丙酮酸对体外循环术后狗心肌细胞凋亡的影响及其对fas、fasL和bcl-2家族蛋白及caspase-3活性的影响

Effects of pyruvate on CPB-induced myocardiocyte apoptosis and on modulation of bcl-2 family protein,fasfasL and caspase-3 pathways in dogs

目的观察丙酮酸对常规体外循环术后狗心肌细胞凋亡和fas、fasL、bcl-2、bcl-xl、bax、以及bad蛋白表达和caspase-3活性的影响。方法10条拟进行常规体外循环(CPB)的成年家狗被随机分为对照组(n=5)和丙酮酸处理组(n=5)。CPB术前、术后和术后3 h分别留取左心室(LV)标本。用原位TUNEL技术检测凋亡的心肌细胞。用免疫组化方法和流式细胞仪测定fas、fasL、bcl-2、bcl-xl、bax和bad蛋白表达。分别用Z-DEVD-AMC底物分解法和TBARS法测定caspase-3活动度和丙二醛(MDA)含量。结果两组中各参数CPB术前与术中在组间和组内均无显著性差异。CPB后,两组的心肌细胞凋亡数明显增高、fas、fasL、bax和bad蛋白表达、caspase-3活性及MDA含量也均较术前和术中明显增高,而丙酮酸(pyruvate)处理组的心肌细胞凋亡数、fas、fasL、bax和bad蛋白表达、caspase-3活性及MDA含量均明显低于对照组。两组bcl-2和bcl-xl表达在CPB术前、术中和术后均无显著性变化。结论pyruvate可以抑制常规CPB术后心肌细胞凋亡,下调fas、fasL、bax和bad蛋白表达,降低caspase-3活性及心肌氧化应激反应。

Objective To investigate the effects of pyruvate on the cardiopulmonary bypass （CPB）-induced myoeardiocyte apoptosis in dog and its role in modulation of fas, fasL, bcl-2, bel-xl, bax, bad and caspase-3 pathways. Methods Ten healthy adult male and female dogs undergoing conventional CPB were randomized into control and pyruvate treated groups （each n - 5）. Pyruvate was used in the latter group to maintain a concentration of 2 nmol/L in plasma and eardioplegic solution. Samples of the left ventricle （LV） were obtained before, during and 3 h after CPB and stored in liquid nitrogen until assay. In situ TUNEL technique was used to detect the apoptotic cells and the number of apoptotic positive cells per 105 myocytes was counted. The expression of fas, fasL bcl- 2, bcl-xl, bax and bad was semi-quantified by flow cytometer technique. The activity of caspase-3 measured by cleavage of Z-DEVD- AMC substrate. Level of Malondiadehyde （MDA） in the myoeardium was measured by thiobarbituric acid reactive substances （TBARS） method. Results All the parameters before and during CPB had no significant difference between intra- and entergroups. After CPB, the number of apoptotic cells in both control and pyruvate treated groups were significantly increased compared to that before and during CPB （P〈0. 003, 0. 003, 0,001 and 0. 001, respectively）, and the number of apoptotic cells in pyruvate treated group was remarkably reduced compared to control group （P=0.019）. Expressions of fas, fasL, bax and bad proteins and the activity of caspase-3 were also significantly upregulated in both groups after CPB compared to those before and during CPB （P〈 0,025, respectively）, and were significantly inhibited in pyruvate treated group （vs control P 〈 0.05, respectively）. While the ex- pressions of bcl-2, and bcl-xl had no significant difference before, during and 3 h after CPB in both groups, The content of MDA in the LV myoeardium after CPB were also elevated in both groups compared to those before and during CPB （P 〈 0.001,0,003,0.001 and 0,001, respectively） and was significantly inhibited in pyruvate treated group compared to control group （P〈 0. 015 ）. Conclusion Treatment with pyruvate can significantly reduce cardiomyocyte apoptosis, down-regulate the expressions of fas, fasL, bax and bad proteins and inhibit the activity of caspase-3 and the production of lipid peroxide in dog hearts following conventional CPB.