二氮嗪预处理对大鼠离体缺血/灌注心肌保护作用的研究

Protective effect of diazoxide preconditioning on ischemia and reperfusion isolated working rat heart

目的探讨二氮嗪预处理(DPC)对心肌缺血/再灌注损伤保护作用的机制。方法Wistar大鼠26只,建立离体心脏Langendorff灌注模型,随机分成4组,即:①缺血/再灌注组(I/R组,n=10):在心脏平衡灌流30 min后,缺血30 min再灌注K-H液1 h。②二氮嗪预处理组(DPC组,n=10):在心脏平衡灌流10 min后,给予含二氮嗪(100μmol/L)的K-H液灌注5 min,再复灌不含二氮嗪的K-H液5 min后,给予含二氮嗪的K-H液灌注5 min;再复灌不含二氮嗪的K-H液5 min,然后缺血30 min,再灌注K-H液1 h。③空白对照组(n=3):用等量盐水代替二氮嗪,过程同DPC组。④二甲基亚砜组(DMSO组,n=3):用DMSO代替二氮嗪,过程同DPC组。取4组大鼠心尖肌制作冰冻切片和电镜标本。前者用于免疫组化染色检测过氧化物酶体增生激活受体γ协同刺激因子1α(PGC-1α)的表达;后者用于对心肌线粒体进行Flameng评分。结果I/R组、DPC组、空白对照组和DMSO组的PGC-1α平均积分吸光度值(IODA),分别为(3.88±1.72)、(8.40±3.64)、(3.40±2.44)和(3.69±1.92),DPC组与其他组比较PGC-1α的表达明显增高(P<0.05)。Flameng评分:I/R组为(1.78±0.14),DPC组为(0.47±0.10),空白对照组为(1.69±0.23)、DMSO组为(1.72±0.17),DPC组较其他组线粒体的损伤明显减轻(P<0.01)。结论DPC后,心肌中PGC-1α的表达明显增加,线粒体的损伤明显减轻,提示DPC对心肌的保护作用与PGC-1α的高表达有关,PGC-1α可能是一种内源性心肌保护物质。

AIM To explore the mechanism of protection against myocardium ischemia/reperfusion （I/R） injury by diazoxide preconditioning （DPC）. METHODS Isolated working heart models of Wistar rats were set up and were divided randomly into four groups. The I/R group （n = 10 ） ： Equilibrium perfusion of 30 minutes was followed by a 60 minutes reperfusion. The DPC group （n = 10） had a 10-rain equilibration and two cycles of 5min of 100 μm diazoxide peffusion followed by a 5-min diazoxide-free period before the 30 rain ischemia and a 60-rain reperfusion. The blank control group （n = 3 ） and the dimethyl sulfoxide （DMSO） group （n = 3） were perfused with the same treatment as in the DPC group, except that diazoxide was replaced with natriichloridum and DMSO. Frozen sections of myoeardium at cardie apex were made and immunohistochemical staining was conducted to detect the expression of peroxisome proliferator-activated receptor-γ coactivator 1±（PGC-1α 70 nm ultrathin sections were made and the mitochondria under each specimen was evaluated according to Flameng score. RESULTS The integrated optical density average （ IODA ） of PGC-1αxpression was （ 8.40 ±3.64 ） in DPC group,（3.88 ±1.72） in I/R group, （3.40 ±2.44 ） in blank control and （ 3.69±1.92 ） in DMSO group. The expression of PGC-1αn DPC group was significantly higher than that in other groups （ P 〈 0. 05 ）. Flameng score of was（ 1.78±0. 14） in I/R group, （0.47 ±0. 10） in DPC group, （ 1.69 ±0. 23 ） in blank control and （1.72 ±0. 17 ） in DMSO group, with significant difference between DPC group and other groups （P 〈 0. 01 ）. CONCLUSION DPC can protect cardiomyocytes from ischemia and reperfu- sion injury, which may be related to the high expression of PGC-1αPGC-1αay be an endogenous defense substance of myocardium.