C/EBPβ Participates In METH-induced Neurotoxicity Through DDIT4/TSC2/mTOR And Trib3/Parkin/alpha-syn Signaling Pathway

Background:Methamphetamine(METH)is a widely abused illicit psychotropic drug.Our previous studies have shown that CCAAT enhancer binding protein beta(C/EBPβ)is an important regulatory mechanism involved in METH-induced neuronal autophagy and apoptosis.However,the detailed molecular mechanism of this process is still unclear.Previous studies have shown that DDIT4,Trib3 and alpha-syn are involved in METH-induced neurotoxicity.We speculated that C/EBPβ was involved in METH-induced DDIT4-mediated autophagy and Trib3-mediated apoptosis.Method:In the first section of the first chapter,the time and concentration gradient models of METH poisoning in vitro were established.Western Blot was used to observe the expression of TSC2 and pTSC2 proteins with the increase of time and concentration of METH.In the second section of the first chapter,DDIT4-specific small interfering RNA was constructed.Western Blot was used to detect the expression of pTSC2 and mTOR autophagy pathway-related proteins after DDIT4 inhibition.In the third section of the first chapter,we constructed specific small interferences to inhibit the expression of C/EBPβ.Western Blot was used to detect the expression of DDIT4,pTSC2 and mTOR autophagy pathway-related proteins after treatment with Si-C/EBPp.In the first section of Chapter 2,we constructed small interferences to inhibit the expression of alpha-syn.Western Blot was used to detect whether METH-induced mitochondrial apoptosis signaling pathway was inhibited after Si-alpha-syn treatment.Section 2 of Chapter 2 establishes the in vitro model of METH poisoning with time and concentration gradient,and observes the changes of Parkin expression by Western Blot.At the same time,we constructed a cell line overexpressing Parkin and detected the expression of alpha-syn,Bax/Bcl-2 and autophagy pathway-related proteins after overexpressing Parkin.In the third section of Chapter II,we constructed small interfering Si-Trib3 to detect the expression of Parkin,alpha-syn,Bax/Bcl-2 and autophagy pathway-related proteins after Trib3 inhibition.The expression of Trib3,Parkin,alpha-syn,Bax/Bcl-2 and apoptotic pathway-related proteins was detected after the inhibition of C/EBPβ.The expression of alpha-syn induced by METH was also detected by immunofluorescence.Section 4 of Chapter 2,after stereotactic injection of recombinant lentivirus interfering with C/EBPβ in SN region,the expression of DDIT4,p-TSC2,mTOR and autophagy-related proteins and the expression of Trib3,Parkin,alpha-syn,Bax/Bcl-2 and apoptosis-related proteins were detected in animal models.Result:Chapter 11.The expression of p-TSC2 increased with the increase of time and concentration of METH.2.Interference with DDIT4 can inhibit the increase of p-TSC2 induced by METH and the autophagy pathway of mTOR.3.Interference with C/EBP(3 can inhibit the increase of DDIT4 and p-TSC2 induced by METH,and also inhibit the mTOR autophagy signaling pathway.Chapter 21.Inhibiting alpha-syn can reverse the mitochondrial apoptotic signaling pathway of SH-SY5Y cells induced by METH.2.In vitro model confirmed that Parkin expression increased after ETH treatment,and overexpression of Parkin could inhibit METH-induced increase of alpha-syn.3.Interference with Trib3 and C/EBPp can inhibit METH-induced Parkin decrease,alpha-syn increase and apoptosis.4.In vivo model validated that C/EBPβ participated in METH apoptosis and autophagy through Trib3/Parkin/alpha-syn and DDIT4/TSC2/mTOR pathways.Conclusion:Based on the above results,we propose a new mechanism of METH-induced cytotoxicity:METH exposure could increase the expression of C/EBP beta protein,trigger DDIT4/TSC2/mTOR signaling pathway,and induce Trib3/Parkin/alpha-syn-related mitochondrial apoptosis signaling pathway.