TRPM7-PI3K在治疗性浅低温(34℃)处理的大鼠离体心肌缺血-再灌注模型中的作用

摘要

目的 在离体大鼠心肌缺血-再灌注模型中,检测常温下心肌缺血-再灌注损伤后TRPM7的表达水平变化以及治疗性浅低温(34℃)在心肌缺血-再灌注中对TRPM7的影响及其与PI3K通路的关系.方法 SPF清洁级健康成年雄性SD大鼠108只,随机分为六组,每组18只,SHAM组:正常大鼠组;I/R组:37℃灌流液灌流大鼠心脏30 min,缺血30 min,37℃灌注液再灌注120 min;I/R+2-APB组:37℃灌流液灌流30 min,缺血30 min,灌注液中加2-APB后行37℃灌注液再灌注120 min;34H+ I/R组:37℃灌流液灌流30 min,缺血30 min,34℃灌注液再灌注120 min;34H+ I/R+ WORT组:37℃灌流液灌流30 min,缺血30 min,在灌注液加wortmannin后行34℃灌注液再灌注120 min;34H+ I/R +2-APB组:37℃灌流液灌流30 min,缺血30 min,在灌注液中加2-APB后行34 ℃灌注液再灌注120 min.以压力换能器连接Medlab生物信号采集系统记录平衡灌流30 min末(T0,基础值),再灌注30 min(T1),60 min(T2),90 min (T3),120 min(T4)各时间点的心率(HR)、左心室收缩压(LVSP)、左心室舒张末压(LVEDP)、左心室内压上升/下降最大速率(±dp/dt max).在再灌注120 min末,取心脏进行HE染色观察心肌损伤,Westernblot测定TRPM7蛋白含量,并测定TTC染色后的心肌梗死面积.结果 I/R组TRPM7的表达量升高(P＜0.05),心肌梗死面积明显增多(P＜0.05);I/R+2-APB组与SHAM组和I/R组比较,TRPM7表达量明显减少(P＜0.05),心肌梗死面积与I/R组比较明显减少(P＜0.05);34H+ I/R组TRPM7的表达量与SHAM组和I/R组相比明显减少(P＜0.05),心肌梗死面积与I/R组相比明显减少(P＜0.05);34H+ I/R+ WORT组TRPM7表达量与34H+ I/R组相比增多(P＜0.05),心肌梗死面积与34H+ I/R组相比有所增加(P＜0.05);34H +I/R +2-APB组TRPM7表达量与34H+ I/R组相比差异无统计学意义(P＞0.05),与34H+ I/R+ WORT组相比其TRPM7表达量减少(P＜0.05);34H+ I/R+2-APB组心肌梗死面积与34H+ I/R组相比差异无统计学意义(P＜0.05),与34+ I/R+ WORT组相比,其心肌梗死面积减小(P＜0.05).各组之间血流动力学变化差异无统计学意义(P＜0.05).结论 在离体心肌缺血-再灌注模型中,心肌缺血-再灌注损伤时TRPM7蛋白呈现高表达;治疗性浅低温(34℃)可以抑制心肌缺血-再灌注中TRPM7的高表达,并且这种保护作用是通过PI3K通路介导产生的.%Objective To investigate the effect of TRPM7 on the isolated myocardial ischemia reperfusion model in rats and to investigate the relationship between TRPM7 and PI3K pathway in the treatment of mild therapeutic hypothermia at 34 ℃.Methods 108 healthy adult male Sprague-Dawley rats were divided into six groups and each group has 18 cases,SHAM group,do not do any processing;I/R group,with 37 ℃ during ischemia for 30 min and reperfusion for 120 min;I/R + 2-APB group,with the addition of TRPM7 inhibitor 2-APB during reperfusion period;34H + I/R group,with 34 ℃ of mild therapeutic hypothermia during reperfusion period;34H + L/R + WORT group,with 34 ℃ of mild therapeutic hypothermia and the addition of PI3K inhibitor wortmannin during reperfusion period;ischemia/reperfusion with mild therapeutic hypothermia and 34H + I/R + 2-APB group,with 34 ℃ of mild therapeutic hypothermia and the addition of TRPM7 inhibitor 2-APB during reperfusion period.Then record the hemodynamics including the level of heart rate (HR),left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure (LVEDP),± dp/dtmax (maximum rate and minimal rate of rise of left ventricular) at the time point of baseline,30 min before ischemia (T0);30 min after reperfusion (T1);60 min after reperfusion (T2);90 min after reperfusion (T3);120 min after reperfusion (T4).At the end of 120 min reperfusion,triphenyltetrazolium chloride staining (TTC) were used to evaluate myocardial infarction size;western blot were used to investigate TRPM7 expression;HE staining was used to observe the myocardial injury.Result In I/R group,the expression of TRPM7 and myocardial infarction area [(52.74 ± 1.25)％] was significantly increased compared with Sham group [(13.87 ± 1.17)％,P ＜0.05];In I/R +2-APB group,the expression of TRPM7 and myocardial infarction area [(42.85 ± 1.19)％] was decreased compared with Sham group and I/R group (P ＜ 0.05);In 34H + I/R group,the high expression of TRPM7 induced by ischemia reperfusion injury was reversed,the expression of TRPM7 was decreased as well as myocardial infarction area [(34.63 ± 0.72) ％] was decreased compared with Sham group and I/R group (P ＜ 0.05);In 34H + I/R + WORT group,the expression of TRPM7 was increased as well as myocardial infarction area [(48.89 ± 0.52) ％] was increased compared with 34H + I/R group (P ＜ 0.05);In 34H + I/R + 2-APB group,the expression of TRPM7 was decreased as well as myocardial infarction area compared with 34H + I/R + WORT group (P ＜ 0.05).There was no significant difference in TRPM7 expression and myocardial infarction area between 34H + L/R group and 34H + I/R + 2-APB group [(33.51 ± 1.31) ％ vs.(34.63 ± 0.72) ％,P ＞ 0.05].There was no significance difference of hemodynamic changes among the groups (P ＞ 0.05).Conclusion TRPM7 is highly expressed in myocardial ischemia reperfusion injury in a rat isolated heart model;Mild therapeutic hypothermia at 34 ℃ can inhibit the high expression of TRPM7 in myocardial ischemia reperfusion,and the protective effect is mediated by PI3K pathway in a rat isolated heart model.