乳化异氟醚对乳鼠缺氧/复氧心肌细胞的保护作用及对caspase-3表达的影响

Emulsified isoflurane protects hypoxia/reoxgenation injury in cultured neonatal rat myocardial cells by suppressing caspase-3 expression

目的观察乳化异氟醚(8%体积比)对培养乳鼠心肌细胞模拟缺血再灌注损伤后心肌细胞的保护作用并探讨其可能作用机制。方法利用原代培养的Wistar乳鼠心肌细胞建立模拟心肌缺血再灌注模型,分正常培养组、H/R组、脂肪乳组、乳化异氟醚(终浓度0.28 mmol/L)组。各组心肌细胞作相应分组处理后,利用倒置显微镜观察心肌细胞的生长状态和搏动频率;XTT法测定细胞活力;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活力,硫代巴比妥酸显色法测定细胞内丙二醛(MDA)含量,并测定缺氧复氧后细胞培养液中乳酸脱氢酶(LDH)活性;Western blot法检测caspase-3蛋白表达。结果与正常培养组比较H/R组细胞SOD下降,MDA、LDH及caspase-3蛋白表达升高(P<0.05)。乳化异氟醚组与H/R组比较,心肌细胞SOD活性提高,MDA、LDH及caspase-3蛋白表达下降(P<0.05),且心肌细胞形态及超微结构改善。结论乳化异氟醚具有明显的抗缺氧复氧损伤、保护心肌细胞的作用,其作用机制可能与抗自由基损伤及抑制caspase蛋白表达水平有关。

Objective To investigate the protective effects of emulsified isoflurane （8% v/v） on myocardial cells after hypoxia/reoxygenation （H/R） in cultured myocardial cells from neonatal rats. Methods Myocardial hypoxia/reoxygenation model was established by culturing myocardial cells primarily isolated from neonatal rats. The myocardial cells were divided into 4 groups, the normal group, model group, fat milk treatment group, and mulsified isoflurane treatment group （0.28 mmol/L）. Cellular morphologic changes and pulsation frequency were observed under inverted microscope. Cell activity was determined by XTF assay. The activities of plasma superoxide dismutase （SOD） was measured by xanthine oxidase method and the contents of serum malonicaldehyde （MDA） by thiobarbituric acid method. The activities of lactate dehydrogenase （LDH） in the culture medium after H/R injury was measured. Western blot analysis was used to detect caspase-3 protein expression in every group. Results Compared with normal group, SOD activity was decreased, and MDA content, LDH activity and the expression of caspase-3 protein were increased in model group （P 〈 0. 05 ）. Compared with model group, SOD activity was increased, while MDA content, LDH activity and the expression of caspase-3 were decreased （P 〈 0. 05 ） after emulsified isoflurane treatment. The morphological and ultrastructural changes of cardiomyocytes were improved after the treatment. Fat milk treatment group showed similar changes, but not significant as emulsified isoflurane treatment did. Conclusion Emulcified isoflurane has protective effects on cultured neonatal rat myocardial cells after H/R injury, which may be related to reducement of oxygen free radical and the inhibition of caspase-3 expression.