二氮嗪强化Celsior液可安全延长供心低温保存时限

Long-term hypothermic preservation of rat hearts with diazoxide-containing celsior solution

目的评价二氮嗪（Diazocide，DE）强化Celsior液对移植鼠供心长时程（10h）低温保存的有效性，并探讨其可能作用机制。方法采用改良Heron法大鼠颈部异位心脏移植模型。SD近亲大鼠随机分为3组，（1）对照组：单纯Celsior液保存鼠心；（2）实验组：30μmol／LDE＋Celsior液保存鼠心；（3）拮抗组：100μmol／L5．HD＋30μmol／LDE＋Celsior液保存鼠心5h；前两组根据保存时间不同分为5、10h组。观察移植心脏复跳情况，并分别于复跳后5min、1周、3个月时留取标本，检测心肌MDA含量、SOD活性、心肌ATP酶、TNF-α、Fas／FasL、Caspase-3、AI等指标以及超微结构检查。结果（1）相同保存时间，实验组与对照组比较，术后5min和1周各指标均有明显优越性；术后3个月时各指标亦较佳，心肌MDA含量低下、SOD和Na^＋．K^＋．ATP酶活性高及FasL、TNF-α基因表达低差异有统计学意义；（2）5-HD消除了实验组中添加DE的优势作用；（3）实验10h组与对照5h组比较，各指标均稍有优越性；（4）DE各组和Celsior5h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；Celsior10h组心肌肌纤维疏松，部分区域肌丝肌节溶解，线粒体肿胀较明显，嵴模糊。结论DE强化Celsior液长时程冷保存鼠供心可较国际公认4～6h延长至10h，仍安全有效。其主要原因可能与DE介导mito-KATP C开放，早期阶段抑制氧化应激引起的细胞凋亡、阻止细胞内钙超载、降低凋亡相关基因表达等对抗心肌缺血再灌注损伤的心肌保护作用有关。

Objective To assess the effectiveness of diazoxide-containing Celsior eardioplegia solution for long-term preservation of the Rat Hearts in vitro, and to study the potential mechanisms of diazoxide, an ATP-sensitive mitechondrial potassium channel opener in reducing the ischemia-reperfusion injury. Methods The modified Heron＇s technique for heterotopic cervical heart transplantation in rat models was used. Before transplantation, SD rats were randomized into control gronps, in which the donor rats＇ hearts were stored in Celsior cardioplegia solution at 4℃ for 5 or 10 hours, experimental goups, in which diazoxide （30 μmol/L）was added to Celsior solution for 5 or 10 hours, and a group of antagonism, in which 5-hydroxydecanoate（5-HD）, a mito-KATPC inhibitor,was added to the solution（ in 100 μmol/L） used in the experimental groups for 5 hours. Samples of the myecardium were collected at 5 min, 1 week and 3 months after heart transplantation and were measured for the level of malondialdchyde（MDA）, the activities of superoxide disrentase（SOD）, Na ^＋ -AT- Pase and Ca^2＋ -ATPase .The gene expression of TNF-α, Fas/FasL and Caspsse-3 was analyzed. Apoptotic cardiomyoeytes were detected by TdT-mediated dUTP nick end labeling （TUNEL） technique. The myocardial ultrastrueture was also observed.Results The experimental groups were much superior to the control groups in the measurements at 5 minutes and 1 week after heart transplantation, for which the donor rats＇ hearts were preserved for the same period. The superiorities were maintained at 3 months. The level of MDA was decressed significantly, the activities of SOD and Na^＋ -K^＋-ATPase were enhanced, gene expression of FasL and TNF-α was reduced with diazoxide. The effects of diazoxide were eliminated by 5-HD. Modest superiorities were observed in one experimental group in which the donor rats＇ hearts were preserved for 10 hours, as compared with one control grenp in which the donor rats＇ hearts were preserved for 5 hours. Histologically, the myocardial ultrastructure was preserved relatively well in the experimental groups and in the 5-honr preserva- tion control group. Conclusion Diazoxide-containing Cdsior solution provides longer protection for the donor rats＇ hearts than well-es- tablished period of 4 to 6 hours. These findings indicate that the mechanisms for diazoxide to alleviate ischemia-repeffusion injury to the rat myoeardium may be associated with its roles in the myocardial protection, which involving early opening of mito-KatpC, a precess providing protectien for mitechondrial structure and function, early in-hibition of apoptosis induced by oxidative stress, prohibition of calcium overload in the cells, reduction of the expression of apoptosis-related genes. The hearts may be preserved safely and effectively in diazoxide-containing Celsior cardioplegia solution for 10 hours.