摘要
目的探讨20-HETE加重大鼠离体心肌缺血再灌注损伤的作用及机制。方法雄性Wistar大鼠随机分为正常对照组(Con组),缺血再灌注模型组(IR组),IR+20-HETE合成酶抑制剂(HET0016)组,Con+HET0016组,每组12只,通过Langendorff灌流系统建立大鼠离体心肌缺血再灌注模型,全心缺血35 min再灌注40 min,Power Lab/8P系统实时监测心肌力学指标;灌流结束后取心肌组织TTC法检测心肌梗死面积;DHE荧光探针法检测活性氧簇(reactive oxygen species,ROS)含量;Western blot检测NADPH氧化酶亚基p47^(phox)磷酸化水平;细胞色素C的还原法检测NADPH氧化酶活性。结果离体心脏缺血再灌注后,加入20-HETE合成酶抑制剂HET0016(1μmol/L)后明显改善了IR导致的心肌力学指标下降,LVDP由IR组的(48.6±3.4)%升至(71.7±3.5)%,+d P/dt_(max)由(51.9±2.1)%升至(69±3.2)%,-d P/dt_(max)由(47.1±3.6)%升至(64.1±3.8)%(P<0.05);IR+HET0016组心肌梗死面积比IR组减小了37.2%(P<0.05)。DHE荧光探针检测发现IR+HET0016处理组心肌组织中ROS含量比IR组降低了45.8%(P<0.05);最后通过对NADPH氧化酶活性检测发现,HET0016显著减少了IR引起的NADPH氧化酶p47^(phox)亚基磷酸化水平及全酶活性的升高(P<0.05)。结论 20-HETE通过激活NADPH氧化酶诱导过量ROS的生成,加重心肌缺血再灌注损伤。
Objective To investigate the effect and mechanism of 20- HETE aggravates myocardial ischemia reperfusion injury in rats. Methods Male Wistar rats were randomly divided into normal control group（ Con）,ischemia- reperfusion group（ IR）,IR ＋ inhibitor of 20- HETE production（ HET0016） group and Con ＋HET0016 group,with 12 rats in each group. Experiments were performed in isolated rat hearts subjected to 35 min of ischemia followed by 40 min of reperfusion in Langendorff preparations. Cardiac contractility was continuously recorded with the Powerlab /8P system. Myocardial infarct size was measured by TTC staining. The level of reactive oxygen species（ ROS） was determined by DHE fuorescence. Phosphorylation of p47phox,a cytosolic component of NADPH oxidase,was detected by Western blot method. NADPH oxidase activity was evaluated by NADPH- dependent superoxide production examined using SOD- inhibitable cytochrome c reduction. Results At the end of reperfusion,administration of HET0016（ an inhibitor of 20- HETE production,1 μmol / l） significantly ameliorated the inhibited cardiac function induced by IR,including augmented LVDP from（ 48. 6 ±3. 4） % to（ 71. 7 ± 3. 5） %, ＋ d P / dt（max） from（ 51. 9 ± 2. 1） % to（ 69 ± 3. 2） % and – d P / dt（max） from（ 47. 1 ±3. 6） % to（ 64. 1 ± 3. 8） %（ P〈 0. 05）. Treatment of the heart with HET0016 significantly attenuated the IR-induced increase in cardiac infarct size by 37. 2%（ P〈 0. 05）. Compared with the IR group,IR ＋ HET0016 treatment significantly reduced the IR- induced increase in DHE fluorescent intensity by 45. 8%（ P〈 0. 05）.Finally,HET0016 also decreased the phosphorylation level of p47phox and depressed NADPH oxidase activity resepectively（ P〈 0. 05）. Conclusion 20- HETE could stimulate NADPH oxidase- derived superoxide production,which aggravated IR- induced myocardial injury.
出处
《合肥医学院学报》
2016年第4期366-371,共6页
Journal of Zunyi Medical University
基金
国家自然科学基金资助项目(NO:81460040)
贵州省科学技术基金资助项目(NO:黔科合LH字[2014]7544)
