缺血预适应对肺缺血-再灌注损伤中细胞凋亡与热休克蛋白70表达的影响

Effect of ischemic preconditioning on pneumocyte apoptosis and the expression of HSP 70 during pulmonary ischemia-reperfusion injury in rats

目的观察缺血预适应（IP）对在体大鼠肺缺血-再灌注（I／R）损伤细胞凋亡及热休克蛋白（HSP70）表达的影响，探讨其作用的可能机制。方法雄性SD大鼠36只，随机分为3组：假手术（SO）组，缺血．再灌注（I／R）组，缺血预适应（IP）组，每组12只。I／R组开胸后用无创微血管钳钳夹肺门远心端，阻断肺门（观察肺无舒缩为阻断标准），建立在体肺脏I／R损伤模型。IP组于缺血开始前，应用3个循环的5min缺血＋5min灌注进行预处理。假手术组仅予开胸术。各组均于2h、5h时结扎肺门取出左肺。用原位末端标记法（TUNEL）检测细胞凋亡指数，免疫组化法测定HSP70表达。计算肺湿干比（W／D），肺泡损伤数定量评价指标（IQA），同时在光镜与电镜下观察肺脏的病理形态学和超微结构的改变。为应用单因素方差分析，组间两两比较采用scheffe检验。结果与SO组比较，I／R组凋亡指数2h点为21．37±4．35、5h点为19．67±3．64，均增加（P=0．000），HSP70表达2h点为0．187±0．019、5h点为0．207±0．021均增加（P=0．000），W/D2h点为6．65±0．85、5h点为7．10±0．94，均增加（P=0．000），IQA 2h点为45．95±2．82、5h点为55．77±3．24均显著升高（P=0．000）。与I／R组比较，IP组凋亡指数2h点为14．02±3．15（P=0．005）、5h点为12．18±2．29（P=0．001），均明显下降，HSP70表达2h点为0．240±0．017（P=0．000）、5h点为0．260±0．022（P=0．002），均增强，W／D2h点为5．39±0．36（P=0．074）、5h点为5．47±0．44（P=0．003），有不同程度降低、IQA 2h点为25．77±3．77、5h点为30．35±3．69，差异具有统计学意义（P=0．000）。肺脏超微结构损害和肺水肿程度明显减轻。结论缺血预适应对肺缺血-再灌注损伤有保护作用，其机制可能是通过上调HSP70的表达而抑制细胞凋亡来实现的。

Objective To investigate the effects of ischemic preconditioning on pneumocyte apoptosis and the expression of HSP70 after lung ischemia-reperfusion（I/R） in rats and discuss its possible mechanism of extenuating ischemia-reperfusion injury. Method Thirty-six male Sprague-Dawley rats were randomly divided into three Groups [sham operation（SO） group, ischemia-reperfusion（I/R） group, and ischemic preconditioning（W） group], twelve in each group. Lung cross-clamping was used to build the I/R model. In IP group, three cycles of 5- minute-ischemia ＋ 5-minute-reperfusion were given to the pulmonary artery before the procedure. Sham operation rats had a thoracotomy only. Two hours（or five hours） reperfusion was given to beth I/R and IP group. Terminal- deoxynucleotidyl Transferase Mediated d-UTP Nick End Labeling（TUNEL） was used to evaluate apoptosis. Expression of HSP70 in lung was observed by immunohistochemical stain and image analysis. Index of quantitative assessment of histologic lung injury（IQA）, wet to dry weight ratio（W/D） were measured. The pathological change of lung tissue was observed under beth light and electron microscopy. Statistical analysis was carried out by One-way Anova. Scheffe test was used for intragroup comparison. Results The apoptosis index and expression of HSP70,W/D,IQA of lung tissue in I/R group were higher than those in the sham operation group （ P 〈 0.01）. Compared with the I/R group, the apoptosis index and expression of HSP70, W/D, IQA of lung tissue significantly decreased （ P 〈 0.01）, the levels of expression of HSP70 increased significantly in IP group（ P 〈 0.01 ）. The pathological and ultrastructure change of lung tissue was better in I/P group than those in I/R group. Conclusions Ischemic preconditioning can extenuate lung I/R injury by the possible mechanism of increasing the expression of HSP70 which inhibits the apoptosis during lung I/R injury.