cAMP—PKA信号转导通路在缺血预处理减轻大鼠离体心脏缺血再灌注损伤中的作用

Role of cAMP-PKA signal transduction pathway in the ischemic preconditioning-induced attenuation of ischemia-reperfusion injury in isolated rat hearts

目的探讨cAMP-PKA信号转导通路在缺血预处理减轻大鼠离体心脏缺血再灌注损伤中的作用。方法成年SD大鼠50只，体重300～350g，采用Langendorff装置建立大鼠离体心脏缺血再灌注模型后随机分为5组（n=10）：缺血再灌注组（IR组）、缺血预处理组（IPC组）、H89组、脯氨酸二硫氨基甲酸组（PDTC组）和双丁酰环磷酸腺苷组（db-cAMP组）。K-H液平衡灌注10min后，R组K-H液继续灌注30min后停灌1h，再灌注30min；IPC组停灌5min，再灌注5rain，反复3次，停灌1h再灌注30min；PDTC和H89组停灌5min，分别用含有PDTC100μmol／L和含有H8910μmol／L的K-H液再灌注5min，反复3次，余同IPC组；db-cAMP组用含db—cAMP200μmol／L的K．H液灌注30min，停灌1h再灌注30rain。于平衡灌注10min、再灌注10、20和30min时记录左心室压力最大变化速率（±dp／dt max）和左心室发展压（LVDP）。于平衡灌注10rain和再灌注30rain时记录冠脉流出量（CF）；测定冠脉流出液中乳酸脱氢酶（LDH）和磷酸肌酸激酶（CK）活性。于再灌注30min时取心肌组织，采用EMSA法检测NF-κB-DNA结合活性，采用RT—PCR法检测TNF-α mRNA表达，采用Western blot法检测磷酸化环腺苷酸反应元件结合蛋白（p-CREB）（Ser133）表达。结果与IR组比较，IPC组和db-cAMP组NF-κB-DNA结合活性和TNF-α mRNA表达降低，±dp／dtmax 和CF升高，CK和LDH活性降低，IPC组、PDTC组和db-cAMP组P—CREB（Serl33）表达升高（P〈0．05或0．01）；与IPC组比较，H89组和PDTC组NF-κB-DNA结合活性和TNF—dmRNA表达升高，±dp／dt max、LVDP和CF降低，CK和LDH活性升高，H89组p-CREB（Ser133）表达降低（P〈0．05或0．01）。结论缺血预处理通过激活cAMP-PKA信号转导通路抑制NF-κB-DNA结合活性，减少炎性因子的基因转录，从而减轻大鼠离体心脏缺血再灌注损伤。

Objective To investigate the role of cAMP-PKA signal transduction pathway in the ischemic preconditioning-induced attenuation of ischemia-reperfusion （I/R） injury in isolated rat hearts. Methods Fifty healthy adult SD rats weighing 300-350 g were anesthetized with intraperitoneal （IP） pentobarbital 50 mg/kg. Their hearts were excised and perfused in a Langendorff apparatus with 37 ℃ oxygenated （95 % O2-5 % CO2 ） modified K-H solution at a constant pressure of 70 cm H20, and randomly divided into 5 groups （n = 10 each） ： group Ⅰ I/R; group Ⅱ ischemic preconditioning （IPC）; group Ⅲ H89 （PKA inhibitor）; group Ⅳ PDTC （NF-κB inhibitor） and groupV db-cAMP. The experiment started after 10 min stabilization. The isolated hearts were first peffused for 30 min, followed by 60 min ischemia and 30 min reperfusion in group I/R （Ⅰ）. Group IPC （ Ⅱ） was subjected to 3 episodes of 5 min ischemia at 5 min intervals before I/R. Group Ⅲ and Ⅳ received 5 min perfusion with H89 10 μmol/L and PDTC 100 μmol/L 3 times at 5 min intervals respectively before I/R. Group V was perfused with db-cAMP 200 μmol/L for 30 min before I/R. Left ventricular developed pressure （LVDP） and ＋ dp/dtm= were measured at stabilization period and 10, 20, 30min of reperfusion. Coronary flow （CF） was measured at stabilization period and 30 min of reperfusion and activities of LDH and creatine kinase （CK） in the coronary effluent were determined. The myocardial specimens were obtained at 30 min of reperfusion for determination of NF-κB-DNA binding activity （by EMSA） and expression of TNF-α mRNA （by RT-PCR ） and p-CREB （Ser133） （by Western blot）. Results Compared with I/R group, NF-κB-DNA binding activity and TNF-α mRNA expression were significantly decreased, ＋ dp/dtmax and CF were significantly increased, CK and LHD activities in the coronary effluent were significantly decreased in group IPC and db-cAMP （group Ⅱ , Ⅴ ） and p-CREB （Ser133） expression was significantly increased in group IPC, PDTC and db-cAMP （group Ⅱ, Ⅳ, Ⅴ ）. Compared with IPC group, NF-κB-DNA binding activity and TNF-α mRNA expression were significantly increased, ± dp/dtmax, LVDP and CF were significantly decreased, CK and LDH activities were significantly increased in group H89 and PDTC （group m, IV ） and p-CREB （Ser133） expression was significantly decreased in group H89 （group m ）. Conclusion Ischemic preconditioning can attenuate I/R injury in isolated hearts by inhibition of NF-κB-DNA binding activity via cAMP-PKA signal transduction pathway which reduces gene transcription of inflammatory eytokine.