肢体缺血再灌注后的肺损伤和细胞凋亡及NO的效应

Effect of nitric oxide on apoptosis in lung injury following ischemia-reperfusion of hind limbs

目的:探讨肢体缺血再灌注(LIR)后肺的损伤性变化以及细胞凋亡在肺损伤发生中的作用;探讨一氧化氮(NO)对LIR后肺组织细胞凋亡的影响。方法:采用本室常规方法复制大鼠LIR模型,给予外源性一氧化氮合酶底物(L-Arg)和一氧化氮合酶抑制剂(L-NAME)处理,采用原位末端标记法(TUNEL)检测缺血4h再灌注4h时各组动物肺组织细胞凋亡情况;采用放免法检测凋亡相关细胞因子TNF-α在肺组织的表达,结合计算机分析系统对结果进行定量分析;采用免疫组织化学方法检测Bcl-2、Bax、caspase-3、TNF-α蛋白表达情况,结合自动图像分析系统对其结果进行定量分析;在光镜下观察肺组织的形态学改变。结果:大鼠LIR后4h,肺泡Ⅱ型上皮细胞、肺血管内皮细胞呈凋亡改变,肺组织TNF-α、caspase-3、Bax明显上调,Bcl-2表达下调。L-Arg处理组,凋亡细胞数明显减少,肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显减弱,Bcl-2表达明显增强;L-NAME处理组动物肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显增强,Bcl-2表达明显减弱。结论:细胞凋亡参与了大鼠LIR后急性肺损伤的发生,且与TNF-α有关;NO可通过减弱细胞凋亡相关因子TNF-α的表达,减轻LIR后肺组织的细胞凋亡。

AIM ： To investigate the injury of lung and the role of cell apoptosis in the pathogenesis of acute lung injury following ischemia - repeffusion of hind limbs and the influence of nitric oxide （NO） to apoptosis. METHODS： Referring to our laboratory normal method, the model rats, which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. L - arginine （ L - Arg） and N - nitro - L - arginine methyl ester （ L - NAME） was adminis- trated respectively to these rats before the experiment. Apoptosis was detected by TdT - mediated dUTP nick end labeling （TUNEL）, respectively. The radioimmunoassay （RIA） was used to detect level in the expression of TNF-α. The immu-nohistochemistry （IHC） method was used to detect the level in the expression of Bcl - 2, Bax, caspase - 3 and TNF-α. The morphologic changes were observed under microscope, respectively. The results of the RIA and the IHC were analyzed quantitatively by relative computer analytical system. RESULTS： After rats＇s hind limbs suffered from ischemia - repeffu- sion, the apoptosis in alveolar epithelial cells and pulmonary vascular endothelial cells was found. The expression of TNF-α, caspase - 3 and Bax increased. Compared with IR rats, the expressions of TNF-α, caspase - 3 and Bax were not obvi- ous in the L - Arg administrated group, but the expression of Bcl - 2 was obvious in that group. Compared with IR rats, the expressions of TNF-α, caspase - 3 and Bax were obvious in the L - NAME administrated group, but the expression of Bcl - 2 was not obvious in that group even weaker than normal ones. CONCLUSION： Apoptosis participated in acute lung in- jury following ischemia - repeffusion of hind limbs. The excess expression of TNF-α related with apoptosis of alveolar epithelial cells and pulmonary vascular endothelial cells. NO may reduce the occurrence of apoptosis and other lung injury through down - regulating the level in the expression of TNF-α.