【摘要】 目的 分析NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎性小体在治疗性浅低温（34℃）后处理的大鼠离体心肌缺血-再灌注模型中的作用并探讨其机制。方法 选择清洁级成年雄性SD大鼠60只，7～10周龄，体重250～300 g。采用随机数字表法将大鼠分为五组：空白对照组（S组）、心肌缺血-再灌注组（IR组）、34℃浅低温后处理心肌缺血-再灌注组（MH组）、34℃浅低温后处理心肌缺血-再灌注+3-TYP组（HT组）和34℃浅低温后处理心肌缺血-再灌注+3-TYP+MCC950组（HTM组）,每组12只。S组在37℃灌流液平衡灌流大鼠心脏180 min; IR组在37℃灌流液平衡灌流大鼠心脏30 min后，缺血30 min, 37℃灌注液再灌注120 min; MH组在37℃灌流液平衡灌流大鼠心脏30 min后，缺血30 min, 34℃灌注液再灌注120 min; HT组在37℃灌流液平衡灌流大鼠心脏30 min后，缺血30 min,在灌注液中加入沉默信息调节因子2同源蛋白3（sirt3）抑制剂3-TYP后行34℃灌注液再灌注120 min; HTM组在37℃灌流液平衡灌流大鼠心脏30 min后，缺血30 min,在灌注液中加入sirt3抑制剂3-TYP和NLRP3抑制剂MCC950后行34℃灌注液再灌注120 min。再灌注120 min后取离体心脏，采用ELISA法测定灌注后心脏漏液中IL-1β、IL-6浓度，Western blot法检测心肌组织中NLRP3和sirt3蛋白相对含量，1%氯化三苯基四氮唑染色计算心肌梗死面积，HE染色观察心肌病理变化。结果 与S组比较，IR组、MH组、HT组和HTM组再灌注30、60、90、120 min时HR明显减慢，LVSP、dp/dt_max明显降低，LVEDP明显升高；心脏漏液中IL-6和IL-1β浓度、心肌梗死面积百分比明显升高（P<0.05）;IR组、HT组和HTM组心肌组织中sirt3蛋白含量明显降低，NLRP3蛋白含量明显升高（P<0.05）;MH组心肌组织中sirt3和NLRP3蛋白含量明显升高（P<0.05）。与IR组比较，MH组和HTM组再灌注30、60、90、120 min时HR明显增快，LVSP、±dp/dt_max明显升高，LVEDP明显降低；心脏漏液中IL-6和IL-1β浓度、心肌梗死面积百分比明显降低（P<0.05）;MH组心肌组织中sirt3蛋白含量明显升高，NLRP3蛋白含量明显降低（P<0.05）;HTM组心肌组织中NLRP3蛋白含量明显降低（P<0.05）。与MH组比较，HT组再灌注30、60、90、120 min时HR明显减慢，LVSP、±dp/dt_max明显降低，LVEDP明显升高；心脏漏液中IL-6和IL-1β浓度、心肌梗死面积百分比、心肌组织中NLRP3蛋白含量明显升高（P<0.05）;HT组和HTM组心肌组织中sirt3蛋白含量明显降低（P<0.05）。与HT组比较，HTM组再灌注30、60、90、120 min时HR明显增快，LVSP、±dp/dt_max明显升高，LVEDP明显降低；心脏漏液中IL-6和IL-1β浓度、心肌梗死面积百分比、心肌组织中NLRP3蛋白含量明显降低（P<0.05）。结论 治疗性浅低温（34℃）可通过改善离体心脏血流动力学参数、降低IL-6、IL-1β浓度、心肌组织中NLRP3蛋白含量、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，其机制可能与线粒体介导sirt3通路抑制炎性小体NLRP3的高表达有关。更多还原

【Abstract】 Objective To analyze the role of NOD-like receptor thermal protein domain associated protein 3（NLRP3） inflammasome in a therapeutic mild hypothermia（34 ℃） treated isolated rat myocardial ischemia-reperfusion model and explore its mechanism. Methods Sixty clean grade adult male SD rats, aged 7-10 weeks, weighing 250-300 g. Using a random number table method, the rats were divided into five groups: blank control group（group S）, myocardial ischemia-reperfusion group（group IR）, 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion group（group MH）, 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion+3-TYP group（group HT）, and 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion+3-TYP+MCC950 group（group HTM）, 12 rats in each group. Group S perfused the rat heart at 37 ℃ with a balanced perfusion solution for 180 minutes. Group IR received balanced perfusion of the rat heart at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes and reperfusion with 37 ℃ perfusion for 120 minutes. Group MH perfused the rat heart at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes and reperfusion with 34 ℃ perfusion solution for 120 minutes. Group HT perfused the hearts of rats at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes, silent mating type information regulation 2 homolog 3（sirt3） inhibitor 3-TYP was added to the perfusate, and then perfused at 34 ℃ for 120 minutes. Group HTM perfused the hearts of rats at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes, sirt3 inhibitor 3-TYP and NLRP3 inhibitor MCC950 were added to the perfusate, and then perfused at 34 ℃ for 120 minutes. The isolated heart was obtained 120 minutes after reperfusion, and the concentrations of IL-6 and IL-1β in the perfused cardiac fluid was measured using ELISA method, Western blot method for detecting the relative content of NLRP3 and sirt3 proteins in myocardial tissue, 1% triphenyl tetrazolium chloride staining for calculating myocardial infarction area, and HE staining for observing myocardial pathological changes. Results Compared with group S, HR were significantly slowed down, LVSP, ±dp/dt_max were significantly decreased, and LVEDP were significantly increased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, and the percentage of myocardial infarction area were significantly increased in groups IR, MH, HT, and HTM（P < 0.05）, the content of sirt3 protein in myocardial tissue were significantly reduced, while the content of NLRP3 protein were significantly increased in groups IR, HT, and HTM（P < 0.05）, the contents of sirt3 and NLRP3 protein in the myocardial tissue were significantly increased in group MH（P < 0.05）. Compared with group IR, HR were significantly increased, LVSP, ±dp/dt_max were significantly increased, and LVEDP were significantly decreased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage and the percentage of myocardial infarction area were significantly decreased in groups MH and HTM（P < 0.05）, the content of sirt3 protein in myocardial tissue was significantly increased, while the content of NLRP3 protein was significantly decreased in group MH（P < 0.05）, the content of NLRP3 protein in myocardial tissue was significantly reduced in group HTM（P < 0.05）. Compared with group MH, HR were significantly slowed down, LVSP, ±dp/dt_max were significantly decreased, and LVEDP were significantly increased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, the percentage of myocardial infarction area, and the content of NLRP3 protein in myocardial tissue were significantly increased in group HT（P < 0.05）, the content of sirt3 protein in myocardial tissue was significantly reduced in groups HT and HTM（P < 0.05）. Compared with group HT, HR were significantly increased, LVSP, ±dp/dt_max were significantly increased, and LVEDP were significantly decreased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, the percentage of myocardial infarction area, and the content of NLRP3 protein in myocardial tissue were significantly reduced in group HTM（P < 0.05）. ConclusionTherapeutic mild hypothermia（34 ℃） can improve hemodynamic parameters of isolated hearts and reduce the concentrations of IL-6 and IL-1β, NLRP3 protein content in myocardial tissue, percentage of myocardial infarction area, improve myocardial pathological changes, and reduce myocardial ischemia-reperfusion injury in rats, the mechanism may be related to the mitochondrial mediated sirt3 pathway inhibiting the high expression of inflammatory corpuscle NLRP3.更多还原