大鼠肾脏缺血再灌注损伤后自噬的初步研究

Preliminary study on autophagy after renal ischemia reperfusion injury in rats

目的研究大鼠肾脏缺血再灌注(I/R)损伤后自噬溶酶体相关蛋白随损伤时间表达的规律,检测自噬体、溶酶体的变化。方法采用肾动脉夹闭法制作大鼠肾脏I/R损伤模型,将实验动物随机分入I/R组和对照组,每组在各时间点各6只。分别于I/R后2、6、12和24h,采用Western印迹法检测肾组织中自噬相关蛋白Belinl和微管相关蛋白1轻链3(LC)的表达情况,在透射电子显微镜下观察I/R后肾组织内自噬小体的形成情况。结果 I/R组大鼠肾组织内Beclin1和LC3蛋白表达水平均自I/R后2h起开始升高,至I/R后24h达到高峰(P值均<0.05)。透射电子显微镜下见,对照组大鼠近端小管上皮细胞核膜完整,染色质结构正常,细胞质中细胞器形态正常,未见自噬小体;I/R组大鼠I/R后2h可见到损伤区域有自噬体形成,溶酶体增多,I/R后24h最为明显。结论大鼠肾I/R损伤后肾组织的自噬活性升高。

Objective To investigate the expression of autophagy/lysosome associated proteins and to detect the changes of autophagy and lysosome after renal ischemia reperfusion injury in rats. Methods Twelve rats were randomly divided into ischemia reperfusion group and control group （n = 6）. The renal ischemia reperfusion injury was achieved by renal artery occlusion. The expression of autophagy/lysosome associated proteins after renal ischemia reperfusion injury were determined by immunoblotting with specific antibody to Beclin 1 and microtubule associated protein 1 light chain 3（LC3）. The formation of autophagosome and the activation of lysosome were observed under transmission electron microscope （TEM）. Results The expression of Beclin 1 and LC3 in nephridial tissue was increased at 2 h after renal ischemia reperfusion and peaked at 24 h （all P^0.05）. Autophagosomes were found and lysosomes were increased under TEM in ischemic cortex at 2 h after renal ischemia reperfusion, and the number of autophagosomes and lysosomes were significantly increased at 12--24 h, while the nuclear membrane was intact and the structure of chromatin and organelles were normal in the epithelial cells of proximal tubule in the control group. Conclusion Autophagy/lysosomal pathway can be activated after renal ischemia reperfusion injury in rats. （Shanghai Med J, 2013, 36： 190-193）