硫氢化钠抑制离体大鼠急性心肌缺血损伤作用的线粒体机制

Mitochondrial mechanism of sodium hydrosulfide induced inhibition in acute myocardial ischemia injury in isolated hearts of rats

目的研究硫氢化钠(Na HS)对离体大鼠急性心肌缺血损伤的线粒体保护途径,并初步探讨其可能机制。方法通过结扎冠状动脉左前降支,制备离体急性心肌缺血损伤模型。心肌缺血2 h后,模型组继续灌注K-H液2 h;Na HS组分别灌注含Na HS 5,10和20μmol·L-1的K-H液2 h。透射电镜观察心肌线粒体超微结构;差速离心法分离线粒体,测定心肌线粒体活力、膜肿胀度及线粒体总ATP酶、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)的活性和丙二醛(MDA)含量;测定冠脉流出液中乳酸脱氢酶(LDH)活性。结果与假手术组相比,模型组线粒体膜肿胀,线粒体活力下降;模型组线粒体内总ATP酶、GSH-Px和SOD活性〔分别为4.76±0.25,68.59±2.00和(23.37±1.13)k U·g-1蛋白〕低于假手术组〔分别为11.23±0.65,133.37±3.47和(74.42±1.97)k U·g-1蛋白〕,MDA含量〔(1.45±0.09)μmol·g-1蛋白〕高于假手术组〔(0.85±0.05)μmol·g-1蛋白〕;冠脉流出液中LDH活性(104±6)U·L-1高于假手术组(50±3)U·L-1(P<0.01)。与模型组相比,Na HS 5,10和20μmol·L-1组明显抑制膜肿胀,线粒体活力有所恢复;线粒体内总ATP酶〔分别为5.06±0.22,7.72±0.37和(10.57±0.44)k U·g-1蛋白〕、GSHPx〔分别为87.94±1.65,106.66±2.14和(125.57±2.12)k U·g-1蛋白〕和SOD〔分别为39.00±1.00,57.46±1.21和(69.56±1.56)k U·g-1蛋白〕活性明显升高,MDA〔分别为1.28±0.09,1.06±0.06和(0.89±0.04)μmol·g-1蛋白〕含量降低(P<0.05或P<0.01);冠脉流出液中LDH活性〔分别为84±3,70±4和(60±4)U·L-1〕显著减低(P<0.01)。结论Na HS对离体急性心肌缺血损伤有保护作用,其机制可能与降低线粒体脂质过氧化水平有关。

OBJECTIVE To investigate the mitochondrial pathway and possible molecular mechanism of sodium hydrosulfide（ Na HS） in acute myocardial ischemia injury in isolated hearts of rats.METHODS The myocardial ischemia injury model was established by ligating of coronary artery. After2 h ischemia,hearts in model group were sequentially subjected to K-H solution for 2 h,while hearts in Na HS groups received 2 h of Na HS 5,10 and 20 μmol·L- 1containing buffer,respectively. The ultrastructural alterations of mitochondria were observed under an electric microscopy. Mitochondria were separated by homogenization and differential centrifugation before their swelling and activity of mitochondria were determined. The activities of ATPase,glutathione peroxidase（ GSH-Px）,superoxide dismutase（ SOD） and lactate dehydrogenase（ LDH）,and the content of malondialdehyde（ MDA）,were respectively measured by spectrophotometry. RESULTS Compared with the sham group,the swelling of mitochondria was distinctly increased in model group. The activities of ATPase,GSH-Px and SOD in myocardial mitochondria of the Na HS group were 4. 76 ± 0. 25,68. 59 ± 2. 00 and（ 23. 37 ± 1. 13） k U·g- 1protein,respectively,which were significantly lower than in the sham group 〔11. 23 ± 0. 65,133. 37 ± 3.47 and（ 74. 42 ± 1. 97） k U·g- 1protein,respectively,P 0. 01〕. The content of MDA in myocardial mitochondria of the model group was（ 1. 45 ± 0. 09） μmol·g- 1protein,which was significantly higher than in the sham group 〔（ 0. 85 ± 0. 05） μmol·g- 1protein,P 0. 01 〕,and the activity of LDH in perfusate was（ 104 ±6） U·L- 1,which was significantly higher than in the sham group（ 50 ± 3） U·L- 1（ P 0. 01）.Compared with the model group,the swelling of mitochondria was distinctly inhibited in Na HS 5,10 and20 μmol·L- 1groups（ P 0. 01）,but the activity of ATPase〔5. 06 ± 0. 22,7. 72 ± 0. 37 and（ 10. 57 ± 0. 44）k U·g- 1protein,respectively〕,GSH-Px〔87. 94 ± 1. 65,106. 66 ± 2. 14 and（ 125. 57 ± 2. 12） k U·g- 1protein,respectively〕and SOD〔39. 00 ± 1. 00,57. 46 ± 1. 21 and（ 69. 56 ± 1. 56） k U·g- 1protein,respectively〕in myocardial mitochondria was significantly increased,the content of MDA〔1. 28 ± 0. 09,1. 06 ±0. 06 and（ 0. 89 ± 0. 04） μmol·g- 1protein,respectively〕in myocardial mitochondria was significantly decreased in Na HS 5,10 and 20 μmol·L- 1groups（ P 0. 05 or P 0. 01）,and the activity of LDH〔83 ± 3,70 ± 4 and（ 60 ± 4） U·L- 1,respectively〕in perfusate was significantly decreased in Na HS 5,10 and 20μmol·L- 1groups（ P 0. 01）. CONCLUSION Na HS prevents acute myocardial ischemia injury in isolated hearts of rats presumably by improving antioxidant defense and attenuating oxidative damage to mitochondria.