Effect And Mechanism Of IP₃R In Autophagy Of Neurons Regulated By TOCP

Objective:To explore the mechanisms relating to the autophagy,the role of inositol 1,4,5 triphosphate receptor?IP₃R?in autophagy regulated by tri-ortho-cresyl phosphate?TOCP?has been investigated in vitro cell experiments.Methods:Human neuroblastoma SK-N-SH cells and mouse neuroma cell N2a cells were used as in vitro models.1.SK-N-SH cells were treated with different concentrations of TOCP?0mmol/L,0.25 mmol/L,0.50 mmol/L,0.75 mmol/L,1.00 mmol/L?for 48 h and N2a cells were treated with different concentrations of TOCP?0?mol/L?25?mol/L?50?mol/L?75?mol/L?100?mol/L?for 24 h.The cell viability was detected by MTT assay and the expression of autophagy-related protein P62 and LC3 II/LC3 I were detected by Western Blot.2.SK-N-SH cells were treated with TOCP?0.75 mmol/L?for 48 h,N2a cells were treated with TOCP?75?mol/L?for 24 h?this treatment concentration and time of TOCP were used in subsequent experiments?.The fluorescence spots of LC3 in cells were detected by indirect immunofluorescence.The number of autophagosomes in two kinds of cells was observed by transmission electron microscope.3.U73122,an inhibitor of phospholipase C?PLC?,was pretreated for 30min and then co-treated with TOCP.The PLC activity was detected by colorimetry and the intracellular free Inositol 1,4,5-trisphosphate?IP₃?was detected by enzyme linked immunosorbent assay.The expression of three subtypes of IP₃R,autophagy-related protein P62 and LC3 II/LC3 I were detected by Western Blot.4.IP₃R inhibitor Xestospongia C and Ryanodine receptor?RyR?inhibitor Ryanodine was pretreated for 30 min and then co-treated with TOCP.The intracellular free calcium ion concentration was detected by fluorescence spectrophotometer.The expression of RyR,three subtypes of IP₃R,autophagy-related protein P62 and LC3 II/LC3 I were detected by Western Blot.5.SK-N-SH cells and N2a cells were treated with TOCP.The co-localization of IP₃R and Beclin 1 were observed by double-labeling immunofluorescence,and the protein interaction between IP₃R and Beclin 1were observed by co-immunoprecipitation.Results:1.After SK-N-SH cells and N2a cells were treated with different concentrations of TOCP,the cell viability was significantly decreased with the increase of TOCP concentration?P<0.001?.The expression levels of autophagy-related protein P62?P<0.05?and LC3 II/LC3 I?P<0.05?were partly increased.2.After SK-N-SH cells and N2a cells were treated with TOCP.Compared to control group,the fluorescence spots of LC3?P<0.05?and the number of autophagosomes were significantly increased.3.U73122 pretreated cells and co-treated with TOCP.The TOCP treatment group was compared with the control group,the PLC activity was significantly increased?P<0.001?and the levels of intracellular free IP₃ were decreased?P<0.05?.The expression levels of IP₃R1?P<0.01?,IP₃R2?P<0.01?,IP₃R3?P<0.01?,autophagy-related protein P62?P<0.01?and LC3 II/LC3 I?P<0.01?were significantly increased.And the combination group was compared with the TOCP treatment group,the PLC activity was decreased?P<0.01?and the levels of intracellular free IP₃ were partly increased?P<0.05?.The expression levels of IP₃R1?P<0.05?,IP₃R2?P<0.05?,IP₃R3?P<0.05?,autophagy-related protein P62?P<0.05?and LC3 II/LC3 I?P<0.05?were partly decreased.4.Xestospongia C and Ryanodine pretreated cells and co-treated with TOCP.The TOCP treatment group was compared with the control group,the intracellular free calcium ion concentration was increased?P<0.001?.The expression levels of RyR?P<0.01?,IP₃R1?P<0.01?,IP₃R2?P<0.01?,IP₃R3?P<0.01?,autophagy-related protein P62?P<0.01?and LC3 II/LC3 I?P<0.01?were significantly increased.And the combination group was compared with the TOCP treatment group,the intracellular free calcium ion concentration was decreased?P<0.001?.The expression levels of RyR?P<0.01?,IP₃R1?P<0.01?,IP₃R2?P<0.01?,IP₃R3?P<0.01?.The expression levels of autophagy-associated protein P62?P<0.01?and LC3 II/LC3I?P<0.01?were all decreased.5.After SK-N-SH cells and N2a cells were treated with TOCP.Compared to control group,the co-localization between the three subtypes of IP₃R and Beclin 1 were significantly reduced and the protein interaction were also significantly reduced?P<0.05?.Conclusions:1.The cell viability of SK-N-SH cells and N2a cells were decreased by TOCP.The autophagic flux and the accumulation of autophagosomes were inhibited by TOCP2.PLC-IP₃-IP₃R pathway were regulated by TOCP,causing the opening of IP₃R and RyR calcium channels,mediating intracellular calcium ion disorder and inhibited the autophagic flux.3.The dissociation of IP₃R-Beclin 1 complex were promoted by TOCP,Beclin 1 was released into the cell and regulated autophagy.4.Compared with SK-N-SH cells,N2a cells were sensitive to the toxic effects caused by TOCP,but the mechanism by which TOCP inhibited autophagic flux was not different between the two kinds of cells.