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

Chapter Ⅰ identification of autophagy and the effect ofmechanical injury on autophagy and apoptosis of neuronal cellsObjective: To identify autophagy of neuronal cells induced by mechanical injury, andobserve the effect of mechanical injury on autophagy and apoptosis.Methods:(1) The mechanical injury model of neuronal cell was established using methodintroduced by HaiPing Que.(2) Cell morphology was observed by hematoxylin-eosin staining，NSE and DAPIdouble-lable fluorescence.(3) The recombinant plasmid pcDNA-3.1(+)-GFP-LC3was constructed, autophagywas evaluated by fluorescent microscopy, monodansylcadaverine (MDC) staining,acridine orange (AO) staining and Western Blot.(4) Cell activity was evaluated by MTT.(5) Cell cycle and apoptosis were evaluated by flow cytometry.(6) The expression of Bcl-2and Bax were evaluated by Western Blot.Results:(1) Many axons and dendrites of neuronal cells were cut off, and a large number offloating cells were observed after3h of mechanical injury.(2) The axons and dendrites of neuronal cells were integrity with purity more than90%.(3) The recombinant plasmid pcDNA-3.1(+)-GFP-LC3was successfully constructedand transfected into neuronal cells.(4) Obvious intracellular green spots were observed after24h of mechanical injury.(5) A large number of autophagic vacuoles were observed after24h of mechanicalinjury by MDC and AO staining. (6) The expression of Beclin-1and LC3Ⅱ/LC3Ⅰreached a peak after24h ofmechanical injury.(7) Cell mortality increased quickly with time of mechanical injury extended, anddecreased slowly24h after injury.(8) The rate of apoptosis increased gradually with time of mechanical injury extended,and significantly increased3h after injury.(9) The expression of Bcl-2increased firstly and decreased3h after injury, while theexpression of Bax increased with time of mechanical injury extended.(10) Obvious cellular debris, apoptotic bodies, uneven fluorescence and larger cellwere observed.Conclusions:(1) Autophagy can be induced by mechanical injury and reaches a peak24h afterinjury.(2) Mechanical injury can lead to apoptosis and necrosis.(3) Apoptosis is inhibited in the early stage of autophagy may through promoting theexpression of Bcl-2and inhibiting the expression of Bax.(4)24h after injury is selected as optimal drug treatment time. chapterⅡ The effect of autophagy regulation on aotuphapy andapoptosis of neuronal cells induced by mechanical injuryObjective: To investigate the effect of autophagy regulation on aotuphapy andapoptosis of neuronal cells induced by mechanical injury.Methods:(1) Autophagy was peomoted and inhibited by rapamycin (Rap) and3-methyladenine (3-MA) respectively.(2) The expression of autophagy and apoptosis-related proteins were detected byWestern Blot.(3) The expression of Beclin-1and LC3were detected by RT-PCR.(4) Cell activity was evaluated by MTT. (5) Cell apoptosis were evaluated by flow cytometry.Results:(1) Rap promoted the expression of Beclin-1and LC3Ⅱ/LC3Ⅰ, while3-MAinhibited the expression of Beclin-1and LC3Ⅱ/LC3Ⅰ.(2) Rap reduced the rate of death and apoptosis of neuronal cells induced bymechanical injury, while3-MA enhanced it.(3) Rap reduced the expression of Bax and promoted the expression of Bcl-2inneuronal cells after injury, while3-MAreduced the expression of Bcl-2andpromoted the expression of Bax in neuronal cells after injury.(4) The expression of p-mTOR and p-AKT decreased significantly in neuronal cellsafter mechanical injury, but the expression of mTOR and AKT increased at acertain extent.(5) Rap reduced the expression of mTOR and AKT significantly.Conclusions:(1) Rap can inhibit apoptosis and necrosis, while3-MA can accelerate apoptosis andnecrosis of neuronal cells at a certain extent.(2) Rap inhibits cell apoptosis may through promoting the expression of Bcl-2andinhibiting the expression of Bax, to the contrary,3-MA accelerates cell apoptosismay through promoting the expression of Bax and inhibiting the expression ofBcl-2.(3) Autohapy activity of neuronal cells increased induced by Rap may throughdown-regulating phosphorylation of mTOR and AKT. ChapterⅢ The role of Beclin-1in the process of autophagy andapoptosis in neuronal cells induced by mechanical injuryObjective: To investigate the role of Beclin-1in the process of autophagy andapoptosis in neuronal cells induced by mechanical injury.Methods: (1) The recombinant plasmid pcDNA-3.1(+)-Beclin-1and pcDNA-3.1(+)-miRNAwere constructed and transfected into neuronal cells, and were identified byWestern Blot.(2) The expression of autophagy and apoptosis-related proteins were detected byWestern Blot.(3) The effect of Beclin-1on cell activity was evaluated by MTT.(4) The effect of Beclin-1on cell apoptosis was evaluated by flow cytometry.Results:(1) The recombinant plasmid pcDNA-3.1(+)-Beclin-1and pcDNA-3.1(+)-miRNAwere successfully constructed.(2) The expression of Beclin-1increased in cell within pcDNA-3.1(+)-Beclin-124hafter mechanical injury, while decreased in cell within pcDNA-3.1(+)-miRNA.(3) The value of LC3Ⅱ/LC3Ⅰ increased significantly in cell withinpcDNA-3.1(+)-Beclin-124h after mechanical injury, while decreased significantlyin cell within pcDNA-3.1(+)-miRNA.(4) The rate of death and apoptosis of neuronal cells within pcDNA-3.1(+)-Beclin-1decreased24h after mechanical injury, while increased of cell withinpcDNA-3.1(+)-miRNA.(5) The expression of Bcl-2increased and Bax decreased in neuronal cells withinpcDNA-3.1(+)-Beclin-124h after mechanical injury, while The expression of Baxincreased and Bcl-2decreased in neuronal cells within pcDNA-3.1(+)-miRNA24h after mechanical injury.Conclusions:(1) Over expression of Beclin-1may promote aotophapy, while silence of Beclin-1may inhibit aotophapy.(2) Over expression of Beclin-1may inhibit cell apoptosis and necrosis, while silenceof Beclin-1may accelerate cell apoptosis and necrosis.(3) Beclin-1inhibits cell apoptosis may through promoting the expression of Bcl-2and inhibiting the expression of Bax, to the contrary, silence of Beclin-1accelerates cell apoptosis may through promoting the expression of Bax andinhibiting the expression of Bcl-2.