心肺复苏大鼠心肌组织CyPA信号通路的研究

Effects of CyPA signal pathway in myocardial tissue after cardiopulmonary resuscitation in rats

目的观察心肺复苏（CPR）后大鼠心肌组织亲环素A（CyPA）信号通路的变化。方法将68只健康雄性SD大鼠按随机数字表法分为假手术组（n=8）及心搏骤停（CA）即刻组、CPR即刻组和CPR后15、30、60、120min组，每个时间点10只。利用呼气末夹闭气管导管模拟窒息后给予机械通气、心脏按压及静脉注射肾上腺素恢复自主循环（ROSC）建立CA—CPR模型。分别于各时间点检测大鼠血流动力学；取腹主动脉血及心肌组织，采用酶联免疫吸附试验（ELISA）检测血清CyPA和CD147水平，采用免疫组化及实时定量反转录-聚合酶链反应（RT—qPCR）检测心肌组织CyPA和CD147的阳性表达及mRNA表达；采用蛋白质免疫印迹试验（WesternBlot）检测心肌组织基质金属蛋白酶（MMP-2、MMP-9）和髓过氧化物酶（MPO）的蛋白表达；苏木素-伊红（HE）染色，光镜下观察心肌组织中性粒细胞浸润情况。结果CA即刻大鼠心率降至0，动脉压降低；CPR即刻动脉压即升高至正常，随时间延长，大鼠心率约在30min时恢复；肾上腺素用量50-60嵋，ROSC时间1～4min。与假手术组比较，随ROSC时间延长，CPR120min内血清CyPA和CD147水平逐渐升高，120min达峰值[CyPA从（786．11±3．93）μg/L升至（2001．80±10．61）倒L，CD147从（2．94±0．02）μg/L升至（5．99±0．02）μg/L，P〈0．05或P〈0．01]；心肌组织CyPA和CD147的mRNA表达（A值）逐渐升高，120min达峰值（以假手术组为1，CyPA的相对表达量达2．42±0．05，CD147的相对表达量达1．88±0．10，均P〈0．01）；镜下观察免疫组化结果显示，胞膜呈褐色的阳性细胞逐渐增多、变深，说明CyPA和CD147表达均增加；心肌组织MMP-2、MMP-9、MPO的蛋白表达逐渐升高，120min时达峰值[MMP-2（灰度值）从0．396±0．021升至0．879±0．020，MMP-9（灰度值）从0．372±0．009升至0．819±0．012，MPO（灰度值）从0．176±0．005升至0．829±0．018，P〈0．05或P〈0．01]；心肌组织中未见中性粒细胞明显浸润。结论CyPA和CD147在CPR大鼠血清及心肌组织中表达上调，是炎症反应的标志物。

Objective To investigate the changes in cyclophilin A （CyPA） signal pathway in rat myocardial tissue after cardiopulmonary resuscitation （CPR）. Methods Sixty-eight healthy male Sprague-Dawley （SD） rats were randomly divided into sham operation group （n = 8 ）, instantancous CPR after cardiac arrest （CA）, immediate CPR after CA （CRP）, and 15, 30, 60 and 120 minutes after CPR groups, respectively, with 10 rats in each group. Asphyxia was simulated by occlusion of the tracheal tube at the end of exhalation. Mechanical ventilation, compression and epinephrine injection were given for restoration of spontaneous circulation （ROSC） in order to reproduce cardiopulmonary resuscitation after cardiac arrest （CA-CPR） models in rats. Hemodynamic changes were recorded at different time points. The blood was collected from abdominal aorta, and myocardial tissue was also harvested. The serum CyPA and CD147 levels were determined by enzyme-linked immunosorbent assay （ELISA）. The protein positive cells and mRNA expression of CyPA and CD147 in myocardial tissue of the rats were determined by immunohistochemical and real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR）. The protein expressions of matrix metalloproteinases （MMP-2, MMP-9 ） and myeloperoxidase （MPO） in rat myocardial tissue were determined by Western Blot. The neutrophil infiltration in rat myocardial tissue was assessed by hematoxylin and eosin （HE） staining. Results The heart rate of rat was lowered to 0 with arterial pressure lowered immediately after CA. Arterial pressure was elevated to normal range immediately after CPR. Heart rate was restored at about30 minutes later, and the dose of epinephrine was 50-60 μg, and ROSC time was 1-4 minutes. Compared with those of the sham group, serum CyPA and CD147 levels were gradually increased along with elongation of ROCS time within 120 minutes, and peaked at 120 minutes, CyPA was increased from （786.11±3.93 ） μg/L to （2 001.80 ±10.61 ）μg/L, and CD147 was increased from （2.94±0.02） μg/L to （5.99± 0.023） μg/L （P 〈 0.05 or P 〈 0.01 ）. CyPA and CD147 mRNA expressions （A value） in rat myocardial tissue were gradually increased, and peaked at 120 minutes. Relative expression of CyPA at 120 minutes was 2.42 ± 0.05 when it was 1 in the control, and relative expression of CD147 at 120 minutes was 1.88± 0.10 （both P 〈 0.01 ）. The immunohistochemical results under light microscope showed that the brown positive cells were gradually increased, which indicated that the expressions of CyPA and CD147 were increased. Expressions of MMP-2, MMP-9 and MPO （gray value） in myocardial tissue were also gradually increased, peaking at 120 minutes, and MMP-2 was increased from 0.396 ± 0.021 to 0.879 ± 0.020, MMP-9 was increased from 0.372± 0.009 to 0.819±0.012, and MPO was increased from 0.176±0.005 to 0.829±0.018 （P 〈 0.05 or P 〈 0.01 ）. No obvious neutrophil infiltration in myocardial tissue was found with HE staining. Conclusion Expressions of CyPA and CD147 were up-regulated in serum and myocardial tissue after CPR in rats, which may be the markers of inflammatory reaction.