Pink1通过调控Drp1参与高氨诱导线粒体异常分裂的机制研究

Study on the mechanism of Pink1 in high ammonia-induced abnormal mitochondrial fission by regulating Drp1

目的阐明线粒体自噬相关蛋白Pink1在高氨诱导线粒体碎片化中的作用及分子机制。方法体外培养人神经母细胞瘤SHSY5Y细胞,给予不同浓度NH_(4)Cl分别处理24、48 h后,利用CCK-8评估细胞活性,结合Hoechst法检测细胞凋亡;选取合适NH_(4)Cl浓度,观察线粒体自噬相关蛋白Pink1在蛋白水平表达变化;随后给予Pink1干扰的慢病毒处理,使用透射电子显微镜(TEM)、免疫荧光、Western blotting以及线粒体ATP检测等方法评估Pink1敲除(Pink1 KD)对线粒体动力相关蛋白Drp1、线粒体数量、密度及ATP合成功能的影响。结果随着氨浓度升高和作用时间延长,细胞活性显著降低(P<0.01),5.0 mmol/L NH_(4)Cl处理24 h细胞即已出现凋亡现象,因此,5.0 mmol/L NH_(4)Cl处理24 h为细胞活性未降低的最大剂量;氨刺激细胞内线粒体相关自噬蛋白比例Pink1/Parkin表达显著升高(P<0.05,P<0.01);Western blotting和免疫荧光染色结果显示Pink1 KD可减少氨诱导的Drp1表达增加(P<0.05);TEM显示Pink1 KD可降低氨诱导的线粒体密度增加(P<0.05),而对线粒体覆盖率以及线粒体平均面积无明显影响;检查线粒体ATP合成功能显示Pink1 KD可改善线粒体ATP合成功能(P<0.01)。结论靶向敲除线粒体Pink1可通过减少Drp1表达而缓解高氨诱导的线粒体碎片化,进而恢复线粒体ATP合成功能。本研究为高氨诱导线粒体形态异常和功能障碍提供了分子机制和潜在治疗靶点。

Objective To elucidate the role and molecular mechanism of mitochondrial autophagy related protein Pink1 in mitochondrial fragmentation induced by high ammonia.Methods Human neuroblastoma SHSY5Y cells were cultured in vitro and treated with different concentrations of NH_(4)Cl for 24 h and 48 h,respectively.CCK-8 was used to evaluate cell activity and Hoechst method was used to detect cell apoptosis.Then,appropriate concentrations of NH_(4)Cl were selected to observe the expression changes of mitochondrial autophagy related protein Pink1 at the protein level.Subsequently,lentivirus treatment with Pink1 interference was given.Transmission electron microscopy(TEM),immunofluorescence,Western blotting and mitochondrial ATP detection were used to detect the effects of Pink1 knockout(Pink1 KD)on mitochondrial dynamic-related protein Drp1,mitochondrial number,density and ATP synthesis.Results With the increase of ammonia concentration and the prolongation of treatment time,the cell activity decreased significantly(P<0.01).Apoptosis occurred in the cells treated with 5.0 mmol/L NH_(4)Cl for 24 h,so 5.0 mmol/L NH_(4)Cl for 24 h was the maximum dose with no decrease in cell activity.Ammonia stimulated the expression of mitochondria-related autophagy protein ratio Pink1/Parkin significantly increased(P<0.05,P<0.01).Western blotting and immunofluorescence staining showed that Pink1 KD could reduce the increase of ammonia-induced Drp1 expression(P<0.05).TEM showed that Pink1 KD could reduce the increase of mitochondrial density induced by ammonia(P<0.05),but had no significant effect on mitochondrial coverage and average area.Examination of mitochondrial ATP synthesis showed that Pink1 KD could improve mitochondrial ATP synthesis(P<0.01).Conclusion Targeted knockout of mitochondrial Pink1 alleviates high ammonia-induced mitochondrial fragmentation by reducing Drp1 expression,thereby restoring mitochondrial ATP synthesis.This study provides molecular mechanisms and potential therapeutic targets for mitochondrial morphological abnormalities and dysfunction induced by high ammonia.