【摘要】 目的 探讨高血糖条件下核因子κB(NF-κB)激活对小鼠心肌缺血再灌注（IR）损伤的影响及其机制。方法 选择雄性C57BL/6J小鼠48只，适应性饲养1周，随机分为NG-Sham组（非DM+假手术）与NG-IR组（非DM+IR）、DM-Sham组（DM+假手术）与DM-IR组（DM+IR）以及DM-IR-NC组（DM+IR+阴性对照）与DM-IR-NF-κB组（DM+IR+NF-κB），每组8只。NG-Sham组与NG-IR组正常饲料喂养，NG-IR组通过结扎冠状动脉左前降支构建IR模型（缺血60 min再灌注24 h）,NG-Sham组开胸暴露冠状动脉左前降支，但不结扎。DM-IR组连续4天腹腔注射链脲佐菌素40 mg/kg诱导DM模型，成模后1周同法构建IR模型。DM-Sham组同法诱导DM模型，成模后1周开胸暴露冠状动脉左前降支，但不结扎。DM-IR-NF-κB组与DM-IR-NC组同法诱导DM模型，成模后1周同法构建IR模型，DM-IR-NF-κB组开胸前30 min尾静脉注射NF-κB重组蛋白0.5 mg/kg,DM-IR-NC组开胸前30 min尾静脉注射等量溶剂。采用TTC染色法评估NG-IR组与DM-IR组心肌缺血面积，采用RT-qPCR法检测NG-IR组与DM-IR组心肌组织NF-κB、IL-6、IL-1β、TNF-α mRNA相对表达量。采用JC-1染色法评估NG-Sham组、NG-IR组、DM-Sham组和DM-IR组心肌细胞线粒体膜电位，采用MitoSOX染色法评估心肌细胞线粒体活性氧（ROS）含量。采用Western blotting法检测NG-IR组与DM-IR组心肌组织AMPK、p-AMPK蛋白相对表达量，计算p-AMPK/AMPK。采用比色法检测DM-IR-NC组与DM-IR-NF-κB组血清AST、LDH含量，采用双抗体夹心ELISA法检测血清CK-MB含量。结果 与NG-IR组比较，DM-IR组心肌缺血面积增加（P<0.05），心肌组织NF-κB mRNA相对表达量上调（P均<0.05），而心肌组织IL-6、IL-1β、TNF-α mRNA相对表达量变化不明显（P均>0.05）。与NG-Sham组比较，NG-IR组与DM-Sham组心肌细胞线粒体膜电位下降，心肌细胞线粒体ROS含量升高（P均<0.05）；与NG-IR组比较，DM-IR组心肌细胞线粒体膜电位下降，心肌细胞线粒体ROS含量升高（P均<0.05）。Pearson相关分析发现，IR小鼠心肌组织NF-κB mRNA相对表达量与心肌细胞线粒体膜电位呈正相关关系（r=0.496,P<0.05）。与NG-IR组比较，DM-IR组心肌组织AMPK蛋白相对表达量升高，p-AMPK蛋白相对表达量降低，p-AMPK/AMPK降低（P均<0.05）。与DM-IR-NC组比较，DM-IR-NF-κB组血清AST、LDH、CK-MB含量均升高（P均<0.05），心肌组织p-AMPK蛋白相对表达量和p-AMPK/AMPK均降低（P均<0.05），而AMPK蛋白相对表达量无明显变化（P>0.05）。结论 高血糖条件下NF-κB激活可增强小鼠心肌IR后缺血心肌的易损性，其机制可能与NF-κB能够诱导AMPK信号通路失活和线粒体质量控制紊乱有关。更多还原

【Abstract】 Objective To investigate the effect and mechanism of the activation of nuclear factor κB(NF-κB) on myocardial ischemia-reperfusion(IR) injury under hyperglycemic condition. Methods Forty-eight male C57BL/6J mice were acclimated for one week and were randomly assigned into six groups: non-diabetic sham-operated group(NG-Sham group), non-diabetic ischemia-reperfusion group(NG-IR group), diabetic sham-operated group(DM-Sham group), diabetic ischemia-reperfusion group(DM-IR group), diabetic ischemia-reperfusion negative control group(DM-IR-NC group), and diabetic ischemia-reperfusion NF-κB recombinant protein intervention group(DM-IR-NF-κB group), with eight mice in each group. Mice in the NG-Sham and NG-IR groups were fed the normal diet. Mice in the NG-IR group underwent myocardial infarction(MI) modeling by ligation of the left anterior descending(LAD) coronary artery, while mice in the NG-Sham group had the LAD artery exposed but not ligated. Mice in the DM-IR group received intraperitoneal injections of streptozotocin(STZ) at 40 mg/kg for four consecutive days to induce diabetes mellitus(DM). One week after the establishment of the DM model, MI was induced by LAD ligation. Mice in the DM-Sham group underwent the same procedure for DM induction and had the LAD artery exposed but not ligated. Mice in the DM-IR-NF-κB and DM-IR-NC groups underwent the same DM induction procedure and MI modeling. Mice in the DM-IR-NF-κB group received an intravenous injection of NF-κB recombinant protein at 0. 5 mg/kg via the tail vein 30 minutes before chest opening, while mice in the DM-IR-NC group did not receive the NF-κB recombinant protein. The myocardial infarct area in the NG-IR and DM-IR groups was assessed by TTC staining. The mRNA expression levels of NF-κB, IL-6, IL-1β, and TNF-α in the myocardial tissues were measured by RT-qPCR. The mitochondrial membrane potential in the NG-Sham, NG-IR, DM-Sham, and DM-IR groups was evaluated using JC-1 staining, and mitochondrial reactive oxygen species(ROS) content was assessed using MitoSOX staining. AMPK and p-AMPK protein expression levels in the myocardial tissues were determined by Western blotting. Serum AST and LDH activities in the DM-IR-NC and DM-IR-NF-κB groups were measured by colorimetric assays, and the content of serum CK-MB was determined using double-antibody sandwich ELISA. Results Compared with the NG-IR group, the myocardial infarct area increased(P<0. 05) and the mRNA expression of NF-κB in the myocardial tissues was up-regulated(P<0. 05), while the mRNA expression levels of IL-6, IL-1β, and TNF-α did not show significant changes in the DM-IR group(all P>0. 05). Compared with the NG-Sham group, the NG-IR and DM-Sham groups exhibited decreased mitochondrial membrane potential and increased ROS content(both P<0. 05); compared with the NG-IR group, the DM-IR group showed further decreased mitochondrial membrane potential and increased ROS content(both P<0. 05). Pearson correlation analysis showed that there was a significant positive correlation between the NF-κB mRNA level and mitochondrial membrane potential in the myocardial tissues of IR mice(r=0. 496, P<0. 05). Compared with the NG-IR group, the DM-IR group exhibited up-regulated AMPK protein expression, down-regulated p-AMPK protein expression, and decreased p-AMPK/AMPK ratio(all P<0. 05). Compared with the DM-IR-NC group, the content of serum AST, LDH, and CK-MB increased(all P<0. 05), the relative expression levels of p-AMPK protein and p-AMPK/AMPK in the myocardial tissues decreased(all P<0. 05), while the relative expression of AMPK protein did not change significantly in the DM-IR-NF-κB group(P>0. 05). Conclusion The activation of NF-κB can promote the susceptibility of the myocardium to IR injury under hyperglycemic condition, and the mechanism may be that NF-κB induces the inactivation of the AMPK signaling pathway and disorder of mitochondrial quality control.更多还原