核受体辅抑制因子1对缺血再灌注损伤小鼠的心肌保护作用

Role of nuclear receptor corepressor 1 in protecting myocardium in mice of myocardial ischemia/reperfusion injury

目的探讨心肌细胞来源的核受体辅抑制因子1(nuclear receptor corepressor 1, NCoR1)对心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MI/RI)小鼠心肌的保护作用及可能机制。方法心肌细胞NCoR1基因特异性敲除小鼠44只,随机分为假手术基因敲除组和MI/RI基因敲除组各22只;具有心脏特异性aMHC-Cre标签的野生型小鼠44只,随机分为假手术野生型组和MI/RI野生型组各22只。MI/RI基因敲除组、MI/RI野生型组结扎左冠状动脉30 min后恢复灌注血流制备MI/RI模型;假手术基因敲除组、假手术野生型组仅穿线不结扎左冠状动脉。造模成功后24 h,各组分别取12只小鼠测定左室射血分数(left ventricular ejection fraction, LVEF)和左心室短轴缩短分数(left ventricular fractional shortening, LVFS),然后处死小鼠取心脏,采用TTC染色法评估心肌梗死面积百分比;各组分别取1只小鼠行MRI检查,评估小鼠心肌收缩情况和水肿程度。造模成功后3 h,各组分别取4只小鼠处死后取心脏组织,行DHE染色观察活性氧(reactive oxygen species, ROS)含量,行免疫组织化学染色观察还原型烟酰胺腺嘌呤二核苷酸磷酸(nacotinamide adenine dinucleotide phosphate, NADPH)、3-硝基酪氨酸(3-nitrotyrosine, 3-NT)阳性细胞;各组分别再取5只小鼠,采用ELISA法检测心肌组织中NADPH、3-NT水平。结果造模后24 h, LVEF、LVFS在MI/RI基因敲除组[(34.16±1.61)%、(16.08±0.87)%]、MI/RI野生型组[(40.61±1.07)%、(19.61±0.60)%]均低于假手术基因敲除组[(58.47±2.12)%、(29.59±1.64)%]、假手术野生型组[(58.23±2.13)%、(30.35±1.47)%](P<0.05),心肌梗死面积百分比在MI/RI基因敲除组[(38.71±2.63)%]大于MI/RI野生型组[(18.34±2.16)%](P<0.05),MI/RI基因敲除组LVEF、LVFS均低于MI/RI野生型组(P<0.05);MI/RI基因敲除组心肌收缩、水肿程度均较MI/RI野生型组严重。ROS含量及NADPH、3-NT水平在MI/RI基因敲除组[(1.61±0.08)%、(3.26±0.19)μg/L、(15.50±1.07)μg/L]、MI/RI野生型组[(0.58±0.03)%、(2.39±0.17)μg/L、(9.96±0.70)μg/L]均高于假手术野生型组[0、(1.12±0.09)μg/L、(3.12±0.14)μg/L]、假手术基因敲除组[0、(0.95±0.04)μg/L、(3.25±0.28)μg/L](P<0.05),MI/RI基因敲除组高于MI/RI野生型组(P<0.05),假手术野生型组与假手术基因敲除组比较差异无统计学意义(P>0.05)。结论心肌细胞来源NCoR1对MI/RI小鼠的心肌具有保护作用,其机制可能缓解MI/RI损伤诱发的氧化应激损伤。

Objective To investigate the role of cardiomyocyte-derived nuclear receptor corepressor 1(NCoR1) in protecting myocardium of mice with myocardial ischemia/reperfusion injury(MI/RI) and its underlying mechanism. Methods Forty-four cardiomyocyte-specific NCoR1 knockout mice were randomly and equally divided into sham-operation NCoR1 knockout group and MI/RI NCoR1 knockout group. Another 44 wild-type mice with aMHC-Cre were randomly and equally divided into sham-operation wild-type group and MI/RI wild-type group. MI/RI mice models were prepared by recovering the left anterior descending(LAD) coronary blood flow after ligating for 30 min in MI/RI NCoR1 knockout group and MI/RI wild-type group. Mice in two sham-operation groups were subjected to the same procedure without alteration to the left coronary artery. Left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS) were measured in 12 mice in each group 24 h after modeling, and these mice were sacrificed. TTC staining was used to evaluate the percentage of myocardial infarct area. The myocardial contractility and edema were evaluated by MRI in one mouse in each group. Four mice in each group were sacrificed to obtain the heart 3 h after modeling to detect the reactive oxygen species(ROS) content by dihydroethidium(DHE) staining. The positive cells of nacotinamide adenine dinucleotide phosphate(NADPH) and 3-nitrotyrosine(3-NT) in myocardial tissue were measured by immunohistochemical staining. And ELISA was used to detect the levels of NADPH and 3-NT in another 5 mice in each group. Results LVFE and LVFS were lower in MI/RI NCoR1 knockout group((34.16 ± 1.61)%,(16.08±0.87)%)and MI/RI wild-type group((40.61±1.07)%,(19.61±0.60)%)than those in sham-operation NCoR1 knockout group((58.47±2.12)%,(29.59±1.64)%)and sham-operation wild-type group((58.23±2.13)%,(30.35±1.47)%)(P<0.05).The percentage of myocardial infarct area was larger in MI/RI NCoR1 knockout group((38.71±2.63)%)than that in MI/RI wild-type group((18.34±2.16)%)(P<0.05),and LVEF and LVFS were lower in MI/RI NCoR1 knockout group than those in MI/RI wild-type group(P<0.05).The myocardial contractility and edema were severer in MI/RI NCoR1 knockout group than those in MI/RI wild-type group.The ROS content and the levels of NADPH and 3-NT were higher in MI/RI NCoR1 knockout group((1.61±0.08)%,(3.26±0.19)μg/L,(15.50±1.07)μg/L)and MI/RI wild-type group((0.58±0.03)%,(2.39±0.17)μg/L,(9.96±0.70)μg/L)than those in sham-operation wild-type group(0,(1.12±0.09)μg/L,(3.12±0.14)μg/L)and sham-operation NCoR1 knockout group(0,(0.95±0.04)μg/L,(3.25±0.28)μg/L)(P<0.05),were higher in MI/RI NCoR1 knockout group than those in MI/RI wild-type group(P<0.05),and showed no significant differences between sham-operation wild-type group and sham-operation NCoR1 knockout group(P>0.05).Conclusion Cardiomyocyte-derived NCoR1 plays a cardioprotective role in MI/RI mice,probably by alleviating MI/RI induced oxidative stress.