Protective Effects Of Sevoflurane Postconditioning On Neonate Rat's Cardiomyocytes Injury Induced By Anoxia/Reoxygenation And Its Mechanism In Vitro

Objective:To investigate whether sevoflurane postconditioning could protect cultured neonate rat's cardiomyocytes from injury induced by anoxia/reoxygenation and whether the mechnism of cardiomyocytes protection is related to p38 mitogen-activated protein kinases pathway (p38 MAPK).Methods:Cardiomyocytes were obtained from Sprague-Dawley (SD) rates'heart aged 1～3days, digested by trypsin, and transported into culture medium. The cultured cells were observed by inverted microscope every day, then identified by a-sarcometric actin immunohistochemically. Anoxia/reoxygenation injury and sevoflurane postconditioning model were made for experiment. Cultured cardiomyocytes were randomly divided into 7 groups:Normal Control group (Group C):cells cultured in incubator for 3h. Anoxia/reoxygenation group (Group A/R):cells suffered from 2h anoxia followed by 1h reoxygenation. Sevoflurane-postconditioning group (Group S-post):cells suffered from 2h anoxia followed by 20min sevoflurane postconditioning and 40min reoxygenation. Sevoflurane-postconditioning+SB203580 group (Group S-post+SB):cells suffered from 2h anoxia and followed by 20min sevoflurane postconditioning and 40min reoxygenation, and SB203580, a specific p38 MAPK inhibitor were used during sevoflurane-postconditioning, Sevoflurane-postconditioning+Dimethyl sulphoxide group (Group S-post+DMSO): cells suffered from 2h anoxia and followed by 20min sevoflurane postconditioning and 40min reoxygenation, and DMSO, the dissolvent of SB203580 were used during postconditioning. SB203580 group (Group SB):cells suffered from 2h anoxia followed by 1h reoxygenation, and SB203580 were used during the first 20min of the reoxygenation. Dimethyl sulphoxide group (Group DMSO):cells suffered from 2h anoxia followed by 1h reoxygenation and DMSO were used during the first 20min of the reoxygenation. At the end of experiment, the activities of lactic dehydrogenase (LDH), creatine kinase (CK), cells'survival and apoptosis rate were measured, and western blot was also used to detect the levels of p38 MAPK and phosphor-p38 MAPK(p-p38).Results:Rat's cardiomyocytes were isolated and cultured successfully in vitro and were identified by a-sarcometric actin immunohistochemically. Compare to Group C, cardiomyocytes survival rate in Group A/R decreased while apoptosis rate, LDH and CK level increased(P<0.05). Compare to Group A/R, cardiomyocytes survival rate in Group S-post increased while apoptosis rate, LDH and CK level decreased (P<0.05). Compare to Group S-post, cardiomyocytes survival rate in Group S-post+SB decreased while apoptosis rate, LDH and CK level increased (P<0.05). There is no statistic significance on the variance mentioned above between Group S-post and Group S-post+DMSO or Group A/R, Group SB and Group DMSO(P>0.05). There is no statistic significance on the level of p38 between groups (P>0.05). However, the level of p-p38 is higher in Group S-post than that of Group A/R (P<0.05), p38 MAPK inhibitor SB203580 decline p-p38 in Group S-post+SB (compared with Group S-post P<0.05).The level of p-p38 in Group SB is also lower than that of Group A/R (P<0.05). There is no statistic significance on the level of p-p38 between Group S-post and Group S-post+DMSO or Group A/R and Group DMSO (P>0.05).Conclusion:Rat's cardiomyocytes were isolated and cultured successfully in vitro. Anoxia/reoxygenation may cause injury in cultured neonatal rat's cardiomyocytes. Sevoflurane postconditioning can lessen the injury of cultured neonatal rat's cardiomyocytes induced by anoxia/reoxygenation. p38 MAPK pathway mediates the cardioprotective effects in sevoflurane- postcondioning.