Amitriptyline Protects Against Lidocaine-induced Neurotoxicity In SH-SY5Y Cells Via Inhibition Of BDNF-mediated Autophagy

Objective To verify the protective effect of amitriptyline(AMI)against the neurotoxicity induced by lidocaine(Lido)and explore its possible mechanism.Methods1.SH-SY5 Y cells were cultured and exposured to lidocaine(2 m M,24 h)to build the neurotoxicity model.Following the pretreatment of AMI(100 n M,24 h),we measured cell viability by CCK-8 assay,observed cell morphology by phase-contrast light microscope,and assessed apoptosis by Terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)Staining.2.Autophagy inhibitor 3-MA(2 m M,2 h)and autophagy agonist rapamycin(200n M,2 h)were used in our study to regulate autophagy activity.MDC autophagy staining was applied to observe the formation of autophagic vacuoles.TUNEL/DAPI fluorescence staining was used to detect the level of apoptosis,and Western blot was adopted to detect the proteins expression levels.3.SH-SY5 Y cells were transfected with lentivirus-mediated sh BDNF and BDNF overexpression recombinant plasmid.Following the AMI pretreatment and Lido intervention,cell viability was measured by CCK-8 and cell apoptosis was assayed by TUNEL/DAPI fluorescence staining.BDNF and the autophagy related proteins was detected by Western blot.4.SH-SY5 Y cells were transfected with lentivirus mediated BDNF knockdown and overexpression to observe the function of BDNF.MDC was used to observe autophagosome,and detection the expression of BDNF,m TOR and P-m TOR proteins was assessed by Western blot.Results1.Amitriptyline protected SH-SY5 Y cells against lidocaine-induced neurotoxicity1)Lido caused cell damage in a time and concentration dependent manner;2)AMI restored cell injury induced by Lido in SH-SY5 Y cells.2.Amitriptyline decreased lidocaine-induced apoptosis via suppression of autophagy in SH-SY5 Y cells1)Lido induced an increase in autophagy activity in SH-SY5 Y cells;2)AMI blocked the autophagy activity caused by Lido;3)3-MA pretreatment improved the cell damage caused by Lido and enhanced the neuroprotection of AMI;4)RAPA exacerbated neurotoxic effect of Lido and reversed the protective effects of AMI in SH-SY5 Y cells.3.Amitriptyline attenuated the neurotoxicity induced by lidocaine through promoting the expression of BDNF in SH-SY5 Y cells1)Lido decreased the expression level of BDNF in SH-SY5 Y cells;2)AMI pretreatment up-regulated BDNF expression in SH-SY5 Y cells treated with Lido;3)Knockdown of BDNF reversed the protective effect of AMI;4)Overexpression of BDNF alleviated cellular injury caused by Lido.4.Amitriptyline inhibited autophagy by upregulation of BDNF expression1)Autophagy regulated by BDNF in SH-SY5 Y cells;2)BDNF inhibited autophagy via activating the m TOR pathway.ConclusionAmitriptyline could effectively improve the neurotoxicity of lidocaine,possibly by up-regulating the expression of BDNF and activating the m TOR pathway to inhibit autophagy.