The protective effect of interleukin-1 receptor antagonist on postischemic reperfused myocardium and its possible mechanism

目的探讨急性心肌梗死(AMI)再灌注过程中白介素-1β(IL-1β)的变化及重组人白介素-1受体拮抗剂(rhIL-1ra)对缺血再灌注心肌的保护作用及其机制.方法 (1)用ELISA法测定了8名健康人和22例接受急诊经皮冠脉介入治疗(PCI)并且梗死相关血管(IRA)血流达心肌梗死溶栓(TIMI)3级的AMI患者血浆IL-1β水平的动态变化.(2)在兔心肌缺血50 min再灌注4 h的模型上,观察不同剂量rhIL-1ra(A组:rhIL-1ra 10 mg/kg;B组:rhIL-1ra 20 mg/kg;C组:rhIL-1ra 40 mg/kg)对心肌梗死面积、心肌髓过氧化物酶(MPO)活性、心肌细胞凋亡程度(TUNEL、Annexin Ⅴ和DNA-梯形图法)及凋亡相关基因Bcl-2/Bax的表达.结果 (1)22例AMI患者再灌注前血浆IL-1β(28 pg/ml±9 pg/ml)已明显高于健康对照组(20 pg/ml±11 pg/ml, P<0.05),IRA再通后12 h血浆IL-1β水平达高峰(86 pg/ml±14 pg/ml),至术后24 h IL-1β血浆水平(56 pg/ml±15 pg/ml)仍明显高于再灌注前 (28 pg/ml±9 pg/ml, P<0.01).(2)兔AMI对照组与rhIL-1ra各组之间心肌缺血面积差异均无显著意义,但各rhIL-1ra组梗死心肌面积均小于对照组,组间差异呈剂量依赖性(47%±7%, 34%±8%, 31%±6%比61%±11%,分别P<0.05, P<0.01和P<0.01).rhIL-1ra呈剂量依赖性地减低心肌组织中MPO活性(16.6 min-1·g·w·w-1±3.6 min-1·g·w·w-1, 10.9 min-1·g·w·w-1±1.9 min-1·g·w·w-1, 7.8 min-1·g·w·w-1±2.2 min-1·g·w·w-1比20.5 min-1·g·w·w-1±4.5 min-1·g·w·w-1, 分别P<0.05, P<0.01和P<0.01)及心肌细胞的凋亡程度,各组TUNEL阳性细胞数及AnnexinⅤ法心肌细胞凋亡率均低于对照组(11.6%±2.7%, 7.7%±2.4%, 4.7%±1.4%比15.6%±3.5%, 分别P<0.05, P<0.01和P<0.01).不同剂量的rhIL-1ra下调了凋亡基因Bax的表达(24.9±8.2, 15.5±3.4, 10.6±2.5比33.3±9.4, P<0.05, P<0.01及P<0.01),对心肌组织抑凋亡基因Bcl-2的表达均无影响.结论 IL-1在心肌缺血再灌注损伤中起重要作用,rhIL-1ra能减轻心肌的炎症反应、降低心肌细胞的凋亡程度、缩小心肌梗死面积.To observe the dynamic changes of plasma inflammatory cytokine interleukin-1 beta (IL-1 beta) in patients with acute myocardiac infarction (AMI) before and after recanalization of the infarct related artery (IRA) and to observe the effect of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the postischemic reperfused myocardium in experimental rabbits.(1) ELISA was used to measure the plasma IL-1 beta of 22 AMI patients, 20 males and 2 females, aged 64 +/- 12, before emergency percutaneous coronary intervention (PCI), and 12 hours and 24 hours after-intervention, and measure the plasma IL-1 beta of 8 healthy controls, 6 males and 2 females, aged 56 +/- 9. (2) Forty rabbits underwent ligation of the left circumflex branch of coronary artery (LCX) for 50 minutes and reperfusion for 4 hours after the ligatures were untied. The rabbits were randomly divided into 4 groups of 10 rabbits to be injected into the left ventricle immediately before the reperfusion with rhIL-1ra 10 mg/kg (group A), 20 mg/kg (group B), or 40 mg/kg (group C), and normal saline (control group) respectively. After reperfusion of 4 hours, the LCX was re-ligated. Evans blue was injected into the left ventricle. 15% KCl was injected intravenously to kill the rabbits. Their hearts were taken out to weigh the non-ischemic, ischemic, and necrotic cardiac muscles so as to calculate the infarct size. The myoperoxidase (MPO) activity was measured by colorimetry. Sections of myocardium were made. The number of apoptotic cardiomyocytes was evaluated by TUNEL method. The apoptotic rate of cardiomyocyte was measured by annexin V method. The DNA expression of myocardium was detected by DNA laddering method. The expressions of Bcl-2 and Bax apoptosis genes were assessed.(1) The average plasma IL-1 beta level of the 22 AMI patients before emergency PCI was significantly higher than that of the controls (28 pg/ml +/- 9 pg/ml vs. 20 pg/ml +/- 11 pg/ml, P < 0.05), and became the highest 12 hours after the intervention (86 pg/ml +/- 14 pg/ml), and the high level lasted to 24 hours after emergency PCI. (2) In the ischemia-reperfusion rabbit model, the infarct size was 47% +/- 7% in the group A, 34% +/- 8% in the group B, 31% +/- 6% in the group C, and 61% +/- 11% in the control group (P < 0.05, 0.01, and 0.01 respectively). The activity of myocardial MPO was 16.6 +/- 3.6 min(-1).g.w.w(-1) in the group A, 10.9 min(-1).g.w.w(-1) +/- 1.9 min(-1).g.w.w(-1) in the group B, 7.8 min(-1).g.w.w(-1) +/- 2.2 min(-1).g.w.w(-1) in the group C, and 20.5 min(-1).g.w.w(-1) +/- 4.5 min(-1).g.w.w(-1) in the control group (P < 0.05, 0.01, and 0.01 respectively). The cardiomyocyte apoptosis evaluated by TUNEL was 38.3 n/HP +/- 7.4 n/HP in the group A, 25.6 n/HP +/- 6.8 n/HP in the group B, 12.2 n/HP +/- 3.3 n/HP in the group C, and 44.4 n/HP +/- 9.5 n/HP in the control group (P < 0.05, and P < 0.01 respectively in comparison between the group B and the control group and between the group C and the control group). The apoptotic rate by annexin V method was 11.6% +/- 2.7% in the group A; 7.7% +/- 2.4% in the group B, 4.7% +/- 1.4% in the group C, and 15.6% +/- 3.5% in the control group (P < 0.05, 0.01, and 0.01 respectively). DNA electrophoresis showed scaling ladder pattern only in the control group. The fluorescent density of the apoptosis gene Bax in myocardium was 24.9 +/- 8.2 in the group A; 15.5 +/- 3.4 in the group B, 10.6 +/- 2.5 in the group C, and 33.3 +/- 9.4 in the control group (P = 0.0298, 0091, and 0052 respectively) and no significant difference in the expression of Bcl-2 was shown among the 4 groups. Myocardial MPO was correlated with cardiomyocyte apoptosis (r = 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05).Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-als; 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05).Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-1.面向21世纪教育振兴行动计划(985计划); 首都医学发展科研项目PubMed中文核心期刊要目总览(PKU)中国科学引文数据库(CSCD)07548-5538