||A rat model of myocardial ischemia and reperfusion injury was used for assessing the antioxidant action of Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from F̲r̲u̲c̲t̲u̲s̲ S̲c̲h̲i̲s̲a̲n̲d̲r̲a̲e̲, in the myocardium. The time-dependent changes in myocardial mitochondrial glutathione antioxidant system and Hsp25/Hsp70 expression in Langendorff-perfused hearts prepared from Sch B-pretreated rats, with or without ischemia-reperfusion challenge, were examined in the present study. To further define the differential role of enhancement of mitochondrial glutathione antioxidant system and induction of HspP25/Hsp70 in the cardioprotection afforded by Sch B, the effect of combined treatment with L-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteinyl synthetase and phorone (PHO), an agent depleting reduced glutathione (GSH), on IR injury in Sch B-pretreated rat hearts was examined. In addition, diphenyl bicarboxylate (DDB), an inactive analogue of Sch B, was also included in this study for comparison. Results obtained from the time course study indicated that Sch B pretreatment at 1.2mmol/kg produced a time-dependent change in mitochondrial glutathione antioxidant system and heat shock proteins (Hsps) expression, with the maximum enhancement observable at 48h post-dosing. In addition, the myocardial protection against IR injury produced by Sch B pretreatment was associated with the enhancement of mitochondrial glutathione antioxidant system and ATP generation capacity. Besides, Sch B pretreatment produced a time-dependent and biphasic change in Hsp25/Hsp70 expression in perfused hearts, with the maxmium enhancement observable at 48h post-dosing. The Sch B-induced increases in Hsp25 and Hsp70 expressions were temporally correlated with the increase in resistance to IR injury. Under IR condition, the significant increase in Hsp 25/Hsp70 expression may be related to the depletion of myocardial ATP level. Consistently, the inhibition of IR-induced increase in Hsps25/Hsp70 expression by Sch B pretreatment was associated with the enhancement in myocardial ATP generation capacity. However, DDB treatment, which neither enhanced myocardial mitochondrial glutathione antioxidant system nor increased tissue ATP generation capacity, did not produce any cardioprotective action. BSO/PHO treatment, which caused the depletion of mitochondrial GSH, abolished the beneficial effects of Sch B treatment on mitochondrial glutathione antioxidant system and ATP generation capacity, as well as almost completely abrogated the cardioprotective action of Sch B against IR injury. Besides, heat shock treatment, which caused a prominent induction of Hsp70 expression but no significant change in mitochondrial glutathione antioxidant system, protected against myocardial IR injury to a lesser extent than that of Sch B treatment. In conclusion, the ability of Sch B to enhance myocardial mitochondrial glutathione antioxidant system and ATP generation capacity rather than Hsps induction mainly contributes to the cardioprotection against IR injury.