脂联素在糖尿病心肌缺血再灌注损伤中的变化作用及分子机制

Effect of Adiponectin Levels With its Related Mechanism on Diabetic Myocardial Ischemia-reperfusion Injury in Experimental Rats

目的:通过建立大鼠糖尿病心肌缺血再灌注模型及缺血后处理模型,探讨脂联素在糖尿病心肌缺血再灌注损伤及缺血后处理中的变化及分子机制。方法:雄性SD大鼠80只,随机分为6组:正常假手术组(n=8)、正常心肌缺血再灌注组(n=16)、正常缺血后处理组(n=16)、糖尿病假手术组(n=8)、糖尿病心肌缺血再灌注组(n=16)和糖尿病缺血后处理组(n=16)。腹腔注射链脲佐菌素(STZ)的方法建立2型糖尿病模型,缺血再灌注模型采用结扎左冠状动脉前降支30 min再灌注120 min的方法建立;缺血后处理于再灌注前给予3个循环再灌注10 s,缺血10 s的处理;假手术模型仅以丝线穿过冠状动脉前降支但不结扎。再灌注120 min后处死大鼠,取心肌组织,三苯基氯化四氮唑法(TTC法)测定梗死面积;采用酶联免疫吸附试验(ELISA)检测血浆中脂联素的含量。免疫印迹方法(Western blot方法)测定心肌组织磷酸化蛋白激酶B(p-Akt)和总蛋白激酶B(Total-Akt)表达。结果:与正常心肌缺血再灌注组比较,正常缺血后处理组心肌梗死面积显著减小(P<0.05),而糖尿病心肌缺血再灌注组及糖尿病缺血后处理组的心肌梗死面积显著增加(P<0.01);与正常假手术组比较,正常心肌缺血再灌注组和正常缺血后处理组血清脂联素和p-Akt蛋白表达显著上调(P<0.05),糖尿病假手术组p-Akt蛋白表达显著下调(P<0.05);与正常缺血后处理组比较,糖尿病假手术组、糖尿病心肌缺血再灌注组及糖尿病缺血后处理组脂联素和p-Akt蛋白表达显著下调(P<0.05),差异均有统计学意义。线性相关分析显示,血浆脂联素的表达水平与心肌梗死面积呈负相关,而与心肌组织中p-Akt表达水平呈正相关,两者的相关系数分别为0.63和0.65(P<0.01)。结论:糖尿病血清脂联素表达降低,可致磷脂酰肌醇3-蛋白激酶B(PI3K/Akt)通路失活,导致糖尿病缺血再灌注损伤加重,缺血后处理不能实现对糖尿病心肌的保护作用。

Objective： To investigate the effect ofadiponectin levels with its related mechanism in diabetic myocardial ischemia-reperfusion injury and ischemia post-conditioning in experimental rats. Methods： A total of 80 male SD rats were randomly divided into 6 groups： Normal sham （NS） group, n=8, Normal ischemia- reperfusion injury （NIRI） group, n=16, Normal ischemia post-conditioning （NIPO） group, n=16 and Diabetic mellitus sham （DMS） group, n=8, Diabetic mellitus ischemia-reperfusion injury （DMIRI） group, n=16, Diabetic mellitus ischemic post-conditioning （DMIPO） group, n=16. DM rats model was established by intraperitoneal injection of streptozotocin; IR model was established by occlusion of left anterior descending （LAD） coronary artery for 30 min followed by reperfusion for 120min; IPO model was established by 3 cycles of ischemia for 10s and reperfusion for 10s; the rats in Sham group received silk line wrapping of LAD without occlusion. The myocardial infarction （MI） area was measured by TTC staining, plasma adiponectin level was examined by ELISA,the protein expressions ofp-Akt and total-Akt were detected by Western blot analysis. Results： Compared with NIRI group, NIPO group had decreased MI area, P〈0.05, while DMIRI group and DMIPO group had increased MI area, P〈0.01; compared with NS group, NIRI group and NIPO group showed up-regulated expression of adiponectin and p-Akt, P〈0.05 and DMS group showed down-regulated p-Akt, P〈0.05. Compared with NIPO group, three DM groups presented down-regulated adiponectin and p-Akt, P〈0.05. Linear correlation analysis indicated that plasma adiponectin expression level was negatively related to MI area and positively related to myocardial tissue p-Akt expression with the correlation coefficient at 0.63 and 0.65 respectively, P〈0.01. Conclusion： Down-regulated plasma adiponectin expression may cause the inactivation of PI3K/Akt signal pathway and therefore aggravate DM ischemia-reperfusion injury which cannot be protected by ischemic post-conditioning in experimental rats.