Smac/Diablo与X-连锁凋亡抑制蛋白在ATP耗竭再恢复诱导肾小管上皮细胞凋亡中的作用

目的 研究线粒体蛋白Smac/Diablo和细胞X-连锁凋亡抑制蛋白(XIAP)在ATP耗竭及再恢复导致肾小管上皮细胞凋亡中的作用和机制。 方法 应用代谢抑制剂暂时性阻断人肾小管上皮细胞(HK-2细胞)内ATP的生成,再换用含糖的培养液使细胞内ATP再恢复,诱导肾小管上皮细胞凋亡。应用Hoechst33342检测肾小管上皮细胞凋亡的发生。用间接免疫荧光检测Smac/Diablo在细胞内的分布。分别提取胞质蛋白和细胞总蛋白,以Western印迹检测胞质中Smac/Diablo、XIAP和活化半胱氨酸天冬氨酸蛋白酶3前体(pro-caspase-3)的蛋白水平。 结果 肾小管上皮细胞内ATP耗竭及再恢复时,Hoechst33342染色可见HK-2细胞核固缩和凋亡小体的形成;间接免疫荧光可见Smac/Diablo由线粒体释放至胞质;Western印迹可见胞质内Smac/Diablo的含量增多( P < 0.01);XIAP和pro-caspase 3的蛋白水平降低(P < 0.05)。 结论 肾小管上皮细胞内ATP耗竭及再恢复时,Smac/Diablo释放至胞质,XIAP蛋白水平降低,进而激活caspase 3,介导肾小管上皮细胞凋亡。

Role of Smac/Diablo and XIAP in ATP depletion/recovery-induced apoptosis in renal tubular epithelial cells

Objective To investigate the role of mitochondrial Smac/Diablo and XIAP in ATP depletion/recovery-induced apoptosis in renal tubular epithelial cells. Methods To induce apoptosis, HK-2 cells were transiently exposed to metabolic inhibitor for 90 rain followed by recovery. Apoptosis was detected by Hoechst33342 staining. The release of Smac/Diablo was examined by indirect immunofluorescence. Cytosolic fraction was extracted to detect the level of Smac/Diablo by Western blot. Total protein was extracted to measure the protein level of XIAP and pro-caspase-3. Results After ATP depletion/recovery in HK-2 cells, Hoechst33342 staining showed nucleus condensation and apoptotic body conformation. Indirect immunofluorescence revealed the release of Smac/Diablo. Western blot showed the increased level of Smac/Diablo in cytosolic fraction and the decreased level of XIAP and pro-caspase-3 after ATP depletion/recovery. Conclusion Smac/Diablo release from mitochondria and decreased level of XIAP in HK-2 cells after ATP depletion/recovery may play an important role in apoptosis through caspase-dependent pathway.