山莨菪碱对心搏骤停家猪线粒体途径心肌细胞凋亡的保护作用

Protective effect of anisodamine against myocardial cell apoptosis through mitochondria impairment in cardiac arrest in pigs

目的探讨山莨菪碱对心搏骤停（CA）家猪心肌线粒体损伤的保护作用及其机制。方法23只雄性家猪按随机数字表法分为肾上腺素组（n=9）、山莨菪碱组（n=9）、对照组（n=5），用交流电刺激法诱发心室纤颤（室颤）建立CA模型。于CA前基础状态、CA8min及自主循环恢复（ROSC）即刻、30min和24h留取血标本，ROSC 24h处死动物留取心肌组织。用酶联免疫吸附试验（ELISA）检测血浆和心肌细胞色素C（CytC）、天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）的蛋白含量；透射电镜下观察心肌超微结构改变；荧光染色法检测心肌细胞凋亡。结果山莨菪碱组ROSC率较肾上腺素组提高了22．22％（77．78％比55．56％，P〉0．05），复苏成功的动物均存活24h。随着ROSC时间的延长，肾上腺素组和山莨菪碱组血浆CytC和caspase-3蛋白含量明显增高，且高于对照组；其中山莨菪碱组ROSC 30min、24h血浆CytC蛋白含量（nmol/L）较肾上腺素组明显减少（48．68±19．50比77．51±29．87，48．98±20．26比82．11±25．09，均P〈0．05）；两复苏组间ROSC后各时间点心肌CytC及血浆和心肌caspase-3蛋白含量比较差异均无统计学意义。肾上腺素和山茛菪碱均能明显减轻心肌细胞线粒体损伤，减少心肌细胞凋亡；以山莨菪碱组作用更好，细胞凋亡率明显下降[（0．15±0．04）％比（0．37±0．04）％，P〈0．01]。结论山莨菪碱可能通过减少CytC蛋白表达量及减轻复苏后心肌线粒体损伤程度来保护心肌细胞、抑制线粒体途径的心肌细胞凋亡。

Objective To investigate the protective effect of anisodamine on myocardial mitochondrial damage in cardiac arrest （CA） in pigs. Methods Twenty-three male pigs were randomly divided into three groups, epinephrine group （n=9）, anisodamine group （ n=9） and control group （ n = 5 ）. CA following ventricular fibrillation （VF） was induced by alternating current. The blood samples were collected before CA, 8 minutes after CA and instantly after recovel3, of spontaneous circulation （ROSC）, and 30 minutes and 24 hours later. Hearts were obtained at 24 hours after ROSC. The changes in Cytochrome C （Cyt C） and caspase-3 in plasma and myocardium were analyzed by enzyme-linked immunosorbent assay （ELISA）. The myocardial specimens were observed by transmission electron microscopy for ultrastructural changes, and apoptosis was assessed with Hoechst 33258 staining. Results The ROSC rate of the anisodamine group was elevated by 22.22% compared with the epinephrine group （77.78% vs. 55.56%, P〉 0.05 ）. All animals with resumption of ROSC survived up to 24 hours. The plasma contents of Cyt C and caspase-3 in the epinephrine group and the anisodamine group gradually increased after ROSC, and were significantly higher than those in the control group. But the plasma Cyt C （nmol/L） level in the anisodamine group was lower than that in the epinephrine group at 30 minutes and 24 hours after ROSC （48.68 ± 19.50 vs. 77.51 ± 29.87, 48.98 ± 20.26 vs. 82.11 ± 25.09, both P〈0.05 ）. There was no significant difference in protein contents of both Cyt C and caspase-3 in plasma and myocardium between two resuscitate groups. Both epinephrine and anisodamine eould mitigate cardiac mitochondrial damage after CA, but the anisodamine showed better effect. The myocardium apoptosis ratio in the anisodamine group was lower than that of the epinephrine group [（0.15 ± 0.04）% vs. （0.37 ± 0.04）%, P〈0.013. Conclusion By decreasing the protein content of Cyt C, and reducing the extent of damage to myocardial mitochondria, anisodamine can protect the myocardial uhrastructure, and restrain the mitochondria-induced cell apoptosis after resuscitation.