Cytological Mechanism Of Addiction To The New Psychoactive Substance 4-MEC

4-Methylecathinone(4-MEC)is a cathinone derivative discovered in recent years.It is a new psychoactive substance to replace the famous "zombie drug" 4-MMC(Methcathinone).It has been found in some "legal stimulants",which have the risk of evading legal supervision and spreading.However,until now,there have few reports on the mechanism of 4-MEC addiction.In the previous stage,our laboratory conducted a comparative study on the proteins in the brain tissues of normal mice and 4-MEC addicted mice by using TMT proteomics technology.Combined with bioinformatics and functional enrichment analysis,a key protein related to 4-MEC addiction--Caveolin-1(CAV1)was discovered.On this basis,the specific molecular mechanism of CAV1 protein involved in 4-MEC addiction was studied in this paper.Using SH-SY5Y as a model cell,the effects of 4-MEC on addiction-related signaling pathway molecules such as CAV1 and BDNF-TrkB as well as neuroplasticity and dopamine system were detected in vitro firstly.It was found that 4-MEC can significantly up-regulate the mRNA and protein levels of CAV1,BDNF-TrkB-related signaling pathway molecules and neuroplasticity markers,and significantly up-regulate the mRNA levels of dopamine receptor DRD1 and significantly down-regulate the mRNA levels of dopamine receptor DRD2 and dopamine transporter DAT.Subsequently,SH-SY5Y cells were transfected with siRNA-CAV1 and CAV1 overexpressing plasmid to knock down or overexpress CAV1 and to explore the molecular mechanism of CAV1’s involvement in 4-MEC addiction.CAV1 knockdown could significantly inhibit 4-MEC-induced up-regulation of mRNA or protein levels of CAV1,BDNF-TrkB related signaling pathway molecules and neuroplasticity markers.Overexpression of CAV1 can significantly promote the up-regulation of protein levels of CAV1,BDNF-TrkB related signaling pathway molecules and the mRNA levels of neuroplasticity markers induced by 4-MEC.Secondly,the interaction between 4-MEC and CAV1 protein was simulated and predicted by molecular docking.It was found that there were potential binding sites between 4-MEC and CAV1 protein;the protein-protein interaction network(PPI)and the simulated docking prediction between proteins showed that CAV1 and TrkB proteins also had the mutual binding effect.Subsequently,Co-Immunoprecipitation(IP)assay was used to verify the interaction between CAV1 and TrkB proteins.Finally,a 4-MEC addiction mouse model was successfully constructed by conditional location preference experiment,and the effects of 4-MEC on CAV1 and BDNF-TrkB addiction-related signaling pathway molecules and neuroplasticity were verified in vivo,which was found to be consistent with the results of in vitro experiments.In conclusion,4-MEC activates the addiction-related BDNF-TrkB signaling pathway mediated by the CAV1 protein,thereby affecting the adaptive changes of neuroplasticity and the expression level of the dopamine system.Therefore,it is speculated that the CAV1 protein is a target protein of 4-MEC addiction and plays an important role in the process of 4-MEC addiction.This study provides basic data and theoretical references for the molecular mechanism of addiction at the cytological level of the new psychoactive substance 4-MEC.