Effect and mechanism of metformin on neuronal apoptosis after traumatic brain injury
ZHU Lei
LI Zhengwei
YIN Hong
WANG Xuecheng
XU Yanyan
WANG Nan
Abstract：Objective To investigate the effect and mechanism of metformin(MET)on neuronal apoptosis after traumatic brain injury(TBI).Methods HT22(mouse hippocampal neurons)cells were selected for the experiment.HT22 cells were randomly divided into the control group,TBI group,MET1 group,and MET2 group.Except for the con-trol group,the in vitro models of TBI were constructed by mechanical scratching in the other groups.We utilized Western blotting to examine the expression of Bcl-2 and adenovirus E1B19kDa-interacting protein 3(BNIP3),LC3,p62,TOMM20,and COX IV.We utilized immunofluorescence double staining to observe the colocalization of mitochondria and lysosome,TUNEL staining to detect apoptosis rate,and LDH kit to detect LDH activity of cell supernatant.The small interfering RNA(siRNA)was used to inhibit the expression of BNIP3.The cells were randomly divided into the control siRNA group,BNIP3-siRNA group,control siRNA+TBI group,and BNIP3-siRNA+TBI group,and the changes of the above parameters were detected at the end of the experiment.Results Compared with the control group,the neu-ronal apoptosis rate increased in the TBI group(P<0.05).Compared with the control group,the expression of BNIP3 and LC3-Ⅱ/Ⅰ increased,while the expression of p62,TOMM20,and COX IV decreased in the TBI group(all P<0.05).Compared with the control group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy increased in the TBI group.Compared with the TBI group,the neuronal apoptosis rates were lower in the MET 1 group and MET 2 group(all P<0.05),and the apoptosis rate was lower in the MET 2 group than in MET 1 group(P<0.05).Com-pared with the TBI group,the expression of BNIP3 and LC3-Ⅱ/Ⅰ decreased in the MET 1 group(all P<0.05),and the expression of BNIP3 and LC3-Ⅱ/Ⅰ was lower in MET 2 group than in MET 1 group(all P<0.05).Compared with the TBI group,the expression of p62,TOMM20,and COX IV increased in the MET 1 group(all P<0.05),and the expres-sion of p62,TOMM20,and COX IV were significantly higher in the MET 2 group than in the MET 1 group(all P<0.05).Compared with the TBI group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy de-creased in the MET 1 group,and the colocalization of mitochondria and lysosomes was lower in the MET 2 group than in the MET 1 group.The neuronal apoptosis rates in the control siRNA+TBI group and BNIP3-siRNA+TBI group were 32.57%±2.68%and 16.59%±1.80%,respectively,with significant difference between the two groups(P<0.05).The expression levels of BNIP3 and the LC3-Ⅱ/Ⅰ value in the control siRNA+TBI group were 0.66±0.01 and 1.46±0.02 respectively,which were significantly higher than those in the BNIP3-siRNA+TBI group(all P<0.05).The expression levels of p62,TOMM20,and COX IV in the control siRNA+TBI group were 0.06±0.01,0.14±0.01,and 0.09±0.01,respectively,which were significantly lower than those in the BNIP3-siRNA+TBI group(all P<0.05).Compared with the BNIP3-siRNA+TBI group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy increased in the control siRNA+TBI group.Conclusion MET can inhibit neuronal apoptosis after TBI,and its mecha-nism may be related to the inhibition of BNIP3-mediated mitophagy.
Keywords：traumatic brain injurymetforminBcl-2/adenovirus E1B19kDa-interacting protein 3mitophagy
Publication Date：2024-12-05
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 43-48 )