424 H 2 O 2 R IN2m,,, (, 510632) : ( Ginkgo biloba extract, EGb761) (Hydrogen peroxide, H 2 O 2 ) RIN2m : 500 mol/l H 2 O 2 RIN2m 6h ; (Control) (H 2 O 2 ) ( Que 100 mol/l) EGb 761 ( EGb 761 100 mol/l), EGb 761 ( EGb 761 10 30 100 g/ml) ; MTT, Hoechst 33258,PI Annexin V2PI :, 500 mol/l H 2 O 2 6h, ( P<0101), EGb 761 H 2 O 2 ( P<0101), : RIN2m, EGb 761 H 2 O 2 RIN2m ;RIN2m ; ; : R28515 : A : 100124454 (2007) 0420424205 Effects of EGb 761 on the Cell Apoptosis Induced by H 2 O 2 in R IN 2m Beta Cells YE Chun2ling, J IN Yong2liang, YE Kai2he, Q IN Liang (Department of Pharmacology, Pharmacy College of J inan University, Guangzhou 510632, China) Abstract O bjevtive: To investigate the effects of Ginkgo biloba extract ( EGb 761) on apop tosis induced by hydrogen peroxide (H 2 O 2 ) in R IN2m beta2cells1 Methods: The apop totic model was made by H 2 O 2 exposed for six hours with a concentration of 500 mol/l1 The cytotoxicitywasmeasured bymtt1 Hoechst 33258 fluorescent staining were used to detect the p rotective effect of EGb 761 on the apop tosis of R IN2m beta2cells induced by H 2 O 2 1 AnnexinV2P I double staining of Flow cytometry were used to detect apop2 to sis quan titively1 Results: Compare to control group, after exposed to 500 mol/l H 2 O 2 for 6 hours, the apop tosis rate incereased and cell survival rate were decreased considerably ( P <0101) 1 Pretreated for 10 hourswith EGb 761, the flow cytometry results showed that the apop to sis rate decreased and cell survival rate were increased considerably ( P<0101, compared to H 2 O 2 con tro l group ) 1 Conclu2 sion: EGb 761 can decrease R IN2m beta2cells damage and apop tosis induced by H 2 O 2 1 Key words EGb 761; R IN2m beta2cells; H 2 O 2 ; Apoptosis,, 1 2,,,,, H 2 O 2 RIN2m,, EGb 761 1 111 ( Extract Ginkgo biloba, EGb 761)( : 20050601) ;515 mmol/l DM EM Gibco ;, ; 2 Hoechst, ; ( Tryp sin), 2( 4, 52,22 ) 22, 52 (MTT), ( p rop idiuiodide, P I) Sigma,Annexin2V PI ( : 20051115) 112 RIN2m 10% ( 100 U /m l) ( 100 g/ml) 515 mmol/l DM EM, 37 5% CO 2 : 3 :,, Tel: 020285223843, E2mail: yechunling2005@1631com
Journal of ChineseMedicinalMaterials 30 4 2007 4 425 113 3111 ( FORMA, U1S1A) ; 5084R ( EPPENDORF, Germ any) ;FACS Aria ( BD ) ; 450 (BIO2RAD, USA ) ;22DI2E2D282 (L E ICA, Ge rm any) 2 211 H 2 O 2 RIN2m 10 5 /m l 96,37,5%CO 2, ( control) ( H 2 O 2 ) ( Que 100 mol/l) EGb 761 ( EGb 761 100 mol/ L),EGb761 ( EGb 761 10 30 100 mol/l), 6 37 10 h, 500 mol/l H 2 O 2 6h, MTT 3 212 MTT 3 4 96, PB S, 012mlPBS 20 l MTT(5mg/ml), 015 mg/ml 37 4h, 150 l DM SO 37, 570nm/630nm (op tical density, OD),, 100%, = / 100% 213 Hoechst 33258, 50 80% 6,,,600 g/min 5min ; 4% 015 ml, 10 m in ( 4 ) ;,PBS 2, 3 min,,, ; 600 g/m in 5min PB S; 015 mlhoechst 33258 5min, ; PB S 2, 3min,,,, ; 350 nm, 460 nm 214 ( FCM ) 21411 RIN2m : 5 10 5 1 10 6 /m l, 0125% 20102% EDTA,3mlPBS, 70%,4 24 h,3 ml PBS 5min, 1% Triton X2100 10 m in,, 0101% RNA 10 m in,, 01005% PI, 4 30 m in, 400, PI, 488 nm, 630 nm,, Cell Quest, ModFit LT 21412 RIN2m : 0125% 20102% EDTA,, 5 10 5 1 10 6 /m l, Annexin2VPI, PI 215 ( gx S), t SPSS10100 3 311 H 2 O 2 500 mol/l H 2 O 2, RIN2 m ( P<0101) 10 100 g/ ml EGb 761 H 2 O 2, H 2 O 2 ( P<0105 P< 0101), 1 Tab 1 Effect of EGb 761 on R IN2m beta2cells in the Presence of 500 M H 2 O 2 ( gx s, n =6) Group s V iability( % ) Control 100 911 H 2 O 2 500 mol/l 5417 819 ## EGb 761 100 g/ml 10117 616 33 Que +H 2 O 2 100 mol/l 9710 719 33 EGb 761 + H 2 O 2 10 g/ml 7310 414 3 30 g/ml 8117 1411 3 100 g/ml 9818 712 33 3 P<0105, 33 P<0101 vs H 2 O 2 ; ## P<0101 vs Control 312,,,, DNA EGb 761,, EGb 761 100 g/ml, H 2 O 2 EGb 761 H 2 O 2 1
426 F ig111 Protection of EGb 761 Aga in st Apoptosis Induced by H 2 O 2 in R IN2m 2cells( 400 ) A1 H 2 O 2 ; B1 H 2 O 2 +10 g/ml EGb761; C1 H 2 O 2 +30 g/ml EGb761; D1 H 2 O 2 + 100 g/m l EGb761; E1 Control; F1 H 2 O 2 + Que Fig12 Protection of EGb 761 Aga inst H 2 O 2 2Induced Apoptosis in RIN2m 2cells( P I) 33 P<0101 vs H 2 O 2 without EGb 761, ## P<0101 vs Control 313 RIN2m 31311 PI : PI 2 3 H 2 O 2 H 2 O 2, RIN2m G 0 /G 1 ( sub2g 1 ), 500 mol/l H 2 O 2 6h RIN2m ( 4312 116% ) ;EGb761,, EGb 761 ( 512 019% ) Que ( 519 110% ),, ( P<0101) 3, EGb 761,, 31312 AnnexinV2PI :Annex2 in V 2PI 4 5 ( PS),AnnexinV PS; PI, DNA, Annexin V2 FITC/PI,H 2 O 2 6h 4, (An2 nexin V 22 2PI + ) (Annexin V 22 2PI 22 ) (Annexin V + 2PI 22 ) (Annexin V + 2PI + ) H 2 O 2 500 mol/l H 2 O 2, RIN2m 6h ( 3312 415 ) %, ( 117 017) %, ( P<0101) ; 100 g/ml EGb 761 (415 017) % Que (515 114) % H 2 O 2, ( P<0101) 4,, 5,
Journal of ChineseMedicinalMaterials 30 4 2007 4 427 F ig131 Apoptosis in R IN2m 2cells A ssessed by FCM A1 H 2 O 2 ; B1 H 2 O 2 +10 g/ml EGb761; C1 H 2 O 2 +30 g/ml EGb761; D1 H 2 O 2 + 100 g/m l EGb761; E1 Control; F1 H 2 O 2 + Que, Annexin V2PI,, PI, Annexin V PI, FCM An2 nexin V2PI,,,, RIN2m H 2 O 2 6 7,PI Annexin V2PI, EGb 761 Fig14 Protection of EGb 761 Aga inst H 2 O 2 2Induced H 2 O 2 RIN2m, Apoptosis in RIN2m 2cells( Annex in V2PI), EGb 761 H 33 P<0101 vs H 2 O 2 without EGB 761, ## 2 O 2 P<0101 vs Control,,,,, ROS,,, 8 H 2 O 2,, H 2 O 2 PC12 RO S 2 9 EGb AnnexinV,AnnexinV, 10 11,EGb,, NO Annexin V,, EGb
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