心肌细胞缝隙连接蛋白43在线粒体敏感性钾通道介导七氟醚预处理减轻大鼠离体心脏缺血再灌注中的作用

Role of cadiocyte connexin 43 in mito=KATP channel mediated cardioprotection against ischemia-reperfusion injury induced by sevoflurane preconditioning in isolated rat hearts

目的评价心肌细胞缝隙连接蛋白43（Cx43）在线粒体敏感性钾（mito—KATP）通道介导七氟醚预处理减轻大鼠离体心脏缺血再灌注中的作用。方法健康成年雄性SD大鼠40只，体重200—250g，采用Langendofff灌注模型进行离体心脏灌注。采用随机数字表法，将心脏随机分为5组（n=8）：对照组（C组）、缺血再灌注组（I／R组）、七氟醚预处理组（S组）、七氟醚预处理＋5-羟葵酸（5-HD）组（SH组）和5-HD组（H组）。采用结扎左冠状动脉前降支（LAD）30min，恢复灌注120min的方法制备心脏缺血再灌注模型。各组平衡灌注10min；然后C组持续灌注，仅于LAD下穿线而不结扎；I／R组继续灌注30min后结扎LAD；S组、S＋H组和H组结扎LAD前30min时分别用3％七氟醚预先饱和的K-H液、3％七氟醚预先饱和的K-H液＋100μmol／L5-HD和K-H液＋100gmol／L5-HD灌注15min，然后用K-H液冲洗15min。分别于给药前（T0）、给药结束即刻（T1）、缺血前即刻（T1）、缺血30min（T3）和再灌注120min（T4）时，记录HR、左心室收缩压（LVSP）、左心室舒张压（LVDP）、左心室最大上升速率（＋dp／dtmax）和左心室最大下降速率（-dp／dtmax）。再灌注结束后，取左心室心肌组织，测定心肌梗死体积，采用免疫组化法测定心肌细胞Cx43表达，采用Western Blot法测定心肌细胞Cx43和磷酸化Cx43（p-Cx43）表达。结果与C组比较，I／R组、S＋H组和H组HR、LVSP、＋dp／dtmax和-dp／dtmax降低，LVDP升高，心肌细胞Cx43和p-Cx43表达下调（P〈0．05）。与I／R组比较，S组HR、LVSP、＋dp／dtmax和-dp／dtmax升高，LVDP和心肌梗死体积降低，心肌细胞Cx43和p-Cx43表达上调（P〈0．05），S＋H组和H组各指标差异无统计学意义（P〉0．05）。结论七氟醚预处理可能通过开放mito—KATP通道，促进心肌细胞Cx43磷酸化，减轻大鼠离体心脏缺血再灌注损伤。

Objective To evaluate the role of cadiocyte connexin 43 （Cx43） in mitO-KATP channel mediated cardioprotection against isehemia-reperfusion（I/R） injury induced by sevoflurane preconditioning in isolated rat hearts. Methods Forty hearts from male adult SD rats weghing 200-250 g were excised and perfused in a Langendorff apparatus with K-H solution with 95% 02-5% CO2 at 36.5-37.5℃. Their hearts were randomly divided into 5 groups （n = 8 each）： control group （group C）, group I/R, sevoflurane preconditioning group （group S）, sevoflurane preconditioning ＋ 5-HD group （group SH） and 5-HD group （group H）, Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery （LAD） for 30 min followed by 120 min reperfu sion. After 10 min of equilibration, group C received continuous perfusion and LAD was exposed but not occluded. Group I/R received continuous perfusion for 30 min and LAD was occluded. In group S, SH and H, 3% sevoflurane, 3 % sevoilurane mixed with 100 μmmol/L 5-HD and 100 μmmol/L 5-HD was added into K-H solution to perfuse for 15 min respectively. After that, the hearts were washed by K-H solution for 15 min. HR, left ventricular systolic pressure （LVSP）, left ventricular diastolic pressure （LVDP） and ＋ dp/dtmax were recorded before administration （T0）, immediately after administration （T1）, immediately before ischemia （T2）, at 30min of ischemia （T3 ） and 120 min of reperfusion （T4）. At the end of reperfusion, left ventricular tissue was removed for determination of myocardial infarct size and expression of Cx43 and phosphor Cx43 （p-Cx43） by immunohistochemistry and Western blot respectively. Results Compared with group C, FIR, LVSP and ±dp/dtmax were significantly de- creased, LVDP was increased and the expression of Cx43 and p-Cx43 were down-regulated in groups I/R, SH and H （P 〈 0.05）. Compared with group I/R, HR, LVSP and ±dp/dtmax were significantly increased, LVDP and myocardial infarct size decreased, and the expression of Cx43 and p-Cx43 up-regulated in group S （ P 〈 0.05）, no significant difference was found in groups S ＋ H and H（ P 〉 0.05）. Conclusion Sevoflurane preconditioning can open mito-KATP channel, promote cadiocyte Cx43 phosphorylation, attenuate I/R injury in isolated rat hearts.