山莨菪碱对心搏骤停猪氧化应激致心肌线粒体损伤的保护作用

Protection of anisodamine on the mitochondrial injury induced by oxidative stress in swine with cardiac arrest

目的通过观察心搏骤停（CA）猪血浆和心肌氧化一抗氧化指标及心肌线粒体结构的变化，探讨山莨菪碱对氧化应激所致心肌缺血／再灌注（I／R）损伤的保护作用。方法23头健康雄性家猪，按随机数字表法分为假手术组（n=5）、肾上腺素组（n=9）和山莨菪碱组（n=9），用交流电刺激法建立CA模型。在CA前、cA8rain及自主循环恢复（ROSC）即刻、30min、24h留取血标本，ROSC24h处死动物留取心肌。采用分光光度法检测丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性；高效液相色谱法测定心肌ATP含量；激光共聚焦显微镜下检测线粒体活性氧簇（ROS）水平；透射电镜下观察心肌组织超微结构，并对线粒体损伤程度进行评分。结果山莨菪碱组ROSC30min和24h时MDA含量（μmol／L）均较肾上腺素组明显减少（30min：43．38±8．12比55．47±10．97，24h：29．96±6．04比37．87±7．85，均P〈0．05）；24h时心肌SOD活性（U／mg）及ATP含量（μmol／g）也均较肾上腺素组显著升高（SOD：1．35±0．50比0．54±0．19，ATP：4．17±1．06比2．95±0．94．P〈0．01和P〈0．05），线粒体ROS含量（RFU）水平降低（88．00±17．67比107．00±21．35，P〈0．05），心肌MDA（μmol／mg）含量有所减少，但差异无统计学意义（16．66±2．89比19．28±3．90，P〉0．05）。透射电镜下观察显示，肾上腺素组心肌细胞排列紊乱，线粒体排列及形态与假手术组有明显差异[线粒体损伤评分（分）：0．41±0．08比0．12±0．01，P〈0．01]；山莨菪碱组线粒体损伤程度较肾上腺素组明显减轻（线粒体损伤评分：0．21±0．05比0．41±0．08，P〈0．05）。结论山莨菪碱可能通过调节氧自由基代谢，从而减轻氧化应激所致心肌线粒体结构和功能的损伤。

Objective To investigate the protection of anisodamine on cardiac ischemia/reperfusion （I/R） injury by oxidative stress by observing the changes in oxidation and antioxidant markers in plasma and myocardium, and the damage of cardiac mitochondria structure in pigs with cardiac arrest （CA）. Methods Twenty-three healthy male swine were divided into three groups using random digits table： sham group （n = 5 ）, epinephrine group （n = 9 ）, and anisodamine group （n = 9 ）. The CA model was reproduced by alternating current. Blood samples were collected before CA, 8 minutes after CA, and 0 minute, 30 minutes, 24 hours after recovery of spontaneous circulation （ROSC）, and hearts were harvested at 24 hours after ROSC. The content of malondialdehyde （MDA） and the enzyme activity of superoxide dismutase （SOD） were analyzed by spectrophotography, the cardiac ATP content was assayed by high performance liquid chromatography, the generation of reactive oxygen species （ROS） was detected by laser confocal microscope, and the myocardial ultrastrnctures were observed with transmission electron microscope to assess mitochondrial damage score. Results At 30 minutes and 24 hours after ROSC, plasma MDA level （μmol/L） of anisodamine group was lower than that of epinephrine group （30 minutes： 43.38± 8.12 vs. 55.47± 10.97, 24 hours： 29.96 ± 6.04 vs. 37.87 ± 7.85, both P〈0.05 ）. Compared with epinephrine group, the cardiac SOD activity （U/mg） and ATP content （μmol/g） of anisodamine group were elevated （SOD： 1.35 ± 0.50 vs. 0.54 ±0.19, ATP： 4.17 ±1.06 vs. 2.95 ±0.94, P〈0.01 and P〈0.05）, and the mitoehondrial ROS level （RFU） was lowered （88.00 ±17.67 vs. 107.00 ±21.35, P〈0.05 ）. Although the cardiac MDA content （txmol/mg） was also reduced, but the difference between two resuscitation groups showed no statistical significance （16.66 ±2.89 vs. 19.28 ±3.90, P〉0.05）. Using electron microscope, in epinephrine group disordered arrangement of cardiac myocyte arrangement was observed, and the mitochondrial alignment and morphology were significantly different from the sham group （mitochondrial damage score ：0.41 ±0.08 vs. 0.12 ±0.01, P〈0.01 ）. The level of mitochondrial injury in anisodamine group was milder than that of epinephrine group （mitochondrial damage score： 0.21 ±0.05 vs. 0.41 ±0.08, P〈0.05）. Conclusion Through regulating oxygen radical metabolism, anisodamine alleviates the injury to myocardial mitochondria structure and function injury as induced by oxidative stress.