C 2 C 12, Mechanisms of heat shock proteins inhibiting C 2 C 12 cell apoptosis induced by hydrogen peroxide

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1 6 ( ) J Cent South Univ(Med Sci) 2004,29 (1) C 2 C 12 3,,,,, (, ) [ ] : C 2 C 12 : C 2 C 12, ; Hoechst33258 ( H 2 O 2 ) C 2 C 12 ; Western2blotting 23,8,9 ; Western2blotting C : C 2 C B2, C, 23,8,9 C 2 C 12 : C 2 C 12, [ ] ; C 2 C 12 ; ; ; ; ; C; [ ] R [ ] A [ ] (2004) Mechanisms of heat shock proteins inhibiting C 2 C 12 cell apoptosis induced by hydrogen peroxide XIAO Wei2min, J IANG Bi2mei,SHI Yong2zhong,et al. ( Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha , China ) Abstract : Objective To explore the mechanisms of C 2 C 12 cell apoptosis induced by hydrogen peroxide (H 2 O 2 ) which is inhibited by heat shock proteins. Methods The expression of heat shock proteins in mouse embryonic myogenic cell line,c 2 C 12 cell,was induced by heat shock response. C 2 C 12 cell apoptosis induced by 0. 5 mmol/ L hydrogen peroxide (H 2 O 2 ) was determined by Hoechst staining. The activities of caspase 23,8,9 were assayed by caspase colorimetric assay kit and Western2blotting. The release of cy2 tochrome C from mitochondria was observed by Western2blotting of cell mitochondria and cytosol fractions. Results The expression of HSP70 and B2crystallin in C 2 C 12 cell significantly increased at 24 hour after the heat shock response. Heat shock response could inhibit the release of cytochrome c from mitochondria to cyto2 plasm,the activation of caspase 23,8,9 and the subsequent apoptosis induced by H 2 O 2 in C 2 C 12 cell. Conclu2 sion HSPs can inhibit the C 2 C 12 cell apoptosis through interference with the activation of both mitochondrial and death receptor pathways,which can provide new clues for the prevention of cardiovascular diseases. Key words : heat shock proteins ; C 2 C 12 cell ; hydrogen peroxide ; apoptosis ; mitochondria pathway ; death receptor pathway ; cytochrome C ; caspase [J Cent South Univ(Med Sci), 2004, 29 (1) : ],, 2, [1 ] : : (19712),,,,, 3 : ( ; ), 973 ( G ) ( )

2 C 2 C 12, 7,, [2 ],, 28 C 29 [3 ] (heat shock protein, HSP) ( ), HSP [4 ] McMillan 1 ( HSF1), HSP [5 ], HSP,, C 2 C 12,, C 2 C 12 H 2 O 2 ; DAB, 23,8,9 C R&D HSP70 B2 Stressgen Gibco Sigma2 Aldrich 1. 2 C 2 C 12 C 2 C % / ml,24 h 1. 3, 42 1 h, 37,5 %CO 2,24 h 1. 4 C 2 C 12 : (Ctrl, n = 6),C 2 C 12, 37,5 % CO 2 4, 8,12,18,24 h ; H 2 O 2 ( H 2 O 2, n = 6),C 2 C mmol/ L H 2 O 2, 37,5 % CO 2 4,8,12,18,24 h H 2 O 2 (HSR + H 2 O 2, n = 6), C 2 C h, 37,5 %CO 2 24 h, 1. 5 :, 50 l, 10 min, ( r/ min, 4, 5 min), ( 5 l Bradford 595 nm ), 50 l 5 l, h,405 nm = OD 405 / OD , 250 mmol/ L,, g 10 min,, r/ min 15 min, ; r/ min 1 h, ( ) 1. 7 Hoechst 33258,, Hoechst 33258,, 1. 8 Western2blotting PBS 3,, min, ( r/ min 10 min),,, g /, 12 % SDS2PAGE, 3 h,, 1h,, 0. 5 h, DAB 25 min,,,, 1. 9 ( gx s), t, P < C 2 C 12 HSPs Western2blotting 24 h,hsp70 B2 ( ) ( 1) 2. 2 H 2 O 2 C 2 C 12 Hoechst 33258, C 2 C 12, H 2 O 2 (0. 5 mmol/ L) 24 h,,,

3 8 ( ),2004,29 (1) ( 2) 2. 3 HSPS H 2 O 2 C 2 C 12 ( 3), C 2 C 12 H 2 O 2 (0. 5 mmol/ L) 4 h, 23,8,9,12 h, 24 h, ( Ctrl, P < 0. 01) ; C 2 C 12 H 2 O 2, 23,8,9 H 2 O 2 ( H 2 O 2, P < 0. 01) Western2blotting ( 4) :,C 2 C 12 23,8, mmol/ L H 2 O 2 4 h, 11,20,10 kd, 12 h, 24 h H 2 O 2 1 (42,1 h, 24 h) C 2 C 12 B2 HSP70 HSR : Fig. 1 Western2blotting showed the effect of heat shock response on the ex2 pression of B2crystallin and HSP70 in C 2 C 12 myogenic cells HSR :heat shock response 3 H 2 O 2 C 2 C 12 23,8,9 HSR : A : 23 ;B : 28 ; C: 29 Fig. 3 Heat shock response (HSR) inhibited the activation of Caspase23,8, 9 in C 2 C 12 cell induced by H 2 O 2 HSR :Heat shock response ;A :ac2 tivity of caspase23 ;B :activity of caspase28 ; C:activity of caspase 29 ( 3 P < vs Ctrl ; # P < vs H 2 O 2 group, n = 5) 2 Hoechst H2O2 C2C12 HSR : Fig. 2 Hoechst staining demonstrated the protective role of heat shock response ( HSR) against C 2 C 12 cell apoptosis induced by H 2 O 2 (0. 5mm,24 h) HSR :heat shock response) 4 Western2blotting H 2 O 2 (0. 5 mmol/ L) C 2 C 12 23,8,9 HSR : ;Procas : ;P11,P20,P10 : 11, kd e23,8,9 Fig. 4 Western2blotting demonstrated heat shock response (HSR) inhibiting the activation of Caspase23,8,9 in C 2 C 12 cell induced by H 2 O 2 (0. 5 mmol/ L) HSR : Heat shock response ; Procas : precursor of Cas2 pase ; P11,P20,P10 :cleaved fragments of Procas23,8,9 with molec2 ular weight of 11,20 and 10 kd respective

4 C 2 C 12, HSPS H 2 O 2 C 2 C 12 C C 2 C 12 Western blotting ( 5), C C 2 C 12, 0. 5 mmol/ L H 2 O 2 C 2 C 12 2 h, C,, C 5 Western blotting H 2 O 2 (0. 5 mmol/ L,2 h) C 2 C 12 C HSR : ;S100 : ;mit : ;Cyt. c : C Fig. 5 Western blotting demonstrates heat shock response (HSR) inhibiting the release of cytochrome C from C 2 C 12 cell induced by H 2 O 2 (0. 5 mmol/ L,2 h) HSR : Heat shock response ;S100 :Cytosol ;mit :Mi2 tochondria ;Cyt : Cytochrome C 3,, (molecular chaper2 one),,,,, [6 ],, [7 ], [1 ], [3 ] C 2 C 12, Hoechst33258,, C 2 C 12 ; Western blotting, 23,8,9 C ( TNF,Fas ), 28 ; C, [8 ],, C 2 C 12,,, Mosser, HSP70 C [9 ], 1 (apoptotic pro2 tease2activating factor21,apaf21), Apaf21 29 [10 ] HSP70 (apoptosis2inducing factor,aif), [11 ] HSP90 Apaf21, C Apaf21 29 [12 ] HSP27/ 25 C, 29, 23 [13 ] B2 23 P24 [14 ], C 2 C 12,, : [1 ] Bromme HJ,Holtz J. Apoptosis in the heart : when and why? [J ]. Mol Cell Biochem,1996,23 (163/ 164 ) : [2 ] Hengartner MO. The biochemistry of apoptosis[j ]. Nature, 2000, 407 (12) : [3 ],,,. [J ].,2003, 11 (3) : [4 ] Samali A,Orrenius S. Heat shock proteins : regulators of stress re2 sponse and apoptosis[j ]. Cell Stress & Chaperones, 1998, 3 (4) : [5 ] McMillan DR, Xiao XZ, Shao L,et al. Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection a2 gainst heat2inducible apoptosis [J ]. J Biol Chem, 1998,273 (13) : [6 ] Xiao XZ, Benjamin IJ. Stress2response proteins in cardiovascular dis2

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