葡萄糖酸镁对离体大鼠心肌缺血再灌注细胞凋亡和Fas及FasL的影响

Effects of magnesium gluconate on the apoptosis as well as the expression of Fas and FasL in cardiomyocytes of rats with ischemia reperfusion injury in vitro

目的观察葡萄糖酸镁对离体大鼠心肌缺血再灌注细胞凋亡和Fas/FasL水平是否存在影响及其可能机制。方法48只雄性SD大鼠随机分为3组:对照组,缺血再灌注组(I/R组),葡萄糖酸镁组。实验1:每组取8只大鼠,对照组用改良的K-H液持续灌注110 min;I/R组用改良的K-H液灌流20 min后,停灌30 min,再灌注60 min;葡萄糖酸镁组改良的K-H液中加入葡萄糖酸镁2.4 mmol/L,余同I/R组。检测心肌灌流液中肌酸激酶(CK),乳酸脱氢酶(LDH)含量,心肌组织中总超氧化物歧化酶(T-SOD)、丙二醛(MDA)含量。实验2:取每组其余8只大鼠,实验过程类似实验1,不同点是其再灌注时间改为120 min。实验结束检测心肌Fas和FasL蛋白表达及心肌细胞凋亡指数。观察实验1、2再灌注20min内心律失常发生情况。结果与I/R组比较,葡萄糖酸镁组心律失常发生率显著下降(P<0.01);再灌流出液中CK、LDH含量明显降低(P<0.01);心肌组织T-SOD活性显著升高(P<0.01),MDA含量、Fas/FasL蛋白表达及细胞凋亡指数明显降低(P<0.01)。结论葡萄糖酸镁可清除氧自由基,干预Fas/FasL的表达从而减少心肌细胞凋亡。

Objective To investigate whether magnesium gluconate has direct influence on the apoptosis and expression of Fas and FasL in the cardiomyocytes of rats with ischemia reperfusion injury in vitro. Methods The hearts of 48 Spragne-Dawely rats were isolated, linked to Langendorff perfusion apparatus, and randomly divided into 3 groups： control group, ischemia/reperfusion（I/R） group and magnesium gluconate group, Experiment 1 ： The hearts of 8 rats in control group were perfused with modified KH buffer for 110 min; the hearts of 8 rats in I/R group were perfused with modified KH buffer for 20 min, exposed to ischemia for 30 min, and then reperfused with modified KH buffer for 60 min; the hearts of 8 rats in magnesium gluconate group were perfused with modified KH buffer with magnesium gluconate for 20 min; exposed to ischemia for 30 min and then reperfused with modified KH buffer with magnesium gluconate for 60 min. Lactate dehydrogenase（LDH） and creatine kinase（CK） in the effluent liquid from the hearts were measured after reperfusion. The activity of total superoxide dismutase（T-SOD）, the concentration of malondialdehyde （MDA） in the left ventricle were detected. Experiment 2 ： The methods were same with experiment 1, but the hearts were reperfused for 120 min. After reperfusion the cardiomyocytes apoptosis was examined with Annexin-V-FITC/PI and the expression of Fas and FasL were investigated with immunohistochemistry. After the two experiments, the incidence of ventricular arrhythmias during reperfusion was assessed. Results Compared with I/R group, the incidence of ven- tricular arrhythmias in magnesium gluconate group was decreased（ P 〈 0.01 ） ; The CK and LDH level in the effluent liquid from the heart in magnesium glueonate group was lower than that in I/R group（ P 〈 0.01 ） ; The MDA level in the left ventricle in magnesium gluconate grout） was decreased than that in I/R group （P 〈0. 01 ） ; The T-SOD activity in the left ventricle in magnesium gluconate group was higher than that in I/R group（ P 〈 0. 01 ） ; The Fas/FasL expression and the cardiomyocytes apoptosis index were significantly lower in magnesium gluconate group than that in I/R group（P 〈0.01 ）. Conclusion Magnesium gluconate has protec- tive effect on myocardial ischemia reperfusion injury in rats. The protective effect may be related to decreasing myocytes apoptosis by descending the expression of Fas/FasL and reducing the production of oxygen-derived free radicals.