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目的研究缺血再灌注损伤对在体窦房结细胞超极化激活环核苷酸门控阳离子通道4(hyperpolarization activated cyclicnucleotide gated cation channel 4,HCN4)表达的影响,探讨缺血再灌注损伤对窦房结组织功能障碍影响的可能机制。方法取家兔85只(体重2～3kg)制作右冠状动脉缺血再灌注损伤模型。将实验动物随机分为3组,实验组A组缺血30min后行再灌注,实验组B组缺血1h后行再灌注,均分别于再灌注后2、4、8、16h取材,每个时间点10只;对照组C组(n=5)未行血流阻断,16h后取材。于各时间点取窦房结组织行HE染色、免疫组织化学染色及透射电镜观察。结果HE染色示A、B组窦房结细胞再灌注2h均发生病理损伤;4、8h窦房结细胞病理损伤随时间延长而逐渐加重;8、16h窦房结细胞病理损伤达高峰;相同再灌注时间,B组病理损伤重于A组。免疫组织化学染色观察示:窦房结细胞HCN4表达主要位于窦房结中央区,向周边表达渐减少。A组再灌注后2、4、8、16hHCN4表达的积分吸光度(IA)值分别为306.20±35.77、216.60±18.59、155.40±19.11、135.00±12.30,B组分别为253.70±35.66、138.70±13.28、79.10±9.60、69.20±8.42,均较C组(397.40±34.11)低(P<0.05);随再灌注时间延长,HCN4表达逐渐降低,至8h时最低,之后无明显变化,再灌注后2、4、8h间比较差异均有统计学意义(P<0.05),再灌注8h与16h比较差异无统计学意义(P>0.05);相同再灌注时间,B组HCN4表达较A组低。透射电镜观察示A、B组窦房结细胞超微结构发生损伤改变,随再灌注时间延长损伤加重,至8h达高峰,再灌注8、16h改变不明显。相同再灌注时间,B组窦房结细胞超微结构损伤重于A组。结论缺血再灌注损伤引起在体窦房结细胞形态结构及HCN4表达改变,可能与缺血再灌注造成窦房结功能障碍和心律失常有关。
Objective To investigate the effect of ischemia-reperfusion injury on the expression of hyperpolarized activated cyclic nucleotide nucleotide-binding channel 4 (HCN4) in vivo and to explore the effect of ischemia-reperfusion injury on sinus Atrial fibrillation dysfunction may affect the mechanism. Methods Totally 85 rabbits (weight 2 ~ 3kg) were used to make a model of right coronary artery ischemia-reperfusion injury. The experimental animals were randomly divided into three groups. The experimental group A was reperfused 30 minutes after ischemia and the experimental group B was reperfused 1 hour after ischemia. All the rats were harvested at 2,4,8,16 h after reperfusion. Time point 10; control group C (n = 5) did not block blood flow, 16h after drawing. At each time point, the atrial node tissues were taken for HE staining, immunohistochemical staining and transmission electron microscopy. Results The HE staining showed that the pathological changes occurred in the sinoatrial node cells of group A and group B at 2h after reperfusion. The pathological changes of sinoatrial node cells increased gradually with the passage of time on day 4 and 8; Perfusion time, group B pathological damage is heavier than the A group. Immunohistochemical staining showed that the expression of HCN4 in sinoatrial node cells was mainly located in the central area of the sinoatrial node and gradually decreased to the periphery. The integral absorbance (IA) values of 2,4,8,16 hHCN4 expression in group A after reperfusion were 306.20 ± 35.77,216.60 ± 18.59,155.40 ± 19.11,135.00 ± 12.30, respectively, while those in group B were 253.70 ± 35.66 and 138.70 ± 13.28, 79.10 ± 9.60,69.20 ± 8.42, which were lower than those in group C (397.40 ± 34.11) (P <0.05). The expression of HCN4 gradually decreased with the time of reperfusion, and reached the lowest level at 8h, (P <0.05). There was no significant difference between 8h and 16h after reperfusion (P> 0.05). At the same time of reperfusion, the expression of HCN4 in group B was lower than that in group A at the same time of reperfusion. Transmission electron microscopy showed that the ultrastructure of sinoatrial node cells in group A and group B changed, with the injury time increasing with the reperfusion time reaching the peak at 8h. The change was not obvious at 8 and 16h after reperfusion. At the same time of reperfusion, the ultrastructure damage of sinoatrial node cells in group B was more severe than that in group A. Conclusion Ischemia-reperfusion injury causes changes of cell morphology and HCN4 expression in the sinus node of the sinuses, which may be related to dysfunction of sinus node and arrhythmia caused by ischemia-reperfusion.