Potential Roles Of Sec22b And Ykt6 In Cerebral Ischemia Reperfusion Injury In Rats: Key Mechanisms

Part ?:Time couse analysis of the proteins of Sec22 b and Ykt6 after MCAO/R.Objective:To investigate the changes of Sec22 b and Ykt6 in vivo and in vitro at different time points after MCAO/R.Methods:In in vivo experiment,30 rats(36 rats were used,30 rats were survived after surgery)were randomly admeasured five groups of six rats each,a sham group and four experimental groups plan in the light of the following time points : 6 h,12 h,24 h and48 h after MCAO/R.All the SD rats in this experiment were executed at the indicated time point after MCAO/R.The brain tissue of all rats in each group was collected and used for western blot analysis and immunofluorescence analysis.The in vitro experiment,which similar to the in vivo experiment,was performed to estimate the effect of OGD/R treatment on the relative protein level and distribution in neuronal cells.Results: 1,According to western blot analysis in vivo,the level of Sec22 b increased with time,peaking at 24 h,and then decreased.While the level of Ykt6 decreased with time.2,In vitro,western blot analysis showed that the protein level of Sec22 b in cultured primary neurons was significantly increased after glucose deprivation/reoxygenation(OGD/R)for 24 h.And the protein level of Ykt6 in cultured primary neurons was increased with time after OGD/R.3,Double immunofluorescence assay of Sec22 b in vivo and vitro verified the MCAO/R-induced increase in the level of Sec22 b.And this result also showed that Sec22 b were aggregated at the end of the axons.Conclusion: The level of Sec22 b increased significantly after MCAO/R,and mainly accumulated at the end of neuronal axons.While the level of Ykt6 decreased gradually.Part ?: Role of Sec22 b and Ykt6 in MCAO/R-induced brain injury.Objective: To investigate the expression and distribution changes of Sec22 b and Ykt6 after intervening the Sec22 b or Ykt6 plasmid or Si RNA.And the potential roles of Sec22 b and Ykt6 in second brain injury by MCAO/R.Methods: In in vivo experiment,144 rats(170 rats were used,144 rats were survived)were randomly assigned to eight groups(n = 18 per group): sham group,MCAO/R group,MCAO/R + vehicle group,MCAO/R + over-Ykt6 group,MCAO/R + over-Sec22 b group,MCAO/R + Ctr Si RNA group,MCAO/R + Si RNA-Ykt6 group and MCAO/R +Si RNA-Sec22 b group.The plasmid or Si RNA,which mixtured with indicated reagent,was injected into intracerebroventricular 48 h before MCAO/R.We chose six rats per group 24 h after MCAO/R and killed them to experimentize immunofluorescence analysis and western blot analysis and their blood was extracted for ELISA.Six rats per group were selected else to performed terminal deoxynncleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining and fluoro-jade B(FJB)staining 24 h after MCAO/R.Then six rats per group were killed and isolated brain tissue to perform 2,3,5-triphenyltet-razolium chloride(TTC)staining.Results: 1,Over expression of Sec22 b increased the expression level and distribution of Sec22 b in brasin tissue,while the level and distribution were significantly decreased after treatment with Si RNA.2,Over expression of Ykt6 markedly increased the expression level of Ykt6 in brain tissue,and the level decreased notably after treatment with Si RNA.3,The results of TUNEL staining,FJB staining and ELISA detection in vivo showed that overexpression of Ykt6 could significantly reduce the apoptosis,necrosis and inflammatory response of neuronal cells after cerebral ischemia and reperfusion.And the expression of Sec22 b remarkably aggravated the the apoptosis,necrosis and inflammatory reaction of neuronal cells.The inhibition of Ykt6 expression could greatly aggravate the apoptosis,necrosis and inflammatory reaction of neuronal cells.Inhibition of Sec22 b expression could reduce apoptosis,necrosis and inflammatory response of neurons.4,The results of TTC staining showed that overexpression of Ykt6 and down-regulation of Sec22 b could reduce the area of cerebral infarction,while overexpression of Sec22 b and down-regulation of Ykt6 increased the area of cerebral infarction.Conclusion: The increase of Sec22 b or the decrease of Ykt6 could aggravate the cerebral ischemia reperfusion injury in some extent.And the decrease of Sec22 b or the riseof Ykt6 could reduce the cerebral ischemia reperfusion injury in a certain degree.Part ?: Roles of Sec22 b and Ykt6 in OGD/R-induced neuronal injury and the effect on autophy.Objective: To investigate the effects of Sec22 b and Ykt6 on oxygen-glucose deprivation and reperfusion injury,we performed TUNEL staining,ELISA and LDH after the intervention of Sec22 b and Ykt6 plasmids and small interference RNA to the primary neurons.The localization of autophagic specific protein LC3 and the degree of binding to Sec22 b and Ykt6 were evaluated by immunofluorescence assay to verfied the extent of the influence of autophagy.Methods: In in vitro experiments,neuronal cells were divided into eight groups :control group,OGD/R group,OGD/R + vehicle group,OGD/R + over-Ykt6 group,OGD/R + over-Sec22 b group,OGD/R +Ctr Si RNA group,OGD/R + Si RNA-Ykt6 group and OGD/R +Si RNA-Sec22 b group.The transfection of indicated plasmid or Si RNA were treated 48 h before OGD/R.These cells were used for immunofluorescence analysis,TUNEL staining.The supernatant were subjected to ELISA and LDH.Results: 1,In vitro,the results of TUNEL staining,ELISA and LDH showed that overexpression of Ykt6 or inhibition of Sec22 b expression could reduce the apoptosis,inflammation and necrosis of neuron cells.While overexpression of Sec22 b or inhibition of Ykt6 could aggravate the apoptosis,inflammation and necrosis of neuron cells.2,When the expression of autophagic protein was increased after oxygen-flucose deprivation reperfusion,the degree of binding with Sec22 b was lower than normal group,and the degree of binding with Ykt6 was significantly higher than normal group.Conclusion: The damage caused by oxygen-glucose deprivation reperfusion in vitro could be aggravated by the rise of Sec22 b level or the decrease of Ykt6 level.But when the level of Sec22 b decreased or the level of Ykt6 increased,the injury after oxgen-glucose deprivation and reperfusion will be relieved.On the other hand,the autophagy was strengthened after oxygen-glucose deprivation reperfusion.According to the degree of binding to autophagy specific proteins LC3,Ykt6 play a significant role in autophagy after oxygen-glucose deprivation reperfusion.