Relation Between Autophagy And Apoptosis Induced By Dexamethasone In Spinal Cord Injury Neuron Cells

Chapter? The relation between autophagy and apoptosis induced by different concentrations of dexamethasone in the mechanical injury model of neuronal cellObjective: To explore the relation between autophagy and apoptosis induced by different concentrations of dexamethasone in the mechanical injury model of neuronal cell.Methods:(1) Primary cultured the spinal cord neurons and were identified by immunocytochemistry with NSE?TUBB3?DAPI.(2) Spinal cord neurons were randomly divided into five groups: Normal ?Control?Dex10-7M?Dex10-6M? Dex10-5M,The mechanical injury model of neuronal cell was established and intervention was administered immediately to 24 hours.(3)Spinal cord neuron cells activity was evaluated by MTT.(4)The expression of autophagy related protein(LC3 and Beclin-1)and apoptosis related protein(Caspase-3)were evaluated by western blot.(5)The expression of autophagy related m RNA(LC3?and Beclin-1)and apoptosis related m RNA(Caspase-3) were evaluated by QPCR.(6)Spinal cord neuron cells apoptosis were evaluated by flow cytometry.(7)The autophagy level was evaluated by fluorescence microscope after staining with acridine orange.(8) The number of autophagosomes and apoptosis bodies was detected by transmission electron microscope.Results:?Spinal cord neurons grew in a well state; the percentage of cells identifiable as neurons(the proportion of cells positive for both NSE and TUBB3) was remainedat high level for all experiments.? MTT:The spinal cord neuron cells activity was obviously decreased compared with normal group after mechanical injury, Dex10-7M group with higher cell activity than the control group, but there was not statistically significant(P>0.05);Dex10-6M group cells activity was obviously increased compared with normal group.However, the Dex10-5M group cells activity were obviously decreased compared with normal group(P<0.05);? Western Blotting:Compared with normal group, the expression of autophagy related protein(LC3?and Beclin-1)were obviously increased after mechanical injury; With increasing the concentration of dexamethasone,the expression of autophagy related protein LC3 ? and Beclin-1 declined gradually( P<0.05);Compared with normal group, The expression of apoptosis related protein(Caspase-3)were obviously increased after mechanical injury; Compared with control group, Dex10-5M and Dex10-6M group Caspase-3 level were obviously decreased(P<0.05),Dex10-5M group Caspase-3 level was higher than Dex10-6M group, but there was not statistically significant(P>0.05);? QPCR: Compared with normal group, The expression of autophagy related m RNA(LC3? and Beclin-1)were obviously increased after mechanical injury;With increasing the concentration of dexamethasone the expression of autophagy related m RNA LC3? and Beclin-1 reduced gradually(P<0.05); Compared with normal group, The expression of apoptosis related m RNA( Caspase-3) was obviously increased after mechanical injury; Compared with control group,Dex10-5M and Dex10-6M group Caspase-3 level were obviously decreased(P<0.05),Dex10-5M group Caspase-3 level was higher than Dex10-6M group, but there was not statistically significant(P>0.05);? Flow cytometry: Compared with normal group, The apoptosis rate was obviously increased after mechanical injury; Compared with control group,Dex10-5M and Dex10-6M group cells apoptosis rate were obviously decreased(P<0.05),Dex10-5M group cells apoptosis rate was higher than Dex10-6M group,but there were not statistically significant(P>0.05);? AO: The red and green fluorescence ratio in the cells of the normal group significantly lower than the mechanical injury groups; With increasing the concentration of dexamethasone red and green fluorescence ratio declined gradually.? Transmission electron microscope: The autophagosomes and apoptotic bodies were rarely observed in the normal group,we can obviously observe the double membrane structure of autophagy and apoptosis body after mechanical injury in the spinal cord neuron. As the dexamethasone concentration increased the number of autophagosomes and apoptotic bodies declined gradually,the number of apoptosis bodies in Dex10-5M group increased than the Dex10-6M group.Conclusions:? Spinal cord neurons autophagy and apoptosis level enhanced in different degrees after mechanical injury;? Dexamethasone can inhibit the level of autophagy in spinal cord neuron cells after mechanical injury, the level of autophagy and dexamethasone were in a concentration dependent manner, The inhibitory effect was more obvious with increasing the concentration of the dexamethasone;? A large dose of dexamethasone protect the mechanical injury in spinal cord neuron cells by inhibiting neuron cell apoptosis; However,excessive the concentration of the dexamethasone will weaken the protective effect on the mechanical injury in spinal cord neuron cells; A low level of dexamethasone does not seem to have a protective effect on the mechanical injury in spinal cord neuron cells? A large dose of dexamethasone reduce the apoptosis of the mechanical injury in spinal cord neuron cells by inhibiting spinal cord neurons autophagic cell death.Chapter? The effect of autophagy regulation on autophagy and apoptosis of mechanical injury in spinal cord neuron cells induced by dexamethasoneObjective: The effect of autophagy regulation on autophagy and apoptosis of mechanical injury in spinal cord neuron cells induced by dexamethasone.Methods:(1)Spinal cord neurons were randomly divided into five groups :Dex10-6M?Dex10-6M+Rap? Dex10-6M+3-MA,the mechanical injury model of neuronal cell was established and intervention was administered immediately to 24 hours.(2)The expression of autophagy related protein(LC3 and Beclin-1)and apoptosis related protein(Caspase-3) were evaluated by western blot.(3)The expression of autophagy related m RNA(LC3?and Beclin-1)and apoptosis related m RNA(Caspase-3) were evaluated by QPCR.(4)Spinal cord neuron Cells apoptosis were evaluated by flow cytometry.(5)The autophagy level was evaluated by fluorescence microscope after staining with acridine orange.(6) The number of autophagosomes and apoptosis bodies was detected by transmission electron microscope.Results:? Western Blotting : Compared with Dex10-6M group, the expression of autophagy related protein(LC3? and Beclin-1)were obviously increased in the Dex10-6M+Rap group;The expression of autophagy related protein(LC3? and Beclin-1)were obviously declined in the Dex10-6M+3-MA group(P<0.05);Compared with other groups, the expression of apoptosis related protein( Caspase-3) was obviously decreased in the Dex10-6M+3-MA group; the Dex10-6M+Rap group showed the highest expression of apoptosis related protein (P<0.05);? QPCR: Compared with Dex10-6M group, the expression of autophagy related m RNA(LC3? and Beclin-1)were obviously increased in the Dex10-6M+Rap group;The expression of autophagy related m RNA(LC3? and Beclin-1)were obviously decreased in the Dex10-6M+3-MA group(P<0.05); Compared with other groups, the expression of apoptosis related m RNA(Caspase-3)was obviously decreased in the Dex10-6M+3-MA group; The Dex10-6M+Rap group showed the highest expression of apoptosis related m RNA(P<0.05);? Flow cytometry : Dex10-6M+3-MA group cell apoptosis was significantly lower than the other groups,the Dex10-6M+Rap group cells showed the highest level of apoptosis.? AO: The red and green fluorescence ratio was significantly lower in Dex10-6M+3-MA group than the other groups,the Dex10-6M+Rap group showed the highest red and green fluorescence ratio.? Transmission electron microscope: The autophagosomes and apoptotic bodies were rarely observed in the Dex10-6M+3-MA group, we can obviously observe the double membrane structure of autophagy and apoptosis body in the Dex10-6M+Rap group.Conclusion: Inhibition the level of autophagy in the mechanical injury model of neuronal cells, cells apoptosis were significantly reduced; activation the level of autophagy in the mechanical injury model of neuronal cells, the level of apoptosis was significantly increased; That led to speculation that large doses of dexamethasone protect the mechanical injury model of neuronal cells by inhibiting autophagy to reduce autophagic cell death, The mechanism may be associated with m TOR signaling pathway.