急性干预α7亚单位的N型乙酰胆碱受体介导的胆碱能抗炎通路对犬快速心房刺激诱发心房颤动的影响

Effect of acute intervention of α7nAChR mediated cholinergic anti-inflammatory pathway on inducibili-ty of atrial fibrillation induced by rapid atrial pacing in canines

目的 探讨α7亚单位的N型乙酰胆碱受体（α7nAChR）介导的胆碱能抗炎通路对犬快速右心房起搏后心房颤动（房颤）诱发的影响及机制.方法 12只比格犬按随机数字表法分为对照组（n=6）和激动组（n=6）.快速右心房刺激（800次/min）6 h,连续3 h刺激后,对照组于4个心房神经节（GP）区域中注射0.9%NaCl溶液（0.5 ml/个）,激动组于4个GP区域注射含PNU-282987（α7nAChR激动剂）的0.9%NaCl溶液（0.5 ml/个）.于实验基线期和每小时刺激末监测心房、肺静脉的有效不应期（ERP）、有效不应期离散度（dERP）和房颤诱发情况.实验基线期、3 h刺激末、终点分别取静脉血检测肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）和乙酰胆碱（ACh）等水平;电生理检查完毕后取心房、右前GP组织检测 TNF-α、IL-6、ACh 及转录激活子3（STAT3）蛋白的表达水平,免疫荧光染色检测α7nAChR蛋白的定位表达.结果 对照组和激动组快速刺激3 h,犬心房、肺静脉的ERP、房颤周长较基线期均明显缩短（P〈0.05）,dERP、房颤诱发率均明显升高（P〈0.05）.对照组3 h刺激末于GP区域注射0.9%NaCl溶液后持续刺激,犬心房、肺静脉的ERP、房颤周长仍明显下降（P〈0.05）,dERP、房颤诱发率及持续时间仍明显升高（P〈0.05）;激动组3 h刺激末于GP区域注射PNU-282987后持续刺激,犬心房、肺静脉的ERP、房颤周长逐渐延长,dERP、房颤诱发率及持续时间逐渐缩短,但与基线期比较差异无统计学意义（P〉0.05）.两组犬静脉血基线期差异无统计学意义,快速刺激6 h后,两组血清TNF-α、IL-6等水平均明显升高、Ach表达均显著降低（P〈0.05）.与对照组相比,激动组犬心房及右前GP组织中TNF-α[（28.5 ± 2.3） pg/ml 对（32.7 ± 2.0） pg/ml,P〈0.05]、IL-6[（192.7 ± 8.9） pg/ml 对（205.8±8.7） pg/ml,P〈0.05]表达水平显著降低,而ACh表达差异无统计学意义[（38.4±3.7） μg/ml对（32.7± 3.0） μg/ml,P〉0.05],STAT3蛋白表达水平明显升高[0.9对0.4,P〈0.05].结论 激动α7nAChR介导的胆碱能抗炎通路通过降低炎性因子水平升高,可抑制心房快速刺激引起的心房肌电重构和房颤的诱发.

Objective To investigate the effect of alpha 7 nicotinic acetylcholine receptor（α7nAChR） mediated cholinergic anti-inflammatory pathway on inducibility of atrial fibrillation（AF） induced by rapid atrial pacing in canines. Methods According to the random number table,twelve beagles were randomized into con-trol group（n=6） and PNU-282987（α7nAChR agonist）group（n=6）.Dogs were subjected to right atrial pacing（RAP）at 800 bpm for 6 hours.After 3 hours pacing,the normal saline was injected into four ganglionated plexis （GPs）（0.5ml/GP）as control,while saline solution containing PNU-282987 was injected into four GP （0.5 ml/GP）in PNU group.Effective refractory period（ERP）of atrial and pulmonary veins（PVs）and the ERP dispersion and induced AF were measured at baseline and every hour in the status of non-pacing.Blood samples were collected at the baseline,3 hours after pacing and endpoint of the protocol to measure the levels of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）and acetylcholine（ACh）.After electrophysiological study,the tissue levels of TNF-α,IL-6,ACh were examined by ELISA,and the expression levels of STAT3 in atrial and anterior right ganglionated plexi（ARGP）tissues were measured by Western blotting.The locations of α7nAChR protein in atrial and ARGP tissues were examined by immunofluorescence staining. Results After 3 hours pa-cing,compared with the baseline,there was a persistent decrease in ERP of atrial and PVs and AF cycle length （P〈0.05）in both groups,while the ERP dispersion,the AF inducibility ratio were significantly increased（P〈0.05）.In control group,after injecting normal saline into GPs,in addition to decresed ERP of atrial and PVs, and AF cycle length（P〈0.05）,the ERP dispersion and the induced times and duration of AF were significantly increased during constant stimulation（P〈0.05）.In PNU group,after injecting PNU-282987 into GPs,the ERP of atrial and PVs,and AF cycle length were gradually prolonged,the ERP dispersion,inducibility ratio and du-ration of AF were gradually decreased during constant stimulation.However these was no significantly difference compared with the baseline（P〉0.05）.The plasma TNF-a and IL-6 concentrations were greatly increased,and ACh levels decreased after 6 hours pacing in both groups.After cessation of pacing for 6 hours,compared with the control group,the tissue levels of TNF-α and IL-6 were significantly decreased[RA：TNF-α：（28.5± 2.3） pg/ml vs. （32.7±2.0） pg/ml,P〈0.05;IL-6：（192.7±8.9） pg/ml vs.（205.8±8.7） pg/ml,P〈0.05],but the level of ACh was no significant difference[RA：（38.4±3.7） μg/ml vs.（32.7±3.0） μg/ml,P〉0.05].The density of signal transducer and activator of transcription-3（STAT3）was significantly increased（RA：0.9 vs.0.4,P〈0.05）in atrial and ARGP tissues in PNU group. Conclusion Activating the α7nAChR mediated cholinergic anti-inflammatory pathway can decreases inflammatory cytokine levels,suppress the atrial electrophysiological remodeling and reduce the incidences of AF by RAP.