| Background and PurposeAcute cerebral ischemia is common in clinic.There are still no effective measures to prevent neuronal death caused by reperfusion injury in cerebral ischemia.When the brain suffers from a nonlethal dose of damage,the endogenous mechanism will be initiated to tolerate subsequent ischemic events.We call this process the ischemic preconditioning.However,the occurrence of cerebral ischemic events is difficult to predict,so the application of preconditioning is limited.After cerebral ischemia,a certain measure of intervention after tCCI will have more clinical application prospects.Our study has confirmed that hypoxia for 2 hours significantly reduced neuronal death in hippocampal CA1 region.The mechanisms involved may have oxidative stress,calcium overload,immunoregulation,apoptosis and immune response.But how to regulate the apoptosis of cells after hypoxia is still unclear.In recent years,many studies have confirmed that the classical Wnt signaling pathway is involved in the pathogenesis of cerebral ischemia.?-catenin,as the core factor of the classical Wnt signaling pathway,taking part in regulating cell differentiation,apoptosis,proliferation,migration and cell cycle.?-catenin has protective effects on neuron after cerebral ischemia.Many transcription factors and signaling mediators are known to participate in the regulation of Wnt signaling pathway.As a histone demethylation enzyme,KDM2A has confirmed that it can promote the degradation of?-catenin in the nucleus.This process regulates embryonic development and the establishment of the anterior-posterior body axis.Until now,it is unclear whether hypoxic postconditioning can play a neuroprotective role in downregulating KDM2A and increasing the expression of?-catenin in the nucleus.In this study,we intend to observe the changes in the expression of KDM2A,?-catenin and H3K36me,H3K36me2 or H3K36me3 in hippocampal CA1 region of adult Wistar rats.To further explore whether hypoxic postconditioning can play a neuroprotective role in hippocampal CA1 region after tGCI by lowering the expression of KDM2A,increasing the expression of?-catenin in the nucleus and regulating the expression of H3K36me,H3K36me2 and H3K36me3.Material and methods:In this experiment,adult male Wistar rats weighing 220 to 270g,were operated by modified classical four vessel occlusion.These rats were randomly divided into sham-operation group,simple hypoxic group,transient global ischemia group and hypoxic postconditioning group.HPC was performed by exposing the rats to a 120 min continuous hypoxia at 1d after tGCI.Rats were placed in a sealed and dampproof box which continuously flowed with humidified mixed gas?8%O2+92%N2?.Nissl staining,NeuN immunohistochemistry and FJB staining were used to observe the survival of neurons in hippocampal CA1region after tGCI-hypoxic postconditioning.Western blot was used to detect the protein expression of?-catenin,KDM2A and H3K36me,H3K36me2 and H3K36me3 in hippocampal CA1 region of sham-operated group,simple hypoxia group,tGCI group and HPC group.Immunohistochemical staining and immunofluorescence were used to observe KDM2A positive cells and intracellular localization in sham-operated group,simple hypoxia group,tGCI group and HPC group.The interaction between KDM2A and?-catenin in sham-operated group,tGCI group and HPC group was observed by using CO-immunoprecipitation.Results:1.Hypoxic postconditioning has protective effects on hippocampal CA1neurons after tGCI in rats.Nissl staining,NeuN immunohistochemistry and FJB staining showed that compared with the sham-operated group,the survival cells in tGCI 7d group were decreased?P<0.05?.But the survival cells in HPC 6d group were increased?P<0.05?,which compared with the tGCI 7d group.2.Hypoxic postconditioning increased the expression of?-catenin in the nucleus of hippocampal CA1 region after tGCI.Compared with sham-operated group,the expression of?-catenin was no significant difference between tGCI group and HPC group in the cytoplasm?P>0.05?.But in the nucleus,compared with sham-operated group,the expression of?-catenin decreased at 4h after tGCI,continued to 50h.And the decrease in tGCI 26h group was the lowest?P<0.05?.The expression of?-catenin was significantly increased in HPC 0h and 6d compared with tGCI 26h and 50h group?P<0.05?.3.Hypoxic postconditioning reduced the expression level of KDM2A in hippocampal CA1 region after tGCI.Compared with sham-operated group,the level of KDM2A protein in the hippocampus CA1 region was increased in t GCI26h group and 50h group?P<0.05?.The KDM2A protein level in HPC group was significantly lower than that in corresponding tGCI group?P<0.05?.The immunohistochemical results showed that the positive cells of KDM2A increased significantly at 26h after tGCI,and the positive cells in HPC 0h group were significantly less than those of the tGCI 26h group.Double Immunofluorescence staining showed that immunoreactivity of KDM2A increased significantly while KDM2A co-localized with NeuN at 26h after tGCI.4.Hypoxic postconditioning attenuated the interaction of KDM2A and?-catenin in the nucleus of hippocampal CA1 region in rats.The results of immunoprecipitation showed that compared with sham-operated group,the interaction between KDM2A and?-catenin in tGCI 26h group was obviously enhanced and was attenuated after hypoxic postconditioning.5.Hypoxic postconditioning increases the level of H3K36me and H3K36me2 in hippocampal CA1 region of rats.Compared with sham-operated group,excepting tGCI 0h group,the protein expression of H3K36me,H3K36me2and H3K36me3 in other groups increased significantly?P<0.05?.Compared with tGCI 26h group,the protein expression of H3K36me and H3K36me2 in HPC 0h group was increased significantly?P<0.05?.However,the protein expression of H3K36me3 was no striking difference between tGCI group and HPC group?P>0.05?.Conclusion:Hypoxia postconditioning can exert neuroprotection against tGCI in Wistar rats,which is achieved by downregulation of KDM2A,increasing the expression of?-catenin in the nucleus.As well as upregulation the monomethylation and dimethylation of H3K36 in hippocampal CA1 region. |
PDF Full Text Request