异氟烷对原代培养的缺氧／复氧损伤心肌细胞活力及线粒体通透性转换的影响

The effect of isoflurane on viability and mitochondrial permeability transition of primary cultured cardiocytes subjected to hypoxia/reoxygenation injury

目的在原代培养SD乳鼠心肌细胞上观察异氟烷（isoflurane，Iso）对缺氧／复氧（hypoxia／reoxygenation，H／R）损伤心肌细胞活力及线粒体通透性转换（mitochondrial permeability transition，MPT）的影响。方法1d-3d龄SD乳鼠心肌细胞原代培养48h后，建立H／R模型。将培养细胞按孔随机分为5组（n=6），在缺氧期予Iso和苍术苷（atractyloside，Atra）处理：①Normal组：在37℃的CO2培养箱中孵育细胞5h（95％空气＋5％CO2）；②H／R组：缺氧（95％N2＋5％CO2）3h，复氧（95％空气＋5％CO2）2h；③Atra组：缺氧3h，缺氧期给予20仙的Atra，复氧2h；④Iso0．4组：缺氧3h，缺氧期给予0．4mmol／L Iso，复氧2h；⑤Atra＋Iso 0．4组：缺氧3h，缺氧期给予0．4mmol／L Iso和20μmol／LAtra，复氧2h。复氧50min后，以Calcein-AM染色心肌细胞，激光共聚焦显微镜观察细胞内线粒体通透性转换孔（mitochond Tial permeability transition pore，MPTP）的开放；复氧2h后，MTT法测定心肌细胞活力。结果复氧2h后，Normal组、H／R组、Atra组、Iso0．4组、Atra＋Iso0．4组吸光度分别为0．442±0．010、0．417±0．006、0．413±0．007、0．462±0．011、0．452±0．008，Normal组、Iso0．4组、Atra＋Iso0．4组明显高于H，R组、Atra组（P〈0．01）；H／R组吸光度与Atra组差异无统计学意义（P〉0．05）。复氧50min后，Normal组、Iso0．4组、Atra＋Iso0．4组Calcein保留在线粒体内，胞浆荧光强度较弱；H／R组、Atra组Calcein从线粒体释放到胞浆，胞浆荧光强度较强。结论①缺氧期Iso处理可以明显抑制心肌细胞MPTP开放，减轻缺血再灌注损伤（ischemia／reperfusioninjury，I／RI）引起的心肌细胞损伤，保存细胞活力；②缺氧期给予Atra不能逆转Iso的心肌保护作用。

Objective To explore whether isoflurane （Iso） conditioning protect cardiocytes against hypoxia/reoxygenation injury by attenuating mitochondrial permeability transition pore （MPTP） open. Methods Cardiocytes were randomly divided into 5 groups （n=6） ：① Normal group, cardiocytes were incubated at 37℃ in a humidified atmosphere of 5% CO2 and 95% air for 5 hours; ② H/R group, cardiocytes were exposed to 3 h of hypoxia （95% N2 and 5% CO2） followed by 2 h of reoxygenation （95% air and 5% CO2） ; ③ Atra group, 3 h of hypoxia followed by 2 h of reoxygenation, atractyloside （20 μmol/L） was added in medium during hypoxia; ④ Iso0.4 group, 3 h of hypoxia followed by 2 h of reoxygenation, 0.4 mmol/L Iso solution was added in mediumuring hypoxia; ⑤ Atra＋Iso0.4 group, 3 h of hypoxia followed by 2 h of reoxygenation, 0.4 mmol/L Iso solution and Atractyloside （20 μmol/L） were added in medium during hypoxia. MTT Cell Viability Assay at 2 hours after reoxygenation were detected by ELISA reader. The onset of MPT was monitored by laser scanning confocal microscope （KSCM） through calcein at 50 minutes after reoxygenation. Results The respective absorbance of Normal group, H/R group, Atra group, Iso0.4 group, Atra＋Iso0.4 group were 0.442±0.010,0.417 ±0.006,0.413±0.007,0.462±0.011, 0.452±0.008, absorbance of Normal group, Iso0.4 group and Atra＋Iso0.4 group were significantly increased compared with H/R and Atra group （P〈0.01）. There was no significant difference between H/R and Atra group （P〉0.05）. After 50 minutes reoxygenation, Calcein of the Normal group, Iso0.4 group, Atra＋Iso0.4 group was remain in the mitochondria, and cytoplasm fluorescence intensity was weak. Caleein of the H/R group and Atra group was released from mitochondria to cytoplasm, and cytoplasm fluorescence intensity was great. Conclusion Iso has protective effect on primary cultured cardiocytes subjected to hypoxia/reoxygenation by attenuating the MPTP open. Atractyloside can not significantly reverse the protective effect of Iso.