肺栓塞复苏后心肌凋亡及卡托普利抗凋亡机制

Myocardial apoptosis and anti-apoptotic mechanism of captopril on cardiac arrest after resuscitation of pulmonary embolism

目的 急性肺栓塞(APE)致猪心脏骤停(CA)和复苏后心肌凋亡及卡托普利抑制心肌凋亡的分子机制.方法 近交系北京长白猪29只,通过颈外静脉注入血栓制作急性肺栓塞致心脏骤停模型,随后给予心肺复苏和溶栓治疗(15000 U/kg尿激酶静脉注射).随机(随机数字法)分为对照组(Control组,5只)、APE致CA(APE-CA组,5只)、19只注射血栓后10只恢复自主循环(ROSC),随机分为自主循环恢复+卡托普利(ROSC-Cap组,5只,ROSC后30 min静脉注射卡托普利22.22 mg/kg)和自主循环恢复+生理盐水(ROSC-SA组,5只,ROSC后30 min静脉注射等量生理盐水).自主循环恢复后6 h处死实验动物取心脏组织,Western blot测定心肌Bax、Bcl-2、Caspase-3、p-Src、p-ERK1/2表达,免疫组化法测定心肌p-Src、p-ERK1/2表达,ELISA检测心肌组织匀浆钠-钾-ATP酶水平.采用SPSS 16.0统计软件分析,计量资料以均数 ± 标准差表示,组间比较采用单因素方差分析.Pearson相关性检验分析p-Src、p-ERK1/2、钠-钾-ATP酶与Bax、Bcl-2、Caspase-3的相关性.结果 与对照组相比,APE-CA和ROSC-SA组心肌Bax(0.25±0.01,0.53±0.01,0.37±0.05,F=14.16,P<0.05)、Caspase-3(0.24±0.01,0.33±0.01,0.34±0.06,F=7.32,P<0.05)表达明显升高,Bcl-2表达明显下降(0.56±0.02,0.19±0.01,0.37±0.10,F=6.68,P<0.05).卡托普利降低Caspase-3、Bax表达,升高Bcl-2表达(均P<0.05).与对照组相比,APE致CA时及ROSC后p-Src、ERK1/2蛋白表达均明显升高、钠-钾ATP酶水平明显下降(均P<0.05);与APE-CA相比,ROSC-Cap组p-Src(0.46±0.01 vs.0.35±0.06,P<0.05)表达明显下降.与生理盐水组相比,卡托普利显著降低心肌ERK1/2蛋白表达(0.41±0.10 vs.0.26±0.07,P<0.05),但对钠-钾ATP酶的水平未产生影响.p-Src、p-ERK1/2分别与Bax正相关,与Bcl-2负相关,钠-钾-ATP酶与Caspase-3负相关.结论 APE致CA和ROSC时钠/钾-ATP酶活性下降、p-Src、p-ERK1/2激活可能为心肌细胞凋亡的分子机制.ROSC后卡托普利可抑制心肌细胞凋亡,其分子机制为降低心肌p-Src、p-ERK1/2表达,为心肺复苏后心肌凋亡的治疗提供新靶点.

Objective To observe the myocardial apoptosis and the molecular mechanism of captopril inhibiting myocardial apoptosis on cardiac arrest (CA) after resuscitation in a porcine acute pulmonary embolism (APE) model. Methods In this study, 29 inbred Beijing Landrace wererandomly (random number)divided into four groups (n=5, each group): control, APE-CA, restoration of spontaneous circulation (ROSC)-captopril, and ROSC-saline. The model of CA and ROSC was induced by APE through injection of thrombus followed by cardiopulmonary resuscitation and thrombolytic therapy (urokinase, 15000 U/kg, iv). Ten of 19 pigs with CA recovered to spontaneous circulation were divided randomly into the ROSC-captopril and ROSC-saline groups. Pigs in the ROSC-captopril group were treated with captopril (22.22 mg/kg) via porcine femoral vein at 30 min after ROSC. Pigs in the ROSC-saline group were treated with equal normal saline at 30 min after ROSC. The myocardial tissues were evaluated at 6 h after ROSC. Western blot was used to evaluate the protein levels of Bax, Bcl-2, Caspase-3, phosphorylated (p)-Src and phosphorylated extracellular regulated protein kinase (p-ERK1/2). Immunohistochemistry was used to evaluate the protein expression of p-Src and p-ERK1/2. Enzyme-linked immunosorbent assay was used to detect myocardial Na+-K+-ATPase levels. Statistical analysis was performed using one-way analysis of variance and pearson correlation test. Results Compared with the control group, the protein expression of Bax (0.25±0.01, 0.53±0.01, 0.37±0.05, F=14.16, P<0.05) and Caspase-3 (0.24±0.01, 0.33±0.01, 0.34±0.06, F=7.32, P<0.05) in the APE-CA and ROSC- saline group were increased significantly, and the Bcl-2 expression was significantly decreased (0.56±0.02, 0.19±0.01, 0.37±0.10, F=6.68, P<0.05). Captopril reduced the protein levels of Caspase-3 and Bax, while stimulated the Bcl-2 expression (all P<0.05). Compared with the control group, the protein expression of p-Src and p-ERK1/2 were higher and the Na+-K+-ATPase level was decreased on CA and ROSC induced by APE (all P<0.05). Compared with the APE-CA group, the p-Src expression in the ROSC-captopril group (0.46±0.01 vs. 0.35±0.06, P<0.05) was decreased significantly. Captopril inhibited the activation of p-ERK1/2 than saline group (0.41±0.10 vs. 0.26±0.07, P<0.05), but has no effect on the Na+-K+-ATPase level. The protein expression of p-Src and p-ERK1/2 were positively correlated with the Bax, and negatively correlated with the Bcl-2 respectively. The myocardial Na+-K+-ATPase level negatively correlated with Caspase-3 protein expression. Conclusions The molecular mechanism of cardiomyocyte apoptosis on CA and ROSC induced by APE might be related to decreased Na+-K+-ATPase level and activation of p-Src and p-ERK1/2. The cardiomyocyte apoptosis were inhibited by captopril through reducing the expression of p-Src and p-ERK1/2 in myocardium.