Effect Of Emulsified Isoflurane On The Neonatal Rat Primary Cardiac Myocytes Hypoxia/Reoxygenation Injury

Objective: To discuss the effect of emulsified isoflurane (EI) with different dosage on neonatal rat hypoxia/reoxygenation (H/R) cardiac myocytes of primary culture in order to provide experiment foundation for its later clinical reseach or application.Methods: To prepare the H/R injury model on the basis of in-vitro neonatal rat cardiac myocytes culture and divide them into 18 groups at random, namely, normal control group (N group), H/R group, H/R+fat emulsion group (F group), The one, two, three, four, five, six, seven,eight,nine and ten time s of 0.28 mmol/L EI are divided into EI1～EI10 group, H/R+Glibenclamide group (G group), and H/R+1.68 mmol·L^-1 EI +10 mmol·L^-1 +Glibenclamide group (EIG group), H/R+100 nmol·L^-1 wortmannin(W group),H/R+1.68 mmol·L^-1 EI +100 nmol·L^-1 wortmannin(EIW group)and H/R+1.68 mmol·L^-1 EI +10 umol·L^-1 Bay K8644(EIB group).Each group extracts the clean liquid on the cellular culture to mensurate LDH and cellular homogenate to mensurate SOD and MDA; place upside down the microscope to observe the characteristics of cardiac myocytes growth and changes of its forms. Dye the calcium probe Fura-2 AM and use the flow cytometry to inspect the calcium concentration in cardiac myocytes. The protein expression of Akt were detected 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 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.3 Both EI group and F group can decrease the H/R injury,but the EI group is better.4 The protein expression of Akt's tendency is P-AktN group < P-AktEIW group P-AktH/R group < P-AktF group 2+ concentration is N group < EI6 group < EIB group < F group 2+ concentration all increase(P<0.05)and SOD declines (P<0.05). Compared to H/R group, LDH, MDA and intracellular Ca²⁺ concentration of each EI dose group declines (P<0.05) and SOD declines (P<0.05); SOD of F group declines (P<0.05), and the differences of its LDH, MDA and intracellular Ca²⁺ concentration have no statistic significanc(eP>0.05). Compared to F group, LDH, MDA and intracellular Ca²⁺ concentration of each EI dose group declines (P<0.05) and SOD increases (P<0.05). Compared to the EI small dose group, the differences of LDH, MDA and SOD of EI medium dose group have no statistic significance(P>0.05)and its Ca²⁺ concentration increases(P<0.05); the LDH of EI large dose group increases(P<0.05), the intracellular Ca²⁺ concentration declines(P<0.05) and the differences of MDA and SOD have no statistic significance(P>0.05).3 EI 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, Thirdly,may be associated with the PI3K/Akt Channel activated by it.