| Ischemic stroke is a dynamic process,and the most effective way to reduce brain damage and dysfunction is to restore the blood perfusion as soon as possible.However,due to the sudden onset of ischemic stroke and the time dependence of thrombolytic therapy,only a few patients can receive thrombolytic therapy within the time window,which severely affects the treatment and prognosis of ischemic stroke.The brain is more sensitive than other organs for ischemic injury.Hippocampus,which is closely related to learning,memory and emotion,has spurred considerable attention.Studies have shown that neurons in the hippocampus undergo different pathological changes after transient ischemia-reperfusion(I/R).Ischemic injury induces delayed death of pyramidal neurons in the CA1 region,yet the neurons in the CA3 and DG are unaffected.However,the molecular mechanism explaining this phenomenon is unknown.The brain,as the most important organ in the body,has a variety of self-protective mechanisms,such as anti-oxidation,anti-excitotoxicity,anti-apoptosis and enhanced expression of prosurvival genes.Brain-derived neurotrophic factor(BDNF),as a member of the neurotrophin family,is involved in the regulation of neurogenesis,neuron differentiation,neuron survival,and synaptic plasticity,and is closely related to ischemic brain damage.However,the mechanism of hippocampal BDNF expression after cerebral ischemia is still unclear.In this study,we first established a transient forebrain ischemia-reperfusion model in rats by using the Puslinelli four-vessel occlusion(4-VO)method.The changes of BDNF protein and mRNA expression in hippocampus were evaluated after ischemia-reperfusion in comparison with the naive group and the sham groups,and to explore the possible mechanism.We found that after 48 h of transient forebrain ischemia,the expression of BDNF protein in the hippocampal CA1 region was decreased,while the expression in the CA3 and DG was increased.Ischemic simulation inhibits the expression of BDNF transcript IV in CA1 but increased the expression of transcription I,IIc,III,IV,VI,and X1 in CA3 and DG.In addition,through the ChIP assay,the changes observed in the promoter1,2,4 and 6 of BDNF gene.Ischemia results in the decrease of H3K27 ac in CA1,the increase of H3K9 ac and H3K14 ac and the increase of H3K9 ac,H3K14ac,and H3K27 ac in CA3.But there was no significant change in DG.These results indicate that the acetylation pattern of histones in the promoter region of BDNF gene may play a critical role in regulating the expression of BDNF protein in hippocampus by altering the expression of different transcripts of BDNF,which will provide a new pathway for the treatment of ischemic brain injury.Part Ⅰ The effect of transient forebrain ischemia-reperfusion on BDNF protein and mRNA expression Objective:After establishing a transient forebrain ischemia-reperfusion model,the changes of BDNF protein and mRNA expression were observed in comparison with the naive group and the sham group.Methods:1.Experimental groups :normal control group(naive group),sham group and transient ischemia-reperfusion model(I/R group).2.Preparation of transient forebrain ischemia-reperfusion model:I/R group: male SD rats(240-260g)were anesthetized with pentobarbital sodium(5mg/100 g,i.p),and fixed on the stereotactic frame through the ear bar.And then,bilateral vertebral arteries were electrocoagulated and bilateral common carotid artery were separated.Bilateral common carotid artery were clamped for 15 min on the second day and then recover the perfusion,resulting in transient forebrain ischemia.Sham group: the operation was the same as the I/R group except that the vertebral artery was not electrocoagulated and the common carotid artery was not occluded.3.Western Blot and RT-qPCR were used to detect the relative expression levels of BDNF protein and mRNA in the hippocampus of different groups.Results:1.Western Blot results showed that there was no significant difference in the relative expression of BDNF in the hippocampus of sham group compared with the naive group.Compared with the sham group,the expression of BDNF protein in the CA1 region of the I/R group was significantly decreased,and the BDNF protein level in the CA3 region and the DG region was significantly increased.2.RT-qPCR results showed that there was no significant difference in the relative expression of BDNF mRNA in hippocampus between the sham group and the naive group.Compared with the sham group,the expression of BDNF transcripts I,IIc,III,IV,VI and X1 in the CA3 and DG regions was upregulated after ischemia-reperfusion.The expression of BDNF transcripts I,III,VI and X1 were upregulated in CA1 region,BDNF transcript IV was downregulated,and BDNF transcript IIc expression was unchanged.Conclusion:After transient forebrain ischemia-reperfusion,the expression of BDNF protein in the hippocampal CA1 pyramidal neurons decreased,and the expression of BDNF transcript IV decreased.The expression of BDNF protein was increased in the CA3 and DG regions after ischemia,and the expression of BDNF transcripts I,IIc,III,IV,VI and X1 was increased.Part Ⅱ The effect of transient forebrain ischemia-reperfusion on the acetylation level of BDNF promoterObjective: Different histones modification can lead to chromatin remodeling,which regulates gene expression.Histone acetylation creates a repulsive power between histone and DNA,leading to open chromatin.This condition contributes to the proximity of transcriptional activator bind to transcription factors to promote gene transcription.Previous studies have shown that BDNF can be regulated by a variety of histone post-translational modifications,such as acetylation,methylation and ubiquitination,and is closely related to many neurological diseases.Therefore,we speculate that histone acetylation also plays a critical role in the expression of BDNF gene after ischemia-reperfusion.In this study,after establishing a transient forebrain ischemia-reperfusion model in rats,compared with the sham group,we observed the changes of acetylation of H3K9,H3K14,and H3K27 ac in BDNF promoter.Methods: 1.Experimental group: sham group,I/R group.2.Ch IP was used to detect the changes of H3K9,H3K14 and H3K27 acetylation patterns in BDNF promoters 1,2,4 and 6 in hippocampal CA1,CA3 and DG areas of rats in sham group and I/R group.Results: Compared with the sham group,the level of H3K27 ac in promoter 1,2,4,and 6 of the BDNF gene in the I/R group decreased in the CA1 region and increased in the CA3 region.H3K9 ac and H3K14 ac levels were upregulated in both CA1 and CA3 regions.In the DG region,there were no significant changes in the levels of H3K9 ac,H3K14ac and H3K27 ac in multiple promoter regions of the BDNF gene.Conclusion: After transient forebrain ischemia-reperfusion,the low H3K27 ac level of histone in the promoter region of BDNF gene in CA1 region may reduce BDNF protein expression by inhibiting BDNF transcript IV transcription.The promoter region of BDNF gene in CA3 region is highly H3K9 ac,H3K14ac and H3K27 ac may increase the expression of BDNF protein by promoting the transcription of BDNF transcripts I,IIc,III,IV,VI and X1.There was no significant relationship between the expression of BDNF protein and the level of H3K9 ac,H3K14ac and H3K27 ac in promoter region in DG region. |
