Study On The Toxic Mechanism Of New Psychoactive Substance 4-methylethylcathinone In Nerve Cells

4-Methylecathinone(4-MEC)is one of new psychoactive substances of cathinones,and its long-term abuse can cause neurotoxicity.Studies have shown that metal ions may participate in the regulatory network related to psychoactive substances.However,there are few reports on how metal ions participate in the neurotoxicity mechanism caused by 4-MEC.In our present study,we firstly found that 4-MEC caused the growth inhibition of human neurotype SH-SY5Y and mouse neurotype HT22 cells by CCK-8 assay.Human neurotype SH-SY5Y cell was chosen as the following experimental model.By useing fluorescence microscopy,flow cytometry and western blot technology,we found that 4-MEC exerted growth inhibition by inducing autophagy and G0/G1cell cycle arrest.Next,we carried out a metalomic study and detected the effect of 4-MEC on the contents,including seven important metal ions of potassium,sodium,iron,calcium,magnesium,zinc and aluminum.The ICP-AES analysis showed that 4-MEC treatment significantly increased the intracellular calcium and magnesium.Q-PCR and western blot detected the genes/proteins expression related to transports of calcium and magnesium.Fluo-3 dying further confirmed that 4-MEC treatment increased intracellular calcium level.The calcium chelator EGTA attenuated the neurotoxicity and increased intracellular calcium ion,alleviated the autophagy and G0/G1 cell cycle arrest caused by 4-MEC,indicate that 4-MEC exerts cytotoxicity by increasing calcium ion,resulting in autophagy and G0/G1 cell cycle arrest.Laser confocal observation revealed that 4-MEC caused calcium overload in the intracellular calcium ion storage mitochondria and endoplasmic reticulum,causing mitochondrial membrane potential disturbance,increased reactive oxygen species and endoplasmic reticulum stress.EGTA/N-acetyl-L-cysteine(NAC)treatment alleviated mitochondrial membrane potential disturbance,increased reactive oxygen species and endoplasmic reticulum stress caused by 4-MEC,indicating that the increased intracellular calcium induced by 4-MEC treatment caused endoplasmic reticulum stress and mitochondrial dysfunction.NAC attenuated the endoplasmic reticulum stress,indicating that the increase in reactive oxygen species induced by 4-MEC through increased intracellular calcium is the stressor of the endoplasmic reticulum stress.The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid attenuated autophagy caused by 4-MEC,indicating that endoplasmic reticulum stress mediated autophagy.These results indicated demonstrate that 4-MEC exerts neurotoxicity through Ca2+-ROS-ER stress-Autophagy pathway.Finally,using proteomics technique,we investigated the effect of 4-MEC on proteins expression in SH-SY5Y cells.The data showed that the levels of eight proteins were changed significantly after treated by 4-MEC.Among them,three(HSPA5,GAPDH and PGAM1)were up-regulated and the other five(ATP5A1,GSR,TALDO1,MED30,and TIMM13)were down-regulated.HSPA5 is related to endoplasmic reticulum stress,ATP5A1,MED30 and TIMM 13 are related to mitochondrial function,and GSR and TALDO1 are related to oxidative stress,further confirming the above-mentioned neurotoxicity mode caused by 4-MEC.In summary,4-MEC exerted neurotoxicity by inducing the increase of intracellular calcium levels to mediate G0/G1cell cycle arrest and ROS-ER stress-Autophagy signaling pathway;NAC may be a potential treatment for 4-MEC-induced cytotoxicity.This study provides a molecular basis for the neurotoxicity mechanism and therapeutic intervention of new psychoactive substances.