| Part one:Involvement of autophagy in methamphetamine-induced hippocampal neural apoptosisMethamphetamine(Meth),a kind of addicted drugs,mainly affects the single amine neurotransmitter transport system,resulting in excitotoxicity and energy homeostasis disturbance,which is linking to several severe adverse effects on the central nerves system.Therefore,Meth abuse has become a serious global public health problem.Meth exposure causes Degradation of neurons and the apoptosis of the neurons is one of the important mechanisms.However,the underlying mechanisms involving Meth induced neural damage remained poorly understood.In the present work,it suggested that Meth can increase the protein level of c-caspase 3,c-PARP and decrease the bcl-2 in time-and dose-dependent manner.Autophagy is a highly conservative evolutionary metabolic process.It can reuse cell components,including the damaged proteins and organelles which are hydrolyzed by the enzyme in lysosome.Our results showed that Meth obviously increased Beclinl,ATG5 and Lc3-Ⅱ/Lc3-Ⅰ expression in time-and dose-dependent manner.The similar result of p62 was also observed.The immunofluorescence result showed that the fluorescence intensity of Lc3 following exposure to Meth significantly enhanced.Since mTOR/p70S6K signal pathway is one of the important pathways in regulating autophagy.Therefore,the mTOR/p70S6K signal pathway was detected.As our western blot results showed that Meth markedly decreased p-p70S6k and p-mTOR level in dose-dependent manner.In order to assess the relationship between autophagy and apoptosis mediated by Meth,the autophagy agonists Rapamycin and the inhibitor 3-MA were used.It revealed that compared with the Meth treatment groups,a significant down-regulated c-caspase 3,c-PARP expression was observed in Rapamycin+Meth treatment group while the c-caspase 3,c-PARP expression in 3-MA+Meth treatment group is obviously increased.Part two:Effects of Methamphetamine on lysosome number,function and LAMP1Lysosome is a membrane bound organelle,and existed in all eukaryotic cells.It is referred to as autophagy terminal digestion station and can eliminate or remove cell metabolic waste,waste material to maintain the balance of biological synthesis reaction.Therefore,in the current work,several fluorescent probes were explored to evaluate the number,pH and activity of lysosome.It showed that compared with control group,the fluorescence intensity of Meth treatment group was obviously enhanced.Meth substantially increases the number of lysosome,decrease pH,increase the activity of cathepsin B and enhance the protein expression of LAMP 1,suggesting that lysosome signaling pathway,the downstream signaling of Autophagy,may be involved in Meth-induced neural damage.Part three:Abnormal fusion of autophagosome and lysosome was involved in Meth induced hippocampal neural damageThe role of fusion between autophagosome and lysosome is very important in the whole flow of autophagy.This process was reported to transfer cell metabolite to lysosome for degrading them.To delineate the fusion process between autophagosome and lysosome mediated by Meth,the virus plasmid mRFP-GFP-Lc3 was used and transfected to the hippocampal neurons.It showed that,compared with the control and rapamycin treatment group,Meth treated group obviously reduced the free red dots and raise the ratio of the yellow spots and free red spots in merged images,suggesting that Meth block the fusion between autophagosome and lysosomes.To detect whether the fusion between autophagosome and lysosome was associated with neurons apoptosis caused by Meth,CQ and bafA1 were used.Our results demonstrated that,compared with Meth treated group,the bafA1+Meth treatment group obviously increase the c-caspase 3 and c-PARP expression levels and CQ+Meth treatment group obviously increase the c-caspase 3 expression levels(P<0.05).However,the changes of bcl-2 and bax were not pronounced with no statistically significance. |
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