ACTA ACADEM IAE MED IC INAE M IL ITAR IS TERTIAE Jan IKK IKB

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1 , No. 1 Vol. ACTA ACADEM IAE MED IC INAE M IL ITAR IS TERTIAE Jan : (2008) HSC2T6 1, 1, 2 ( 1, ; 2, ) : HSC2T6, CCK28 ; AnnexinV2F ITC /P I HSC2T6 ; AO /EB,W estern blot NF2 B P65 P2IKK P2I B, P65 HSC2T6 ; AO /EB AnnexinV /P I HSC2T6 ; 52 HSC2T6 W estern blot 52 IKK I B NF2 B P65 ( P < 0. 05),, P65 HSC2T6, Rel/NF2 B / I B / IKK : ; HSC2T6; ; ; : R ; R965; R : A Apoptosis of hepa tic stella te cells induced by sulfa sa laz ine ZHU Hui2li 1, SHEN Wei 1, CHEN Zhong2Yong 2 ( 1 Department of Gastroenterology, Second Affiliated Hospital of Chongqing Medical Uni2 versity, Chongqing ; 2 Department of Hepatobiliary Surgery, Second People s Hosp ital of L iangshan, Xichang , Sichuan, China) Abstract: O b jec tive To determ ine whether sulfasalazine stimulates hepatic stellate cell (HSC2T6) apop2 tosis and its possible mechanism. M e tho d s CCK28 assay, acridine orange /ethidium brom ide (AO /EB ) and Annexin F ITC /P I were used to determ ine cell growth and cell apop tosis. The exp ression of NF2 B P65, phospho2ikk and phospho2i B was detected byw estern blotting. observed with laser confocal m icroscopy. The nuclear translocation of HSC2T6 P65 was R e su lts Sulfasalazine disp layed a strong growth inhibition and p ro2 moting apop tosis effect on HSC2T6 cells in a dose and tim e2dependent manner. Sulfasalazine, but not 52am in2 osalicylic acid or sulfapyridine, inhibited the activation of NF2 B by down2regulating the exp ressions of P2IKK, P2I B and the nuclear translocation of P65. Co nc lu sio n Sulfasalazine can inhibit NF2 B activity and p romote apop tosis in HSC2T6 cells, where the Rel/NF2 B / I B / IKK pathway p lays an important role in HSC survival. Key words: sulfasalazine; HSC2T6; apop tosis; p roliferation; NF2 B ( Sulfasalazine, SASP) (52am inosalicylic acid, 52ASA ) ( sulfapyridine, SPY), IKB ( IKK IKK ) NF2 B [ 1 ], HSC2 T6, ; L02 :,,,, : , E2mail: zhuhuili2006@ sohu. com :, E2mail: shenwei315@163. com : ; : HSC2 T6,, HSC2T6 ; 52 Sigma, (DMSO), 0. 1 mmol/l, - 20, DMS0 < 0. 1% Cell Counting Kit28 A ssay(cck28 ) ; Annex2 inv2f ITC /P I ; NF2 B P65 IKK IKB W estern B lot Santa cruz ( )

2 1,. HSC2T HSC2T6,, 10% U /L RPM I1640, 37 5% CO 2,, 0. 25% % EDTA 3 m in,,,, /m l, ( 2. 5% ), 0. 1%DMSO ( 2. 5% 0. 1% DMSO), ( SASP) : mmol/l SASP; 52 (52ASA) : mmol/l 52ASA; ( SPY) : mmol/l SPY; 52 + ( 52ASA + SPY) : 2 mmol/l 52ASA + 2 mmol/l SPY Cell Counting Kit28 A ssay (CCK28 ) :, / 6 96 (, PBS, ) 100 l,, (, ) h,,, 100 l, 37 5% CO 2 24 h 24 h, 10 l CCK28, 60 m in, 450 nm [D (490) ] (Annexin2V) / ( P I), PBS 2, 10 l An2 nexin2v 5 l P I, 10 m in, 488 nm FACS Calibur, , ModFit LT 3 :, ( phosphatidylserine, PS), Annexin2 V - /P I - ;, PS, Annex2 in2v + /P I - ;, PS, Annexin2V + /P I AO /EB,, 0. 1%DMSO ( ), 24 h, 1 m l 100 g/m l AO EB, /L, 95 l, 5 l AO /EB,,, 3 m in,, 510 nm W estern blot NF2 B P65 IKK IKB2 Satus Cruzs WB, BCA, g, SDS2PAGE,, NF2 B P65 IKK IKB ( 1 10 ), ( ), W estern L ightning, 2action,, NF2 B 6 A : ; B : TNF2 ( TNF2 150 U /m l 30 m in) ; C : SASP ( 2 mmol/l ) ; D : TNF2 + SASP ( SASP 60 m in, TNF2 30 m in) 4% 10 m in, PBS 2, 0. 5% Triton X2100, m in, PBS 3, 10 m in m in, 10 l NF2 B P65 ( ) (1 100 ), 2 h, PBS 2, 5 l 2 IgG2F ITC ( ), 30 m in, PBS 2,, 90%,, : p inhole: 1. 0; : 10; gx s, SPSS Dunnett2t HSC2T6 CCK28, HSC2T6,, SASP mmol/l D ( 490 ) ( ) ( ) ( ), ( ), DMSO ( ), ( P < 0. 05) 52 HSC2T6 24 h, HSC2T6 52ASA mmol/l D ( 490) ( ) ( ) ( ) ; SPY mmol/l D (490) ( ) ( ) ( ) ; 52ASA + SPY D ( 490 ) ( ), DMSO, ( P > 0. 05) 2. 2 Annexin V /P I 52 HSC2T6 24 h, mmol/l ( % ) ( ) ; ( ) ; ( ) ; ( ) ; DMSO ( ) ; DMSO, DMSO ( P < 0. 05) 2 mmol/l (Annexin2V + /P I + ), 52 HSC2T6, ( P > 0. 05) ( 1) 2. 3 ( 400),, ( 2A ),,, 1 mmol/l HSC2T6 24 h, ( 2B)

3 : ; 2: DMSO ; mmol/l SASP ; mmol/l 52ASA ; mmol/l SPY ; 12: 52ASA + SPY a: P < 0. 05, b: P < 0. 01, DMSO 1 HSC2T6 A: ; B: 1 mmol/l SASP ; : ; g : 2 AO /EB HSC2T6 ( 400) 2. 4 IKK IKB 2 W estern blot, TNF2 20 m in, IKK IKB2, 60 m in, ( 3) 60 m in IKK IKB2 60 m in, TNF2 60 m in, IKK IKB2, 52 IKK IKB2 ( 4A B)

4 1,. HSC2T HSC2T6 P65 ( ), ( 6A ), TNF2 HSC2T6 60 m in,, P65 ( 6B), 30 m in, P65 ( 6C) ; 30 m in, TNF2 60 m in, TNF2 HSC2T6P65 ( 6D) 3 A: ; B: TNF2 ; C: SASP ; D: SASP TNF2 6 HSC2T6 P65 ( 400) ( hepatic stellate cells, HSC),, HSCs, NF2 B, HSCs, HSCs, [ 2 ] NF2 B HSCs, HSCs,,, 30%, 70% 52, 52,, NF2 B, 52, IKK, IKB, IKB, NF2 B [ 1, 3, 7 ] CCK28 (An2 nexin2v) / ( P I) AO / EB, HSC2T6,, 52 HSC2T6 Oakley [ 6 ] 24 h AO /EB,, 52 HSC T [ 4, 5 ], NF2 B,, 52 HSC2T6 NF2 B [ 4 ] W estern blot HSC2T6 IKK IKB P65 TNF2 HSC2T6, 20 m in, IKK IKB, 60 m in,, P65 HSC2T6 TNF2 NF2 B P65 HSC2T6 30 m in, TNF2 IKK IKB NF2 B P65, P65 52, TNF2 HSC2T6 IKK IKB, P65 NF2 B, HSC2T6 TNF2 HSC2T6 IKK IKB P65, NF2 B, HSC2T6 Oakley [ 6 ] Oakley [ 6 ] DNA 45 ( GADD45 ), JNK, JNK SP600125, GADD45, NF2 B NF2 B JNK GADD45 NF2 B JNK, MKK7 JNK,, HSC : IKK, I B, NF2 B NF2 B

5 74 30 GADD45, JNK, [ 8 ], NF2 B ; [ 9 ],,, : [ 1 ] Weber C K, L iptay S,W irth T, et al. Supp ression of NF2kappa B activi2 ty by sulfasalazine is mediated by direct inhibition of Ikappa B kinases alpha and beta[ J ]. Gastroenterology, 2000, 119 (5) : [ 2 ] Lang A, Schoonhoven R, Tuvia S, et al. Nuclear factor kappa B in p roliferation, activation and apoptosis in rat hepatic stellate cells[ J ]. J Hepatol, 2000, 33 (1) : [ 3 ] W ahl C, L ip tay S, Adler G, et al. Sulfasalazine: a potent and specific inhibitor of nuclear factor kappa B [ J ]. J Clin Invest, 1998, 101 (5) : [ 4 ] L ip tay S, Fulda S, Schanbacher M, et al. Molecular mechanism s of sulfasalazine2induced T2cell apop tosis[ J ]. B r J Pharmacol, 2002, 137 (5) : [ 5 ] Salh B, A ssi K, Huang S, et al. D issociated ROS production and ce2 ram ide generation in sulfasalazine2induced cell death in Raw cells[ J ]. J Leukoc B iol, 2002, 72 (4) : [ 6 ] Oakley F, Meso M, Iredale J P, et al. Inhibition of inhibitor of kappab kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis[ J ]. Gastroenterology, 2005, 128 (1) : [ 7 ] Habens F, Srinivasan N, Oakley F, et al. Novel sulfasalazine ana2 logues with enhanced NF2kappaB inhibitory and apoptosis p romoting activity[ J ]. Apoptosis, 2005, 10 (3) : [ 8 ] Herm isson M, W eller M. NF2kappaB2independent actions of sul2 fasalazine dissociate the CD95L2 and Apo2L / TRA IL2dependent death signaling pathways in human malignant glioma cells [ J ]. Cell Death D iffer, 2003, 10 (9) : [ 9 ] Rust C, Bauchmuller K, Bernt C, et al. Sulfasalazine reduces bile acid induced apoptosis in human hepatoma cells and perfused rat livers [ J ]. Gut, 2006, 55 (5) : ( ) ( 66 ), > 60 MC ( P < 0. 05) 52 MC, 11, 21. 1%, 2 46, 40, 30, 4 ( P > 0. 05), ( P > 0. 05) MC, B illiarrd 1825, 1857, V irchow,,, MC 0. 06% 5. 90% [ 1 ] MC,,,,, [ 2 ],,,, 3. 2 MC,,, [ 3 ],,,,,,,,,,, ,,,,,,,, MC, MC [ 4 ], MC,MC,, 60,, [ 5 ],, 46, 6,, MC :, : R : B : [ 1 ],,,. [ J ]., 1994, 14 (1) : [ 2 ],. [M ]. :, 1999: 470. [ 3 ],. [ J ]., 2001, 21 (5) : [ 4 ] Byers R J, Marsh P, Parkinson D, et al. Melanosis coli is associated with an increase in colonic ep ithelial apop tosis and notwith laxative use [ J ]. H istopathology, 1997, 30 (2) : [ 5 ] Lestina L S. An unusual case of melanosis coli[ J ]. Gastrointest En2 dosc, 2001, 54 (1) : ( )