| Objective: To investigate the protective effects and possible mechanism of emulsified isoflurane on myocardial cells after hypoxia-/ reoxygenation(H/R) in cultured myocardial cells from neonatal rat in order to provide experiment foundation for its later clinical reseach or application.Methods: Myocardial hypoxia/reoxygenation model was established by culturing primary myocardial cell from neonatal rat in vitro. The myocardial cells were divided into four groups, the control group(cultured in normoxia 6h), H/R group(cultured in hypoxia 3h and reoxgenation 3h) , fat milk group(fat milk pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h), EI group(EI pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h). Every group cellular morphologic changes and pulsatile frequence were observed under inverted microscope and the ultrastructure of myocardial cells was observed under transmission electron microscope(TEM);Myocardial cell's survival rate were determined by XTT;Cardiomyocytes were collected and ultrasonic-ccation for the later analysis of SOD and the content of MDA,the activities of plasma superoxide dismutase (SOD) were measured by method of xanthine oxidase and the contents of serum Malonicaldehyde (MDA) by the method of thiobarbituric acid;The activities of lactate dehydrogenase (LDH) in cell-cultured solution after H-R injury were measured according to relevant kits; The apoptotic rate of myocardial cells was determined by flow cytometry; The expression of bcl-2 mRNA and bax mRNA were detected by using RT-PCR method; The expression of Bcl-2/Bax protein were observed by Western blot.Results:1 The morphologic changes of cardiomyocytes: After H/R procedure cardiomyocytes beated unregularily and powerlessly,sells contracted and tended to get round in shape ,cellular pseudopods decreased in H/R,EI ,fat milk groups compared with control group. The ultrastrctural changes of cardiomyocytes were improved in EI,fat milk groups compared with H/R.2 The survival rate of myocardial cell:Compared with control group,the rate decreased in H/R,EI,fat milk groups(p<0.05).but compared with H/R group,the rate increased in EI and fat milk groups.3 SOD activities and LDH,MDA concentration: Compared with control group,SOD activity decreased and MDA,LDH increased in H/R group (p<0.05). Compared with H/R group, SOD activity increased, MDA and LDH decreased (p<0.05)in EI and fat milk groups.4 The apoptotic rate and the expression of correlated apoptotic gene Both EI and fat milk groups could significantly decrease the apoptotic rate (P< 0.05),while EI shoud have stronger effect(P<0.05). EI group signify -cantly up-regulated Bcl-2 expression and down-regulated Bax expression (P<0.05) while fat milk group can't(P< 0.05).Conclusion1 H/R injured cultured myocardial cells from neonatal rat markedly,including the decreasing of myocardial cell's survival rate, the increasing of serum myocardial enzyme leakage and myocardial MDA,the elevation of apoptotic rate ,injury of myocardial ultrastructure.2 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R.,which included reduction of myocardial cell's survival rate,the reduction of the leakage of myocardial enzyme,reduction of oxygen-derived free radials,protection of myocardial ultrastructure,inhibition of apoptosis, better recovery of cardiac function during reoxygenation.3 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R. The possible mechanism may be that: one is by reducing Oxygen Free Radical,the other is by inhibiting myocardial apoptosis through up-regulating Bcl-2 expression and down- regulating Bax expression .
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