PI3K和ERK信号通路在吗啡预处理减轻心力衰竭大鼠离体心脏缺血再灌注损伤中的作用

Roles of PI3K and ERK signaling pathways in reduction of ischemia-reperfusion injury to isolated hearts by morphine preconditioning in rats with chronic heart failure

目的 评价1-磷脂酰肌醇3-激酶（PI3K）和细胞外信号调节激酶（ERK）信号通路在吗啡预处理减轻心力衰竭大鼠离体心脏缺血再灌注损伤中的作用.方法 雄性成年SD大鼠,体重200～230 g,尾静脉注射盐酸多柔比星2 mg/kg,1次/周,连续6周,制备大鼠慢性心力衰竭模型.第8周末将成功制备慢性心力衰竭模型的大鼠42只,采用随机数字表法分为7组（n=6）：假手术组（S组）、缺血再灌注组（I/R组）、吗啡预处理组（MP组）、ERK抑制剂PD98059＋吗啡预处理组（PD＋ MP组）、PI3K抑制剂渥曼青霉素＋吗啡预处理组（WT＋ MP组）、PD98059组（PD组）和渥曼青霉素组（WT组）.采用Langendorff离体心脏灌注及结扎左冠状动脉前降支（LAD）法制备大鼠缺血再灌注损伤模型,缺血30min,再灌注120 min.S组只缝线,不结扎LAD,持续灌注K-H液195 min.I/R组先灌注K-H液45 min,再制备大鼠缺血再灌注损伤模型.MP组先灌注K-H液15 min,再灌注含1μmol/L吗啡的K-H液进行预处理,灌注5 min,再次灌注K-H液5min,共3个循环,然后制备心脏缺血再灌注损伤模型.PD＋ MP组和WT＋ MP组于吗啡预处理前10 min,分别用含ERK抑制剂PD98059（10 μmol/L）和PI3K抑制剂渥曼青霉素（100 nmol/L）的K-H液进行灌注,持续至缺血5 min.PD组和WT组于缺血前40 min持续至缺血5 min分别灌注含PD98059和渥曼青霉素的K-H液.各组分别于心脏稳定灌注15 min、再灌注5和10 min时,收集冠脉流出液,采用化学比色法检测LDH活性.再灌注120 rin时,取下大鼠心脏,测量缺血危险区体积（AAR）、梗死区体积（IS）及IS/AAR比值.结果 与S组比较,I/R组再灌注5和10min时LDH活性升高,IS和IS/AAR均增加（P＜0.05）,AAR差异无统计学意义（P＞0.05）;与I/R组比较,MP组再灌注5min时LDH活性降低,IS和IS/AAR减小（P＜0.05）,AAR差异无统计学意义,WT组和PD组LDH活性、IS、AAR和IS/AAR差异无统计学意义（P＞0.05）;与MP组比较,PD＋ MP组再灌注5和10 min时LDH活性升高,IS和IS/AAR减小（P＜0.05）,WT＋ MP组LDH活性、IS、AAR和IS/AAR差异无统计学意义（P＞0.05）.结论 ERK信号通路的激活参与了吗啡预处理减轻心力衰竭大鼠离体心脏缺血再灌注损伤,而PI3K信号通路无此作用.

Objective To evaluate the roles of 1-phosphatidylinositol 3-kinase （PI3K） and extracellular signal-regulated kinase （ERK） signaling pathways in reduction of ischemia-reperfusion （I/R） injury to the isolated hearts by morphine preconditioning in the rats with chronic heart failure.Methods Adult male Sprague-Dawley rats,weighing 200-230 g,in which doxorubicin 2.0 mg/kg was injected via the tail vein once a week for 6 weeks to induce chronic heart failure,were studied.At the end of 8th week,42 rats with chronic heart failure were randomly divided into 7 groups （n =6 each） using a random number table：sham operation group （group S）,I/R group,morphine preconditioning group （group MP）,PD98059 （ERK inhibitor） ＋ morphine preconditioning group （group PD ＋ MP）,wortmannin （PI3K inhibitor） ＋ morphine preconditioning group （group WT ＋ MP）,PD98059 group （group PD） and wortmannin group （group WT）.The hearts were quickly excised and passively perfused in a Langendorff apparatus and subjected to 30 min of occlusion of the left coronary artery followed by 2 h of reperfusion to establish the model of I/R injury.In group S,the hearts were only sutured,but not ligated and were continuously perfused with K-H solution for 195 min.In group I/R,the hearts were perfused with K-H solution for 45 min before ischemia.In group MP,the hearts were perfused with K-H solution for 15 min,with K-H solution containing morphine 1 μmol/L for 5 min and then with K-H solution for 5 min （3 cycles in total） before ischemia.In PD ＋ MP and WT ＋ MP groups,the hearts were perfused with K-H solution containing PD98059 （10 μmol/L） and wortmannin （100 nmol/L）,respectively,starting from 10 min before morphine preconditioning until 5 min of ischemia.In PD and WT groups,the hearts were perfused with K-H solution containing PD98059 （10 μmol/L） and wortmannin （100 nmol/L）,respectively,starting from 40 min before ischemia until 5 rin of ischemia.At 15 min of equilibration （baseline） and 5 and 10 min of reperfusion,the coronary flow was collected to detect the activity of lactate dehydrogenase （LDH）.Infarct size （IS） and area at risk （AAR） were measured at the end of reperfusion and IS/AAR ratio was calculated.Results Compared with group S,LDH activity was significantly increased at 5 and 10 min of reperfusion,IS and IS/AAR ratio were also increased （P 〈 0.05）,and no significant change was found in AAR in group I/R （P 〉 0.05）.Compared with group I/R,LDH activity was significantly decreased at 5 min of reperfusion,IS and IS/AAR ratio were also decreased （P 〈 0.05）,and no significant change was found in AAR in group MP,and no significant change was found in LDH activity,IS,AAR and IS/AAR ratio in WT and PD groups （P 〉 0.05）.Compared with group MP,LDH activity was significantly increased at 5 and 10 min of reperfusion （P 〈 0.05）,and IS and IS/AAR ratio were decreased in group PD ＋ MP,and no significant change was found in LDH activity,IS,AAR and IS/AAR ratio in group WT ＋ MP （P 〉 0.05）.Conclusion Activation of ERK signaling pathway is involved in reduction of I/R injury to isolated hearts by morphine preconditioning in rats with chronic heart failure,however,PI3K signaling pathway has no such effect.