L型钙通道参与乳化异氟醚对离体心肌细胞缺氧/复氧损伤的保护作用

目的探讨乳化异氟醚(EI)对原代培养心肌细胞缺氧/复氧损伤的保护作用及其与钙通道的关系。方法用原代培养乳鼠离体心肌细胞,建立缺氧/复氧(H/R)损伤模型,随机分为5组(n=8),正常组(N组)、缺氧/复氧组(H/R组)、H/R+脂肪乳组(F组)、H/R+1.68 mmol.L-1EI组(EI组)和H/R+1.68 mmol.L-1EI+10 umol.L-1BayK8644组(EIB组)。各组取细胞培养上清液测定LDH活力与cTnI含量,心肌细胞匀浆后测定SOD活力与MDA含量;用倒置显微镜观察心肌细胞生长特性及形态的变化。钙离子探针Fura-2 AM染色后用流式细胞术检测心肌细胞内钙离子浓度。结果与N组比较,各组的LDH、MDA、cTnI、钙离子浓度均升高(P<0.05),SOD下降(P<0.05);与H/R组比较,F组的SOD下降(P<0.05),LDH、MDA、cTnI、钙离子浓度的差异均无统计学意义(P>0.05);EI组和EIB组的LDH、MDA、cTnI、钙离子浓度均降低(P<0.05),SOD升高(P<0.05)。与F组比较,EI组的LDH、MDA、cTnI、钙离子浓度均下降(P<0.05),SOD升高(P<0.05)。与EI组比较,EIB组的LDH、MDA、cTnI、钙离子浓度均升高,SOD降低(P<0.05)。结论乳化异氟醚对原代培养心肌细胞缺氧/复氧损伤时的保护作用与其抑制L型钙通道,降低细胞内钙超载有关。

L-type calcium channel participates in the protective effect of emulsified isofluraneon the primary cardiac myocytes hypoxia/reoxygenation injury in neonatal rats

Aim To discuss the mechanism of protective effect of emulsified isoflurane(EI) on neonatal rat hypoxia/reoxygenation(H/R) cardiac myocytes of primary culture and the association with reduction of intracellular calcium overload.Methods The H/R injury model on the basis of in vitro neonatal rat cardiac myocytes culture was prepared and divided into 5 groups randomly,namely,normal control group(N group),H/R group,H/R+fat emulsion group(F group),H/R+1.68 mmol·L-1 EI group(EI group),H/R+1.68 mmol·L-1 EI +10 umol·L-1 Bay K8644 group(EIB group).The clean liquid on the cellular culture was extracted in each group to mensurate LDH and cellular homogenate was made to mensurate SOD and MDA;the microscope was placed upside down to observe the characteristics of cardiac myocytes growth and changes of its forms;the calcium probe Fura-2 AM was and dyed the flow cytometry was used to inspect the calcium concentration in cardiac myocytes.Results Compared to N group,the LDH,MDA,cTnI and intracelluar Ca2+ concentration of other groups all increased(P<0.05) and SOD declined(P<0.05).Compared to H/R group,SOD of F group declined(P<0.05),and the differences of the LDH,MDA,cTnI and intracelluar Ca2+ concentration had no statistic significance(P>0.05);LDH,MDA,cTnI and intracelluar Ca2+ concentration of EI and EIB group declined(P<0.05) and SOD increased(P<0.05).Compared to F group,the LDH,MDA,cTnI and intracelluar Ca2+ concentration of EI group declined(P<0.05) and SOD increased(P<0.05).Compared to EI group,the LDH,MDA,cTnI and intracelluar Ca2+ concentration of EIB group increased(P<0.05) and SOD declined(P<0.05).Conclusions EI has the protective effect on the neonatal rat cardiac myocytes with H/R injury,and its mechanism may be associated with blocking L-type calcium channel and reduction of intracellular calcium overload.