The Effects Of Neuroserpin On Rat Cortical Astrocytes Undergoing Hypoxia/Reoxygenation

Ischemia Stroke is one of the main diseases that cause unnature death and disability. Neuroserpin(NSP), mainly expressed in centrol nervers system, is one of the members of serpins super family. NSP is a inhibitor of neurogenic tissue plasminogen activator(tPA),and participate in the regulation of the activity of proteolysis of extracellular tPA and plasmase. Some researches show that NSP can protect neuron from death in ischemic stroke. In the early stage of ischemic stroke,the application of NSP can reduce the volume of infarction,the degree of edema,the leakage of the blood-brain barrier(BBB) extremely. And finally alleviate the apopotosis of neuron after reperfusion. In our previous research,we demonstrated that NSP can alleviate the toxic injury of neuron after oxygen and glucose deprivation (OGD) in vitro. We also found that, NSP can inhibit the activation of microglia after OGD,and reduced the secretion of IL-1β,NO. In this research, we observed the change of activity of astrocytes after OGD injury.We also investigated that after NSP pre-disposal,whether the activity of astrocytes changed after OGD and what mechanisms involved. After this research,we want to go to a step further to identify the neuroprotection effect of NSP.PartⅠThe subcultured astrocytes and the build up of the hypoxia/reoxygenation modelObjective:To build up the hypoxia/reoxygenation(H/R) model.Methods:We used subcultured cortical astrocytes of SD rats and verificated it with immunofluorescence double label,then built up the H/R model.Results:1. The purity of subcultured astrocytes reach 95% with the immunofluorescence double label of GFAP. 2. Six hours after OGD,the activity of astrocytes begin to descend gradually. And 12 hours after OGD,the activity below normal group obviously(P<0.05). After OGD 12h, the activity of astrocytes descend gradually along with the time of reoxygenation. And,when the reoxygenation last for 24 hours, the activity of astrocytes remains about 50% compared with the activity of Astrocytes before OGD treatment.Conclusions:The hypoxia/reoxygenation(H/R) model of subcultured cortical astrocytes is effective.PartⅡThe effect of NSP on Astrocytes undergoing Hypoxia/ReoxygenationObjective:To investigate the effect of NSP on the activity and the release of inflammatory mediators of Astrocytes undergoing Hypoxia/Reoxygenation.Methods:1. To subcultured astrocytes and build up the H/R model.2. Investigate the effect of NSP,with different concentration, on the activity of astrocytes after H/R. The cells were randomly divided into three groups.The first group is normal group;The second group is H/R group(OGD12h and then reoxygenation 24h).The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 24h. The final concentration of NSP was 1.25,2.5,5,10,20,40ng/ml.). We tested the activity and the injury of astrocytes with the method of MTT and LDH respectively.3. The effect of NSP(5ng/ml) predisposal on astrocytes after H/R. The cells were randomly divided into three groups. The first group is normal group;The second group is H/R group(OGD12h and then reoxygenation 0,2,4,6,8,10,12,24h);The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 0,2,4,6,8,10,12,24h. The final concentration of NSP was 5ng/ml.).At each time pot,we tested the activity of astrocytes with the method of MTT. And then,we tested the apoptosis rate with Annexin V-FITC/PI(Flow cytometry).4. The effect of NSP(5ng/ml) predisposal on the secretion of NO,IL-1β,TNF-αof astrocytes after H/R. The cells were randomly divided into three groups. The first group is normal group;The second group is H/R group(OGD12h and R0,6,12h);The third group is H/R with NSP predisposal(This group of cell was treated with NSP for 1 hour and then OGD12h R0,6,12h. The final concentration of NSP was 5ng/ml.). At each time pot,we tested the concentration of NO,IL-1βand TNF-a in the culture Supernatant of astrocytes.Results:1. After the predisposal of NSP with different concentration,the activity of astrocytes(H/R) enhance extremely, while the toxic substances release of astrocytes reduced. At the low concentration stage, the activity of astrocytes enhance with the increase of the concentration of NSP. And, at the concentration of 5ng/ml, NSP exhibit the best protection effect.2. After the predisposal of NSP, the activity of astrocytes elevated compared with the control group at each time pot of reoxygenation. As to the results of Flow cytometry, we found that the apoptosis rate of astrocytes(H/R) reduced after the predisposal of NSP.3. After the predisposal of NSP, the secretion of NO,IL-1βand TNF-αof astrocytes reduced at the the stage of reoxygenation.Conclusions:1. The NSP predisposal enhance the activity and exhibit the anti-apoptosis effect of astrocytes undergoing H/R.2. The NSP predisposal inhibits the inflammatory reaction of astrocytes undergoing H/R.PartⅢThe mechanisms of the protective effect of neuroserpinObjective:To study the signal transduction mechanism of astrocytes and to investigate the possible mechanism of the protective effects of NSP by H/R which can activate Astrocytes.Methods:We used subcultured astrocytes and built the H/R model. The cells were randomly divided into three groups:normal group, H/R group (OGD12h reoxygenation 24h), and NSP+H/R group (This group of cell was treated with NSP for 1 hour and then OGD12h reoxygenation 24h. The final concentration of NSP was 5ng/ml). Expression of MAPK,AKT and NF-κB was determined by Western-blot.Results:1. The western-blot show that small quantity expression of phospho-ERK, phosphor-JNK and phospho-P38 in normal astrocytes and the level increased obviously under the stimulation of H/R. While,the activation of MAPK was not influenced by the predisposal of NSP.2. The western-blot show that small quantity expression of p-AKT in normal astrocytes and the level increased obviously under the stimulation of H/R. While,the activation of MAPK was not influenced by the predisposal of NSP.3. The western-blot show that small quantity expression of p-IKKα/βand NF-κB P65 (nuclear) in normal astrocytes and the level increased obviously under the stimulation of H/R. After the predisposal of NSP, the activation of NF-κB was inhibited extremely.Conclusions:The signal transduction of MAPK, PI3K/Akt and NF-κB of astrocytes was activated after H/R. And the predisposal of NSP inhibite the activation of NF-κB extremely.Conclusions:1. The NSP predisposal enhance the activity and exhibit the anti-apoptosis effect of astrocytes after H/R.2. The NSP predisposal inhibit the inflammatory reaction of astrocytes after H/R.3. The signal transduction of MAPK, PI3K/Akt and NF-κB of astrocytes was activated after H/R. And the predisposal of NSP inhibite the activation of NF-κB extremely.