右美托咪定对大鼠离体肺缺血再灌注损伤时自噬的影响

Effect of dexmedetomidine on autophagy during ischemia-reperfusion injury in isolated rat lungs

目的评价右美托咪定对大鼠离体肺缺血再灌注损伤时自噬的影响。方法SPF级健康雄性SD大鼠,体重250~320g,制备成功离体肺灌注模型30个,采用随机数字表法分成3组(n=10):对照组(C组)、肺缺血再灌注组(I/R组)和右美托咪定组(DEX组)。C组持续灌流150min;I/R组灌流15min后,停止通气和灌流60min,继续通气和灌流75min,制备大鼠离体肺缺血再灌注损伤模型;DEX组用含10nmol/L右美托咪定的K-H液灌流15min后,停止通气和灌流60min,继续通气和灌流含10nmol/L右美托咪定的K-H液75min。分别于灌流10min、复灌15、45和75min时记录肺血管阻力(PVR)及肺动脉氧分压(PaO2)。于复灌75min时留取肺组织,称湿重(W)和干重(D),计算W/D比值,光镜下观察肺组织形态学并计算肺组织损伤评分,电镜下观察肺组织细胞自噬空泡。采用Westernblot法测定哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)和LC3Ⅱ的表达。结果与C组比较,I/R组和DEX组PVR、W/D比值、肺组织损伤评分和LC3Ⅱ表达水平升高,PaO2降低(P<0.05);与I/R组比较,DEX组PVR、W/D比值、肺组织损伤评分和LC3Ⅱ表达水平降低,PaO2增高,mTOR和p-mTOR表达上调(P<0.05)。I/R组肺组织大量自噬空泡形成,DEX组肺组织少量自噬空泡形成。结论右美托咪定减轻大鼠离体肺缺血再灌注损伤的机制与抑制自噬有关。

Objective To evaluate the effect of dexmedetomidine on autophagy during ischemia-reperfusion(I/R)injury in isolated rat lungs.Methods SPF healthy male Sprague-Dawley rats,weighing 250-320 g,were anesthetized with atropine and pentobarbital sodium,and their lungs were excised to establish the isolated lung perfusion model.Thirty lungs in which isolated lung perfusion models were successfully established were divided into 3 groups(n=10 each)by a random number table method:control group(C group),lung I/R group(I/R group)and dexmedetomidine group(DEX group).The isolated rat lungs were continuously perfused for 150 min in C group.After 15 min of perfusion,the isolated lungs were subjected to 60 min of ischemia and apnea followed by 75 min of ventilation and reperfusion to establish the model of isolated lung I/R injury in I/R group.In DEX group,the isolated lungs were perfused for 15 min with K-H perfusion fluid containing 10 nmol/L dexmedetomidine,and then subjected to 60 min of ischemia and apnea followed by 75 min of ventilation and reperfusion with K-H perfusion fluid containing 10 nmol/L dexmedetomidine.Pulmonary vascular resistance(PVR)and partial pressure of arterial oxygen(PaO2)were recorded at 10 min of perfusion and 15,45 and 75 min of reperfusion.Lung tissues were collected at 75 min of reperfusion for measurement of lung wet weight(W)and dry weight(D)and for examination of morphological changes of lung tissues(with a light microscope)and autophagic vacuoles of lung cells(under an electron microscope).The W/D ratio and lung injury score were calculated.The expression of mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR)and microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ)in lung tissues was detected by Western blot.Results Compared with C group,the PVR,W/D ratio and lung injury score were significantly increased,the expression of LC3Ⅱ was up-regulated,and PaO2 was decreased in I/R group and DEX group(P<0.05).Compared with I/R group,the PVR,W/D ratio and lung injury score were significantly decreased,the expression of LC3Ⅱ was down-regulated,PaO2 was increased,and the expression of mTOR and p-mTOR was up-regulated in DEX group(P<0.05).A lot of autophagic vacuoles of lung cells were found in I/R group,and a few autophagic vacuoles of lung cells were observed in DEX group.Conclusion The mechanism by which dexmedetomidine reduces isolated lung I/R injury is related to inhibiting autophagy in rats.