schisandrin b ( sch b ) is the most abundant , active dibenzocyclooctadiene derivative isolated from the fruit of schisandra chinensis ( turcz ) baillon ( schisandraceae ) , a traditional chinese herb commonly used as astringent and clinically used for the treatment of viral and chemical hepatitis . early studies in our laboratory have demonstrated the ability of sch b to protect against myocardial ischemia / reperfusion ( i / r ) injury . the cardioprotection afforded by sch b pretreatment was associated with the enhancement in tissue glutathione antioxidant status , particularly in the mitochondrion.[24 ] recent studies also showed that heat shock protein ( hsp)25 and hsp70 expression contributes at least partly to the cardioprotection afforded by sch b pretreatment against i / r injury . in regard to the action on mitochondria , sch b treatment was found to decrease the sensitivity of ca - induced mitochondrial permeability transition and mitochondrial membrane potential in hypoxia / reoxygenation - challenged h9c2 cardiomyocytes . however , the biochemical mechanism(s ) underlying the sch b - induced glutathione antioxidant and/or heat shock responses in cardiomyocytes remains to be established . in this connection , it has been suggested that reactive oxygen species ( ros ) generated from cytochrome p-450 ( cyp)-catalyzed reaction with sch b may trigger the glutathione antioxidant responses in rat hearts . the sch b preparation used in our previous animal and cell studies was found to ( )-schisandrin [ ( )g - sch ] and ( )schisandrin b [ ( )sch b ] in a ratio of 2:8 ( w / w ) . ( )sch b [ figure 1 ] was found to be more potent than ( )-sch in eliciting the glutathione antioxidant response and protecting against hypoxia / reoxygenation - induced apoptosis in h9c2 cardiomyocytes . to define the role of cyp - mediated ros generation in the cytoprotective action of ( )sch b , we here examined the effects of 1-aminobenzotriazole ( abt , a broad spectrum inhibitor of cyp ) and antioxidant ( dimethylthiouracil , dmtu ) on ( )sch b - induced ros production and the subsequent glutathione antioxidant and heat shock responses in h9c2 cardiomyocytes . the effects of abt and dmtu pretreatment on ( )sch b - induced protection against hypoxia / reoxygenation - induced apoptosis were also investigated . chemical structure of ( )schisandrin b cell culture medium and fetal bovine serum ( fbs ) were obtained from gibco brl life technologies ( grand island , ny ) . reduced glutathione ( gsh ) , oxidized glutathione ( gssg ) , glutathione reductase ( gr ) , abt and dmtu were purchased from sigma ( st . louis , mo ) . h9c2 cells , cultured as monolayers in dulbecco 's modified eagle 's medium ( dmem ) ( gibco brl ) , is a permanent cell line derived from cardiac myoblast , which was supplemented with 10% ( v / v ) fbs . the medium contained glucose ( 4.5 g / l ) and glutamine ( 4.5 mm ) , supplemented with nahco3 ( 17 mm ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) . the cells were grown under an atmosphere of 5% ( v / v ) co2 in air at 37c . a stock of cells was grown in a 75 cm culture flask and split before confluence at a subcultivation ratio of 1:10 . cells used for experiments were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and cells in each well were allowed to grow to achieve 60 - 80% confluence within 24 h prior to drug treatment . to investigate the effects of cyp inhibitor / antioxidants on ( )sch b - induced ros production , cells were pre - incubated with abt ( 10 mm final concentration , for 2 h ) or dmtu ( 20 mm , 1 h ) ) prior to the addition of ( )sch b at 7.5 or 15 m . vehicle [ phosphate - buffered saline ( pbs ) ] was added for the respective control . then cells were coincubated with abt / dmtu and ( )sch b , and the ros generation was monitored in situ for 6 h at 37c . to investigate the effects of ( )sch b on cellular gsh and hsp 25/70 levels , cells were incubated with ( )sch b at 7.5 or 15 m for 6 h. then the drug - containing medium was removed , and the cells were cultured in fresh medium ( without drug ) for 16 h. preliminary time - course studies indicated that 6 h of drug exposure followed by 16 h of drug - free culture caused maximum increases in cellular gsh and hsp 25/70 levels in h9c2 cells . exposure of cells to ( )sch b at concentrations higher than 15 m produced cytotoxic effect . cells ( 110 ) were seeded on a 96-well black multi - titer plate with clear bottom , and they were allowed to grow overnight . the cells were washed twice with pbs and then incubated with 0.1% bsa in pbs containing 5 m ( final concentration ) dcfda ( dissolved in 0.2% ( v / v ) ethanol ) for 30 min at 37c . the cells were washed twice with pbs again and then incubated with fresh medium containing 500 m nadph ( final concentration ) , 7.5 or 15 m ( )sch b , with or without abt or dmtu pre- and co - treatment , as described above . the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm by victor v multi - label counter ( perkin elmer , turku , finland ) every minute for 6 h at 37c . the fluorescence intensity of drug - treated cells was normalized with reference to the respective time - matched vehicle ( i.e. , dmso ) control . the extent of drug - induced ros production was estimated by computing the area under the curve ( auc ) plotting normalized fluorescence intensity against time ( min ) , and expressed in arbitrary unit . for the measurement of cellular gsh level , cells were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and they were allowed to grow overnight . after the drug treatment , cells were centrifuged at 540 g for 15 min at 4c . an aliquot of 100 l 3% ( v / v ) 5-sulfosalicyclic acid ( ssa ) were added to cells . after shaking for 10 min at 4c gsh levels were determined enzymatically using 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb ) and gr , in a protocol modified from griffith . to measure gssg levels , aliquots ( 50 l ) of ssa supernatants were mixed with 5 l of 20% ( w / v ) 2-vinylpyridine and 5 l 60% ( v / v ) triethanolamine in microcentrifuge tubes . each tube was allowed to stand at room temperature for 1 h. a reaction mixture containing 0.63 mm dtnb and 0.053 mm nadph in phosphate buffer ( 0.1 m , with 5 mm na2edta ; ph 7.5 ) was pre - incubated at 30c for 5 min . an aliquot ( 30 l ) of ssa sample supernatant ( total glutathione ) or an aliquot ( 30 ml ) of gssg sample was added to a well of a 96-well microtiter plate , and 180 l of prewarmed reaction mixture , containing 0.525 u / ml gr , was next added . the concentrations of total glutathione and gssg were estimated from calibration curves using gsh and gssg [ dissolved in 3% ( w / v ) ssa ) as standards , and expressed as nmol / mg protein . gsh levels were estimated by subtracting twice the amount of gssg from that of total glutathione . cells ( 2.5  10 cells / well ) were seeded on 6-well plate , and they were allowed to grow overnight . after drug treatment the cells were then incubated with 300 l of sds - lysis buffer ( 20 mm tris - hcl , 2 mm edta , 3 mm egta , 1% triton x-100 , 10% glycerol , 2 mm dithiothreitol , 5 g / ml leupeptin , 5 g / ml aproptinin , 5 g / ml pepstain , 1 m phenylmethylsulphonyl fluoride , ph 7.5 ) for 20 min at 4c on a shaker . the cell lysates were further processed by passing through a 26 g  syringe for 10 times , and the protein concentrations in cell lysates were determined by using the dc protein assay kit ( bio - rad laboratories , hercules , ca ) . hsp 25 and hsp 70 levels were estimated by western blot analysis using specific antibodies ( anti - hsp 25 , catalog # spa-801 ; anti - hsp 70 , catalog # spa-812 ) from stressgen ( vancouver , bc , canada ) following sds - page analysis of the cell lysates , using a separating gel of 10% ( w / v ) acrylamide . hsp 25/27 and hsp 70 ( human recombinant proteins from stressgen ) and actin ( prepared from bovine muscle , sigma ) were used as markers . the immuno - stained protein bands were revealed by an enhanced chemiluminescence reaction ( amersham ecl+ ) . all immuno - blots were analyzed by densitometry , and the amounts ( arbitrary units ) of hsp were normalized with reference to actin levels ( arbitrary units ) in the sample . cells used for this experiment were seeded at 2.5  10 cells on a 40 mm culture plate , and they were allowed to grow overnight . after drug treatment , the cells were washed twice with krebs - ringer bicarbonate buffer ( krb , 115 mm nacl , 4.7 mm kcl , 2.5 mm cacl2 , 1.2 mm kh2po4 , 1.2 mm mgso4 , 24 mm nahco3 , 10 mm hepes , ph 7.4 ) . aliquots ( 2.5 ml ) of krb supplemented with 0.01% bovine serum albumin were added to the cells immediately prior to the hypoxia . a billups - rothenberg modular incubator chamber ( billups - rothenberg , inc . , del mar , ca ) in essence , cells were placed in the sealed chamber , and the chamber was flushed with nitrogen for 15 min at flow rate of 20 ml / min . after closing all sealable connectors , the chamber was transferred to an incubator and the cells in the chamber were subjected to a 2-h period of hypoxia at 37c . reoxygenation was initiated by opening the chamber and then replacing the krb with fresh dmem medium . the cells were then cultured in the incubator under an atmosphere of 5% co2 in air at 37c for 16 h. preliminary time course studies showed that a 2-h period of hypoxia followed by 16 h of reoxygenation was optimal for achieving a 1 - 2-fold increase in apoptotic cell death in h9c2 cells . apoptotic cell death was measured by annexin v - fitc - fluos ( roche , penzberg , germany ) and propidium iodide ( pi ) ( sigma , st . louis , mo ) stainings , in which annexin v bound to the exposed phosphatidylserine on the plasma membrane of the apoptotic cells . briefly , cells were rinsed with ice - cold pbs and then resuspended in 100 l of binding buffer containing 140 mm nacl , 2.5 mm cacl2 , 1.5 mm mgcl2 and 10 mm hepes , ph 7.4 . one - hundred microliters of annexin v staining solution ( 1 l of annexin v stock solution being mixed with 100 l of binding buffer ) was added to the cells and incubated in the dark for 15 min at room temperature . the cells were washed with ice - cold pbs and resuspended in 600 l of binding buffer . immediately after mixing with 3 l pi solution ( 50 g / ml ) , the cells were analyzed by flow cytometry using a coulter epics xl flow cytometer ( beckman coulter , inc . , the fluorescence of annexin v and pi were monitored at emission wavelengths of 525 nm and 575 nm , respectively , with the excitation wavelength being at 488 nm for both probes . approximately 10,000 cells were counted in each of the samples and data were statistically analyzed using an expo32 adc analysis program . the percentage of early apoptotic cells was estimated by noting the annexin v positive but pi negative cells , while the percentage of late apoptotic cells was estimated by noting the number of cells which were both annexin v and pi positive . the total apoptotic cell death was calculated by summing up the percentages of early apoptosis and late apoptosis . post - hoc multiple comparisons were performed using least significant difference ( lsd ) test . cell culture medium and fetal bovine serum ( fbs ) were obtained from gibco brl life technologies ( grand island , ny ) . reduced glutathione ( gsh ) , oxidized glutathione ( gssg ) , glutathione reductase ( gr ) , abt and dmtu were purchased from sigma ( st . louis , mo ) . h9c2 cells , cultured as monolayers in dulbecco 's modified eagle 's medium ( dmem ) ( gibco brl ) , is a permanent cell line derived from cardiac myoblast , which was supplemented with 10% ( v / v ) fbs . the medium contained glucose ( 4.5 g / l ) and glutamine ( 4.5 mm ) , supplemented with nahco3 ( 17 mm ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) . the cells were grown under an atmosphere of 5% ( v / v ) co2 in air at 37c . a stock of cells was grown in a 75 cm culture flask and split before confluence at a subcultivation ratio of 1:10 . cells used for experiments were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and cells in each well were allowed to grow to achieve 60 - 80% confluence within 24 h prior to drug treatment . to investigate the effects of cyp inhibitor / antioxidants on ( )sch b - induced ros production , cells were pre - incubated with abt ( 10 mm final concentration , for 2 h ) or dmtu ( 20 mm , 1 h ) ) prior to the addition of ( )sch b at 7.5 or 15 m . vehicle [ phosphate - buffered saline ( pbs ) ] was added for the respective control . then cells were coincubated with abt / dmtu and ( )sch b , and the ros generation was monitored in situ for 6 h at 37c . to investigate the effects of ( )sch b on cellular gsh and hsp 25/70 levels , cells were incubated with ( )sch b at 7.5 or 15 m for 6 h. then the drug - containing medium was removed , and the cells were cultured in fresh medium ( without drug ) for 16 h. preliminary time - course studies indicated that 6 h of drug exposure followed by 16 h of drug - free culture caused maximum increases in cellular gsh and hsp 25/70 levels in h9c2 cells . exposure of cells to ( )sch b at concentrations higher than 15 m produced cytotoxic effect . cells ( 110 ) were seeded on a 96-well black multi - titer plate with clear bottom , and they were allowed to grow overnight . the cells were washed twice with pbs and then incubated with 0.1% bsa in pbs containing 5 m ( final concentration ) dcfda ( dissolved in 0.2% ( v / v ) ethanol ) for 30 min at 37c . the cells were washed twice with pbs again and then incubated with fresh medium containing 500 m nadph ( final concentration ) , 7.5 or 15 m ( )sch b , with or without abt or dmtu pre- and co - treatment , as described above . the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm by victor v multi - label counter ( perkin elmer , turku , finland ) every minute for 6 h at 37c . the fluorescence intensity of drug - treated cells was normalized with reference to the respective time - matched vehicle ( i.e. , dmso ) control . the extent of drug - induced ros production was estimated by computing the area under the curve ( auc ) plotting normalized fluorescence intensity against time ( min ) , and expressed in arbitrary unit . for the measurement of cellular gsh level , cells were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and they were allowed to grow overnight . after the drug treatment , cells were centrifuged at 540 g for 15 min at 4c . an aliquot of 100 l 3% ( v / v ) 5-sulfosalicyclic acid ( ssa ) were added to cells . after shaking for 10 min at 4c gsh levels were determined enzymatically using 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb ) and gr , in a protocol modified from griffith . to measure gssg levels , aliquots ( 50 l ) of ssa supernatants were mixed with 5 l of 20% ( w / v ) 2-vinylpyridine and 5 l 60% ( v / v ) triethanolamine in microcentrifuge tubes . each tube was allowed to stand at room temperature for 1 h. a reaction mixture containing 0.63 mm dtnb and 0.053 mm nadph in phosphate buffer ( 0.1 m , with 5 mm na2edta ; ph 7.5 ) was pre - incubated at 30c for 5 min . an aliquot ( 30 l ) of ssa sample supernatant ( total glutathione ) or an aliquot ( 30 ml ) of gssg sample was added to a well of a 96-well microtiter plate , and 180 l of prewarmed reaction mixture , containing 0.525 u / ml gr , was next added . the concentrations of total glutathione and gssg were estimated from calibration curves using gsh and gssg [ dissolved in 3% ( w / v ) ssa ) as standards , and expressed as nmol / mg protein . gsh levels were estimated by subtracting twice the amount of gssg from that of total glutathione . cells ( 2.5  10 cells / well ) were seeded on 6-well plate , and they were allowed to grow overnight . after drug treatment the cells were then incubated with 300 l of sds - lysis buffer ( 20 mm tris - hcl , 2 mm edta , 3 mm egta , 1% triton x-100 , 10% glycerol , 2 mm dithiothreitol , 5 g / ml leupeptin , 5 g / ml aproptinin , 5 g / ml pepstain , 1 m phenylmethylsulphonyl fluoride , ph 7.5 ) for 20 min at 4c on a shaker . the cell lysates were further processed by passing through a 26 g  syringe for 10 times , and the protein concentrations in cell lysates were determined by using the dc protein assay kit ( bio - rad laboratories , hercules , ca ) . hsp 25 and hsp 70 levels were estimated by western blot analysis using specific antibodies ( anti - hsp 25 , catalog # spa-801 ; anti - hsp 70 , catalog # spa-812 ) from stressgen ( vancouver , bc , canada ) following sds - page analysis of the cell lysates , using a separating gel of 10% ( w / v ) acrylamide . hsp 25/27 and hsp 70 ( human recombinant proteins from stressgen ) and actin ( prepared from bovine muscle , sigma ) were used as markers . the immuno - stained protein bands were revealed by an enhanced chemiluminescence reaction ( amersham ecl+ ) . all immuno - blots were analyzed by densitometry , and the amounts ( arbitrary units ) of hsp were normalized with reference to actin levels ( arbitrary units ) in the sample . cells used for this experiment were seeded at 2.5  10 cells on a 40 mm culture plate , and they were allowed to grow overnight . after drug treatment , the cells were washed twice with krebs - ringer bicarbonate buffer ( krb , 115 mm nacl , 4.7 mm kcl , 2.5 mm cacl2 , 1.2 mm kh2po4 , 1.2 mm mgso4 , 24 mm nahco3 , 10 mm hepes , ph 7.4 ) . aliquots ( 2.5 ml ) of krb supplemented with 0.01% bovine serum albumin were added to the cells immediately prior to the hypoxia . a billups - rothenberg modular incubator chamber ( billups - rothenberg , inc . , del mar , ca ) in essence , cells were placed in the sealed chamber , and the chamber was flushed with nitrogen for 15 min at flow rate of 20 ml / min . after closing all sealable connectors , the chamber was transferred to an incubator and the cells in the chamber were subjected to a 2-h period of hypoxia at 37c . reoxygenation was initiated by opening the chamber and then replacing the krb with fresh dmem medium . the cells were then cultured in the incubator under an atmosphere of 5% co2 in air at 37c for 16 h. preliminary time course studies showed that a 2-h period of hypoxia followed by 16 h of reoxygenation was optimal for achieving a 1 - 2-fold increase in apoptotic cell death in h9c2 cells . apoptotic cell death was measured by annexin v - fitc - fluos ( roche , penzberg , germany ) and propidium iodide ( pi ) ( sigma , st . louis , mo ) stainings , in which annexin v bound to the exposed phosphatidylserine on the plasma membrane of the apoptotic cells . briefly , cells were rinsed with ice - cold pbs and then resuspended in 100 l of binding buffer containing 140 mm nacl , 2.5 mm cacl2 , 1.5 mm mgcl2 and 10 mm hepes , ph 7.4 . one - hundred microliters of annexin v staining solution ( 1 l of annexin v stock solution being mixed with 100 l of binding buffer ) was added to the cells and incubated in the dark for 15 min at room temperature . the cells were washed with ice - cold pbs and resuspended in 600 l of binding buffer . immediately after mixing with 3 l pi solution ( 50 g / ml ) , the cells were analyzed by flow cytometry using a coulter epics xl flow cytometer ( beckman coulter , inc . , the fluorescence of annexin v and pi were monitored at emission wavelengths of 525 nm and 575 nm , respectively , with the excitation wavelength being at 488 nm for both probes . approximately 10,000 cells were counted in each of the samples and data were statistically analyzed using an expo32 adc analysis program . the percentage of early apoptotic cells was estimated by noting the annexin v positive but pi negative cells , while the percentage of late apoptotic cells was estimated by noting the number of cells which were both annexin v and pi positive . the total apoptotic cell death was calculated by summing up the percentages of early apoptosis and late apoptosis . post - hoc multiple comparisons were performed using least significant difference ( lsd ) test . cell culture medium and fetal bovine serum ( fbs ) were obtained from gibco brl life technologies ( grand island , ny ) . reduced glutathione ( gsh ) , oxidized glutathione ( gssg ) , glutathione reductase ( gr ) , abt and dmtu were purchased from sigma ( st . louis , mo ) . h9c2 cells , cultured as monolayers in dulbecco 's modified eagle 's medium ( dmem ) ( gibco brl ) , is a permanent cell line derived from cardiac myoblast , which was supplemented with 10% ( v / v ) fbs . the medium contained glucose ( 4.5 g / l ) and glutamine ( 4.5 mm ) , supplemented with nahco3 ( 17 mm ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) . the cells were grown under an atmosphere of 5% ( v / v ) co2 in air at 37c . a stock of cells was grown in a 75 cm culture flask and split before confluence at a subcultivation ratio of 1:10 . cells used for experiments were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and cells in each well were allowed to grow to achieve 60 - 80% confluence within 24 h prior to drug treatment . to investigate the effects of cyp inhibitor / antioxidants on ( )sch b - induced ros production , cells were pre - incubated with abt ( 10 mm final concentration , for 2 h ) or dmtu ( 20 mm , 1 h ) ) prior to the addition of ( )sch b at 7.5 or 15 m . vehicle [ phosphate - buffered saline ( pbs ) ] was added for the respective control . then cells were coincubated with abt / dmtu and ( )sch b , and the ros generation was monitored in situ for 6 h at 37c . to investigate the effects of ( )sch b on cellular gsh and hsp 25/70 levels , cells were incubated with ( )sch b at 7.5 or 15 m for 6 h. then the drug - containing medium was removed , and the cells were cultured in fresh medium ( without drug ) for 16 h. preliminary time - course studies indicated that 6 h of drug exposure followed by 16 h of drug - free culture caused maximum increases in cellular gsh and hsp 25/70 levels in h9c2 cells . exposure of cells to ( )sch b at concentrations higher than 15 m produced cytotoxic effect . cells ( 110 ) were seeded on a 96-well black multi - titer plate with clear bottom , and they were allowed to grow overnight . the cells were washed twice with pbs and then incubated with 0.1% bsa in pbs containing 5 m ( final concentration ) dcfda ( dissolved in 0.2% ( v / v ) ethanol ) for 30 min at 37c . the cells were washed twice with pbs again and then incubated with fresh medium containing 500 m nadph ( final concentration ) , 7.5 or 15 m ( )sch b , with or without abt or dmtu pre- and co - treatment , as described above . the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm by victor v multi - label counter ( perkin elmer , turku , finland ) every minute for 6 h at 37c . the fluorescence intensity of drug - treated cells was normalized with reference to the respective time - matched vehicle ( i.e. , dmso ) control . the extent of drug - induced ros production was estimated by computing the area under the curve ( auc ) plotting normalized fluorescence intensity against time ( min ) , and expressed in arbitrary unit . for the measurement of cellular gsh level , cells were seeded at a density of 3.75  10 cells / well in a 12-well culture plate , and they were allowed to grow overnight . after the drug treatment , cells were centrifuged at 540 g for 15 min at 4c . an aliquot of 100 l 3% ( v / v ) 5-sulfosalicyclic acid ( ssa ) were added to cells . after shaking for 10 min at 4c gsh levels were determined enzymatically using 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb ) and gr , in a protocol modified from griffith . to measure gssg levels , aliquots ( 50 l ) of ssa supernatants were mixed with 5 l of 20% ( w / v ) 2-vinylpyridine and 5 l 60% ( v / v ) triethanolamine in microcentrifuge tubes . each tube was allowed to stand at room temperature for 1 h. a reaction mixture containing 0.63 mm dtnb and 0.053 mm nadph in phosphate buffer ( 0.1 m , with 5 mm na2edta ; ph 7.5 ) was pre - incubated at 30c for 5 min . an aliquot ( 30 l ) of ssa sample supernatant ( total glutathione ) or an aliquot ( 30 ml ) of gssg sample was added to a well of a 96-well microtiter plate , and 180 l of prewarmed reaction mixture , containing 0.525 u / ml gr , was next added . the concentrations of total glutathione and gssg were estimated from calibration curves using gsh and gssg [ dissolved in 3% ( w / v ) ssa ) as standards , and expressed as nmol / mg protein . gsh levels were estimated by subtracting twice the amount of gssg from that of total glutathione . cells ( 2.5  10 cells / well ) were seeded on 6-well plate , and they were allowed to grow overnight . after drug treatment , cells were washed once with pbs . the cells were then incubated with 300 l of sds - lysis buffer ( 20 mm tris - hcl , 2 mm edta , 3 mm egta , 1% triton x-100 , 10% glycerol , 2 mm dithiothreitol , 5 g / ml leupeptin , 5 g / ml aproptinin , 5 g / ml pepstain , 1 m phenylmethylsulphonyl fluoride , ph 7.5 ) for 20 min at 4c on a shaker . the cell lysates were further processed by passing through a 26 g  syringe for 10 times , and the protein concentrations in cell lysates were determined by using the dc protein assay kit ( bio - rad laboratories , hercules , ca ) . hsp 25 and hsp 70 levels were estimated by western blot analysis using specific antibodies ( anti - hsp 25 , catalog # spa-801 ; anti - hsp 70 , catalog # spa-812 ) from stressgen ( vancouver , bc , canada ) following sds - page analysis of the cell lysates , using a separating gel of 10% ( w / v ) acrylamide . hsp 25/27 and hsp 70 ( human recombinant proteins from stressgen ) and actin ( prepared from bovine muscle , sigma ) were used as markers . the immuno - stained protein bands were revealed by an enhanced chemiluminescence reaction ( amersham ecl+ ) . all immuno - blots were analyzed by densitometry , and the amounts ( arbitrary units ) of hsp were normalized with reference to actin levels ( arbitrary units ) in the sample . cells used for this experiment were seeded at 2.5  10 cells on a 40 mm culture plate , and they were allowed to grow overnight . after drug treatment , the cells were washed twice with krebs - ringer bicarbonate buffer ( krb , 115 mm nacl , 4.7 mm kcl , 2.5 mm cacl2 , 1.2 mm kh2po4 , 1.2 mm mgso4 , 24 mm nahco3 , 10 mm hepes , ph 7.4 ) . aliquots ( 2.5 ml ) of krb supplemented with 0.01% bovine serum albumin were added to the cells immediately prior to the hypoxia . a billups - rothenberg modular incubator chamber ( billups - rothenberg , inc . , del mar , ca ) in essence , cells were placed in the sealed chamber , and the chamber was flushed with nitrogen for 15 min at flow rate of 20 ml / min . after closing all sealable connectors , the chamber was transferred to an incubator and the cells in the chamber were subjected to a 2-h period of hypoxia at 37c . reoxygenation was initiated by opening the chamber and then replacing the krb with fresh dmem medium . the cells were then cultured in the incubator under an atmosphere of 5% co2 in air at 37c for 16 h. preliminary time course studies showed that a 2-h period of hypoxia followed by 16 h of reoxygenation was optimal for achieving a 1 - 2-fold increase in apoptotic cell death in h9c2 cells . apoptotic cell death was measured by annexin v - fitc - fluos ( roche , penzberg , germany ) and propidium iodide ( pi ) ( sigma , st . louis , mo ) stainings , in which annexin v bound to the exposed phosphatidylserine on the plasma membrane of the apoptotic cells . briefly , cells were rinsed with ice - cold pbs and then resuspended in 100 l of binding buffer containing 140 mm nacl , 2.5 mm cacl2 , 1.5 mm mgcl2 and 10 mm hepes , ph 7.4 . one - hundred microliters of annexin v staining solution ( 1 l of annexin v stock solution being mixed with 100 l of binding buffer ) was added to the cells and incubated in the dark for 15 min at room temperature . the cells were washed with ice - cold pbs and resuspended in 600 l of binding buffer . immediately after mixing with 3 l pi solution ( 50 g / ml ) , the cells were analyzed by flow cytometry using a coulter epics xl flow cytometer ( beckman coulter , inc . , the fluorescence of annexin v and pi were monitored at emission wavelengths of 525 nm and 575 nm , respectively , with the excitation wavelength being at 488 nm for both probes . approximately 10,000 cells were counted in each of the samples and data were statistically analyzed using an expo32 adc analysis program . the percentage of early apoptotic cells was estimated by noting the annexin v positive but pi negative cells , while the percentage of late apoptotic cells was estimated by noting the number of cells which were both annexin v and pi positive . the total apoptotic cell death was calculated by summing up the percentages of early apoptosis and late apoptosis . post - hoc multiple comparisons were performed using least significant difference ( lsd ) test . ( )sch b caused a time- ( 0 - 360 min ) and dose- ( 7.5 and 15 m ) dependent increase in ros production in h9c2 cardiomyocytes under the present experimental condition [ figure 2 ] . when the amount of ros production was estimated by computing the area under the curve plotting the percent increase in fluorescence ( with respect to the time - matched vehicle control ) against time ( up to 360 min ) , ( )sch b at 7.5 and 15 m were found to increase the ros production by 24 and 32% ( p<0.05 ) , respectively , when compared with the vehicle control [ figure 3 ] . pre- and co - incubations with abt or dmtu almost completely abrogated the ( )sch b - induced ros production in h9c2 cardiomyocytes , with the inhibitory effect of abt being more potent [ figure 3 ] . ( )sch b - induced reactive oxygen species ( ros ) production in h9c2 cardiomyocytes . fluorescence intensity was monitored every minute for 360 minutes , as described in materials and methods . data , which were normalized with time - matched control , were expressed as a percentage of value at time zero . values given are the mean of triplicate data effects of cyp inhibitor and antioxidant on ( )sch b - induced ros production in h9c2 cardiomyocytes . 1-aminobenzotriazol ( abt ) and dimethylthiouracil ( dmtu ) were added at final concentrations of 10 mm and 20 mm , respectively , as described in materials and methods . the ( )sch b ( 7.5 or 15 m)-induced ros production was monitored for 360 min . values given are the meansd , with triplicate data . # significantly different from the respective dmso group ( p<0.05 ) exposure of h9c2 cardiomyocytes to ( )sch b ( 7.5 and 15 m ) for 6 h caused a dose - dependent increase ( 40 and 47% , p<0.05 ) in cellular gsh level at 16 h post-()sch b exposure [ figure 4a ] . pre- and co - treatment with abt or dmtu during the ( )sch b exposure strongly suppressed the stimulation of cellular gsh level [ figure 4a ] . similar exposure of h9c2 cardiomyocytes to ( )sch b - induced hsp 25 and hsp 70 production in a dose - dependent manner ( 12 and 25% , 33 and 42% , respectively , p<0.05 ) at 16 h post-()sch b exposure [ figure 4b ] . pre- and co - treatment with abt or dmtu during the ( )sch b exposure completely abrogated the induction of hsp 25 , with the inhibition by abt being more significantly [ figure 4b ] . similarly , the induction of hsp 70 by ( )sch b were also strongly suppressed by pre- and co - treatment with abt or dmtu [ figure 4c ] . effects of cyp inhibitor and antioxidant pre / co - treatment on ( )sch b - induced increases in cellular - reduced glutathione ( gsh ) level and heat shock protein ( hsp ) 25/70 production in h9c2 cardiomyocytes . cells were pre / co - treated with abt or dmtu , as described in materials and methods . cells were exposed to ( )sch b ( 7.5 or 15 m ) for 6 h. cellular gsh and hsp 25/70 levels were measured at 16 h post-()sch b exposure , as described in materials and methods . values given are the meansd , with triplicate data . # significantly from the respective dmso group ( p<0.05 ) ; * significantly different from the control ( )sch b 7.5 m group ( p<0.05 ) exposure to ( )sch b ( 15 m ) for 6 h , in the absence or presence of abt or dmtu , did not affect the extent of apoptosis in cultured h9c2 cardiomyocytes [ figure 5 ] . hypoxia / reoxygenation induced apoptosis in h9c2 cardiomyocytes , with the extent of apoptosis being increased by 1-fold ( p<0.05 ) , as compared to the non - hypoxia / reoxygenation control [ figure 5 ] . ( )sch b strongly suppressed the hypoxia / reoxygenation - induced apoptosis at 16-h post - drug exposure , with the degree of protection being 48% ( p<0.05 ) . the cytoprotection afforded by ( )sch b against hypoxia / reoxygenation - induced apoptosis was almost completely abrogated by pre- and co - treatment with abt or dmtu during the 6-h exposure to ( )sch b. effects of cyp inhibitor and antioxidant pre / co - treatment on ( )sch b protection against hypoxia / reoxygenation - induced apoptosis in h9c2 cardiomyocytes . cells were pre / co - treated with abt or dmtu as described in figure 4 . they were exposed to ( )sch b ( 15 mm ) for 6 h followed by hypoxia / reoxygenation ( hypo / reoxy ) challenge ( hypoxia for 2 h and reoxygenation for 16 h ) at 16-h post-()sch b exposure . # significantly differently from the respective non - hypo / reoxy group ( p<0.05 ) ; * significantly different from the respective dmso - treated hypo / reoxy group ( p<0.05 ) a recent study in our laboratory has shown that cyp - catalyzed reaction is likely involved in the sch b - induced glutathione antioxidant response in rat hearts . presumably , the protective response is triggered by ros arising from the cyp - catalyzed metabolism of sch b. in the present study , ( )sch b , an active stereoisomer of sch b , was found to stimulate ros production in h9c2 cardiomyocytes in situ , which could be completely suppressed by abt or antioxidant . the exposure of h9c2 cardiomyocytes to ( )sch b for 6 h resulted in an optimal stimulation of cellular gsh level and hsp 25/70 production at 16 h post - exposure . the ( )sch b - induced glutathione antioxidant and heat shock responses were associated with the cytoprotection against hypoxia / reoxygenation - induced apoptosis . both glutathione antioxidant / heat shock responses and cytoprotection against hypoxia / reoxygenation - induced apoptosis were abrogated by abt or antioxidant pre / co - treatment during the 6-h ( )sch b exposure . taken together with the observation that the extent of ( )sch b - induced ros production was greatly reduced by abt or dmtu treatment , our results strongly suggested the causal involvement of cyp - mediated ros production in triggering cellular protective responses in h9c2 cardiomyocytes . in this regard , the cumulative amount of ros generated within 6-h exposure ( but not 3 h ) to ( )sch b culminated in optimal glutathione antioxidant and heat shock responses in h9c2 cardiomyocytes ( data not shown ) . conceivably , the sustained production of ros from up to 6 h of cyp - catalyzed metabolism of ( )sch b elicits a redox signaling , possibly through mitogen - activated protein kinases ( mapk ) pathway , with resultant transcription activation of antioxidant / heat shock response element.[1214 ] a recent study indicated that ( )sch b could activate all three mapk pathways , namely , extracellular signal - regulated protein kinase , p38 mapk and c - jun - nh2-terminal kinases , in h9c2 cardiomyocytes within 6 h of treatment . it is well established that oxidative stress is involved in the pathogenesis of ischemia / reperfusion injury , and this pathological condition can be mimicked by subjecting cultured cardiomyocytes to hypoxia / reoxygenation challenge.[1820 ] a burst of ros production arising from the restoration of oxygen to the previous hypoxic cardiomyocytes increases intracellular oxidative stress , which can eventually contribute to necrotic and/or apoptotic cell death . consistently , our results indicated that a 2-h period of hypoxia followed by 16 h of reoxygenation caused significant apoptotic cell death in cultured h9c2 cardiomyocytes . signs of cytotoxicity such as depletion of gsh and alteration of calcium homeostasis have been observed in hypoxia / reoxygenation - challenged cells . the cytoprotection afforded by ( )sch b against hypoxia / reoxygenation - induced apoptosis may mainly be related to the enhanced cellular glutathione status and the subsequent increased resistance of mitochondria to ca - induced permeability transition . as regards the role of hsp 25 and hsp 70 in cytoprotection , an earlier study in our laboratory has demonstrated that the cardioprotection afforded by sch b against myocardial i / r injury is partly contributed by the induction of hsp 25 and hsp 70 in rats . taken together , the results suggest that ( )sch b can elicit cellular glutathione antioxidant and heat shock responses through the intermediacy of cyp - catalyzed ros production , thereby protecting against oxidant - induced apoptosis in h9c2 cardiomyocytes .
objective : schisandrin b ( sch b ) is the most abundant , active dibenzocyclooctadiene derivative isolated from the fruit of schisandra chinensis ( turcz ) baillon ( schisandraceae ) . 
 ( )sch b was found to be the most potent stereoisomer of sch b in producing cytoprotective action in h9c2 cardiomyocytes . 
 the elucidation of biochemical mechanism underlying the cytoprotection of ( )sch b has attracted much interest in the area of preventive medicine . here , we examined whether the ( )sch b - induced enhancement of glutathione antioxidant and heat shock responses and the associated cytoprotection against hypoxia / reoxygenation - induced apoptosis are mediated by reactive oxygen species ( ros ) arising from cytochrome p-450 ( cyp)-catalyzed metabolism of ( )sch b in h9c2 cardiomyocytes.materials and methods : the effects of cyp inhibitor ( 1-aminobenzotriazole , abt ) and antioxidant ( dimethylthiouracil , dmtu ) on ( )sch b - induced ros production and associated increases in cellular - reduced glutathione ( gsh ) level as well as heat shock protein ( hsp ) 25/70 production were investigated in h9c2 cardiomyocytes . 
 the ( )sch b - induced ros generation was monitored with or without abt / dmtu for 6 h in situ , while ( )sch b - induced cellular gsh level and hsp 25/70 production , as well as cytoprotection were measured at 16 h post-()sch b exposure.results:the results indicated that ( )sch b caused a dose - dependent increase in ros production in h9c2 cardiomyocytes , which was completely suppressed by pre- and co - treatment with abt or dtmu . 
 the incubation with ( )sch b for 6 h caused dose - dependent increases in cellular gsh level and hsp 25/70 production , as well as protection against hypoxia / reoxygenation - induced apoptosis at 16-h post - drug exposure in h9c2 cardiomyocytes . 
 all these cellular responses were abrogated by treatment with abt or dmtu.conclusion:the results suggest that ros arising from the cyp - catalyzed metabolism of ( )sch b elicit glutathione antioxidant and heat shock responses , thereby protecting against oxidant - induced apoptosis in h9c2 cardiomyocytes .