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目的 观察亚低温对心搏骤停(CA)猪心肺复苏(CPR)后心肌β-肾上腺素能受体(β-AR)信号通路的影响,探讨其心肌保护作用的机制.方法 选择健康雄性长白猪复制CA-CPR模型(右心室致颤, 8 min后进行CPR),并按随机数字表法分为两组(n=8):亚低温组于自主循环恢复(ROSC)后20 min内使用亚低温治疗仪将动物血温诱导为33 ℃并维持6 h;对照组则用冷热毯将动物体温维持在(38.0±0.5)℃.实验过程中持续监测动物心率(HR)、平均动脉压(MAP)、左室内压上升或下降最大速率(±dp/dt max);分别在CA前及ROSC后0.5、1、3、6 h用热稀释法测量心排血量(CO),并收集静脉血检测心肌肌钙蛋白I(cTnI)水平;于CA前及ROSC后6 h用心脏超声测量左室射血分数(LVEF).ROSC后6 h处死动物并留取心肌组织标本,用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测β1-AR mRNA表达,用酶联免疫吸附试验(ELISA)检测腺苷酸环化酶(AC)、环磷酸腺苷(cAMP)含量,用蛋白质免疫印迹试验(Western Blot)检测G蛋白耦联受体激酶2(GRK2)蛋白表达.结果 两组动物复苏后HR较基础值明显升高,CO、±dp/dt max明显下降,MAP无明显变化,血清cTnI水平明显升高.与对照组比较,亚低温组动物ROSC后0.5、1、3 h HR明显降低(次/min:142.80±12.83比176.88±15.14,115.80±11.48比147.88±18.53,112.60±7.40比138.50±12.02,均1<0.01), ROSC后1 h和3 h CO明显升高(L/min:3.97±0.40比3.02±0.32,4.00±0.11比3.11±0.59,均1<0.01),ROSC后3 h和6 h ±dp/dt max明显升高〔+dp/dt max(mmHg/s):3402.5±612.7比2130.0±450.6,3857.5±510.4比2562.5±633.9;-dp/dt max(mmHg/s):2935.0±753.2比1732.5±513.6,3520.0±563.6比2510.0±554.3,均1<0.05〕,ROSC后3 h和6 h血清cTnI水平明显下降(μg/L:1.39±0.40比3.24±0.78,1.46±0.35比3.78±0.93,均1<0.01).心脏超声显示,两组ROSC后6 h LVEF均较CA前明显降低,但亚低温组LVEF明显高于对照组(0.52±0.04比0.40±0.05,1<0.05).ROSC后6 h,亚低温组心肌组织β1-AR mRNA表达和AC、cAMP含量明显高于对照组〔β1-AR mRNA(2-ΔΔCT):1.18±0.39比 0.55±0.17,AC(ng/L):197.0±10.5 比162.0±6.3,cAMP (nmol/L):1310.58±48.82比891.25±64.95,均1<0.05〕,GRK2蛋白表达明显低于对照组(GRK2/GAPDH:0.45±0.05比0.80±0.08,1<0.05).结论 亚低温可以减轻CA-CPR猪复苏后心肌的损伤程度,其作用机制可能与减轻β-AR信号通路受损有关.

Objective To observe the effect of mild hypothermia on myocardial β-adrenergic receptor (β-AR) signal pathway after cardiopulmonary resuscitation (CPR) in pigs with cardiac arrest (CA) and explore the mechanism of myocardial protection. Methods Healthy male Landraces were collected for reproducing the CA-CPR model (after 8-minute untreated ventricular fibrillation, CPR was implemented). The animals were divided into two groups according to random number table (n = 8). In the mild hypothermia group, the blood temperature of the animals was induced to 33 ℃ and maintained for 6 hours within 20 minutes after return of spontaneous circulation (ROSC) by using a hypothermia therapeutic apparatus. In the control group, the body temperature of the animals was maintained at (38.0±0.5)℃ with cold and warm blankets. The heart rate (HR), mean arterial pressure (MAP), the maximum rate of increase or decrease in left rentricular pressure (+dp/dt max)were measured during the course of the experiment. The cardiac output (CO) was measured by heat dilution methods before CA (baseline), and 0.5, 1, 3, 6 hours after ROSC respectively, the venous blood was collected to detect the concentration of cTnI. Left ventricular ejection fraction (LVEF) was measured with cardiac ultrasound before CA and 6 hours after ROSC. Animals were sacrificed at 6 hours after ROSC and the myocardial tissue was harvested quickly, the mRNA expression of β1-AR in myocardium was detected by reverse transcription-polymerase chain reaction (RT-PCR), the contents of adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) were detected by enzyme linked immunosorbent assay (ELISA), the protein content of G protein-coupled receptor kinase 2 (GRK2) was detected by Western Blot. Results After successful resuscitation, the HR of both groups were significantly higher than the baseline values, CO, ±dp/dt max were significantly decreased, MAP were not significantly changed, serum cTnI levels were significantly increased. Compared with the control group, HR at 0.5, 1, 3 hours after ROSC were significantly decreased in mild hypothermia group (bpm: 142.80±12.83 vs. 176.88±15.14, 115.80±11.48 vs. 147.88±18.53, 112.60±7.40 vs. 138.50±12.02, all 1 < 0.01), CO was significantly increased at 1 hours and 3 hours after ROSC (L/min: 3.97±0.40 vs. 3.02±0.32, 4.00±0.11 vs. 3.11±0.59, both 1 < 0.01), +dp/dt max at 3 hours and 6 hours was also significantly increased after ROSC [+dp/dt max (mmHg/s): 3 402.5±612.7 vs. 2 130.0±450.6, 3 857.5±510.4 vs. 2 562.5±633.9; -dp/dt max (mmHg/s): 2 935.0±753.2 vs. 1 732.5±513.6, 3 520.0±563.6 vs. 2 510.0±554.3, all 1 < 0.05], the cTnI was significantly decreased at 3 hours and 6 hours afher ROSC (μg/L: 1.39±0.40 vs. 3.24±0.78, 1.46±0.35 vs. 3.78±0.93, both 1 < 0.01). The left at 6 hours after ROSC in both groups was decreased as compared with that before CA. The LVEF in the mild hypothermia group was higher than that in the control group (0.52±0.04 vs. 0.40±0.05, 1 < 0.05). The mRNA expression of β1-AR, and concentrations of AC and cAMP in hypothermia group were significantly higher than those in control group [β1-AR mRNA (2-ΔΔCT): 1.18±0.39 vs. 0.55±0.17, AC (ng/L):197.0±10.5 vs. 162.0±6.3, cAMP (nmol/L): 1 310.58±48.82 vs. 891.25±64.95, all 1 < 0.05], GRK2 was lower than that in the control group (GRK2/GAPDH: 0.45±0.05 vs. 0.80±0.08, 1 < 0.05). Conclusion Mild hypothermia can reduce the degree of cardiac function injury after CPR, and its mechanism may be related to the reduction of impaired myocardial β-AR signaling after CPR.