乳化异氟醚后处理对糖尿病大鼠心肌缺血-再灌注损伤的影响

Effect of emulsified isoflurane postconditioning on myocardial ischemia-reperfusion injury in diabetic rats

目的 观察乳化异氟醚后处理对糖尿病大鼠心肌缺血-再灌注(IR)损伤的影响,并探讨可能的分子机制。方法 选择SPF级雄性SD大鼠45只,2月龄,体重240~260 g。采用随机数字表法分成五组:假手术组(S组,n=5)、心肌IR损伤组(IR组,n=10)、糖尿病+心肌IR损伤组(DM组,n=10)、糖尿病+心肌IR损伤+乳化异氟醚后处理组(EI组,n=10)和糖尿病+心肌IR损伤+钙/钙调蛋白激酶Ⅱ(CaMKⅡ)抑制剂(KN93)组(KN93组,n=10)。S组仅开胸穿线但不结扎左冠状动脉前降支(LAD);IR组穿线稳定后30 min阻断LAD,缺血45 min,再灌注2 h建立IR损伤模型;DM组接受腹腔注射2%链脲佐菌素溶液建立糖尿病模型,4周后建立心肌IR损伤模型;EI组建立糖尿病心肌IR损伤模型时,于再灌注前3 min微量输液泵给药8%乳化异氟醚2 mg/kg,持续8 min;KN93组建立糖尿病心肌IR损伤模型时,于缺血前30 min左心室心内局部注射KN93 5μg并平衡30 min。于再灌注2 h后,采用TTC染色法测定心肌梗死面积百分比,ELISA法测定血清CK-MB浓度,比色法测定血清LDH、MDA和SOD浓度,Western bolt法检测CaMKⅡ、p-CaMKⅡ、信号转导和转录催化因子-3(STAT3)及p-STAT3蛋白含量,并计算p-CaMKⅡ/CaMKⅡ及p-STAT3/STAT3。结果 与S组比较,IR组、DM组、EI组和KN93组血清CK-MB、MDA浓度明显升高(P<0.05),IR组和DM组血清SOD浓度明显降低(P<0.05),IR组、DM组和EI组血清LDH浓度和p-CaMKⅡ/CaMKⅡ明显升高(P<0.05),IR组、EI组和KN93组p-STAT3/STAT3明显升高(P<0.05)。与IR组比较,DM组心肌梗死面积百分比、血清CK-MB、LDH、MDA浓度和p-CaMKⅡ/CaMKⅡ明显升高(P<0.05),血清SOD浓度和p-STAT3/STAT3明显降低(P<0.05);EI组和KN93组心肌梗死面积百分比和血清CK-MB、LDH、MDA浓度明显降低(P<0.05),p-STAT3/STAT3明显升高(P<0.05);KN93组血清SOD浓度明显升高(P<0.05),p-CaMKⅡ/CaMKⅡ明显降低(P<0.05)。与DM组比较,EI组和KN93组心肌梗死面积百分比、血清CK-MB、LDH、MDA浓度和p-CaMKⅡ/CaMKⅡ明显降低(P<0.05),SOD浓度和p-STAT3/STAT3明显升高(P<0.05)。结论 乳化异氟醚后处理可减轻糖尿病大鼠心肌缺血-再灌注损伤,其机制与抑制CaMKⅡ蛋白的磷酸化有关。

Objective To observe the effect of emulsified isoflurane postconditioning on myocardial ischemia-reperfusion(IR) injury in diabetic rats,and explore the possible molecular mechanism.Methods Forty-five adult male SPF SD rats,aged 2 months,weighing 240-260 g,were randomly divided into five groups:sham control group(group S,n = 5),IR group(group IR,n = 10),diabetes + IR group(group DM,n = 10),diabetes + IR + emulsified isoflurane postconditioning group(group EI,n = 10) and diabetes + IR + Ca^(2+)/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ) inhibitor(KN93) group(group KN93,n = 10).Group S:rats were only threaded without ligation of the left anterior descending coronary artery(LAD).Group IR:rats were thread for 30 minutes,and the LAD of rats were ligated for 45 minutes,reperfused for 2 hours.Group DM:rats were induced by intraperitoneal injection of 2% streptozotocin solution,then received myocardial IR after 4 weeks.Group EI:rats with diabetes received myocardial IR,and 3 minutes before reperfusion 8% emulsified isoflurane 2 ml/kg was intravenously infused for 8 minutes.Group KN93:rats with diabetes received myocardial IR,and KN93 5 μg was injected into the left ventricle before ischemia and then balanced for 30 minutes.Rats were sacrificed 2 hours after reperfusion,and the myocardial infarction area was detected by TTC staining.Serum creatinine kinase-MB(CK-MB) level was measured by enzyme linked immunosorbent assay(ELISA).Serum lactate dehydrogenase(LDH),malondialdehyde(MDA) and superoxide dismutase(SOD) levels were measured by colorimetry.The expression levels of CaMKⅡ,p-CaMKⅡ,signal transduction and transcription catalytic factor-3(STAT3) and p-STAT3 were detected by Western blot,and p-CaMKⅡ/CaMKⅡ and p-STAT3/STAT3 were calculated.Results Compared with group S,the serum concentrations of CK-MB and MDA were significantly increased in groups IR,DM,EI,and KN93(P < 0.05),the serum concentrations of SOD were significantly decreased in groups IR and DM(P < 0.05),the serum concentrations of LDH and p-CAMKⅡ/CaMKⅡ were significantly increased in groups IR,DM and EI(P < 0.05),p-STAT3/STAT3 was significantly increased in groups IR,EI and KN93(P < 0.05).Compared with group IR,the percentage of myocardial infarction area,the serum concentrations of CK-MB,LDH,and MDA,and p-CaMKⅡ/CaMKⅡ were significantly increased in group DM(P < 0.05),the serum concentrations of SOD and p-STAT3/STAT3 were significantly decreased in group DM(P < 0.05);the percentage of myocardial infarction area,the serum concentrations of CK-MB,LDH,and MDA were significantly decreased in groups EI and KN93(P < 0.05),p-STAT3/STAT3 was significantly increased in groups EI and KN93(P < 0.05);the serum concentrations of SOD was significantly increased in group KN93(P < 0.05),and p-CaMKⅡ/CaMKⅡ was significantly decreased in group KN93(P < 0.05).Compared with group DM,the percentage of myocardial infarction area,the serum concentrations of CK-MB,LDH,and MDA,and p-CaMKⅡ/CaMKⅡ were significantly decreased in groups EI and KN93(P < 0.05),the serum concentrations of SOD and p-STAT3/STAT3 were significantly increased in groups EI and KN93(P < 0.05).Conclusion Emulsified isoflurane postconditioning could protect against myocardial IR injury in diabetic rats possibly through the inhibition of CaMKⅡ phosphorylation.