Xg for 15 min 끼1e supematants were collec
‘
ed from the lysates and the protein concentrations were detennined Aliquots of the lySates (40 앵of protein) were boiled for 5 min and e\ectrophoresed in 10% sodium dodecylsulfate-polyacrylamidegel η1e blots in the gels were transferred。
nto nitrocellulose membranes (Bio-Rad. Hercules. CA. USA). which were then incubated with the primary antibody π113 membranes were further incubated with the secondary imrnunoglobulin-G-horseradish peroxidase conjugates(Pierce Rockford. IL,USA). Protein bands were detected using anenhanced chemi\uminescenceWesLem blotting detection kit (Amersham‘LitUe Cha\font,Buckinghamshire. UK). and then exposed onto X -ray film
[~~- ---'F
용굶과글써딛웰웰둔~-~~→←
j
Figure 4,Effect of isoacteoside on phospho ErK expression. Western blol analysis WD.S performed using phospho grK specific anlibody‘
NR다
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톨~"•••--_ •••_--ß--AclinI ..
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j) Cooke MS. MistIγ N. Wood C. Herbert KE. Lunec J Immunogenicity of DNA damaged by reactive oxygen species-Implications for anti-DNA antibodies in \upus
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6h삐
p"",야mE<K Oxidative stresses are involved in the process ofmulti-stage carcinogenesis. F1avonoid has polyphenol groups which are found in cofTee. beer. wine and fruits and vegetables Polyphenols have reported to reduce the risk of cardiovascular disease and increase the antioxidant capacity in KB cells.18. 19) AIU10ugh many studies have reported the antioxid없t effects of f1avonoids‘there is no report on the antioxidant effects via HO-l induction of isoacteoside ,which
belongs to flavonoid g:roup. in lung fibroblast cells. HO-l is a defensive enzyme against oxidative stress. HO-l degrades heme inlo carbon monoxide (CO)‘iron. and biliverdin. As shown Fig 2. u-eatmenl of isoacæoside increased the HO-l protein expression in a time dependent manner. Moreover lhe transcription faclor‘Nrf2. regulates the antioxidant response elemenl (ARE) of lhe phase 2 detoxifying and anlioxidant enzymes. resu\ting in induction of HO-l expression. Isoacteoside increased Nrf2 expression up to 48h as shown in :Fig 3,FurU1.ermore‘isoacteoside increased Lhe phospho Erk expression. which is upslream of Nrf2,at
6h(Fig 4)
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