摘要：
Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in urocortin postconditioning-induced protection of rat cardiomyocytes.Methods Clean-grade healthy male Sprague-Dawley rats,aged 16-20 weeks,weighing 200-250 g,were used in this study.Cardiomyocytes of rats were isolated,cuhured and divided into 4 groups (n =28 each) using a random number table method:control group (group C),H/R group (group HR),urocortin postconditioning group (group U) and 5-hydroxydecanoate (5-HD,mito-KAw channel blocker) plus urocortin postconditioning group (group HU).In group C,the cells were continuously cultured for 150 min in an incubator filled with 95％ O2-5％ CO2 at 37 °C.In group HR,the cells were exposed to 40-min hypoxia in an incubator filled with 95％ N2-5％ CO2 at 37 °C,followed by 110-min reoxygenation.In group U,the cells were exposed to 40-min of hypoxia,followed by 10-min reoxygenation,and then cultured in a cuhure medium containing 10-8mmol/L urocortin for 30 min,followed by 70-min reoxygenation.In group HU,the cells were cultured for 10 min in a culture medium containing 10-4mmol/L 5-HD,and the other treatments were similar to those previously described in group U.At the end of reoxygenation,the opening of mitochondrial permeability transition pore (mPTP) and mitochondrial membrane potential (MMP) were measured by fluorescence spectrophotometry.The expression of Bax and Bcl-2 was determined by Western blot,and the cell viability was measured by CCK-8 assay.Results Compared with group C,the viability of cardiomyocytes and MMP were significantly decreased,the opening of mPTP was increased,the expression of Bcl-2 was down-regulated,and the expression of Bax was up-regulated in the other 3 groups (P＜0.05).Compared with group HR,the viability of cardiomyocytes and MMP were significantly increased,the opening of mPTP was decreased,the expression of Bcl-2 was up-regulated,and the expression of Bax was down-regulated in group U,and the opening of mPTP was decreased (P ＜ 0.05),and no significant change was found in the other parameters in group HU (P＞0.05).Compared with group U,the viability of cardiomyocytes and MMP were significantly decreased,the opening of mPTP was increased,the expression of Bcl-2 was down-regulated,and the expression of Bax was up-regulated in group HU (P＜0.05).Conclusion The mechanism by which urocortin postconditioning attenuates H/R-induced damage to rat cardiomyocytes is associated with promoting mito-KATP channel opening and inhibiting mPTP opening.%目的 评价线粒体ATP敏感性钾(mtio-KATP)通道在尿皮质素后处理对大鼠心肌细胞保护效应中的作用.方法 清洁级健康雄性SD大鼠,16～20周龄,体重200～250 g,分离培养心肌细胞,采用随机数字表法将心肌细胞分成4组(n=28):对照组(C组)、缺氧复氧组(HR组)、尿皮质素后处理组(U组)和mito-KATP通道阻滞剂5-羟葵酸(5-HD)+尿皮质素后处理组(HU组).C组:在37°C95％ O2-5％ CO2培养箱中持续培养150 min;HR组:在37°C95％ N2-5％ CO2培养箱中缺氧40min后,复氧110 min;U组:缺氧40 min后先复氧10 min,然后在含10-8 mmol/L尿皮质素的培养基中孵育30 min,再复氧70 min;HU组:采用含10-4 mmol/L 5-HD的培养基孵育10 min,随后处理同U组.于复氧结束时,采用荧光法检测线粒体通透性转换孔(mPTP)开放程度以及线粒体膜电位(MMP).采用Western blot法检测Bax和Bcl-2表达,采用CCK-8法检测细胞活力.结果 与C组比较,其余3组心肌细胞活力和MMP降低,mPTP开放程度升高,Bcl-2表达下调,Bax表达上调(P＜0.05);与HR组比较,U组心肌细胞活力和MMP升高,mPTP开放程度降低,Bcl-2表达上调,Bax表达下调,HU组mPTP开放程度降低(P＜0.05),其余指标差异无统计学意义(P＞0.05);与U组比较,HU组心肌细胞活力和MMP降低,mPTP开放程度升高,Bcl-2表达下调,Bax表达上调(P＜0.05).结论 尿皮质素后处理对大鼠心肌细胞保护效应的全部机制与促进mito-KATP通道开放有关.
