氧化应激对心房颤动犬心房肌细胞凋亡及凋亡相关蛋白影响的研究

The effects of oxidative stress on atrial myocardial cells apoptosis and expression of apoptosis protein in atrial fibrillation canines

目的观察普罗布考（probucol）对长期心房快速起搏诱发心房颤动（房颤）犬心房肌细胞凋亡及凋亡相关蛋白表达的影响，探讨氧化应激在房颤心房结构重构中的作用。方法杂种犬20只，随机分为假手术组（n=6）、对照组（n=7）和普罗布考组（n=7）。无菌条件下开胸后在犬右心房缝植4对心外膜记录电极，电极尾端经皮下由犬背部穿出；在右心耳缝植螺旋型起搏电极，连接实验用AOO高频起搏器（400次／min），心房快速起搏6周，建立房颤犬模型；假手术组犬仅缝植心外膜记录电极和起搏电极但不起搏；对照组及普罗布考组犬心房快速起搏6周；普罗布考组于起搏前一周开始服用普罗布考（100mg·kg^-1·d^-1），直至起搏结束。TUNEL法检测心房肌细胞凋亡情况；免疫组化方法及免疫印记法检测凋亡相关蛋白caspase-3、bcl-2和bax表达情况；免疫组化方法检测calpainⅠ表达；比色法检测心房肌总抗氧化能力（T-AOC）、丙二醛（MDA）和抗超氧阴离了（抗O2^-）水平；于起搏前、起博6周后，经心外膜电极记录各组犬房颤诱发情况。结果与假手术组犬相比，对照组犬左、右心房肌凋亡细胞数量显著增加[（44．3±9．7）％vs（1．36±0．70）％，（42．1±11．9）％vs（1．07±0．50）％，P〈0．01]，心房肌caspase-3、bax和calpainⅠ表达明显上调（P〈0．01），bcl-2表达显著下调（P〈0．01）。与对照组犬相比，普罗布考组犬左、右心房肌凋亡细胞数量明显减少[（21．4±5．8）％vs（44．3±9．7）％，（20．1±6．1）％vs（42．1±11．9）％，P〈0．01]，calpainⅠ、caspase-3和bax表达显著下调（P〈0．01），bcl-2表达增加（P〈0．05）。与假手术组犬相比对照组犬心房肌MDA水平明显增加（P〈0．01），T-AOC、抗O2^-水平明显降低（P〈0．01）；与对照组犬相比，普罗布考组犬心房肌MDA水平显著降低（P〈0．05），T-AOC、抗O2^-水平显著增加（P〈0．01）。对照组和普罗布考组起搏后房颤诱发率和平均持续时间均较起搏前显著增加（P〈0．05）；起搏后普罗布考组房颤诱发率较对照组降低（P〈0．05），房颤平均持续时间显著减少（P〈0．01）。结论普罗布考可能通过抑制氧化应激，增加bcl-2表达，降低calpainⅠ、caspase-3和bax表达，阻止长期心房快速起搏诱发房颤犬心房肌细胞凋亡，对房颤心房结构重构防治有益，能够减少房颤发生。

Objective The aim of this study was to determine the effect of probueol on oxidative stress in atrial myoeyte apoptosis of long-term rapid pacing-induced artrial fibrillation（AF）. Methods tn this study, 20 canines were randomly divided in 3 groups, sham-operated group （n = 6） , control group （n = 7 ） and probueol group （ n = 7）. One thin silicon plaque containing 4 pairs of electrodes were sutured to right atrium. A pacemaker was implanted in a subcutaneous pocket and attached to a screw-in epicardial lead in the right atrial appendage. The canines in probucol group were fed with probucol （ 100 mg·kg^-1·d^-1 ） 1 week before rapid atrial pacing until pacing stop. We attempted to identify myocardial cell apoptosis by terminal transferase nick end-labelling （TUNEL）. Immunohistochemistry and Western-blot were used to examine the expression of a final effector of programmed cell death, caspase-3 and of regulatory proteins from the bcl-2 family （bax and bcl- 2）. Immunohistochemistry was also used to examine the expression of calpain Ⅰ. The indexes of oxidative stress were also measured by color comparison. The inducibility and duration of AF were measured. Results To compare with the sham-operated group, the positive percentage of TUNEL measurment was dramatically increased in left and right atrium of eontrol group [ （44. 3 ± 9. 7 ） % vs （ 1.36 ± 0. 70 ） %, （ 42. 1 ± 11.9 ） % vs （1.07 ±0. 50）% ,P 〈0. 01 ]. To compare with the control group, the positive percentage of TUNEL measurment was dramatically decreased in left and right atrium of probucol group [ （21.4 ± 5.8 ）% vs （44. 3 ± 9.7） % , （20. 1± 6. 1 ） % vs （42.1 ± 11.9 ） % , P 〈 0. 01 ]. Probucol can reduce the protein expression of caspase-3, bax and calpain Ⅰ and increase the expression of bcl-2 and dramatically decrease the inducibility and duration of AF. The indexes of oxidative stress in probucol group were lower than the level in control group. Conclusions As an antioxidant, probucol can prevent the atrial myocytes apoptosis of AF in chronic rapid atrial pacing canines. There may be a relationship between oxidative stress and atrial structure remodeling of AF.