亚甲蓝预处理对大鼠离体肺缺血再灌注损伤的影响

Effect of methylene blue preconditioning on ischemia-reperfusion injury in isolated rat lungs

目的 评价亚甲蓝预处理对大鼠离体肺缺血再灌注损伤的影响.方法 清洁级健康成年雄性SD大鼠18只,3月龄,体重240～320 g,采用随机数字表法分为3组（n=6）：假手术组（Sham组）、肺缺血再灌注组（I∕R组）和亚甲蓝预处理组（MB组）.制备离体肺缺血再灌注损伤模型.MB组停灌前2 h腹腔注射亚甲蓝2 mg∕kg,I∕R组和MB组灌流20 min后,停灌45 min后恢复灌注.于再灌注60 min 时检测灌流液 LDH 活性,称肺湿重（W）和干重（D）,计算 W∕D 比值,检测MDA、ATP、ROS和SOD水平.分离肺组织线粒体和胞浆,检测线粒体膜电位（MMP）和肿胀程度,测定胞浆细胞色素c（Cyt c）含量.采用 TUNEL法检测肺组织凋亡细胞,计算凋亡指数.结果 与Sham组比较,I∕R组和MB组灌流液LDH、肺W∕D比值、ROS和MDA、胞浆Cyt c水平和细胞凋亡指数升高,线粒体肿胀程度加重,ATP含量和MMP降低,I∕R组肺组织SOD活性降低（P〈0.05）;与I∕R组比较,MB组灌流液LDH、肺W∕D比值、ROS和MDA、胞浆Cyt c水平和细胞凋亡指数降低,线粒体肿胀程度减轻,肺组织SOD活性、线粒体ATP含量和MMP升高（P〈0.05）.结论 亚甲蓝预处理可减轻大鼠离体肺缺血再灌注损伤,其机制与改善线粒体功能,抑制细胞凋亡有关.

 Objective To evaluate the effect of methylene blue（MB）preconditioning on ischemi-a-reperfusion（I∕R）injury in isolated rat lungs. Methods Eighteen pathogen-free healthy male Sprague-Dawley rats, aged 3 months, weighing 240-320 g, were divided into 3 groups（n=6 each）using a ran-dom number table： sham operation group（group Sham）, lung I∕R group（group I∕R）and methylene blue preconditioning group（group MB）. A model of isolated lung I∕R injury was established in pentobarbi-tal sodium-anesthetized rats. MB 2 mg∕kg was intraperitoneally injected at 2 h before stopping perfusion in group MB. Isolated lungs were perfused for 20 min, followed by 45-min ischemia, and then reperfused for 60 min in I∕R and MB groups. At 60 min of reperfusion, the activity of lactic dehydrogenase（LDH）in the perfusate was detected, wet weight（W）and dry weight（D）was determined, W∕D ratio was calcu-lated, and the levels of malondialdehyde（MDA）, ATP, reactive oxygen species（ROS）and superoxide dismutase（SOD）were measured in lung tissues. Mitochondria and cytoplasm were isolated from lung tis-sues for determination of mitochondrial membrane potential（MMP）, degree of mitochondrial swelling and content of cytochrome C（Cyt c）in cytoplasm. Apoptotic cells in lung tissues were detected using TUNEL, and apoptotic index was calculated. Results Compared with group Sham, the activity of LDH in perfu-sate, W∕D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly in-creased, the degree of mitochondrial swelling was aggravated, and the content of ATP and MMP were de-creased in I∕R and MB groups, and the SOD activity was significantly decreased in group I∕R（P〈0.05）. Compared with group I∕R, the activity of LDH in perfusate, W∕D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly decreased, the degree of mitochondrial swelling was attenu-ated, and the activity of SOD, content of ATP and MMP were increased in group MB（P〈0.05）. Con-clusion Methylene blue preconditioning can reduce I∕R injury in isolated rat lungs, and the mechanism may be related to improving mitochondrial function and inhibiting cell apoptosis.