Study on the mechanism of Pink1 in high ammonia-induced abnormal mitochondrial fission by regulating Drp1
BAI Yunhu
WANG Quanhui
YUE Wei
XIE Jun
WU Feifei
WANG Yayun
YANG Yanling
ZHANG Chunxu
Abstract：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 NH4C1 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 NH4 Cl were selected to observe the expression changes of mitochondrial autophagy related protein Pink1 at the protein level.Subsequently,lentivirus treatment with Pinkl 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 NH4C1 for 24 h,so 5.0 mmol/L NH4C1 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 Pink1KD could reduce the increase of ammonia-induced Drp1 expression(P＜0.05).TEM showed that Pink1KD 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 Pink1KD could improve mitochondrial ATP synthesis(P＜0.01).Conclusion Targeted knockout of mitochondrial Pinkl 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.
Keywords：high ammoniamitochondriaPink1Drp1ATP
Publication Date：2024-01-28
Online Publishing Date：2026-08-26(First online date of this platform, not the publication date of the document)
Pages：7( 59-65 )