2型糖尿病大鼠急性心肌缺血对心肌新生血管的影响

Effect of acute myocardial ischemia on myocardial angiogenesis in type 2 diabetic rats

目的探讨糖尿病急性心肌缺血对心肌新生血管生成的影响及其机制。方法清洁级健康雄性8周龄sD大鼠52只，任意选取其中20只以高脂喂养联合小剂量链脲佐菌素建立糖尿病大鼠模型；以糖尿病建模成功的大鼠与16只正常大鼠以结扎冠状动脉前降支制作急性心肌梗死模型；另外16只大鼠只开胸不造模、不给药作为假手术组。心肌梗死造模术后2周，处死各组动物，取左心室梗死区及相邻区域心肌，采用免疫组化方法检测各组大鼠缺血周边区微血管密度（MVD），Western blotting法检测血管内皮生长因子（VEGF）及其信号转导蛋白[磷酸化蛋白激酶B（p-Akt）、内皮型一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS）]、内皮抑素蛋白的表达。组间数据比较采用单因素方差分析，样本均数间两两比较采用q检验，两组计量资料比较采用t检验。结果3组成活大鼠分别为：糖尿病心肌梗死组12只，非糖尿病心肌梗死组（对照组）13只，假手术组14只。与对照组相比，糖尿病组缺血心肌新生血管明显减少（19．7±3．8比14．2±3．6，q=2．98，P〈0．05）。糖尿病组、假手术组VEGF表达显著低于对照组（分别为0．89±0．12、0．65±0．23和1．53±0．20，F=6．52，P〈0．01）。糖尿病组和对照组p-Akt和eNOS蛋白表达差异无统计学意义（t值分别为3．02、2．78，P〉0．05），但糖尿病组p-eNOS蛋白表达显著减少（0．49±0．09比1．16±0．12，t=5．68，P〈0．05）。糖尿病组内皮抑素表达显著高于对照组（4．6±0．6比2．3±0．4，t=8．63，P〈0．05）。结论糖尿病大鼠急性缺血心肌的新生血管生成低下，VEGF转导通路在多个水平上参与其中，并与内皮抑素共同作用。

Objective To investigate the effect of acute myocardial ischemia on myocardial angiogenesis in diabetic rats and its possible mechanisms. Methods A total of 52 clean male 8-week SD rats were enrolled in this study, twenty of the rats were fed with high lipid food stuff and intravenously injected with streptozotocin to establish type 2 diabetic model. The left anterior descending coronary artery in the successfully-established diabetes rats models and 16 healthy rats （as control group） were ligated to establish acute myocardial infarction model. And another 16 rats were given thoracotomy only （as sham group）. As a result, 12 rats survived in the diabetic group, 13 in the control group, 14 in the sham group. Two weeks after artery ligation, the rats were sacrificed to harvest the cardiac muscle in the left ventricular infarct zone and adjacent regions. The immunohistochemistry was applied to detect microvascular density （MVD） around ischemic zones in the three groups. Western blotting was used to detect the expression of vascular endothelial growth factor （VEGF）, VEGF signaling mediators （phosphorylated protein kinase B （p- Akt）, endothelial nitric oxide synthase （ eNOS ）, and phosphorylated eNOS （ p-eNOS ） ） and endostatin. Statistical significance between groups was determined with one-way analysis of variance （ANOVA）, SNK q test was performed to compare the means between either two test groups. Results Compared with the control group, diabetic group had a significantly reduced MVD （19. 7 ±3.8 vs 14. 2 ±3.6, q =2. 98, P 〈 0. 05） around the ischemic territory. The protein expression of VEGF in the control diabetic group were all significantly lower than that in the sham group（ 1.53±0. 20, 0. 89 ± 0. 12, and 0. 65 ± 0. 23, respectively; F = 6.52,P 〈 0. 01 ）. Compared with those in the control group, the p-eNOS expression was down-regulated （0.49 ± 0. 09 vs 1.16 ± 0. 12, t = 5.68, P 〈 0. 05 ） and the endostatin expression was up-regulated significantly （4. 6 ±0. 6 vs 2. 3 ± 0. 4, t = 8.63, P 〈 0. 05 ） in the diabetic group. There were similar protein expression of p-Akt and eNOS between the control and diabetic groups（ t values were 3, 02 and 2. 78, respectively, both P 〉 0. 05）. Conclusion Together with endostatin, VEGF signaling pathway may play an important role in reduced angiogenesis at multiple levels in diabetic myocardium.