Fas/FasL和Caspase-3在二氮嗪强化Celsior液超时冷保存鼠心移植中的表达及其意义

Expression of Fas/FasL and Caspase-3 proteins in rat myocardium during long-term hypothermic pre-servation by Celsior solution supplemented with diazoxide

目的研究心肌线粒体三磷腺苷敏感性钾离子通道(mitochondrial ATP-sensitive potassium channel,MitoKATPC)开放剂二氮嗪(diazoxide,DE)添加入Celsior液中对大鼠供心超时冷保存的效果,并探讨其抗心肌凋亡作用的可能机制。方法SD大鼠112只,随机分成7组,每组16只(供者、受者各8只)。(1)Celsior组:保存液为单纯Celsior液;(2)DE组:保存液为30μmol/LDE+Celsior液;上述两组又以保存时间不同分为5 h、8 h、10 h组。(3)5-HD组:保存液为Celsior液+DE(30μmol/L)+5-HD(100μmol/L),保存时间为5 h。利用改良Heron法建立大鼠颈部异位心脏移植模型,观察移植术后5 min内心脏恢复跳动、心率表现;采用原位末端标记染色法检测心肌细胞凋亡,用免疫组织化学方法检测心肌Fas/FasL和Caspase-3蛋白的表达情况;以及用电镜观察心肌超微结构等。结果(1)除5 h保存时间的心脏外,DE各组心脏移植术后30 s复跳率较相应Celsior液组显著提高;DE组心率明显较Celsior液组快(P<0.05),DE 10 h组与Celsior 5 h组无明显区别(P>0.05);(2)与Celsior液组相比,DE处理明显降低各组心肌细胞凋亡指数(P<0.05),减少Fas/FasL和Caspase-3蛋白的表达(均为P<0.05);DE10 h组与Celsior5 h组各指标比较差异无统计学意义(P>0.05);(3)5-HD可取消上述DE的作用;(4)DE各组和Celsior 5 h组心肌超微结构保护相对较佳:肌纤维排列整齐,肌节清,线粒体肿胀不明显,嵴结构较为清楚;Celsior 10 h组心肌结构尚清楚,但肌纤维疏松,个别区域肌丝肌节溶解,线粒体肿胀较明显,嵴模糊。结论DE强化Celsior液冷保存的鼠供心,可能通过激活MitoKATPC,明显抑制Fas/FasL和Caspase-3蛋白的表达,保护线粒体功能和结构的完整,从而使心肌细胞凋亡减少,减轻缺血再灌注损伤,使得DE强化Celsior液超时(10 h)冷保存的鼠供心移植安全有效。

Objective The purpose of this study was to study the effect of a mitochondrial ATP-sensitive potassium channel （MitoKATPC） opener diazoxide （DE） on Fas/FasL and Caspasc-3 proteins expression in rat heart during long-term hypothermic preservation, and to explore the possible mechanism of DE against the myocardic apoptosis during ischemia-reperfusion. Methods Rat heterotopic cervical heart transplantation models were established with the modified Heron＇s technique. The rat donor hearts were stored in 4℃ Celsior cardioplegia solution with or without DE （30 μmol/L） for 5, 8, 10 hours respectively before transplantation. The restoration of heartbeat and heart rate were observed. Apoptotic cardiomyocytes were detected using TUNEL technique. The expression of Fas/FasL and Caspase-3 proteins were also detected with immunohistochemical method. The myocardial uhrastructure was examined under electron microscope. Results （ 1 ） The rate of heart resuscitation 30 seconds after transplantation in DE treatment groups was much higher than that of Celsior groups except 5 hours preservation group. （2） Compared with Celsior solution preservation groups, significantly accelerated the donor＇s heart rate, reduced the apoptosis index and the expression of Fas/FasL and Caspase-3 proteins were observed in DE treated groups. （3） The protection effect of DE were attenuated by a MitoKAwpC inhibitor 5-hydrnxydecanoate （5-HD）. （4） Minor histological changes of myocardium was observed in DE groups and Celsior 5 h group. Conclusions These results indicate that DE eould alleviate rat myocardial injury induced by ischemia-reperfusion through reducing the expression of Fas/FasL and Caspase-3 proteins and protecting the mitochondrial structure and function via opening of MitoKATPC. And the Celsior cardioplegia solution supplemented with diazoxide could safely and effectively preserve the hear1 for 10 hours.