文章摘要
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
  
DOI：10.12089/jca.2022.08.016
中文关键词: 乳化异氟醚  糖尿病  心肌缺血-再灌注损伤  钙/钙调蛋白激酶Ⅱ  大鼠
英文关键词: Emulsified isoflurane  Diabetes  Myocardial ischemia-reperfusion injury  CaMK Ⅱ  Rat
基金项目:国家自然科学基金面上项目（81770824）
作者单位E-mail
杨正超 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科（现在武汉市第一医院麻醉科）  
高素敏 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科（现在南京医科大学附属淮安第一医院急诊科）  
董嗣伟 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科  
王蓉 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科  
夏瑞 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科  
袁世荧 430022,武汉市,华中科技大学同济医学院附属协和医院重症医学科  
姚尚龙 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科  
王婷婷 430022,武汉市,华中科技大学同济医学院附属协和医院麻醉科 wangtt201307@163.com 
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中文摘要:
      
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 5 μg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。
英文摘要:
      
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 + Ca2+/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.
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