The Role Of Mitochondrial Ferritin In Neuronal Apoptosis Induced By Methamphetamine In Hippocampus Of Mice And HT-22 Cell Line

Objectives:Methamphetamine(MA)was a highly addictive drug that could cause serious damage to the central nervous system for long time abuse.The aim of this experiment was to clarify that neuronal apoptosis could be induced by MA and explore whether mitochondrial ferritin(FtMt)played a role in neuronal apoptosis which induced by MA.Methods:1.Expressions of apoptosis-related proteins(Cyt-c,Caspase-3,Bax,and Bcl-2)and FtMt in hippocampus of mice which treated with MA.(1)Establishment of animal model: 20 adult male C57BL/6J mice were randomly divided into control group and MA group.MA group was injected intraperitoneally with 2 mg/kg MA twice a day,with an interval of 12 hours,for 5 consecutive days.The control group was intraperitoneally injected with corresponding dose of normal saline twice a day,with an interval of 12 hours,for 5 consecutive days.(2)Behavioural test: the mice were tracked in the open field test(OFT)to observe the abnormal behaviour.(3)Histological observation: Hematoxylin and eosin(H-E)staining was used to observe the histopathology of hippocampus.(4)Western blot was used to detect the expressions of apoptosis-related proteins(Cyt-c,Caspase-3,Bax,and Bcl-2)and FtMt.2.Expressions of apoptosis-related proteins(Cyt-c,Caspase-3,Bax,and Bcl-2)and FtMt in cells of hippocampal neurons of mice(HT-22 cell line)which treated with MA.(1)HT-22 cell line was cultured to establish a neurotoxicity model induced by MA.(2)CCK-8 kit was used to detect the viability of cells at different potency and time of MA administered.(3)The morphology of cells was observed by inverted microscope.(4)Western blot was used to detect the expressions of apoptosis-related proteins(Cyt-c,Caspase-3,Bax,and Bcl-2)and FtMt.3.The effect of silencing FtMt on MA induced apoptosis of HT-22 cell line.(1)The sh RNA was used to silence FtMt and establish the cell model of transfected with sh RNA.(2)CCK-8 kit was used to detect the viability of cells after FtMt was silenced.(3)Western blot was used to detect the expressions of apoptosis-related proteins(Cyt-c,Caspase-3,Bax,and Bcl-2)after FtMt was silenced.Results:1.The behaviour of mice in MA group was changed and apoptosis could be induced by MA and the expression of FtMt was increased in the hippocampus of mice which treated with MA.Open field test(OFT)data showed that the movement trajectory of mice in the MA group was complex and their activities increased.The results of hematoxylin and eosin(H-E)staining showed that the hippocampal tissue of mice in MA group has obvious morphological changes,including neuronal degeneration,nuclear lysis and cytoplasmic hyperchromia.The appearance suggested which MA caused neuronal damage in the hippocampus of mice.Compared with the control group,the expressions of Cyt-c and Caspase-3 were increased in MA group,while the expression of Bcl-2 was decreased,which suggested that MA could cause apoptosis in the hippocampus of mice.Compared with the control group,the expression of FtMt was increased in the hippocampus of mice in MA group.2.MA could induce apoptosis in HT-22 cell line and increase the expression of FtMt.The result of CCK-8 showed that the viability of cells was decreased gradually with the increase of the potency of MA and the prolongation of time.According to the result of CCK-8,the optimal potency of MA was 2 m M,and the optimal time was 24 h.Compared with the control group,the morphology of cells in MA group was changed.The apophyses became shorter or even disappeared,and the cells were shrunk,which suggested that MA could cause cellular damage.Compared with the control group,the expressions of Cyt-c,Caspase-3,and Bax in MA group was increased,and the expression of Bcl-2 was decreased,which indicated MA could induce apoptosis.The result also showed that the expression of FtMt in MA group was higher than that in control group.3.Silencing FtMt could aggravate apoptosis induced by MA in HT-22 cell line.After silencing FtMt,the viability of HT-22 cell line which treated with MA was decreased,the expressions of Cyt-c,Caspase-3,and Bax were increased,and the expression of Bcl-2 was decreased.These results showed that FtMt might play a protective role in MA-induced neuronal apoptosis.Conclusions:1.Methamphetamine abuse could induce neuronal apoptosis in hippocampus of mice and HT-22 cell line,the expression of FtMt was increased.2.The apoptosis induced by methamphetamine was increased after silencing FtMt gene,the data suggested that FtMt might play a protective role in neuronal apoptosis which induced by methamphetamine.