The Effects Of Neuroserpin On The Neurons And Microglias Undergoing Hypoxia/Reoxygenation

PartⅠThe expression of Neuroserpin and tPA in cultured neurons and their changesafter H/RObjective:To investigate the expression of NSP and tPA in cultured neurons innormal condition and after H/R.Methods:We used primary rat cultured cortical neurons and built thehypoxia/reoxygenation (H/R) model.Appling the immunofluorescence double labelmethod to observe the change of NSP in neurons,using western blot to examine thechanges of NSP protein levels and using the ELISA Kit to investigate the changes oftPA protein levels.The time point chosen is before-hypoxia,hypoxia 90 min,reoxygenation 2h,4h,6h,8h,12h and 24h.Results:1.The cell body of neuronal cell was swelling after 90min hypoxia and6h reoxygenation insult,MAP-2 immunofluorescence revealed some neuraxonappeared as string-of-beads or broken,CCK-8 assay suggested the neuronal survivalrate deceased 50% compared to normal condition.2.Immunofluorescence revealedthat the expression of NSP is high in both normal condition and after H/R in neurons.Western blot results revealed that there is a high expression of NSP protein in normalcondition,however,the expression still increased after H/R and NSP protein levelsreached its peak at the time point of 6h after reoxygenation and then decreased.3.ELISA Kit revealed that there is very low expression of tPA protein in normalcondition.It could also be increased by H/R and tPA protein expression reached itspeak at the time point of 6h after reoxygenation and then decreased.Conclusion:The H/R model was effective.The expression of NSP and tPAprotein levels both increased after H/R.And the changes of the two proteins almostkept consistent. PartⅡThe Protective effects of Neuroserpin on Neuronal damage fromHypoxia/ReoxygenationObjective:To study the neuronal damage of the primary cultured neuronscaused by tPA in the different cultured conditions and then to investigate that if NSPcan decrease these injuries caused by tPA.Methods:We used primary rat cultured cortical neurons and built the H/R model.First,we tested the neuronal damage of the primary cultured neurons caused by tPA.1.TPA with Different densities were used to intervent normal neurons in different time(0.5h,1h,4h,6h,8h and 24h).The cells were randomly divided into normal group,PBS group and tPA group.2.Interventing neurons with tPA before H/R.The cellswere randomly divided into normal group,hypoxia 90 min reoxygenation 4h group(H/R group),PBS+H/R group,tPA+H/R group.The drugs would intervent theneurons for 4h before H/R.3.Interventing neurons with tPA after H/R.The cellswere randomly divided into hypoxia 90 min and reoxygenation group,H/R+PBSgroup,H/R+tPA group.PBS and tPA were added in hypoxia 90 min.The time ofreoxygenation was 0.5h,1h,4h and 6h.The final concentration oftPA was 0.01,0.05,0.075 and 0.1 ug/μL.CCK-8 was used to test the neuronal survival.Then we tested the effects of NSP on neurons.1.Interventing neurons with NSPbefore H/R.The cells were randomly divided into hypoxia 90 min and reoxygenation4h group (H/R),PBS+H/R group (intervented neurons 4h before H/R),tPA+H/Rgroup (intervented neurons 4h before H/R) and NSP+tPA+H/R group (pretreatedneurons with NSP for 0.5h,then intervented them with tPA 4h before H/R).2.Interventing neurons with NSP after H/R.The cells were randomly divided intohypoxia 90 min reoxygenation 4h group (H/R),H/R+PBS group (added PBS whenhypoxia 90 min,and then reoxygenated 4h),H/R+tPA group (added tPA whenhypoxia 90 min,and then reoxygenated 4h),H/R+tPA+NSP group (added tPA whenhypoxia 90 min and reoxygenated 0.5h,and added NSP to cultivate 4h),H/R+NSP(added NSP when hypoxia 90 min,and then reoxygenated 4h).Then we observed thechanges of morphology by inverted microscopy with the method ofimmunofluoscence,the neuronal survival rate by CCK-8,the cytotoxicity by LDHrelease assay and the apoptosis ratio of neurons by TUNEL assay.Results:1.TPA had harmful effects on normal cultured neurons.2.TPA could increase the neuronal injurys before H/R.3.The neuronal survival had a tendencywith the time of intervention when neurons were intervented by tPA after H/R.Andthe effects of different time of intervention was different with the differentconcentration of the drugs.So tPA could increase the injuries after H/R.4.NSP couldinhibit the effects oftPA both before H/R and after H/R.Conclusion:TPA had a harmful effect on normal neurons and the neuronalsurvival had a negative correlation with the concentration of tPA and the time ofintervention.TPA could increase the injuries of neurons undergoing H/R and theneuronal survival still had a negative correlation with the concentration of tPA and thetime of intervention.NSP had protective effects on neurons by decreasing the harmfuleffects oftPA in different conditions.PartⅢThe effects of Neuroserpin on neurons through microglias1.The effects of Neuroserpin on the Microglias undergoingHypoxia/ReoxygenationObjective:To study the effects of tPA on microglias in the different culturedconditions and then to investigate that ifNSP can decrease these effects.Methods:We used primary rat cultured cortical microglia and built the H/Rmodel.First,we tested the tPA protein levels released by Microglias undergoing H/Rby the way of using the ELISA kit.Secondly,we studied the effects of NSP on themicroglias undergoing H/R.The cells were randomly divided into three groups.Thefirst group was divided into normal group,PBS group and hypoxia 90 minreoxygenation group.The time of reoxygenation was 1h,3h,5h,7h and 24h.Thesecond group was divided into normal group,PBS group and tPA group.The finalconcentration of tPA was 0.01,0.025,0.05,0.075 and 0.1μg/μl.The time ofintervention was 24h.The third group was divided into normal group,PBS group,tPAgroup (0.05μg/μl,24h),NSP+tPA group (pretreated microglias with NSP for 0.5h andthen intervented them with tPA 24h),OGD3h group,tPA+OGD3h group (interventedmicroglias with tPA 24h before OGD3h),NSP+tPA+OGD3h group (pretreatedmicroglias with NSP for 0.5h and then intervented them with tPA 24h before OGD3h).Then we observed the changes of morphology by inverted microscopy,the rate of proliferation by CCK-8 and the inflammatory cytokines IL-1βand NO in culturesupernatants by ELISA kit.Results:1.Microglias could release tPA undergoing H/R.TPA proteinexpression reached its peak at the time point of 3h after reoxygenation.2.Themicroglias could be activated by tPA or H/R.IL-1βand NO can be releasedundergoing the intervention of tPA or H/R.NO but not IL-1βhad a time dependentincrease after OGD in cultured microglia.3.The intervention of NSP could impactthese effects which was caused by tPA or hypoxia/reoxygenation.Conclusion:Microglias could release tPA undergoing H/R.TPA could induce theproliferation and activation of the microglias.H/R and tPA could make microgliasrelease IL-1βand NO.NSP could reduce the effects of the tPA or H/R on themicroglias.NSP could decrease the liberation of IL-1βand NO released bymicroglias.2.The effects of the microglial conditioned medium on neuronsObjective:To investigate that if the protective effects of NSP on neurons arecaused by the effects on the microglias.Methods:We used primary rat cultured cortical microglias and built H/R model.Preparing the microglial conditioned medium (CM),and it was divided into N-CM(normal CM),PBS-CM,H/R-CM (OGD 3h and then reoxygenated 1h,3h,5h,7hand 24h),NSP+H/R-CM (pretreated microglias with NSP for 0.5h before H/R),tPA-CM (the final concentration of tPA was 0.01,0.025,0.05,0.075 and 0.1μg/μl.)and NSP+tPA-CM (pretreated microglias with NSP for 0.5h and intervented it withtPA).Then we observed the changes of morphology by inverted microscopy with themethod of immunofluorescence,the neuronal survival rate by CCK-8,the cytotoxicityby LDH release assay,the cellular necrosis and the apoptosis by Kai Ji kit and theapoptosis ratio of neurons by TUNEL assay.Results:The H/R-CM had more harmful effects on the neurons and it couldcause the cellular necrosis.These injuries were obvious while OGD 3h and with thetime of reoxygenation,the injuries aggravated (P＜0.05).The tPA-CM also hadharmful effects on neurons.The effects were light when the final concentration of tPAwas 0.01μg/μL and it mainly caused the cellular apoptosis.With the increase of theconcentration of tPA,the harmful effects increased (P＜0.05).The injuries of the NSP group were lighter than the other groups (P＜0.05).Conclusion:The H/R-CM and tPA-CM both had harmful effects on neurons.NSP could decrease these injuries.PartⅣThe protective mechanism of NeuroserpinObjective:To study the signal transduction mechanism ofmicroglia and toinvestigate the possible mechanism of the protective effects of NSP by inhibiting tPAwhich can activate microglias.Methods:We used primary rat cultured cortical microglia and built the H/Rmodel.The cells were randomly divided into five groups:normal group,tPA group(the final concentration was 0.05μg/μl,24h),OGD3h group,NSP+tPA group(pretreated microglias with NSP for 0.5h and intervented it with tPA 24h) andNSP+OGD3h group.Expression of MAPK was identified by immunocytochemistrystaining and was determined by Western-blot.The activation of NF-κB wasdetermined by the immunofluorescence double label method.Results:The immunofluorescence double label method showed that there was asmall quantity expression of phospho-ERK and phospho-P38 in normal microgliasand the level increased obviously under the stimulation of tPA or OGD3h.Westernblot showed the same results.The immunofluorescence double label method showedthat there were high expressions of phospho-JNK both in normal microglias and thecells stimulated by tPA or OGD3h.Western blot showed that the expressions ofphospho-JNK in the cells stimulated by tPA or OGD3h were little higher than thenormal ones.The immunofluorescence double label method showed that NF-κB wasactivated by tPA or OGD.NSP group inhibited the signal transduction.Conclusion:TPA and OGD could activate ERK1/2 pathway,P38 pathway andJNK pathway of microglias.And ERK 1/2 pathway and P38 pathway were moreimportant.NSP could inhibit the signal transduction by inhibiting tPA.TPA and OGDcould als0 activate NF—κB.NSP could inhibit the activation of NF-κB. CONCLUSIONS:1.The expressions of NSP and tPA protein levels both increased after H/R.And thechanges of the two proteins almost kept consistent.2.TPA had harmful effects on normal cultured neurons.TPA could increase theneuronal injuries before H/R or after H/R.3.NSP could inhibit the effects oftPA both before H/R and after H/R.4.Microglias could release tPA undergoing hypoxia/reoxygenation.5.TPA could induce the proliferation and activation of the microglias.H/R and tPAcould make microglias release IL-1βand NO.6.NSP could reduce the effects of the tPA or H/R on the microglias.It could decreasethe liberation of IL-1βand NO released by microglias.7.The H/R-CM and tPA-CM both had harmful effects on neurons.NSP coulddecrease these injuries.8.TPA and OGD could activate ERK1/2 pathway,P38 pathway and JNK pathway ofmicroglias.ERK 1/2 pathway and P38 pathway were more important.NSP couldinhibit these signal transduction by inhibiting tPA.TPA and OGD could alsoactivate NF-κB.NSP could inhibit the activation of NF-κB.