缺血后处理对大鼠肺缺血再灌注损伤中自噬的影响

Effects of postconditioning on autophagy of lung ischemic reperfusion injury in rats

目的 探讨缺血后处理对大鼠在体肺缺血再灌注损伤中自噬的影响.方法 取健康雄性SD大鼠24只,按随机数字表法均分为假手术组、缺血再灌注组、缺血后处理组各8只.所有大鼠开胸后游离左侧肺门,用阻断带控制灌注时间.假手术组组持续灌注120 min;缺血再灌注组缺血30 min,再灌注120 min;缺血后处理组缺血30 min、灌注30 s、缺血30 s,反复3次,然后全面恢复灌注120 min.应用Western印迹法测定各组肺组织中哺乳动物雷帕霉素靶蛋白（mTOR）及其磷酸化（p-mTOR）、微管相关蛋白（LC3-Ⅱ）水平;光镜下观察各组肺组织病理学改变并计算肺损伤评分,电镜下观察各组细胞自噬空泡.结果 缺血再灌注组肺组织中mTOR、p-mTOR相对表达水平（0.40±0.03、0.33±0.10）与假手术组（0.37±0.07、0.31 ±0.10）差异均无统计学意义（均P＞0.05）,而缺血后处理组（0.46±0.09、0.55 ±0.07）均显著高于假手术组及缺血再灌注组（均P＜0.05）;缺血再灌注组、缺血后处理组LC3-Ⅱ相对表达水平及肺损伤评分均显著高于假手术组（0.53±0.08、0.38±0.03比0.25±0.06及15.79±1.33、11.67±1.55比5.58±0.39）,而缺血后处理组显著低于缺血再灌注组（均P ＜0.05）.光镜下可见缺血再灌注组肺组织中中性粒细胞浸润、间质水肿、肺不张及透明膜形成,电镜下可见自噬空泡形成;缺血后处理组上述改变减轻.结论 缺血后处理可通过减轻细胞内自噬而保护肺缺血再灌注组织,可能与增强mTOR作用有关.

Objective To explore the effects of postconditioning on autophagy of lung injury in situ during lung ischemic reperfusion.Methods Twenty-four male Sprague-Dawley rats were randomly divided into 3 groups of sham-operated （S）,ischemic-reperfusion （I/R） and ischemic postconditioning （IpostC） （n =8 each）.All underwent left thoracotomy after anesthesia.In the S group,a line was only placed around left hilum but not fastened.In the I/R group,a line was fastened to block the blood flow of left lung for 30 min and then loosened for reperfusion for 120 min.In the IpostC group,after blocking the blood flow of left lung for 30 min,left hilum was fastened for 30 sec and loosened for 30 sec.Lung tissues were measured by Western blot.Histopathological changes of lung tissues were observed,lung injury scores calculated and autophagic vacuoles determined by electron microscope.Results The relative expression levels of mammalian target of rapamycin （mTOR） and phosphorylated mTOR （p-mTOR） in group I/R （0.40 ± 0.03,0.33 ±0.10） were not different with those of group S （0.37 ±0.07,0.31 ±0.10） （both P 〉0.05）.However,both significantly increased in group IpostC （0.46 ± 0.09,0.55 ± 0.07） （both P 〈 0.05）.As compared with group S,the relative expression level of LC3-Ⅱ and lung injury score significantly increased in groups I/R and IpostC （0.53 ± 0.08,0.38 ± 0.03 vs 0.25 ± 0.06 ; 15.79 ± 1.33,11.67 ±1.55 vs 5.58 ± 0.39） while obviously decelined in group IpostC versus group I/R （all P 〈 0.05）.In group I/R,neutrophil infiltration,interstitial edema,atelectasis and hyaline membrane formation were observed microscopically in lung tissues and the formation of autophagic vacuoles was evident under electron microscope.The changes of group IpostC were milder than those of group I/R.Conclusions Ischemic postconditioning has protective effects on lung ischemic reperfusion injury by attenuating autophagy.It may be related with strengthening mTOR.