蛋白激酶A在缺血预处理抑制心肌细胞核因子-κB-DNA结合活性中的作用

Role of Protein Kinase A in Ischemia Preconditioning for Inhibiting Binding Activation of Nuclear Factor-κB-DNA in Cardiomyocyte

目的:探讨蛋白激酶A在缺血预处理抑制心肌细胞核因子-κB-脱氧核糖核酸(DNA)(NF-κB-DNA)结合活性中的作用。方法:采用Langendorff离体心脏灌注模型。40只SD大鼠随机分为4组,缺血/再灌注组、缺血预处理组、蛋白激酶A抑制剂组(H89组)和脯氨酸二硫氨基甲酸组(PDTC组,核因子-κB抑制剂)。在平衡期、复灌10 min、20 min、30 min记录心率、左心室发展压,在平衡期、复灌30 min时记录冠状动脉流量,测定冠状动脉流出液中乳酸脱氢酶和肌酸激酶含量。复灌30 min后留取心肌用蛋白质免疫印迹法(Western blot)检测磷酸化环腺苷酸反应元件结合蛋白[p-CREB(Ser133)]含量,凝胶电泳迁移率法(EMSA)检测核因子-κB-DNA结合活性。结果:与缺血/再灌注组相比,缺血预处理组和PDTC组心肌细胞核内p-CREB(Ser133)含量升高了2.31倍和1.65倍;与缺血预处理组相比,H89组p-CREB(Ser133)含量显著降低,差异均有统计学意义(P均<0.01)。与缺血/再灌注组相比,缺血预处理组心肌细胞核内核因子-κB-DNA结合活性明显降低;与缺血预处理组相比,H89组和PDTC组核因子-κB-DNA结合活性显著升高,差异均有统计学意义(P<0.05～0.01)。复灌30min时,与缺血预处理组相比,缺血/再灌注组、H89组和PDTC组心率、左心室发展压和冠脉流量明显降低,乳酸脱氢酶和肌酸激酶活性显著升高,差异均有统计学意义(P均<0.05～0.01)。结论:缺血预处理可通过激活蛋白激酶A抑制核因子-κB-DNA结合活性减轻心肌缺血再灌注损伤。

Objective ：To investigate the role of protein kinase A（PKA） in ischemia preconditioning for inhibiting binding activation of nuclear factor-κB-DNA（NF-κB-DNA） in cardiomyocyte. Methods：Forty SD rats were divided into four groups and each group contained 10 rats. Ischemia/reperfusion （I/R） group, Ischemia preconditioning（ IPC ） group, Pretreatment with PKA inhibitor H89 （ H89 ） group, and NF-κB inhibitor PDTC （ PDTC ） group. The isolated hearts were Langendorff-perfused by K-H solution for 10 minute as stabilization period in each group. Then, additional treatment was followed in I/R group：perfusion 30 min,rest 1 hour,and re-perfusion 30 min; in IPC group：rest 5 min, reperfusion 5 min,repeated this procedure for 3 times,then,rest 1 hour and reperfusion 30min; in H89 group and PDTC group： rest 5 min,then reperfused by 10 μM H89 and 100 μM PDTC for 5 min respectively,and repeated this procedure for 3 times. Heart rate（HR） and left ventricular developed pressure（ LVDP）, difference between left ventricular end systolic pressure and end diastolic pressure were recorded at baseline,post-reperfusion at 10 min,20 min and 30 min. At baseline and 30 min of reperfusion, coronary flow（CF） was measured, and contents of lactate dehydrogenase （LDH） and creatine kinase （CK） in the coronary effluent were analyzed. The heart tissues were sampled at 30 min of reperfusion and then rapidly stored in liquid nitrogen. The levels of phosphorylated cAMP response element binding protein on Ser133 [ p-CREB（Ser133） ] were measured using Western blot analysis, NF-κB-DNA activation was analyzed using electrophoretic mobility shift assay（EMSA）. Results：Compared with I/R group,the levels of p-CREB（Ser133） in IPC and PDTC groups were increased by 2. 31 folds and 1.65 folds, respectively （P 〈 0. 01 ）, compared with IPC group, the levels of p-CREB （Ser133 ） in H89 group was significantly lower（P 〈0. 01 ）. Compared with L/R group,the binding activity of NF-κB-DNA was significantly lower in IPC group（P 〈0. 01 ）, compared with IPC group, the binding activity of NF-κB-DNA in H89 and PDTC groups were significantly higher（ P 〈 0. 05 ）. Compared with IPC group,in I/R, H89 and PDTC groups, at 30min of reperfusion, HR, LVDP and CF were improved significantly （P 〈0. 05） and the contents of LDH and CK were decreased significantly in IPC group（P 〈0. 05 or 0. 01 ）. Conclusion ：The results demonstrated that the activated PKA inhibited the binding activity of NF-κB-DNA and attenuated I/R injury in the cardioprotection of IPC.