细胞凋亡和程序性坏死在心肌缺血再灌注损伤中的作用研究

The Role of Apoptosis and Necroptosis in Myocardial Ischemia-Reperfusion Injury

目的通过使用特异性抑制剂ZVAD和KN93分别抑制细胞凋亡和程序性坏死的发生,来比较在心肌缺血再灌注损伤中哪一种细胞死亡方式占比更大。方法选择雄性C57/BL6小鼠40只,随机分为假手术(sham)组、缺血再灌注(I/R)组、ZVAD组和KN93组,在结扎前15 min,sham组不做处理,I/R组注射二甲基亚砜(DMSO)溶液,ZVAD组和KN93组则分别注射ZVAD和KN93溶液,建立心肌缺血再灌注损伤模型。氯化三苯基四氮唑(TTC)染色测心肌梗死面积,酶联免疫吸附测定(ELISA)法检测心肌肌钙蛋白I(cTnI),炎性因子肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)水平,用蛋白印迹(WesternBlot)检测特异蛋白受体相互作用蛋白激酶-3(RIP3)和胱天蛋白酶-3(caspase-3)的表达。结果(1)ZVAD组和KN93组较I/R组心肌梗死面积明显缩小(P<0.05),且KN93组相比ZVAD组,心肌梗死面积更小(P<0.05);(2)ZVAD和KN93处理后相比I/R组,cTnI表达明显减少(P<0.05),而KN93组cTnI低于ZVAD组(P<0.05);(3)IL-1β和TNF-α在I/R组中明显升高(P<0.01),而在ZVAD组和KN93组中明显降低(P<0.05),KN93组IL-1β和TNF-α明显低于ZVAD组(P<0.05);(4)caspase-3和RIP3的表达在I/R组显著升高(P<0.05),而ZVAD和KN93组相对于I/R组二者的表达有所减少。其中RIP3的表达,KN93组相比I/R组显著下降(P<0.05),ZVAD组相比I/R组有所下降,但无统计学意义(P>0.05);caspase-3的表达,ZVAD组相比I/R组显著下降(P<0.05),KN93组相比I/R组无统计学意义(P>0.05)。结论(1)ZVAD和KN93干预可以减轻I/R损伤,起到心肌保护作用;(2)KN93相比ZVAD在减少心肌细胞死亡方面存在较大优势,提示在I/R损伤中,相比细胞凋亡,程序性坏死可能起更大作用。

Objective To investigate the role and weight of apoptosis and necroptosis induced by specific inhibitors ZVAD and KN93 in myocardial ischemia-reperfusion injury(MIRI).Methods A total of 40 male C57/BL6 mice were randomized into sham,I/R,ZVAD and KN93 group.Sham group kept untreated at 15 minutes before ligation,I/R group was injected with DMSO solution as well as ZVAD and KN93 groups injected with ZVAD and KN93 solutions respectively.Myocardial infarction area was measured by TTC staining.Myocardial troponin cTnI,inflammatory factors TNF-αand IL-1βwere detected by ELISA.Specific proteins RIP3 and caspase-3 were detected by Western blot.Results Compared with I/R group,ZVAD and KN93 groups showed significantly smaller infarct area(P<0.05)just as the smaller area in KN93 group than ZVAD group(P<0.05),cTnI expressions were decreased in ZVAD and KN93 groups(P<0.05)with further reduced in KN93 group compared with ZVAD group(P<0.05).IL-1βand TNF-αwere significantly increased in I/R group(P<0.01),decreased in ZVAD and KN93 groups(P<0.05)and further decreased in KN93 group compared with ZVAD group(P<0.05).The expressions of caspase-3 and RIP3 were higher in I/R group(P<0.05)and lower in ZVAD and KN93 groups than I/R group.Meanwhile,the RIP3 in KN93 group and caspase-3 in ZVAD group were decreased(P<0.05)compared with those in I/R group(P<0.05).Conclusion(1)ZVAD and KN93 could decrease I/R injury and protect the myocardium.(2)KN93 exerts great advantages in reducing myocardial cell deaths compared with ZVAD,which suggests that necroptosis may play a more important role in I/R injury than apoptosis.