LAS-1000(Fuji,Honshu,Japan)으로 검출하였다.
확인하였다.
1.5×105cells/well의 bEnd.3세포를 실험에 이용하기 전 날 6wellplate에 가하
IFN-β
배양하였다. 염색된 시료는 VECTASHIELDⓇ(VectorLab, Burlingame, CA, USA)로 slide glass위에 고정하였고 confocal laser scanning microscope와 exciting 480nm 및 emission 540nm(FV-300,Olympus,Tyoko,Japan)에서 형광 을 측정하였다.
7.Transfection andluciferaseassay
3×104cells/well의 bEnd.3 세포를 24well의 plate에 가하고 1일 동안 37°,5%
CO2배양 조건의 세포 배양기에서 배양 하였다.TransIT-LT1(MirusMadison, WI,USA)를 이용하여 NF-kB binding site(2X)-luciferasereporterplasmid 와 β-galactosidaseplasmid를 co-transfection하였다.4시간 동안 co-transfection한 후 completemedia로 교체하고 24시간동안 안정화 시켰다.이 후 1% penicillin streptomycin 만 첨가된 DMEM 500㎕를 넣어 12시간 동안 serum starvation처 리한 후 LPS,MALP-2또는 poly[I:C]와 IBC(0.1,1,5,10μM)를 동시 처리하여 12시간 동안 배양하였다.
배양이 끝난 세포는 차가운 (4°C)1 x PBS(phosphate buffered saline)로 2회 세척 후 각 well당 reporterlysis1x buffer를 100㎕ 씩 넣은 후 70°에 얼린 후 녹여 완전히 용해 시켰다.냉장 원심 분리기에서 14,000rpm 으로 20분간 원심분 리 하여 얻은 상층액은 Luciferase Assay System 과 β-galactosidaseEnzyme System(Promege,Madison,WI,USA)을 이용하여 activity를 확인하였다.
8.통계처리
모든 자료는 평균 ± S.E.M.으로 나타내었고 Student’st-test로 통계처리 하 여 P<0.05수준에서 유의성을 검증하였다.
Ⅳ.결과
sample은 12시간동안 배양하였다.IBC는 LPS에 의해 유도된 ICAM-1 발현을
(P<0.05)있는 저해효과를 확인 하였다(Fig.6).
경로가 아닌 TRIF의존적인 경로에서도 IBC의 영향이 관여되는지를 확인하기 위
Fig .1.Effect of IBC on the viability of cerebrovascular endothelial cells
bEnd.3cellswereincubated with IBC(0.1,1,5μM)for12,24or48hour in 5% CO2 incubator.Cellviability was determined by MTT assay.
MTT assay was performed as described in Material and Methods.
Values are expressed as mean ±S.E.M of3 samples.* P <0.05,** P
<0.001comparedwith control(CTL).
Fig .2. IBC suppressed LPS-induced adhesion of leukocytes to cerebrovascularendothelialcells.
bEnd.3 cells were incubated with LPS(1㎍/㎖) and varying concentration(0.1,1,5μM)ofIBC for12hourand furtherincubated with CMTMR pre-labeld U937 cells at37 °C for1hour.Adhered cells were imaged under confocalmicroscope.(A). Fluorescence intensity was measured with a spectrofluorometer(B).Valuesareexpressed asmean
±S.E.M of3 samples.# P < 0.01 compared with control(CTL),*P <
0.05comparedwith LPS alone.
Fig .3.IBC reduced LPS-induced ICAM expression in cerebrovascular endothelialcells.
bEnd.3 cells were exposed to LPS in presence ofIBC(0.1,1,5μM)for 4houror12hourto assessthelevelofmRNA and protein,respectively.
Theprotein and mRNA levels ofICAM-1 were determined by western blotting and RT-PCR as described in Materials and Methods.Values areexpressed asmean ±S.E.M of3samples.#P < 0.01compared with control(CTL),*P < 0.05comparedwith LPS alone.
Fig .4. IBC inhibited LPS-induced NF-kB transcription activity in cerebrovascularendothelialcells.
bEnd.3cellswereexposed toLPS(1㎍/㎖)in presenceofIBC(0.1,1,5μ M)to assessIkB-α phosphorylation(A),NF-kB translocation(B :green, Alexa FluorⓇ 488;blue,Hoechst33258),NF-kB luciferase activity(C). Luciferase and β-galactosidase enzyme activities were measured as described in Materialsand Methods.Luciferaseactivity wasnormalized with β-galactosidaseactivity.Valuesareexpressed asmean ±S.E.M of 3 samples.# P < 0.001 compared with control (CTL),* P < 0.05 comparedwith LPS alone.
Fig .5. Effect of IBC on LPS-induced IFN-β expression in cerebrovascularendothelialcells.
bEnd.3 cells were exposed to LPS in presence ofIBC(0.1,1,5μM)for 4hour to assess the levelof mRNA and protein,respectively.The mRNA levels of ICAM-1 were determined by western blotting and RT-PCR as described in Materials and Methods.Values are expressed as mean ±S.E.M of 3 samples.# P < 0.01 compared with control (CTL),*P < 0.05comparedwithLPS alone.
Fig .6. IBC inhibited LPS-induced IFN-β transcriptional activity in cerebrovascularendothelialcells.
bEnd.3 cells were co-transfected with IRF3 binding site(IFNβ
PRDⅢ-Ⅰ) -luciferase reporter plasmid and the expression plasmid of β -galactosidase.after24hour,cells were treated with LPS(1㎍/㎖)and IBC(0.1,1,5μM) for 12 hour,Luciferase activities were measured as described in Materials and Methods and luciferase activity was normalized with β-galactosidase activity.Values are expressed as mean ±S.E.M of3 samples.#P < 0.001 compared with control(CTL),
*P<0.01comparedwithLPS alone.
Fig .7.IBC suppressed MALP-2-induced ICAM-1 in cerebrovascular endothelialcells.
bEnd.3 cells wereexposed to MALP-2(10ng/㎖)in presenceofIBC(0.1, 1,5μM)for4houror12hourtoassessthelevelofmRNA andprotein, respectively.Theprotein and mRNA levelsofICAM-1weredetermined by western blotting and RT-PCR as described in Materials and Methods.Values are expressed as mean ±S.E.M of3 samples.# P <
0.001 compared with control(CTL),* P < 0.05,** P<0.01 compared with MALP-2alone.
Fig .8.IBC inhibited MALP-2-induced NF-kB transcription activity in cerebrovascular endothelialcells.
bEnd.3 cells were co-transfected with NF-kB binding site(2x)-luciferase reporter plasmid and the expression plasmid of β -galactosidase.After24hour,cellsweretreated with MALP-2(10ng/㎖) and IBC(0.1,1,5μM)for12 hour,Luciferase activities were measured as described in Materials and Methods and luciferase activity was normalized with β-galactosidase activity.Values are expressed as mean ±S.E.M of3 samples.#P < 0.001 compared with control(CTL),
*P < 0.05comparedwith MALP-2alone.
Fig .9. IBC reduced MALP-2 induced IkB-α phosphorylation in cerebrovascularendothelialcells.
bEnd.3 cells wereexposed to MALP-2(10ng/㎖)in presenceofIBC(0.1, 1,5μM)for15min toassessIkB-α phosphorylation.ThelevelsofI kB-α phosphorylation were determined by western blotting as described in Materialsand Methodsand Valuesareexpressed asmean ±S.E.M of3 samples.#P < 0.001compared with control(CTL),** P < 0.01and * P <0.05comparedwith MALP-2alone.
Fig .10. IBC suppressed on poly[I:C]-induced IFN-β expression in cerebrovascularendothelial cells.
bEnd.3cellswereexposedtopoly[I:C](1㎍/㎖)in presenceofIBC(0.1,1, 5μM)for4hourto assessthelevelofmRNA and protein,respectively.
Theprotein and mRNA levels ofICAM-1 were determined by western blotting and RT-PCR as described in Materials and Methods.Values are expressed as mean ±S.E.M of 3 samples.# P < 0.001 compared with control(CTL),* P < 0.05,** P<0.01 compared with poly[I:C]
alone.
Fig .11.IBC inhibited on poly[I:C]-induced IFN-β transcription activity on cerebrovascular endothelialcells.
bEnd.3 cells were co-transfection with IRF-3 binding site(2x)-luciferase reporter plasmid and the expression plasmid ofβ -galactosidase.after24hour,cells were treated with poly[I:C](1㎍/㎖) and IBC(0.1,1,5μM)for12hour,Luciferaseactivitiesweremeasureas described in Materials and Methods and luciferase activity was normalized with β-galactosidase activity. Values are expressed as mean ±S.E.M of3 samples.#P < 0.001 compared with control(CTL),
*P < 0.05,**P<0.01comparedwith poly[I:C]alone.
Fig .12.IBC reduced on poly[I:C]-induced ICAM-1 in cerebrovascular endothelialcells.
bEnd.3 cells were exposed to poly[I:C]in presence ofIBC(0.1,1,5μM) for 4 hour or 12 hour to assess the level of mRNA and protein, respectively.Theprotein and mRNA levelsofICAM-1weredetermined by western blotting and RT-PCR as described in Materials and Methods.Values are expressed as mean ±S.E.M of3 samples.# P <
0.001compared with control(CTL),*P < 0.05compared with poly[I:C]
alone.
Fig .13.IBC inhibited poly[I:C]-induced NF-kB transcription activity in cerebrovascular endothelialcells.
bEnd.3 cells were co-transfection with NF-kB binding site(2x)-luciferase reporter plasmid and the expression plasmid ofβ -galactosidase.After24 hour,cells were treated with poly[I:C](1㎍/㎖) and IBC(0.1,1,5μM)for12 hour,Luciferase activities were measured as described in Materials and Methods and luciferase activity was normalized with β-galactosidase activity.Values are expressed as mean ±S.E.M of 3 samples. # P < 0.001 compared with control (CTL),**P<0.01comparedwith poly[I:C]alone.
Fig .14.IBC reduced on poly[I:C]-induced IkB-α phosphorylation in cerebrovascularendothelialcells.
bEnd.3cellswereexposedtopoly[I:C](1㎍/㎖)in presenceofIBC(0.1,1, 5μM)for15min toassessIkB-α phosphorylation.ThelevelsofIkB-α phosphorylation were determined by western blotting as described in Materialsand Methodsand Valuesareexpressed asmean ±S.E.M of3 samples.# P < 0.001 compared with control(CTL),*** P < 0.001 comparedwith poly[I:C]alone.
Ⅴ.고 찰
비만세포,NK세포,regulatory T 세포와 같은 다양한 면역반응에 관련된 세포에
과 ICAM-1의 발현에서 농도의존적인 억제효과를 보였다 (Fig.2,Fig.3).TLR 4
해 공유되어진 중심적인 연결기이다.MyD-88이 결핍된 세포는 다양한 TLRs
을 담당하는 TANK-binding kinase1(TBK1)은 IKKi의 인산화를 유발해 IRF3 의 transcription을 통한 typeⅠIFN의 발현을 유도한다.TBK1은 typeⅠIFN의 발현을 유도할 뿐만 아니라 NF-kB promoteractivity를 증가시키는 또 다른 신 호전달계를 가지고 있다.TBK1의 두가지의 신호전달과정은 targetmolecule에 따라 반응하는 민감성이 다르며,서로 경쟁적인 역할로서 신호전달에 관여한다.
IBC를 처리하였을 때 ICAM-1의 발현과 IFN-β의 발현에 억제효과의 차이를 보인 것이 TBK1의 신호전달과정에서 보여 지는 targetmolecule에 따라 약물에 대한 민감성의 차이로 인해 억제효과의 차이가 발생한 것으로 예상되어진다.
하지만 TLR 4의 신호전달계는 MyD-88보다 TRIF에 의존적인 경로를 통해 거 의 대부분의 염증매개인자들이 경유하는 것으로 알려져 있다(Bjorkbackaetal., PhysioGenomics,2004).
본 연구에서는 IBC가 MyD-88과 TRIF에 의존적인 경로를 통해 억제함으로써 ICAM-1의 발현이 저해됨을 확인 할 수 있었으며,비록 두 경로에 대한 억제자 용에 차이가 있음에도 불구하고 IBC는 TLR4를 경유하여 발생되는 뇌 염증반응 조절에 효과적일 것으로 예상된다.
Ⅵ.결론
Isobavachalcone(IBC)이 LPS에 의해 유도된 세포부착분자 ICAM-1의 발현에 미 치는 영향에 대해 확인한 결과 다음과 같은 결론을 얻었다.
1.LPS에 의해 유도된 내피세포와 백혈구의 부착현상이 IBC에 의해 농도 의존 적으로 감소되었다.
2.LPS에 의해 유도된 세포부착분자 ICAM-1의 발현이 IBC에 의해 농도 의존적 으로 억제되었다.
3.IBC는 LPS에 의해 유도된 NF-kB 활성화를 농도 의존적으로 억제하였다.
4.MyD-88에 의존적인 경로에 특이적인 MALP-2에 의해 유도된 ICAM-1의 발 현이 IBC의 처리에 의해 농도 의존적인 억제 효과를 확인하였다.
5.TRIF에 의존적인 경로에 특이적인 poly[I:C]에 의해 유되된 ICAM-1및 I FN-β의 발현이 IBC에 의해 유의적으로 억제되었으나 농도의존적 억제 양상에는 차 이가 있었다.
따라서 IBC는 TLR 4신호전달계를 억제함으로써 세포부착분자의 발현 및 백혈 구-내피세포부착을 억제함으로써 뇌 염증반응을 개선할 수 있을 것으로 추정되 었다.
참고문헌
1.AdachiO,KawaiT,TakadaK,MatsumotoM,TsutsuiH,SakagamiM, NakanishK andAkiraS:TargeteddistruptionoftheMyD88generesults inlossofIL-1andIL-18-mediatedfunction.Immunity9(1):143-150,1998
2. Akira S,Uematsu S and Takeuchi O:Pathgen recognition and innate immunity.Cell124(4):783-801,2006
3.AlbeldaS and Buck C:Integrin and othercelladhesion molecules.FASEB Jounal4(11):2868-2880,1990
4.Alexopoulou L and Holt AC:Recognition of double-stranded RNA and activation ofNF-kappaB by Toll-like receptor3.Nature 413(6857):732-8.
2001
5.Amor S and Puentes F:Inflammation in neurodegenerative diseases. Immunology129(2):154-69.2010
6.Balyasnikova IV,Pelligrino DA,Greenwood J,Adamson P,Dragon S, Raza H, Galea E: Cyclic adenoosine monophosphate regulates the expression ofthe intercellularadhesion molecule and the inducible nitric oxide synthase in brain endothelialcells.J Cereb Blood Flow Metab 20:688-699,2000
7.BjörkbackaH,Fitzgerald KA,HuetF,LiX,Gregory JA,LeeMA,Ordija CM, Dowley NE, Golenbock DT, Freeman MW: The induction of
macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades. Physiol Genomics 19:319-307, 2004
8.Chen K andHuang J:CD40/CD40L dyadintheinflammatory andimmune responses in the centralnervous system.CellMolImmunol3(3):163-9, 2006
9.Clercka LSD,Bridtsa CH,Mertensa AM,Moensa MM and Stervens WJ: Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelialcells and the effectoffluorochromes on cellular function.JounalofImmunologicalMethods172(1):115-124,1994
10.Cusson-Hermance N,Lee TH,Fitzerald KA and Kelliher MA: Rip1 encephalomyelitis.Journalofneuroimmunology66(1-2):103-114,1996
13.Fitzgerald KA,Rowe DC,Barnes BJ,Caffrey DR,Visintin A,Latz E, MonksB,PithaPM,GolenbockDT:LPS-TLR4signaling toIRF-3/7and NF-kappaB involvesthetolladaptersTRAM andTRIF.JExpMed 198:
1043-1055,2003
14. Glass CK, Saijo K, Crotti A: Nuclear receptors, inflammation, and neurodegenerativediseases.AdvImmunol:106:21-59,2010
15.Hermance NC,Khurana S,Lee TH,Fitzgerald KA and Kelliher MA:
Rip1 mediates the TRIF-dependent Toll-like receptor3-and 4-induced NF-kB activation butdoesnotcontributetointerferon regulatory factor3
Rip1 mediates the TRIF-dependent Toll-like receptor3-and 4-induced NF-kB activation butdoesnotcontributetointerferon regulatory factor3