国际麻醉学与复苏杂志   2013, Issue (10): 9-9
    
二氮嗪后处理对缺氧/复氧成年大鼠心肌细胞存活率及磷酸化糖原合成激酶-3β、Bcl-2和Bax表达的影响
叶英, 龚国丽, 曾因明, 赵其宏1()
1.徐州医学院附属医院
Effects of diazoxide postcongditioning on the cell viability，phospho glycogen synthase kinase-3β，Bcl-2，Bax protein expressionin in cultured adult rat cardiac myocytes suffered from hypoxia/reoxygenation injury
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摘要：

目的　研究线粒体ATP-敏感性钾通道（mitochondrial ATP-sensitive potassium channel， mitoKATP通道）开放剂二氮嗪（diazoxide， DZ）后处理对成年大鼠心肌细胞缺氧/复氧（hypoxia/reoxygenation， H/R）后细胞存活及对磷酸化糖原合成激酶-3β（phospho glycogen synthase kinase-3β， pGSK-3β），Bcl-2，Bax表达的影响。　方法　体外培养原代成年大鼠（30只）心肌细胞建立H/R损伤模型，按随机数字表法随机分为5组（每组6只）：① Normal组：在二氧化碳（CO2）培养箱中持续培养6 h组；② H/R组：缺氧3 h复氧3 h组；③ DZ组：缺氧3 h复氧3 h，复氧5 min时给予100 μmol/L DZ组；④ DZ+5-HD组：缺氧3 h复氧3 h，缺氧末给予100 μmol/L 5-羟葵酸盐（5-hydroxydecanoate， 5-HD），复氧5 min时给予100 μmol/L DZ组；⑤ 5-HD组：缺氧3 h复氧3 h，缺氧末给予100 μmol/L 5-HD组。复氧3 h末通过计数细胞长杆率测定存活率，免疫印迹法测定心肌细胞内pGSK-3β， Bcl-2 和Bax的表达。　结果　复氧3 h末，Normal组单个心肌细胞收缩幅度为（13.12±0.19）%，与Normal组比较，其他各组单个心肌细胞收缩幅度明显降低［H/R组为（7.97±0.22）%，DZ组为（10.48±0.20）%，DZ+5?-HD组为（7.97±0.19）%，5-HD组为（8.22±0.22）%］（P>0.05），心肌细胞内Bcl-2表达水平降低，Bax表达水平升高（P<0.05）。DZ后处理组心肌细胞存活率为（64±5）%，与H/R组比较明显增高（P<0.05），心肌细胞内Bcl-2、pGSK-3β表达水平升高，Bax表达水平降低，而缺氧末即刻给予5?蛳HD可以逆转DZ后处理的这些作用（P<0.05）；5-HD组与H/R组比较差异无统计学意义（P>0.05）。　结论　DZ后处理可能通过激活mitoKATP 通道、上调pGSK-3β及Bcl-2，下调Bax的表达增加H/R后成年大鼠心肌细胞的存活。
关键词： 二氮嗪；后处理；线粒体ATP敏感性钾通道；GSK-3β；Bcl-2；Bax
Abstract:

Ｏｂｊｅｃｔｉｖｅ　To investigate the effect of diazoxide（DZ） postconditioning mediating the expression of phospho glycogen synthase kinase-3β，Bcl-2，Bax and the cell viability during hypoxia/reoxygenation（H/R） in cultured adult rat cardiac myocytes.　Methods　The model of isolated adult rat cardiac myocytes was established and randomly divided into 5 groups（n=6）：① Normal group：caridocytes were incubated at 37 ℃ in a humidified atmosphere of 5% carbon dioxide（CO2） and 95% air for 6 h； ② H/R group：cardiocytes were exposed to 3 h of hypoxia followed by 3 h of reoxygenation； ③ DZ group： cardiocytes were exposed to 3 h of hypoxia followed by 3 hof reoxygenation， while 100 μmol/L DZ was added in medium 5 min after reoxygenation； ④ DZ+5-hydroxydecanoate（5-HD） group： cardiocytes were exposed to 3 h of hypoxia followed by 3 h of reoxygenation， while 100 μmol/L 5-HD was added in medium rightly after 3 h' hypoxia and 100 μmol/L DZ was added in medium 5 min after reoxygenation； ⑤ 5-HD group： 3 h of hypoxia followed by 3 h of reoxygenation， while 100 μmol/L 5-HD was added in medium after 3 h' hypoxia. The cell viability was assayed by the rate of Rod-shaped cells； the expression of pGSK-3β， Bcl-2 and Bax were assessed by western blot 3 h after reoxygenation.　Results　After 3 h reoxygenation， contraction of the single myocyte is（13.12±0.19）% in normal group. Compared with the normal group， the cell viability was greatly decreased in the other 4 groups［（7.97±0.22）% for H/R group，（10.48±0.20）% for DZ group， （7.97±0.19）% for the DZ+5?-HD group， （8.22±0.22）% for the 5-HD group］（P<0.05）. The cell viability in DZ group was（64±5）%. Compared with the H/R group， it was significantly increased（P<0.05）. The content of Bcl-2 and pGSK-3β in DZ group were higher（P<0.05）， while the expression of Bax was lower（P<0.05）. But these effects were abolished with administration of 5-HD rightly after hypoxia（P>0.05）； There was no statistical difference between H/R group and 5-HD group（P>0.05）.　Conclusions　DZ could alleviate hypoxia/reoxygenation injury through mediating the expression of pGSK-3β， Bax， Bcl-2 proteins via opening of mitoKATP channel.
Key words: Diazoxide；　Postconditioning；　Mitochondrial ATP-sensitive potassium channel；　glycogen synthase kinase-3β；　Bcl-2；　Bax