摘要目的 探讨内源性孤啡肽(N/OFQ)是否通过Raf激酶抑制蛋白(RKIP)影响急性心肌缺血大鼠心律失常及心肌细胞膜表面 β1-肾上腺素能受体(β1-AR)的表达.方法 ①实验一:按随机数字表法将30只6周龄成年雄性SD大鼠分为假手术组〔Sham组,只开胸不结扎冠状动脉(冠脉)〕、心肌缺血模型组(结扎冠脉左前降支)、内源性N/OFQ拮抗剂UFP-101预处理组(UFP-101组,术前10 min经尾静脉注射1 mL/kg UFP-101),每组10只.记录各组大鼠术后15 min内心律失常结果;术后15 min取缺血心肌,采用蛋白质免疫印迹试验(Western Blot)检测磷酸化RKIP(p-RKIP)表达.②实验二:按随机数字表法将30只4周龄雄性SD大鼠分为UFP-101对照组、RKIP过表达组、RKIP拮抗组,每组10只.UFP-101对照组每日腹腔注射玉米油,其余两组注射RKIP上调剂香蜂草苷(Didymin),3组大鼠饲养4周后均行冠脉结扎,并均于术前10 min经尾静脉注射UFP-101.其中RKIP拮抗组术前2 h腹腔注射RKIP特异性拮抗剂洛法他丁(locostatin).记录各组大鼠术后15 min内心律失常结果;Western Blot法检测术后15 min时心肌组织p-RKIP表达及心肌细胞膜表面 β1-AR表达.结果 ①实验一:与Sham组比较,模型组和UFP-101组室性期前收缩(VEB)、室性心动过速(VT)、心室纤颤(VF)发生明显增多,心律失常评分明显升高;此外,与Sham组相比,模型组心肌组织p-RKIP表达升高,UFP-101组心肌组织p-RKIP表达下降.与模型组比较,UFP-101预处理可显著减少心律失常发生〔心律失常评分(分):1.5(0.3,5.0)比4.0(2.0,5.0),P<0.05〕,心肌组织p-RKIP表达明显下降(p-RKIP/总RKIP:0.20±0.11比0.43±0.11,P<0.05).说明拮抗N/OFQ能降低RKIP的磷酸化,减少心律失常发生.②实验二:与UFP-101对照组相比,RKIP过表达可显著增加心律失常事件发生,心肌细胞膜表面β1-AR表达也明显增多;而拮抗RKIP过表达可使心律失常的发生有所缓解〔心律失常评分(分):3.0(2.0,3.0)比4.0(2.0,5.0),P<0.05〕,并且明显减少心肌细胞膜表面β1-AR的表达(β1-AR/Na+-K+-ATP酶:0.88±0.09比1.02±0.08,均P<0.05),而总RKIP表达差异无统计学意义(总RKIP/GAPDH:5.40±0.21比5.36±0.19,P>0.05).说明内源性N/OFQ通过RKIP影响大鼠缺血性心律失常和心肌细胞膜表面β1-AR表达.结论 内源性N/OFQ可通过增加RKIP磷酸化表达来影响大鼠缺血心肌细胞膜表面β1-AR的表达及心律失常.

abstractsObjective To investigate whether endogenous nociceptin/orphanin FQ (N/OFQ) can inhibit arrhythmia and expression of β1-adrenergic receptor (β1-AR) on the surface of myocardial cell membrane in acute myocardial ischemia rats by Raf kinase inhibitory protein (RKIP). Methods ① Experiment one: according to random number table method, 30 adult male Sprague-Dawley (SD) rats with only 6 weeks of age were divided into Sham group (open the chest but do not ligate the coronary artery), myocardial ischemia model group (coronary ligation of left anterior descending branch), and endogenous N/OFQ antagonists UFP-101 pretreatment group (UFP-101 group, preoperative 10 minutes after tail vein injection of 1 mL/kg UFP-101), with 10 rats in each group. Arrhythmia was recorded within 15 minutes after operation. The expression of phosphorylated RKIP (p-RKIP) was detected by Western Blot. ② Experiment two: according to the random number table method, 304-week-old male SD rats were divided into UFP-101 control group, RKIP over expression group and RKIP antagonism group, with 10 rats in each group. The UFP-101 control group was intraperiton eally injected with corn oil every day, while the other two groups were injected with up adjuster of RKIP (Didymin). The rats in the three groups were all ligated after 4 weeks of feeding, and UFP-101 was injected through the tail vein 10 minutes before the operation. The RKIP antagonist group received intraperitoneal injection of the RKIP-specific antagonist locostatin 2 hours before surgery. Arrhythmia results were recorded within 15 minutes after operation. Western Blot was used to detect the expression of p-RKIP in myocardial tissue and&nbsp;expression of β1-AR on the surface of myocardial cell membrane 15 minutes after surgery. Results ①Experiment one: compared with Sham group, ventricular ectopic beat (VEB), ventricular tachycardia (VT) and ventricular fibrillation (VF) increased significantly in the model group and UFP-101 group, and arrhythmia score increased significantly. In addition, compared with the Sham group, p-RKIP expression was increased in the model group and decreased in the UFP-101 group. Compared with the model group, preconditioning with UFP-101 significantly reduced the occurrence of arrhythmia [arrhythmia score: 1.5 (0.3, 5.0) vs. 4.0 (2.0, 5.0), P < 0.05], and the expression of p-RKIP in myocardial tissue significantly decreased (p-RKIP/total RKIP: 0.20±0.11 vs. 0.43±0.11, P < 0.05). This indicated that antagonistic N/OFQ could reduce the phosphorylation of RKIP and the occurrence of arrhythmia. ② Experiment two:compared with the UFP-101 control group, overexpression of RKIP significantly increased the occurrence of arrhythmia events, and the expression of β1-AR on the surface of the myocardial cell membrane significantly increased. And antagonism RKIP overexpression could make the occurrence of arrhythmia eased [arrhythmia score: 3.0 (2.0, 3.0) vs. 4.0 (2.0, 5.0), P < 0.05], and significantly reduce the expression of myocardial cell membrane surface β1-AR (β1-AR/Na+-K+-ATPase: 0.88±0.09 vs. 1.02±0.08, P < 0.05), while there was no significant difference in total RKIP expression (total RKIP/GAPDH: 5.40±0.21 vs. 5.36±0.19, P > 0.05). This indicated that endogenous N/OFQ affected the expression of plasma β1-AR on the surface of myocardial cell membrane and ischemic arrhythmia in rats through RKIP. Conclusion Endogenous N/OFQ can affect the expression of plasma β1-AR on the membrane surface of ischemic myocardium and arrhythmia in rats via increased expression of RKIP phosphorylation.