A HaCaT p38mapk. caspase23

Transcription

1 ca spa se23 A HaCaT p38mapk 1, 2, 1, 3, 3, 4, 13 (11, ; 21, ; 31, ; 41, ) : p38 (p38 MAPK) 23 ( caspase23), (poly2 peptide from Chlam ys farreri,pcf) A (UVA ) HaCaT 6 : UVA UVA mmol L -1 CUVA mmol L -1 PCFUVA mmol L -1 PCF UVA mmol L -1 PCF UVA HaCaT ; PCF p38 MAPK ( SB203580) caspase23 (Ac2DEVD2CHO ) ; p38mapk p38 MAPK ; caspase23 PCF UVA HaCaT ; SB Ac2 DEVD2CHO UVA HaCaT ; mmol L -1 PCF UVA p38 MAPK caspase23 PCF UVA HaCaT, p38 MAPK caspase23 : ; A ; ; 23; ;HaCaT : R967; R :A : (2007) Inh ib ition of Polypeptide from Chlamys farreri on UVA 2Induced Apoptosis of HaCaT Cells D epend ing on p38mapk Pathway and Caspase23 LIJin2lian 1,YAN Zhou2ping 2,CHEN Xue2hong 1,WANG Yue2jun 3,SUNMi 3,SHIYu2xi 4,WANG Chun2bo 13 ( 11 M edical College, Q ingdao U niversity, Q ingdao , China; 21 Haici M edical Treatm ent Conglom erate, Q ingdao , China; 31 Yellow Sea Fisheries Research Institute, Chinese Academ y of Fishery Sciences, Q ingdao , China; 41 D epartm ent of C lin ica l Medicine, BinzhouMedicalCollege, Binzhou , China) ABSTRACT: OBJECTIVE To investigate the inhibiton of polypep tide from Chlam ys farreri ( PCF) on UVA 2induced HaCaT cells apoptosis through p38 mitogen activated protein kinase (MAPK) pathway and caspase231methods Experiments were divided into six group s: con tro l group, UVA mode l group, UVA mmol L -1 vitam ine C positive control group, UVA mmol L -1 PCF group, UVA mmol L -1 PCF group, UVA mmol L -1 PCF group1 UVA 2induced apop totic model of HaCaT cells was established by orthogonal design1 U sing agarose gel electrophoresis, the effects of PCF, p38 MAPK inhibitor SB and caspase23 inhibitor Ac2DEVD2CHO on UVA2induced apop tosis were investigated1 Exp ression levels of p38 MAPK and phosphorylated p38 MAPKwere determined bywestern blot analysis1 Caspase23 activitywas assayed by flow cytometry1results PCF significantly p rotected UVA2induced apop tosis1 SB and Ac2DEVD2CHO had inhibitory effects on UVA2induced apop tosis of HaCaT cells1 PCF inhibited UVA2induced phosphorylation of p38mapk and activation of caspase23 onadose2dependented manner1co NCL US IO N PCF can p rotect HaCaT cells from UVA2induced apop tosis1 Its inhibitory effect on apop tosis may attribute to inhibition of activation of p38 MAPK and caspase231 KEY WO RD S:polypep tide from Chlam ys farreri; ultroviolet A; m itogen activated protein kinase; caspase23; apop tosis; HaCaT A ( nm ) B ( nm), [1] A (UVA ) 90% 99%,,, UVA (polypep tide from Chla2 : ( ) ; ( Y2003c02) :,, 3 :,,, Tel: ( 0532) Fax: ( 0532) E2 mail: cbwang666@1261com 116 Chin Pharm J, 2007 January, Vol142 No

2 m ys farreri, PCF), 879,PCF UVA HeLa [224] HaCaT, PCF (ROS) (MDA ), ( SOD ) ( GSH2Px) [5] p38 (mitogen activated protein kinase, MAPK) caspase23 PCF UVA HaCaT PCF (, >96%, mmol L -1,4 ) ;DMEM ( Gibco ) ;p38 caspase23 (Ac2 DEVD2CHO )(Santa C ruz B iotechnology) ; p38 ( Cell Signaling Technology) ; caspase23 (BD B iosciences) ; 2 ( ) ;p38 SB ( Calbiochem ) ; ( Propidium iod ide, P I) NP - 40 ( Sigma ) ; ( ) ;FACS- vantage ( Becton Dickinson ) 112 HaCaT( ) DM EM ( 10%, u L mg L -1 ),37,5%CO 2, 6 : UVA UVA mmol L -1 C (VitC ) UVA mmol L -1 PCF ( PCF1 ) UVA mmol L -1 PCF ( PCF2 ) UVA mmol L -1 PCF ( PCF3 ) 80% 90%,, PB S 2, PB S 2 ml, 113, PB S 2, 1L , 50 mg L -1 PI ( NP240 RNA ) 200 L,,4 30 m in 400 FACS DNA, nm, CV <2%,FSC/SSC , Modfit LT 114 UVA HaCaT 3, :A2UVA ( J cm -2 ) ;B2 ( h) L 9 ( 3 4 ),, 1 1 Tab 1 Factors and levels of orthogonal test Factors Levels A B % 90%, PCF 2h, p38 MAPK SB ( 10 mol L -1 ) caspase23 Ac2DEVD2CHO ( 100 mol L -1 ) 1h, UVA 18 h,pbs 2, 500 L [150 mmol L -1 NaCl, 10 mmol L -1 Tris2HCl (ph 715 ) 10 mmol L -1 ED TA, 015% SDS, 500 mg L -1 K],,50 ; / (1 1), 1, g 5min, ; 1/10 3 mol L -1 2, - 20 DNA; g 10 m in,, 70% 1 ; 50 L TE [10mmol L -1 Tris2HC1 (ph 810),10mmol L -1 ED TA ], RNA, 0102 g L -1,37 30 m in; 15 g L -1, DNA ladder 116 p38 80% 90%, 2h PCF, 30 m in : PB S 3, PB S, L [20mmol L -1 Tris2HC1 ( ph 715 ),150mmol L -1 NaCl, 1 mmol L -1 ED TA, 1 mmo l L -1 EGTA, 1% Triton X2 100, 215 mmol L -1 ( sodium pyrophos2 phate),1 mmol L -1 2 ( 2glycerophos2 phate),1mmol L -1 Na 3 VO 4,1mg L -1 ( leupep tin),1mmol L -1 (phenylmethyl2 sulfonyl fluoride) ], 30 m in; 117 Chin Pharm J,2007 January, Vol142 No12

3 115 ml, r min m in, ; (BCA, ), 40 g,10% SDS2 PA GE ;, (1 TB S, 011% Tween220, 5% BSA ) 1h; ( ) ( ) p38 MAPK,4 ;TBST 3, 5min; IgG, 1h;TBST 3, 5min; (DAB ) Quantity one 117 caspase23 UVA 2h PCF, 12 h , PB S 2, 4% 2mL 30 m in;, TritonX2100, 10 m in; PBS 2, caspase23, 30 m in 2, caspase ;SPSS Q UVA HaCaT L 9 (3 4 ) 9,, 2, 3 ( P <0101), A 2 B 2, 8J cm -2 UVA HaCaT 18 h 2 Tab 2 O rthogonal design and apop totic rate No. A B Error Error Apoptotic rate /% PCF UVA HaCaT 1,UVA DNA, C PCF 3 Tab 3 Variance analysis of orthogonal design Sou rce of va ria tion l MS F P A < B < Error : F (2, 22) =5. 72 Note: F (2, 22) =5. 72 C mmol L -1 PCF UVA HaCaT 1 PCF UVA HaCaT DNA M-marker;1-UVA mmol L -1 ;2-UVA mmol L -1 ;3-UVA mmol L -1 ;4-UVA+ C (5168 mmol L -1 ) ;5-UVA ;6- Fig 1 Effect of PCF on UVA 2induced DNA fragmentation in HaCaT cells M-marker; 1-UVA+ PCF5169 mmol L -1 ;2-UVA + PCF2184 mmol L -1 ;3-UVA + PCF1142 mmol L -1 ; 4 - UVA +Vit C(5168 mmol L -1 ) ; 5 - UVA model ; 6 - control 213 SB Ac2DEVD2CHO UVA HaCaT p38 MAPK caspase23 UVA HaCaT, p38 MAPK SB caspase23 Ac2DEVD2CHO, SB DNA ladder, p38 MAPK UVA HaCaT ; Ac2DEVD2CHO DNA ladder, UVA HaCaT caspase PCF p38 p38 3 UVA HaCaT p38 p38 (p2p38) p38 p38 2, p38 p2p38 p38 C PCF p38 ( P >0105) ( 3B ) ; UVA 30 m in, p2p38, C PCF p2p Chin Pharm J, 2007 January, Vol142 No

4 2 SB Ac2DEVD2CHO UVA HaCaT DNA M - marker; 1 - UVA + SB203580; 2 - UVA + Ac2DEVD2CHO; 3 - UVA ;4- Fig 2 Effect of SB and Ac2DEVD2CHO on UVA 2in2 duced DNA fragmentation in HaCaT cells M - marker; 1 - UVA + SB203580; 2 - UVA + Ac2DEVD2CHO; 3 - UVA model; 4-control ( P <0101), PCF p38, ( 3C) PCF UVA caspase23, ( P <0101) ;PCF caspase23 caspase23 : ( % ) = [ (UVA caspase23 - caspase23 ) /UVA caspase23 ] PCF UVA HaCaT caspase23 1n =3, gx s Tab 4 Inhibitory effect of PCF on activities of caspase23 in UVA 2radiated HaCaT cells1n =3, gx s Caspase23 activity Inhibition Groups ( fluorescence intensity) ratio / % Control/mmol L UVA ) - UVA +V it C /5. 68 mmol L ) UVA + PCF1 / ) UVA + PCF2 / ) 4) UVA + PCF3 / ) 3) 5) : 1) P <0101 ; 2) P <0101 UVA ; 3) P <0101, 4) P < 0105 UVA + PCF1 ; 5) P <0105 UVA + PCF2 Note: 1) P <0101, vs control; 2) P <0101, vs UVA; 3) P <0101, 4) P <0105, vs UVA + PCF1; 5) P <0105, vs UVA + PCF2 3 PCF UVA p38 MAPK A-p38 p38 ;B - p38 ;C- p38 ;Ctr- ; UVA - UVA ;VitC - C ( 5168 mmol L -1 ) ;PCF13- (5169, 2184, 1142 mmol L -1 ) ; 1) P <0101 ; 2) P <0101 UVA ; 3) P <0101 UVA + PCF1 ; 4) P <0105 UVA + PCF2 Fig 3 Effect of PCF on UVA 2induced p38 MAPK p rotein ex2 p ression A - p rotein expressions of p38 and phosphorylated p38; B - exp ression level of p38; C - exp ression level of phosphorylated p38; Ctr - control; UVA - UVA model; VitC - vitamin C ( 5168 mmol L -1 ) ; PCF13 - PCF ( 5169, 2184, 1142 mmol L -1 ) ; 1) P <0101, vs control; 2) P <0101, vsuva model; 3) P <0101, vs UVA + PCF1; 4) P <0105, vs UVA + PCF2 215 PCF UVA caspase23 HaCaT caspase23, 4 UVA 12 h, caspase23, ( P <0101) ; ,Miyachi [6] 1983 ( sunburn cell), HaCaT, C E PCF,PCF UVA [5], :PCF p38 MAPK caspase23,pi PI, 8J cm -2 UVA HaCaT 18 h DNA , (DNA ladder), PCF UVA HaCaT,, mmol L -1 PCF UVA DNA ladder (MAPK) 119 Chin Pharm J,2007 January, Vol142 No12

5 p38 MAPK, p38 MAPK,, p38 MAPK :Assefa [7],p38 MAPK C UVB HaCaT ;,Chouinard [8], UVB p38 MAPK p53,p38mapk, ( ) p38 MAPK p38 p38 MAPK, p38 SB DNA ladder, p38 MAPK UVA HaCaT UVA HaCaT 30 m in, p38, PCF p38, PCF p38 MAPK HaCaT caspase Caspase23, caspase23,, caspase23 Ac2DEVD2CHO, UVA HaCaT DNA ladder, UVA HaCaT caspase23 caspase23 caspase23, caspase23 PCF, UVA caspase23,, PCF caspase23 UVA HaCaT Assefa [7], p38 MAPK UVB caspase23, caspase23 p38 MAPK,,p38MAPK caspase23,p38mapk caspase23, UVA 30 m in p38,12 h caspase23,18 h, p38 MAPK caspase23 UVA HaCaT PCF UVA HaCaT, p38 MAPK caspase23 PCF UVA HaCaT, PCF,,p38MAPK p53, AP21,NF2 B, PCF p38mapk, PCF REFERENCES [ 1 ] DE GRU IJL F R1 Skin cancer and solar UV radiation [ J ]1EurJ Cancer, 1999, 35 (14) : [ 2 ] WANG C B, YAO R Y, L IU ZT, et al1 Protective effect of poly2 peptide from Chlamys farreri on hairlessm ice damaged by ultravi2 oleta [J ]1Acta Pharmacol Sin ( ), 2002, 23 (9) : [ 3 ] YAO R Y, WAN G C B 1 Protective effects of polypep tide from Chlam ys farreri on Hela cells damaged by ultraviolet A [ J ]1Acta Pharm acol S in ( ), 2002, 23(11) : [ 4 ] HAN Y T, HAN Z W, YU G Y, et al1 Inhibitory effect of poly2 peptide from Chlam ys farreri on ultraviolet A induced oxidative damage on human skin fibroblastsin vitro [J]1Pharm acol Res, 2004, 49 (3) : [ 5 ] DOU M, HAN Y T, HAN Z W, et al1 Inhibitory effect of poly2 peptide from Chlamys farreri on UVA induced apop tosis in human keratinocytes [ J ]1Invest N ew D rugs, 2004, 22 (4) : [ 6 ] MIYACHIY, HORIO T, IMAMURA S1 Sunburn cell formation is p revented by scavenging oxygen intermediates [ J ] 1Clin Exp Dermatol, 1983, 8 (3) : [ 7 ] ASSEFA Z, VANTIEGHEM A, GARMYN M, et al1 p38 Mito2 gen2activated p rotein kinase regulates a novel, caspase2independ2 ent pathway for the m itochondrial cytochrome C release in ultravi2 oletb radiation2induced apop tosis[ J ] 1J BiolChem, 2000, 275 (28) : [ 8 ] CHOU INARD N, VALER IE K, ROUABH IAM, et al1 UVB 2me2 diated activation of p38 m itogen2activated p rotein kinase enhances resistance of normal human keratinocytes to apoptosis by stabili2 zing cytop lasm ic p53 [ J ] 1B iochem J, 2002, 365 ( Pt1) : ( : ) 120 Chin Pharm J, 2007 January, Vol142 No