七氟醚后处理减轻大鼠心肌缺血-再灌注损伤

Effects of sevoflurane postconditioning on necroptosis during myocardial ischemia-reperfusion injury in rats

目的分析七氟醚后处理对大鼠心肌缺血-再灌注损伤的影响并探讨其机制。方法清洁级成年雄性SD大鼠72只,3月龄,体重180~230 g。采用随机数字表法分为四组:假手术组(S组)、单纯七氟醚组(Sev组)、缺血-再灌注组(IR组)和七氟醚后处理组(SP组),每组18只。S组:穿线不阻断左冠状动脉前降支(LAD);Sev组:穿线不阻断LAD,穿线稳定后60 min吸入2.4%七氟醚15 min;IR组:穿线稳定后30 min阻断LAD,缺血30 min,再灌注2 h;SP组:穿线稳定后30 min阻断LAD,缺血30 min,再灌注2 h,同时在穿线稳定后60 min吸入2.4%七氟醚15 min。于再灌注后2 h,采用ELISA法检测血清LDH浓度,Western Blot法检测心肌受体相互作用蛋白1(RIPK1)、p-RIPK1、受体相互作用蛋白3(RIPK3)、p-RIPK3、混合系激酶结构域样蛋白(MLKL)、p-MLKL含量,荧光显微镜观察程序性坏死心肌细胞的荧光强度,1%氯化三苯基四氮唑(TTC)染色法检测心肌梗死面积百分比,HE染色后光镜下观察心肌病理学形态。结果与S组比较,IR组和SP组血清LDH浓度明显升高(P<0.05),心肌RIPK1、p-RIPK1、RIPK3、p-RIPK3、MLKL、p-MLKL含量明显增加(P<0.05),程序性坏死心肌细胞荧光强度明显增强(P<0.05),心肌梗死面积百分比明显升高(P<0.05),心肌细胞排列更为紊乱。与IR组比较,SP组血清LDH浓度明显降低(P<0.05),心肌RIPK3、p-RIPK3、MLKL和p-MLKL含量明显减少(P<0.05),程序性坏死心肌细胞荧光强度明显减弱(P<0.05),心肌梗死面积百分比明显降低(P<0.05),心肌细胞排列更为整齐。S组和Sev组间各指标差异无统计学意义。结论七氟醚后处理可通过升高血清LDH浓度、降低心肌梗死面积百分比、改善心肌病理学改变减轻大鼠心肌缺血-再灌注损伤,其机制可能与抑制缺血-再灌注损伤后心肌细胞程序性坏死相关。

Objective To evaluate the effects of sevoflurane postconditioning on necroptosis during myocardial ischemia-reperfusion injury rats.Methods Seventy-two adult male Sprague-Dawley rats,aged 3 months,weighing 180-230 g,were randomly divided into 4 groups:sham control group(group S),purely administration of sevoflurane group(group Sev),ischemia-reperfusion group(group IR)and sevoflurane postconditioning group(group SP),18 rats in each group.Group S was only threading but not blocking the left anterior descending coronary artery(LAD).Group Sev was only threaded but LAD was not blocked,2.4%sevoflurane was inhalated continued for 15 minutes after threading 60 minutes.Group IR was threaded 30 minutes,LAD ischemia was blocked for 30 minutes,and reperfusion was performed for 2 hours.Group SP was threaded 30 minutes,LAD ischemia was blocked for 30 minutes,reperfusion was performed for 2 hours,and 2.4%sevoflurane was inhalated continued for 15 minutes after threading 60 minutes..After reperfusion,the serum LDH concentration was detected by ELISA method,the expression levels of receptor interacting protein kinase 1(RIPK1),p-RIPK1,receptor interacting protein kinase 3(RIPK3),p-RIPK3,mixed lineage kinase domain-like protein(MLKL),and p-MLKL were measured by western blot,the fluorescence signal value of necroptosis cardiomyocyte was observed by fluorescence microscopy,myocardial infarct size was measured by 1%2,3,5-triphenyltetrazolium chloride(TTC),and the myocardial pathological slices of rats in each group were observed under the light microscope after HE staining.Results Compared with group S,serum LDH concentration risen(P<0.05),the expression of RIPK1,p-RIPK1,RIPK3,p-RIPK3,MLKL,and p-MLKL was up-regulated(P<0.05),the fluorescence intensity of necrotic myocardial cells was enhanced(P<0.05),myocardial infarction area increased(P<0.05)and cardiomyocytes arranged disorderly in groups IR and SP.Compared with group IR,serum LDH concentration reduced(P<0.05),the expression of RIPK3,p-RIPK3,MLKL,and p-MLKL was down-regulated(P<0.05),the fluorescence intensity of necrotic myocardial cells was inhibited(P<0.05),myocardial infarction area decreased(P<0.05)and cardiomyocytes lined up neatly in the group SP.There were no statistical differences between groups S and Sev in these indicators.Conclusion Sevoflurane postconditioning could reduce myocardial ischemia-reperfusion injury in rats,improve hemodynamics,cardiac function indicators and myocardial pathology changes,and its mechanism relates to necroptosis.